Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 618432 14 pageshttpdxdoiorg1011552013618432

Research ArticleModulation of Pineal Melatonin Synthesis by GlutamateInvolves Paracrine Interactions between Pinealocytes andAstrocytes through NF-120581B Activation

Darine Villela12 Victoria Fairbanks Atherino1 Larissa de Saacute Lima3

Anderson Augusto Moutinho1 Fernanda Gaspar do Amaral2 Rafael Peres2

Thais Martins de Lima2 Andreacutea da Silva Torratildeo2 Joseacute Cipolla-Neto2

Cristoacuteforo Scavone3 and Solange Castro Afeche1

1 Laboratory of Pharmacology Butantan Institute Avenida Vital Brasil 1500 05503-900 Sao Paulo SP Brazil2 Department of Physiology and Biophysics Institute of Biomedical SciencesUniversity of Sao Paulo Avenida Professor Lineu Prestes 1524 05508-900 Sao Paulo SP Brazil

3 Department of Pharmacology Institute of Biomedical Sciences University of Sao Paulo Avenida Professor Lineu Prestes 152405508-900 Sao Paulo SP Brazil

Correspondence should be addressed to Solange Castro Afeche soafechebutantangovbr

Received 26 April 2013 Accepted 28 June 2013

Academic Editor Alejandro Lomniczi

Copyright copy 2013 Darine Villela et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The glutamatergic modulation of melatonin synthesis is well known along with the importance of astrocytes in mediatingglutamatergic signaling in the central nervous system Pinealocytes and astrocytes are the main cell types in the pineal gland Theobjective of this work was to investigate the interactions between astrocytes and pinealocytes as a part of the glutamate inhibitoryeffect on melatonin synthesis Rat pinealocytes isolated or in coculture with astrocytes were incubated with glutamate in thepresence of norepinephrine and the melatonin content was quantified The expression of glutamate receptors the intracellularcalcium content and the NF-120581B activation were analyzed in astrocytes and pinealocytes TNF-120572rsquos possible mediation of the effect ofglutamate was also investigated The results showed that glutamatersquos inhibitory effect on melatonin synthesis involves interactionsbetween astrocytes and pinealocytes possibly through the release of TNF-120572 Moreover the activation of the astrocytic NF-120581Bseems to be a necessary step In astrocytes and pinealocytes AMPA NMDA and group I metabotropic glutamate receptors wereobserved as well as the intracellular calcium elevation In conclusion there is evidence that the modulation of melatonin synthesisby glutamate involves paracrine interactions between pinealocytes and astrocytes through the activation of the astrocytic NF-120581Btranscription factor and possibly by subsequent TNF-120572 release

1 Introduction

The mammalian pineal gland is a component of the pho-toneuroendocrine system and is involved in the regulationof circadian rhythms through the nocturnal synthesis ofmelatonin The gland consists mainly of pinealocytes butalso presents interstitial cells the majority of which areastrocytes [1 2] The pinealocytes are the neuroendocrinecells that synthesize melatonin and secrete it into thebloodstream In mammals this process is controlled by

the retinohypothalamic pathway and sympathetic innerva-tion with norepinephrine being released in the perivascularspace of the gland at night [3ndash5] Norepinephrine interactswith its proper 1205731 and 1205721 receptors and stimulates the tran-scription and translation of the key enzyme arylalkylamineN-acetyltransferase AANAT (EC 23187) resulting in mela-tonin synthesis and secretion [6ndash10]The role of astrocytes onmelatonin production however remains unclear

Several neurotransmitters can modulate melatonin syn-thesis [10ndash12] Acetylcholine and glutamate are two important

2 BioMed Research International

modulators that inhibit melatonin synthesis by decreasingthe gene expression and activity profile of both AANAT andhydroxyindole-O-methyltransferase (HIOMT) (EC 2114)Glutamate is stored in synaptic-like microvesicles in pinealo-cytes and is released upon cholinergic stimulation [13 14]The interaction of glutamate with AMPA-type ionotropicreceptors in the pinealocytes induces greater glutamate exo-cytosis amplifying the cholinergic action [15 16] Recentlyan investigation demonstrated that the glutamate vesiculartransporter could be responsible for pinealocyte depolariza-tion and glutamate release [17] The final effect of glutamateresults from its binding to mGluR3 a class II metabotropicglutamate receptor which is coupled to the inhibitory G-protein (Gi) leading to reductions in the amount of cAMPand the synthesis of melatonin [12]

Studies have shown the presence of many types ofglutamate receptors in the pineal gland In addition to theknown AMPA and mGluR3 (40 42) expression in the pinealgland Yatsushiro and colleagues showed that rat pinealocytesexpress mGluR5 [18] a class I metabotropic receptor relatedto inositol triphosphate (IP

3) formation and intracellular

calcium mobilization whose role on pineal metabolism hasnot yet been defined The presence of glutamate receptors inpineal astrocytes has also been demonstrated NMDA andAMPA ionotropic receptors were characterized in the ratpineal gland [19] while mGluR2 mGluR3 andmGluR5 wereidentified in the gerbil pineal gland [20] These data mayindicate that glutamatergic signaling in the pineal gland ismore complex than previously thought and involves bothastrocytes and pinealocytes similar to the pattern observedin the central nervous system (CNS)

Indeed in the CNS astrocytes are important partners atthe synapses Over the past decade an increasing number ofobservations have progressively challenged the classical viewthat astrocytes are passive participants in synaptic functionIn fact there is a dynamic two-way communication betweenglia and neurons called tripartite synapse inwhich astrocyticprocesses are associated with the presynaptic and postsy-naptic neuronal elements [21] Neurotransmitters released bypresynaptic neurons evoke a calcium increase in adjacentastrocytes which in turn can release several transmitterscalled gliotransmitters including glutamate D-serine ATPand large molecules like atrial natriuretic factor and tumornecrosis factor-120572 (TNF-120572) [21ndash24] These gliotransmittersfeed back onto the presynaptic neuron either to enhance orto depress further release of the neurotransmitters [25] orthey can act on postsynaptic neurons causing excitatory orinhibitory responses [26]

Glutamate signaling in the CNS is classically relatedto the activation of nuclear factor 120581B (NF-120581B) Thus glu-tamate induces the transcription of genes such as nitricoxide synthase cyclooxygenase 2 Mn-superoxide dismu-tase calbindin glutamate receptors Bcl-2 and brain-derivedneurotrophic factor [27ndash29] Additionally recent studieshave shown that astrocytes use NF-120581B as a mediator oftranscriptional responses to a variety of stimuli includingtissue injury and diseases [30] NF-120581B can induce or repressgene expression by binding to DNA sequences known as

120581B elements [31 32] Activation of NF-120581B typically involvesthe phosphorylation of proteins that inhibit NF-120581B (I120581Bs)by the I120581B kinase (IKK) complex which results in I120581Bdegradation [33] NF-120581B is then released from the inhibitorycomplex and translocated to the nucleus In mammaliancells there are five NF-120581B family membersmdashRel A (p65) RelB c-Rel p50p105 (NF-120581B1) and p52p100 (NF-120581B2)mdashanddifferent NF-120581B complexes are formed by diverse homo- andheterodimerization processes [34 35] In the pineal glandtwo independent studies showed that NF-120581B is involved inthe inflammatory response and exhibits a circadian rhythm[36 37]

Based on these data we aimed to investigate the inhib-itory action of glutamate on melatonin synthesis analyzingthe crosstalk between astrocytes and pinealocytes NF-120581Bactivation glutamate receptor expression and intracellularcalcium responses were analyzed in both cell types In thepresent work we demonstrated that the paracrine interac-tions between pinealocytes and astrocytes are fundamentalfor the glutamate inhibitory effect on melatonin synthesisand that it involves the activation of astrocytic NF-120581B tran-scription factor and the release of a soluble factor possiblyTNF-120572

2 Materials and Methods

21 Animals Male Wistar rats (200ndash220 g) were kept undera 12 12 h light-dark cycle (lights on at 06 00 h) in atemperature-controlled room (21plusmn 2∘C) with water and foodad libitum The animals were euthanized by decapitationbetween 09 00 and 10 00 h All experimentswere performedin accordance with the guidelines of the Brazilian Collegefor Animal Experimentation (COBEA) and approved bythe Committee of Ethics in Animal Experimentation of theInstitute of Biomedical Sciences (CEUAmdashPermit number091) University of Sao Paulo (Sao Paulo Brazil)

22 Pineal Gland Culture Pineal glands were cultured aspreviously described [38] Briefly after decapitation the ratpineal glands were removed and immediately placed in ice-cold BGJb medium (Fitton-Jackson Modification GibcoGrand Island NY USA) with phenol red modified by theaddition of bovine serum albumin (1mgmL) 2mM glu-tamine 01mgmL ascorbic acid and penicillin (100UmL)-streptomycin (100120583gmL) Pineal glands were cultivated(37∘C 95 O

2 5 CO

2) in the same BGJb medium in

24-well plates (2 glandswell 200120583Lwell) for 48 h (themedium was changed after the first 24 h) After this periodall glands were placed in fresh medium for 1 h beforetreatment For melatonin analysis the glands were incubatedwith a combination of the following drugs norepinephrine(NE) (1120583M) glutamate (100 and 600 120583M) and pyrrolidinedithiocarbamate (PDTC) (300 120583M) After 5 h the glandswere frozen on dry ice and kept at minus80∘C until they wereassayed All culture media components and drugs used werepurchased from Sigma (St Louis MO USA)

BioMed Research International 3

23 Isolated Pinealocyte Culture Pinealocytes were obtainedby papain digestion (Papain Dissociation System Wor-thington Biochemical Corporation Freehold NJ USA) Theglands were removed and immediately placed in ice-coldDulbeccorsquos Modified Eaglersquos Medium (DMEM) (glucose1000mgL HEPES 59 g and sodium bicarbonate 37 g)(Sigma St Louis MO USA) The tissue was incubatedat 37∘C for 45min in papain (001) and DNase (001)solution After removal of papain and its blockage withovomucoid (2mgmL) the cells were mechanically dispersedand resuspended in DMEM supplemented with 10 fetal calfserum and 1 penicillin-streptomycin to a final concentra-tion of 2 times 105 cellsmL Fifteen milliliters of cells in culturemedium was cultivated in 75 cm2 culture flasks at 37∘C in 5CO295 air for 24 h After 24 h the cells in suspension were

predominantly pinealocytes which were separated after theremoval of all the culture medium content The cells werecentrifuged and resuspended in DMEM at a concentrationof 2 times 105 cellsmL transferred to a 24-well plate (1mL perwell) and kept at 37∘C in 5 CO

295 air for 1 h prior to the

pharmacological treatments For melatonin determination inthe culturemedium the cells were stimulated withNE (1 120583M)alone or in associationwith glutamate (100 or 600120583M) for 5 h

24 Isolated Astrocyte Culture After the removal of thepinealocytes in suspension the astrocytes remained attachedto the culture flasks and were kept in culture for one weekAfter this period the cells were washed twice with Hankssolution and 6mL of trypsin 025 was added for 5minTrypsin action was blocked with DMEM supplemented with10 fetal calf serum The cells were centrifuged at 300 g for5min at 20∘C The pellet was resuspended according to thedifferent assays

25 Pinealocyte and Astrocyte Coculture After the dissocia-tion of the pineal gland cells by papain digestion pinealocytesand astrocytes were cocultivated in the same well (24-wellplates) in DMEM medium with 10 fetal calf serum at 37∘Cin 5 CO

295 air at a concentration of 4 times 105 cellsmL

(1mL per well) for 60 h After that the medium was replacedby fresh medium without fetal calf serum and the cellswere kept for 1 h at 37∘C in 5 CO

295 air prior to the

pharmacological treatments For melatonin determination inthe culturemedium the cells were stimulated withNE (1 120583M)alone or in associationwith glutamate (100 or 600120583M) for 5 hWhenBB1101 (10120583M) [39] the inhibitor of TNF-120572 convertingenzyme (TACE ADAM17) was used it was added to themedium 30min before the treatments described previously

The cells in co-culture were also physically separated byinserts (transwell with 04120583mpore Corning NY USA) withastrocytes in the bottom of the wells and pinealocytes withinthe inserts The incubation procedures described previouslywere reproduced

26 Cell Culture Characterization by Immunocytochem-istry Suspensions of pineal gland cultured cells (astrocytespinealocytes or co-culture) were washed twice in phosphatebuffer (PB 01M pH 74) for medium removal and fixed

with 2 paraformaldehyde in PB for 15min After twoadditional washes in PB cells were spread upon gelatin-and chromoalumen-coated slides and allowed to dry andadhere using a hot plate (37∘C) Cells were then incubatedwith mouse monoclonal antibodies either against the glialfibrillary acidic protein (GFAP Sigma USA) or CR3 com-plement receptor (OX-42 BD Biosciences USA) diluted1 1000ndash1 2000 in PB containing 001 Triton X-100 in awet chamber for 14ndash18 h at room temperature Following 3 times10min washes in PB cells were then incubated with a wheatgerm agglutinin-rhodamine label (WGA Vector Laborato-ries USA) mixed with a donkey anti-mouse IgG conjugatedto fluorescein isothiocyanate (Jackson Labs USA) diluted1 200 and 1 100 respectively for 2 h Slideswere thenwashed3 times 10min and coverslipped with glycerol-carbonate bufferThe material was analyzed using a fluorescence microscopeequipped with standard filters and digital images werecollected and mounted using Adobe Photoshop software(Adobe Systems Inc USA) Controls for the experimentsconsisted of the omission of primary antibodies or agglutininfrom the procedure No staining was observed in these cases

27 Intracellular Calcium Measurement Isolated astrocytesand pinealocytes were obtained as described previously Thecells were placed on a thin glass coverslip and incubated inDMEMwith 16 nMof Fluo-4 (Molecular Probes Inc EugeneOR USA) kept at 37∘C for 1 h and then washed three timeswith the same medium Isolated astrocytes and pinealocyteswere treated with glutamate (600 120583M) or glutamatergicagonists (N-methyl-D-aspartic acidmdashNMDA 100 120583M120572-amino-3-hydroxyl-5-methyl-4-isoxazole-propionatemdashAMPA 50 120583M (S)-35-dihydroxyphenylglycinemdashDHPG50120583M) (Tocris Bioscience Ellisville MO USA) A laserscanning microscope (LSM 510 Carl Zeiss Jena Germany)equipped with the Attofluor ratio imaging system (AttoInstruments Rockville MD USA) was used Measurementswere performed using an excitation filter of 505 nm and theemission was monitored at 530 nm

28 Determination of Melatonin Content Melatonin con-tent in pineal glands and culture medium was determinedby high performance liquid chromatography (HPLC) withelectrochemical detection using Empower software (WatersSystem Milford MA USA) [38] Melatonin was separatedon a resolve C18 column (5 120583m 150 times 39mm) The chro-matographic system was isocratically operated with thefollowing mobile phase 01M sodium acetate 01M citricacid and 015mM EDTA 30 methanol pH 37 at a flowrate of 1mLmin The electrochemical detector potential wasadjusted to +900mV The elution time for melatonin wasapproximately 6min Each gland was sonicated (MicrosonXL 2005 Heat System Inc Farmingdale NY USA) in asolution of 01M perchloric acid containing 002 EDTAand 002 sodium bisulfate Culture medium was dilutedin the same solution of perchloric acid (1 1 volvol) Aftercentrifugation (2min 13000 g) 40120583L of the supernatantwas injected into the chromatographic system (InjectorMod7125 20120583L loop Rheodyne Inc San Francisco CA USA)

4 BioMed Research International

All the salts and reagents used were obtained from Merck(Frankfurter Darmstadt Germany)

29 Electrophoretic Mobility Shift Assay

291 Pineal Gland Nuclear Protein Extracts Nuclear extractswere obtained from pineal gland homogenates as previouslydescribed [37] after incubation for 30min with or withoutglutamate (600 120583M) Briefly the glands were homogenizedin lysis buffer (10mM HEPES 10mM KCl 01mM EDTA10 glycerol and 01mM PMSF) and after 15min nonylphenoxypolyethoxylethanol (NP)-40 (10) was added Thesamples were vigorously mixed centrifuged (12000 g 1min4∘C) twice and resuspended in extraction buffer (10mMHEPES 05M KCl 1mM EDTA 10 glycerol and 01mMPMSF) After incubation on ice for 15min and centrifugation(20000 g 5min 4∘C) the supernatant containing the nuclearproteins was collected and stored at minus80∘C Protein concen-tration was determined using the BioRad protein reagent(BioRad Hercules CA USA)

292 Astrocytes and Pinealocytes Nuclear Protein ExtractsNuclear extracts were obtained from isolated astrocytes andpinealocytes homogenates as previously described [40] afterincubation for 30min with or without glutamate (600120583M)Briefly the cells were homogenized in lysis buffer (10mMHEPES 15mM MgCl

2 10mM KCl 05mM PMSF 01mM

EDTA and 3mMorthovanadate) After addingNP-40 (10)sampleswere vigorouslymixed centrifuged (11000 g 20min4∘C) twice and resuspended in extraction buffer (20mMHEPES 15mMMgCl

2 300mMNaCl 025mM EDTA 25

glycerol 05mM PMSF 3mM orthovanadate 2 120583gmL leu-peptin and 2 120583gmL antipain) After a 20min incubation onice and centrifugation (11000 g 20min 4∘C) the supernatantcontaining the nuclear proteins was collected and stored atminus80∘C The protein concentration was determined using theBioRad protein reagent

293 Gel Shift Assay An electrophoretic mobility shift assayfor NF-120581B was performed utilizing the gel shift assay kitfrom Promega (Madison WI USA) as described previ-ously [41] A 32P-NF-120581B double-stranded consensus oligonu-cleotide probe (51015840-AGTTGAGGGGACTTTCCCAGGC-31015840)and nuclear extracts were used DNA-protein complexeswere separated by electrophoresis through a 6 non-denaturing acrylamidebisacrylamide (375 1) gel in 053Tris-borate-EDTA (TBE) for 2 h at 150V Gels were vac-uum dried and analyzed by autoradiography For compe-tition experiments NF-120581B and transcription initiation fac-tor II (TFIID 51015840-CAGAGCATATAAGGTGAGGTAGGA-31015840)unlabeled double-stranded consensus oligonucleotide wereincluded in ten-fold excess of the molar concentration of 32P-NF-120581B probe to detect specific and nonspecific DNA-proteininteractions respectively Unlabeled oligonucleotides wereadded to the reaction mixture 20min before the addition ofthe radioactive probeThe composition of the complexes wasdetermined by supershift assays adding antibodies (SantaCruzBiotechnologies SantaCruz CAUSA) against different

NF-120581B subunits (p50 p52 c-Rel and p65 1 20 dilution)either before or after the incubation of nuclear extracts withthe labeled oligonucleotide

210 RNA Extraction and RT-PCR Total RNA was extractedfrom isolated astrocytes and pinealocytes usingTRIzol (Invit-rogen Carlsbad CA USA) according to the manufacturerrsquosinstructions cDNA synthesis was performed using Super-Script III Reverse Transcriptase (RT) (Invitrogen CarlsbadCA USA) from 1120583g of total RNA After the RT reaction 2-120583L aliquots were used as a cDNA template for polymerasechain reaction (PCR) amplifications using GoTaq DNAPolymerase (Promega Madison WI USA) The cyclingparameters used were 30 s at 95∘C 30 s at each primerrsquosannealing temperature and 30 s at 72∘C using a MyGeneSeries Gradient Thermocycler (LongGene China) PCRproducts were separated on 12 ethidium bromide-agarosegels and the bands were visualized by digital scanning Theprimers used in this assay are presented in supplementaryTable 1 (see Table 1 in Supplementary Material available athttpdxdoiorg1011552013618432)

211 Flow Cytometry Pineal glands in culture were stimu-lated with NE 1 120583M in the absence or presence of glutamate100 120583M or 600120583M After 5 h of incubation the cells weredissociated via proteolytic digestion with papain In an assayof membrane integrity 50120583L of a propidium iodide solution(20120583g permL in saline buffer)was added to the cells (500120583L)Propidium iodide is a highly water-soluble fluorescent com-pound that cannot pass through intact membranes as it isunable to enter viable cells After membrane disruption itbinds to DNA by intercalating between the bases with littleor no sequence preference After 5min of incubation at roomtemperature the cells were evaluated in FACScalibur flowcytometer equipment (BectonDickinson San Jose CAUSA)using Cell Quest software (Becton Dickinson San Jose CAUSA) Fluorescence was measured using the FL2 channel(orange-red fluorescencemdash58542 nm) DNA fragmentationwas analyzed after DNA staining with propidium iodideaccording to the previously described method [42] Cellswere resuspended in a solution with detergents (300120583Lhypotonic solution containing 20120583gmL propidium iodide01 sodium citrate and 01TritonX-100) that permeabilizethe cells which promptly incorporate the dye into DNAThe pellet was gently resuspended and the cells were thenincubated for 2 h at room temperature Fluorescence wasmeasured and analyzed by flow cytometry as describedpreviously

212 Statistical Analysis Data were always presented asthe mean plusmn SEM Statistical analyses (GraphPad Prism 50software San Diego CA USA) were performed using one-way ANOVA followed by a Bonferroni post hoc test Whenappropriate Studentrsquos 119905-test was applied The graphs relatedto confocal microscopy were prepared utilizing Origin 50software (MicroCal Software Inc Northampton MA USA)Autoradiographs of NF-120581Bwere quantified using ScionImagesoftware (Scion Corp Frederick MD USA)

BioMed Research International 5

GFAP

(a)

WGA

(b)

GFAP

(c)

WGA

50120583M

(d)

Figure 1 Cell cultures immunocytochemistry Digital images of isolated cultures of astrocytes (a) and pinealocytes (b) showing specific celltype staining using a mouse monoclonal antibody against the glial fibrillary acidic protein (GFAP) and a wheat germ agglutinin-rhodaminelabel (WGA) to identify pinealocytes The pictures at the bottom illustrate the distribution of glial cells (c) and pinealocytes (d) in a mixedcoculture The insets show photomicrographies of the cell cultures in contrast phase microscopy astrocytes (a) pinealocytes (b) and co-culture (c and d) In isolated pinealocytes and in co-culture many cells are clustered Magnification 200x (astrocytes and pinealocytes) and100x (co-culture)

3 Results

The cultures used in this study were characterized and val-idated by immunostaining with GFAP a specific marker forastroglial cells and OX-42 a marker for microglia (specificfor activated microglia as the antibody reacts with the CR3complement) This OX-42 antibody is suitable for pinealgland staining because the predominant type of microglia inthis tissue expresses complement type 3 receptors [43] WGAwas used as a pinealocyte marker because it labels photore-ceptors [44 45] and pinealocytes aremodified photoreceptorcells [46 47]This technique proved to be efficient in labelingthese pineal cells (Figure 1)

Cultured isolated astrocytes showed fluorescence forGFAP (Figure 1(a)) but not for OX-42 and WGA (data notshown) Cultured pinealocytes lacked immunostaining forthe two glial markers GFAP and OX-42 (data not shown)and reacted withWGA demonstrating the absolute predom-inance of pinealocytes (Figure 1(b)) Figures 1(c) and 1(d)show the distribution of pinealocytes and astrocytes in themixed culture (co-culture) The round appearance of thelabeled cells in Figure 1 different from that in the insets is dueto the fixation process with paraformaldehyde In Figure 1(d)pinealocytes appear smaller than those in Figure 1(b) most

likely due to the use of trypsin to obtain the cells furtherdissociating the cell clusters

In intact cultured pineal glands the known effect ofglutamate in reducing NE-stimulated melatonin synthesis(119875 lt 005) was observed (Figure 2(a)) On the other handwhen isolated cultured pinealocytes were incubated withglutamate (100 and 600 120583M) in the presence of NE (1 120583M)this effect was abrogated (Figure 2(b)) Glutamatersquos inhibitoryeffect on melatonin synthesis as observed in intact culturedpineal glands was only reproduced when pinealocytes werecultivated in the presence of astrocytes in co-culture (119875 lt001) (Figure 2(c))

To investigate if the contact between astrocytes andpinealocytes is necessary for the inhibitory effect of gluta-mate the two cell types were cocultivated in the same wellbut separated by inserts It was observed that glutamatersquosinhibitory effect on NE-stimulated melatonin synthesis wasexpressed in the same way as when the cells were cultivatedwith physical contact indicating that a diffusible factorreleased by astrocytes acted on the pinealocytes (119875 lt 001)(Figure 2(d))

The possibility of TNF-120572 as a gliotransmitter that medi-ates the glutamate effect was tested using BB1101 a preferredinhibitor of metalloproteinases with sheddase activity that

6 BioMed Research International

18

15

12

9

6

3

0

Mela

toni

n (n

ggl

and)

lowast

NE 1120583MGlu 100120583MGlu 600120583M

+ +

+

+

+

minus minus

minus minus

(a)

0

Mela

toni

n (n

gm

L)

NE 1120583MGlu 100120583MGlu 600120583M

+ +

+

+

+

minus minus

minus minus

120

80

40

(b)

Mela

toni

n (n

gm

L)

NE 1120583MGlu 100120583MGlu 600120583M

+ +

+

+

+

minus minus

minus minus

50

40

30

20

10

0

lowastlowast lowastlowast

(c)

NE 1120583MGlu 600120583M

Mela

toni

n (n

gm

L)

+ +

+minus

lowastlowast

100

80

60

40

20

0

(d)

Figure 2 Glutamate effects on melatonin synthesis (a) Pineal gland culture stimulated with norepinephrine (NE 1120583M) in the absence orpresence of glutamate (Glu 100 and Glu 600120583M) Melatonin content is expressed in nggland as the mean plusmn SEM of 3 experiments One-wayANOVA Bonferroni multiple comparison test lowast119875 lt 005 versus NE 1 120583M 119899 = 12 for each group (b) Isolated rat pinealocyte culture underNE 1120583M stimulation in the absence or presence of Glu 100 or Glu 600120583M 119899 = 6 for each group (c) Pinealocyte and astrocyte co-culturestimulated with NE 1 120583M in the absence or presence of Glu 100 or Glu 600 120583M One-way ANOVA Bonferroni multiple comparison testlowastlowast119875 lt 001 versus NE 1 120583M 119899 = 8 for each group (d) Astrocytes and pinealocytes were cultured together but with physical separation by

inserts Astrocytes were cultivated in the bottom of the plate and pinealocytes were cultivated within the inserts and stimulated with NE1 120583M associated or not associated with Glu 600 120583M 119879 test lowastlowast119875 lt 001 119899 = 4 for each group For the cell cultures melatonin was evaluatedin the culture medium and expressed in ngmL as the mean plusmn SEM of 3 experiments

includes the TACE It was verified that when the cells inco-culture were incubated with BB1101 (10 120583M) prior to thetreatments with glutamate (600 120583M) and NE (1 120583M) it wasable to completely revert the inhibitory effect of glutamate onmelatonin synthesis (119875 lt 005) (Figure 3)

RT-PCR analyses showed mRNA expression of classIII metabotropic glutamate receptors (mGluR1mGluR5 andmGluR2mGluR3 resp) as well as ionotropic NMDA andAMPA-type receptors (GluN1 GluA1 GluA2 and GluA3subunits) in astrocytes and pinealocytes (Figures 4(a) and4(b)) Contrarily mGluR7 mRNA (class III metabotropicreceptors) was not detected in any of the cell types(Figure 4(a)) To determine whether class I mGluRs NMDA

and AMPA ionotropic receptors are functional and incor-porated into the astrocytic and pinealocytic membranes anintracellular calcium analysis by confocal microscopy wasused It was observed that the class I metabotropic receptoragonist DHPG (50120583M) and the ionotropic receptor agonistsNMDA (100120583M) and AMPA (50 120583M) were able to increasethe astrocytes and pinealocytes intracellular calcium contentin a similar way as that observed for glutamate (600 120583M)(Figure 5 Table 1)

Electrophoretic mobility shift assays revealed that gluta-mate (100 and 600120583M) activates NF-120581B in cultured rat pinealglands The result of the competition study showed that theupper complex 1 was displaced by an excess of unlabeled

BioMed Research International 7

60

40

20

0NE 1120583M

Mela

toni

n (n

gm

L)

lowast

+ +

+ +

+

+

+

+minus minus

minus minus

lowastlowast

BB1101 10120583MGlu 600 120583M

Figure 3 (a) Ablation of the inhibitory glutamatergic effect onmelatonin synthesis by the TACE inhibitor BB1101 Pinealocytesand astrocytes were maintained in co-culture and stimulated withnorepinephrine (NE 1 120583M) in association with glutamate (Glu600 120583M) and BB1101 (10120583M) One-way ANOVA Bonferroni mul-tiple comparison test lowastlowast119875 lt 001 versus NE 1 120583M lowast119875 lt 005 versusNE 1 120583M + Glu 600 120583M 119899 = 4 for each group 3 experiments

Table 1 Summary of intracellular [Ca2+] changes in astrocytes andpinealocytes after stimulation

Cell typeagents Number of cellsexamined [Ca2+]

119894response

Isolated astrocytesGlu 600 120583M 68 79AMPA 50 120583M 97 80NMDA 100 120583M 49 90DHPG 50 120583M 80 74

Isolated pinealocytesGlu 600 120583M 66 80AMPA 50 120583M 78 85NMDA 100 120583M 53 90DHPG 50 120583M 60 76

Intracellular [Ca2+] in fluo-4-loaded astrocytes and pinealocytes was mea-sured as described in Section 2 The majority of the stimulated cellsresponded with an increase in intracellular [Ca2+] after the addition of eitherglutamate (600120583M) ionotropic glutamatergic agonists (AMPA 50120583M andNMDA 100120583M) or a class I metabotropic glutamatergic agonist (DHPG50120583M)

NF-120581B but not by a TFIID double-stranded oligonucleotideconsensus sequence demonstrating the specificity of the NF-120581BDNA interactionThe lower complex 2 was less efficientlydisplaced by unlabeledNF-120581B probe (Figure 6(a)) Supershiftanalysis indicated that the antibody against the p50 subunitwas able to shift the DNAprotein interaction present incomplex 1 The presence of antibodies against the p52 p65and c-Rel subunits did not affect DNA-protein complexes(Figures 6(b) and 6(c)) Taken together these results indicatethat at these concentrations glutamate activates the p50p50

homodimer Complex 2 was not displaced by the antibodiesand was not considered to be related to the NF-120581B family

Astrocyte and pinealocyte isolated cultures were used todetermine the cell type in which NF-120581B was being activatedGlutamate (600120583M)was shown to activateNF-120581B exclusivelyin astrocytes and not in pinealocytes (119875 lt 001) (Figure 7)

The involvement of NF-120581B activation in glutamatersquosinhibitory effect on melatonin synthesis was analyzed usinga specific inhibitor of NF-120581B PDTC PDTC (300 120583M) pre-vented the inhibitory effect of glutamate in cultured rat pinealglands stimulated by NE (1 120583M) (119875 lt 001) (Figure 8)

Glutamate cytotoxicity was evaluated because it is knownthat this neurotransmitter in high concentrations couldinduce cell death by cytotoxicity [48] Cell membraneintegrity and DNA fragmentation were assessed by flowcytometric analysis using propidium iodide (PI) Supple-mentary Figure 1 is representative of the results obtainedafter cell incubation with NE (1120583M) in the absence (controlgroup) or presence of glutamate (100 and 600120583M) for 5 hThe histograms show the fluorescence intensity in the FL-2channel which corresponds to the PI emission wavelengthWhen the cell membrane is intact the fluorescence signal islow and is related to autofluorescence (M1) A high signalmeans that PI was able to enter the cell and bind to DNA(M2) Glutamate treatments for 5 h were not able to inducea loss of membrane integrity (Supplementary Figure 1(a))The percentage of cells with ruptured membranes in thecontrol group was 453 whereas it was 502 and 577 inthe 100 and 600 120583M glutamate-treated groups respectively(Supplementary Figure 1(c)) Histograms in SupplementaryFigure 1(b) represent the results of DNA fragmentationanalysis After PI treatment the DNA content in live cellspresented a high fluorescence signal (M2) DNA fragmenta-tion induces the formation of small DNA pieces that exhibitlow fluorescence intensity (M1) The percentages of cellswith DNA fragmentation after glutamate stimulation 100and 600120583M were 286 and 335 respectively which weresimilar to the value of control cells (366) (SupplementaryFigure 1(d))

4 Discussion

Isolated pinealocytes isolated astrocytes and pineal-ocyteastrocyte co-culture were used in this study Theimmunocytochemistry characterization of the cell typespresent in these different cultures demonstrated the pre-dominance of pinealocytes in isolated pinealocyte culturewith the absence of glial cells In the same way astrocytecultures do not contain a significant number of microglialcells or pinealocytes In co-culture pinealocytes are thepredominant cells as astrocytes are present in minorquantities reproducing the proportion present in intactpineal glands (51) Thus the cultures were considered to beappropriate for this study

Data from the literature have shown that glutamate acts asan inhibitory neurotransmitter on melatonin synthesis [12]According to Yamada et al [13 14 16] and Yatsushiro et al[16] glutamate is stored in microvesicles and released from

8 BioMed Research International

mGluR1 mGluR2 mGluR3 mGluR5 mGluR7

M H2O As Pi Hi As Pi Hi As Pi Hi As Pi Hi As Pi Hi

200 bp100 bp

(a)

M H2O As Pi Hi As Pi Hi As Pi Hi As Pi Hi

200 bp100 bp

GluA1 GluA2 GluA3 GluN1

(b)

Figure 4 RT-PCR detection of metabotropic and ionotropic glutamate receptors in isolated astrocytes and pinealocytes (a) RT-PCRdetection of transcripts for mGluR1 (79 bp) mGluR2 (198 bp) mGluR3 (196 bp) mGluR5 (165 bp) and mGluR7 (70 bp) in astrocytes (As)pinealocytes (Pi) and hippocampus (Hi) Molecular weight marker (M) and water (H

2O) are presented in lanes 1 and 2 respectively (b)

RT-PCR detection of transcripts for GluA1 (203 bp) GluA2 (155 bp) GluA3 (193 bp) and GluN1 (201 bp) in astrocytes (As) pinealocytes(Pi) and hippocampus (Hi) The molecular weight marker (M) and PCR product generated with water (H

2O) are presented in lanes 1 and 2

respectively

pinealocytes upon cholinergic stimulation It interacts withionotropic and metabotropic glutamate receptors presentin pinealocyte membranes releasing additional glutamateand inhibiting melatonin synthesis respectively Recently amechanism involving the glutamate transporter GLT-1 wasproposed to be responsible for glutamate release [17] Inthese studies involving pinealocytes an autocrineparacrineprocess was suggested for the influence of glutamate onmelatonin synthesis In the presentworkwe demonstrated animportant role for astrocytes in glutamatersquos modulatory effecton melatonin synthesis that includes a paracrine interactionwith pinealocytes

Whereas in isolated pinealocytes glutamate does notexhibit the typical inhibitory effect on melatonin synthesisas observed in intact pineal glands its association withastrocytes restores this effect These results demonstrate thatastrocytes act as partners that collaborate to produce theglutamate effect In addition the fact that the effect occurredeven when inserts were used preventing the physical contactbetween the cells in co-culture suggests that a solublefactor is a mediator of this process The hypothesis that gapjunctions mediated the interactions between astrocytes andpinealocytes as occurs in the pineal gland [49] was excludedfrom this observationThe cytokine TNF-120572 a gliotransmitterthat is released by astrocytes could be the soluble factorthat is involved in the interactions between astrocytes andpinealocytes In fact the absence of a soluble form of TNF-120572 interferes with the glutamate response

The possibility that the glutamate effects were due to celldeath was investigated by flow cytometry As the cell viabilityand DNA fragmentation levels were similar between controland glutamate-stimulated groups showing no damage topineal cells even when treated with 600120583M of glutamate itis possible to exclude a glutamate cytotoxic effect as a causeof melatonin reduction

Although glutamate receptor expression had been firstdescribed in pinealocytes its expression was also laterdemonstrated in astrocytes mGluR3 and AMPA (GluA1subunit) were shown to be present in pinealocytes [12 16]

and NMDA and AMPA (GluA23) receptors were present inrat pineal gland astrocytes [19] Ionotropic receptor mRNAwas similarly observed in astrocytes in the present studyin addition to the novel observation of metabotropic recep-tor (mGluR1 mGluR2 mGluR3 mGluR5) expression Inpinealocytes the expression of mGluR1R2R3R5 AMPAand NMDA receptors was shownThis evidence supports thepossibility of astrocyte participation in rat pineal glutamater-gic signaling

Confocal microscopy assays using NMDA AMPA andDHPG agonists in isolated astrocytes and pinealocytesrevealed increased intracellular calcium content in both celltypes for all the agonists used PCR and confocal microscopyanalyses suggest both gene and protein expression in classI mGluR AMPA and NMDA ionotropic receptors reveal-ing the importance of the glutamate receptors as signaltransducers in rat pineal gland astrocytes and pinealocytesThus glutamate seems to interact with its receptors inboth cell types but its effect on melatonin synthesis nec-essarily involves astrocyte glutamate receptors The possibleparacrine interactions between pinealocytes and astrocytesseem to be the key in understanding the role of astrocytes inthe rat pineal gland

Transcription factors exert a great influence on pinealphysiology CREB and ICER are two transcription factorsthat intimately control the transcription of AANAT [50]Additionally there are other transcription factors in the ratpineal gland whose roles in pineal melatonin synthesis arenot known [51] NF-120581B is present in the rat pineal glandshowing a circadian rhythm and involvement in inflamma-tory responses [36 37] NF-120581B is a classic possible glutamatemediator and its activation in glial cells was demonstrated toplay a role in inflammatory processes [30]The present resultsdemonstrated NF-120581B activation by glutamate in culturedisolated astrocytes supporting its role as a mediator ofglutamate effects Supershift assay in cultured pineal glandsstimulated with glutamate showed the displacement of thep50 subunit emphasizing the importance of p50p50 homod-imer formationThis is an astrocyte-dependent event because

BioMed Research International 9

0 50 100 150 200 250 300 350

10

20

30

40

50

60

Time (s)

Isolated astrocytes

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

Glu 600120583M

(a)

0 30 60 90 120 150

155

160

165

170

175

180

185

Time (s)

Isolated pinealocytes

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

Glu 600120583M

(b)

0 100 200 300 400 500

10

20

30

Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

AMPA 50120583M

(c)

0 30 60 90 120 150 180

102

104

106

108

110

112

114

Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

AMPA 50120583M

(d)

0 50 100 150 200 250 300

400

600

800

1000

1200

1400

Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

NMDA 100120583M

(e)

0 50 100 150 200 250

900

1200

1500

Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

NMDA 100120583M

(f)

100 150 200 250

126

128

130

132

134

136

Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity) DHPG 50120583M

(g)

0 100 200 300 400

80

90

100

110

120

130

Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

DHPG 50120583M

(h)

Figure 5 Analysis of intracellular calcium in isolated astrocyte and pinealocyte cultures Traces of Fluo-4 fluorescence indicate theintracellular calcium transients from single cells Additions of exogenous glutamate or glutamatergic agonists were performed as indicatedby the arrows

10 BioMed Research International

ControlGlu 100120583MGlu 600120583MUnlabeled NF-120581BUnlabeled TFIID

1

(NS) 2

Free probe

+

minus

minus

minus

minus +

minus

minus

minus

minus

+

minus

minus

minus

minus +

+

minus

minus

minus +

+

minus

minus

minus+

+

minus

minus

minus

+

+

minus

minus

minus

(a)

(NS) 2

Free probe

(p50p50) 1

(NS) 3

+

+

minus

minus

minus

minus

+

minus

minus

minus

minus

minus

+

minus

minus

minus

minus

minus

+

+

minus

minus

minus

minus

+

+

minus

minus

minus

minus

+

+

minus

minus

minus

minus

ControlGlu 100120583MAnti p50Anti p52Anti p65Anti c-Rel

(b)

Free probe

Anti c-Rel

(NS) 2

(p50p50) 1

(NS) 3

ControlGlu 600120583MAnti p50Anti p52Anti p65

+minus minus minus minus minus

+

+ +

+

+ + +

+

+minus minus

minus

minus

minus

minus minus minus minus minus

minus

minus minus minus

minus minus

minus minus minus

minus minus

(c)

Figure 6 Electrophoretic mobility shift assays for NF120581B in the pineal gland (a) Competition studies were performed using nuclear extract(5120583g) from cultured pineal glands treated with glutamate (Glu) 100 and 600 120583M in the absence or presence of unlabeled specific (NF-120581Bconsensus sequence 10-fold molar excess) or nonspecific oligonucleotide (TFIID consensus sequence 10-fold molar excess) as indicated(b c) Supershift assays were performed with the same nuclear extract (5120583g) incubated in the absence or presence of antibodies against thesubunits p50 (1 10 dilution) p65 (1 20 dilution) p52 and c-Rel (1 10 dilution) as indicated Antibodies were added 20min prior to theaddition of the radiolabeled NF-120581B consensus oligonucleotide The position of specific NF-120581BDNA binding complexes p50p50 (band 1 bc) is indicated NS represents no specific binding (bands 2 and 3 a b c) The localization of the free probe is also indicated

BioMed Research International 11

ControlGlu 600120583M

minusminus

minus +

NF-120581B

(a)

20000

15000

10000

0Control Glu 600120583M

5000

lowastlowast

(au

)

(b)

ControlGlu 600120583M

minusminus

minus +

NF-120581B

(c)

(au

)

Control Glu 600120583M

16000

12000

8000

4000

0

(d)

Figure 7 Electrophoretic mobility shift assays for NF120581B in isolated astrocytes and pinealocytes (a) Nuclear proteins (5 120583g) were extractedfrom astrocytes treated with glutamate (Glu) 600120583M (b)Densitometric analysis (arbitrary units) of theNF-120581B band is presented on panel (a)One-way ANOVA Studentrsquos test lowastlowast119875 lt 001 versus control group mean plusmn SEM (c) Nuclear proteins (5120583g) were extracted from pinealocytestreated with Glu 600 120583M (d) Densitometric analysis (arbitrary units) of the NF-120581B band is presented in (c) The position of the specificNF-120581BDNA binding complex is indicated 119899 = 5 for each group 3 experiments

its activation was not observed in pinealocytes Astrocyteand pinealocyte supershift assays were not performed due totechnical limitations related to the amount of nuclear protein

The p50 subunit is synthesized from its precursor p105The processing of p105 and the resulting generation ofmature p50 subunits seem to be a constitutive event as p50homodimers are not regulated by I120581B proteins [34] The p50subunit formation seems to be regulated by cotranslationaland posttranslational mechanisms In the posttranslationalmechanism the p50 subunit is synthesized from its precursorp105 by phosphorylation at its 927 and 932 serine residueswhich allows the ubiquitinization and proteolytic degrada-tion of p105 by the 26S proteasome [52] In the cotranslationalmechanism p50 and p105 can be generated from a singlemRNA [53] There is also evidence that the 20S protea-some constitutively processes p105 to p50 in a ubiquitin-independent manner [52] The p50p50 homodimer wasoriginally considered to be a transcriptional repressor butnow there is substantial evidence that it can also inducegene transcription after a complex formation with the BCL3nuclear protein an atypical member of the I120581B family ofproteins that is considered to be a coactivator of p50p50homodimers [34]

The link between NF-120581B activation by glutamate and thereduction of melatonin synthesis was evidenced with the useof PDTC a NF-120581B translocation inhibitor [54] PDTC pre-vented glutamatersquos inhibitory effect on melatonin synthesisTaken together the crucial presence of astrocytes for theinhibitory effect of glutamate the exclusive activation of NF-120581B by glutamate in the same cell type and the demonstrationof NF-120581Brsquos role inmelatonin synthesis indicate that astrocytesunder glutamate stimulation possibly release a factor thatpromotes a decrease in pinealocyte melatonin synthesisTNF-120572may be a good candidate Indeed TNF-R1 the TNF-120572 receptor that interacts with soluble TNF is present in thepineal gland reinforcing the importance of this signalingpathway [55] Acting as an inhibitor of TACE to prevent TNF-120572 release BB1101 was thus adequate in demonstrating the roleplayed byTNF-120572 In addition the hypothesis that the p50p50homodimer may induce TNF-120572 gene expression and releaseneeds to be clarified

In the CNS astrocytes are known to release gliotransmit-ters such as glutamate D-serine and ATP [21] Moreoverastrocytes can also synthesize and release proinflammatorycytokines in response to NF-120581B activation such as TNF-120572 and interleukin-1 and induce gene expression of iNOS

12 BioMed Research International

NE 1120583MGlu 100120583MGlu 600120583MPDTC 300120583M

Mela

toni

n (n

ggl

and)

20

15

10

5

0

lowastlowast

lowastlowast

+ +

+ + +

++

+

+

+

+

+ +

minus minus minusminus

minus minus minusminus

minus minus minus

Figure 8 Effect of PDTC an inhibitor of NF-120581B on melatoninsynthesis Pineal glands in culture were incubated with PDTC(300120583M) and stimulated by norepinephrine (NE 1120583M) in thepresence of glutamate (100 and 600120583M) andmelatonin content wasevaluated One-way ANOVA Bonferroni multiple comparison test119875 lt 001 versus NE 1 120583M lowastlowast119875 lt 001 versus NE + Glu 100 120583Mand NE + Glu 600 120583M plotted as the mean plusmn SEM 119899 = 12 for eachgroup 3 experiments

PinealocyteAstrocyte

NF-120581Bactivation

AMPANMDAmGluR15

Release of solublefactor

TNF-120572

Glu

TNF-120572 alone (recruiting AMPA)or in association with Glu

mGluR3TNF-R1 AMPA[iCa+2]

Melatoninsynthesis

Figure 9 Diagram illustrating the putative paracrine interactionbetween pinealocytes and astrocytes in the modulation of pinealmelatonin synthesis by glutamate Glutamate (Glu) induces intra-cellular Ca2+ increase in astrocytes with the subsequent activationof NF120581B and release of a soluble factor possibly TNF-120572 This factoralone or in association with Glu promotes melatonin synthesisreduction in pinealocytes

resulting in nitric oxide formation [56] In this work wesuggested that TNF-120572 is the factor that is released byastrocytes under glutamate stimulation because the TACEinhibitor antagonized glutamatersquos effects Reinforcing thisidea recent preliminary data obtained in our laboratorywith the L929 tumor cell lineage which is sensitive to TNF-120572 showed that astrocytes release TNF-120572 when stimulatedby glutamate (data not shown) The literature states that

TNF-120572 decreases the serotonin content and AANAT mRNAexpression resulting in the reduction of N-acetylserotonin[57 58] the immediate precursor of melatonin synthesis Itis not clear whether TNF-120572 acts by itself or in combinationwith glutamate because there are glutamate receptors in thepinealocyte membrane [12 15 16 18] Recent studies havereported the control of hippocampal synapses by TNF-120572 dueto the increased insertion of AMPA receptors in the surfaceof the neuronal soma [59]The same effect could take place inthe pineal gland in which TNF-120572 released by the astrocytesin response to NF-120581B activation could increase the numberof AMPA receptors in the pinealocyte membrane resultingin the inhibition of melatonin synthesis

A model of the putative paracrine interactions betweenastrocytes and pinealocytes in the modulation of pinealmelatonin synthesis by glutamate is shown in Figure 9

5 Conclusions

In summary according to our results we propose thatglutamate acts on glutamate receptors (AMPA NMDAandor mGluR15) in astrocytes increasing the intracellularcalcium concentration promoting NF-120581B activation anddriving the release of a soluble factor possibly TNF-120572 whichacts independently or with glutamate in the pinealocytesresulting in inhibition of melatonin synthesis

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

This work was supported by a grant from FAPESP (0404328-1) Darine Villela was supported by a master fellowshipfrom FAPESP (0556943-4) Anderson Augusto Moutinhoand Victoria Fairbanks Atherino were supported by CNPq(PIBIC)

References

[1] J Calvo J Boya A Borregon and J E Garcia-MaurinoldquoPresence of glial cells in the rat pineal gland a light andelectronmicroscopic immunohistochemical studyrdquoAnatomicalRecord vol 220 no 4 pp 424ndash428 1988

[2] L Vollrath The Pineal Organ Springer New York NY USA1981

[3] R Y Moore J C Speh and J P Card ldquoThe retinohypothalamictract originates from a distinct subset of retinal ganglion cellsrdquoJournal of Comparative Neurology vol 352 no 3 pp 351ndash3661995

[4] D Sugden ldquoMelatonin biosynthesis in the mammalian pinealglandrdquo Experientia vol 45 no 10 pp 922ndash932 1989

[5] A F Afeche D CMVillelaM V Abrahao R Peres J Cipolla-Neto et al ldquoMelatonin and pineal glandrdquo in New Researchon Neurosecretory Systems E Romano Ed pp 151ndash177 NovaScience Publishers Hauppauge NY USA 2008

[6] J Arendt Melatonin and the Mammalian Pineal Gland Chap-man amp Hall London UK 1995

BioMed Research International 13

[7] M Ehret P Pevet and M Maitre ldquoTryptophan hydroxylasesynthesis is induced by 3101584051015840-cyclic adenosine monophosphateduring circadian rhythm in the rat pineal glandrdquo Journal ofNeurochemistry vol 57 no 5 pp 1516ndash1521 1991

[8] D C Klein G R Berg and J Weller ldquoMelatonin synthesisadenosine 3101584051015840-monophosphate and norepinephrine stimulateN-acetyltransferaserdquo Science vol 168 no 3934 pp 979ndash9801970

[9] D C Klein N L Schaad M A A Namboordiri L Yu and JL Weller ldquoControl of N-acetyltransferaserdquo Biochemical SocietyTransactions vol 20 no 2 pp 299ndash304 1992

[10] V Simonneaux and C Ribelayga ldquoGeneration of the melatoninendocrine message in mammals a review of the complexregulation of melatonin synthesis by norepinephrine peptidesand other pineal transmittersrdquoPharmacological Reviews vol 55no 2 pp 325ndash395 2003

[11] S Ishio H Yamada C M Craft and Y MoriyamaldquoHydroxyindole-O-methyltransferase is another target forL-glutamate-evoked inhibition of melatonin synthesis in ratpinealocytesrdquo Brain Research vol 850 no 1-2 pp 73ndash78 1999

[12] H Yamada S Yatsushiro S Ishio et al ldquoMetabotropic gluta-mate receptors negatively regulate melatonin synthesis in ratpinealocytesrdquo Journal of Neuroscience vol 18 no 6 pp 2056ndash2062 1998

[13] H Yamada A Yamamoto M Takahashi H Michibata HKumon and Y Moriyama ldquoThe L-type Ca2+ channel isinvolved in microvesicle-mediated glutamate exocytosis fromrat pinealocytesrdquo Journal of Pineal Research vol 21 no 3 pp165ndash174 1996

[14] H Yamada A Yamamoto S Yodozawa et al ldquoMicrovesicle-mediated exocytosis of glutamate is a novel paracrine-likechemical transduction mechanism and inhibits melatoninsecretion in rat pinealocytesrdquo Journal of Pineal Research vol 21no 3 pp 175ndash191 1996

[15] H Yamada A Ogura S Koizumi A Yamaguchi and YMoriyama ldquoAcetylcholine triggers L-glutamate exocytosis vianicotinic receptors and inhibits melatonin synthesis in ratpinealocytesrdquo Journal of Neuroscience vol 18 no 13 pp 4946ndash4952 1998

[16] S Yatsushiro H Yamada M Hayashi A Yamamoto andY Moriyama ldquoIonotropic glutamate receptors trigger micro-vesicle-mediated exocytosis of L-glutamate in rat pinealocytesrdquoJournal of Neurochemistry vol 75 no 1 pp 288ndash297 2000

[17] M-HKim SUehara AMuroyama BHille YMoriyama andD-S Koh ldquoGlutamate transporter-mediated glutamate secre-tion in the mammalian pineal glandrdquo Journal of Neurosciencevol 28 no 43 pp 10852ndash10863 2008

[18] S Yatsushiro H Yamada M Hayashi S Tsuboi and YMoriyama ldquoFunctional expression of metabotropic glutamatereceptor type 5 in rat pinealocytesrdquo NeuroReport vol 10 no 7pp 1599ndash1603 1999

[19] C Kaur V Sivakumar and E A Ling ldquoExpression ofN-methyl-D-aspartate (NMDA) and 120572-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) GluR23 receptors inthe developing rat pineal glandrdquo Journal of Pineal Research vol39 no 3 pp 294ndash301 2005

[20] H Pabst and P Redecker ldquoInterstitial glial cells of the gerbilpineal gland display immunoreactivity for the metabotropicglutamate receptors mGluR23 and mGluR5rdquo Brain Researchvol 838 no 1-2 pp 60ndash68 1999

[21] M M Halassa T Fellin and P G Haydon ldquoThe tripartitesynapse roles for gliotransmission in health and diseaserdquoTrends in Molecular Medicine vol 13 no 2 pp 54ndash63 2007

[22] S M Finkbeiner ldquoGlial calciumrdquo Glia vol 9 no 2 pp 83ndash1041993

[23] E A Newman ldquoNew roles for astrocytes regulation of synaptictransmissionrdquo Trends in Neurosciences vol 26 no 10 pp 536ndash542 2003

[24] J T Porter and K D McCarthy ldquoAstrocytic neurotransmitterreceptors in situ and in vivordquo Progress in Neurobiology vol 51no 4 pp 439ndash455 1997

[25] V Parpura T A Basarsky F Liu K Jeftinija S Jeftinija and PG Haydon ldquoGlutamate-mediated astrocyte-neuron signallingrdquoNature vol 369 no 6483 pp 744ndash747 1994

[26] T D Hassinger P B Atkinson G J Strecker et al ldquoEvidencefor glutamate-mediated activation of hippocampal neurons byglial calcium wavesrdquo Journal of Neurobiology vol 28 no 2 pp159ndash170 1995

[27] R H Lipsky K Xu D Zhu et al ldquoNuclear factor 120581B is a criticaldeterminant in N-methyl-D-aspartate receptor-mediated neu-roprotectionrdquo Journal of Neurochemistry vol 78 no 2 pp 254ndash264 2001

[28] M P Mattson C Culmsee Z Yu and S Camandola ldquoRoles ofnuclear factor 120581B in neuronal survival and plasticityrdquo Journal ofNeurochemistry vol 74 no 2 pp 443ndash456 2000

[29] C DMunhoz B Garcıa-Bueno J L MMadrigal L B LepschC Scavone and J C Leza ldquoStress-induced neuroinflammationmechanisms and new pharmacological targetsrdquo Brazilian Jour-nal of Medical and Biological Research vol 41 no 12 pp 1037ndash1046 2008

[30] S Camandola andM PMattson ldquoNF-ΚB as a therapeutic targetin neurodegenerative diseasesrdquo Expert Opinion on TherapeuticTargets vol 11 no 2 pp 123ndash132 2007

[31] B Kaltschmidt D Widera and C Kaltschmidt ldquoSignaling viaNF-120581B in the nervous systemrdquo Biochimica et Biophysica Actavol 1745 no 3 pp 287ndash299 2005

[32] P T Massa H Aleyasin D S Park X Mao and S W BargerldquoNF120581B in neurons the uncertainty principle in neurobiologyrdquoJournal of Neurochemistry vol 97 no 3 pp 607ndash618 2006

[33] H Hacker and M Karin ldquoRegulation and function of IKK andIKK-related kinasesrdquo Sciencersquos STKE vol 2006 no 357 p re132006

[34] S Beinke and S C Ley ldquoFunctions of NF-120581B1 and NF-120581B2 inimmune cell biologyrdquo Biochemical Journal vol 382 no 2 pp393ndash409 2004

[35] N D Perkins ldquoIntegrating cell-signalling pathways with NF-120581Band IKK functionrdquo Nature Reviews Molecular Cell Biology vol8 pp 49ndash62 2007

[36] E Cecon P A Fernandes L Pinato Z S Ferreira and R PMarkus ldquoDaily variation of constitutively activated nuclear fac-tor 120581B (NFKB) in rat pineal glandrdquoChronobiology Internationalvol 27 no 1 pp 52ndash67 2010

[37] Z S Ferreira P A C M Fernandes D Duma J Assreuy MC W Avellar and R P Markus ldquoCorticosterone modulatesnoradrenaline-induced melatonin synthesis through inhibitionof nuclear factor 120581Brdquo Journal of Pineal Research vol 38 no 3pp 182ndash188 2005

[38] S C Afeche R Barbosa J H Scialfa I M Terra A CCassola and J Cipolla-Neto ldquoEffects of the blockade of highvoltage-activated calcium channels on in vitro pineal melatoninsynthesisrdquo Cell Biochemistry and Function vol 24 no 6 pp499ndash505 2006

14 BioMed Research International

[39] B Barlaam T G Bird C Lambert-Van Der Brempt DCampbell S J Foster and R Maciewicz ldquoNew 120572-substitutedsuccinate-based hydroxamic acids as TNF120572 convertaseinhibitorsrdquo Journal of Medicinal Chemistry vol 42 no 23 pp4890ndash4908 1999

[40] I Glezer C D Munhoz E M Kawamoto T MarcourakisM C Werneck Avellar and C Scavone ldquoMK-801 and 7-Ni attenuate the activation of brain NF-120581B induced by LPSrdquoNeuropharmacology vol 45 no 8 pp 1120ndash1129 2003

[41] Y Rong and M Baudry ldquoSeizure activity results in a rapidinduction of nuclear factor-120581B in adult but not juvenile ratlimbic structuresrdquo Journal of Neurochemistry vol 67 no 2 pp662ndash668 1996

[42] I Nicoletti G Migliorati M C Pagliacci F Grignani and CRiccardi ldquoA rapid and simple method for measuring thymocyteapoptosis by propidium iodide staining and flow cytometryrdquoJournal of Immunological Methods vol 139 no 2 pp 271ndash2791991

[43] Y F Jiang-Shieh C H Wu M L Chang J Y Shiehand C Y Wen ldquoRegional heterogeneity in immunoreactivemacrophagesmicroglia in the rat pineal glandrdquo Journal ofPineal Research vol 35 no 1 pp 45ndash53 2003

[44] K-O Soderstrom ldquoLectin binding to the human retinardquoAnatomical Record vol 220 no 2 pp 219ndash223 1988

[45] F Uehara M Sameshima T Muramatsu and N OhbaldquoLocalization of fluorescence-labeled lectin binding sites onphotoreceptor cells of the monkey retinardquo Experimental EyeResearch vol 36 no 1 pp 113ndash123 1983

[46] H-W Korf ldquoThe pineal organ as a component of the biologicalclock phylogenetic and ontogenetic considerationsrdquo Annals ofthe New York Academy of Sciences vol 719 pp 13ndash42 1994

[47] E Maronde and J H Stehle ldquoThe mammalian pineal glandknown facts unknown facetsrdquo Trends in Endocrinology andMetabolism vol 18 no 4 pp 142ndash149 2007

[48] X-M Zhang and J Zhu ldquoKainic acid-induced neurotoxicitytargeting glial responses and glia-derived cytokinesrdquo CurrentNeuropharmacology vol 9 no 2 pp 388ndash398 2011

[49] D Stellwagen and R CMalenka ldquoSynaptic scalingmediated byglial TNF-120572rdquo Nature vol 440 no 7087 pp 1054ndash1059 2006

[50] E Maronde M Pfeffer J Olcese et al ldquoTranscription factorsin neuroendocrine regulation rhythmic changes in pCREB andICER levels framemelatonin synthesisrdquo Journal of Neurosciencevol 19 no 9 pp 3326ndash3336 1999

[51] D M Bustos M J Bailey D Sugden et al ldquoGlobal dailydynamics of the pineal transcriptomerdquoCell and Tissue Researchvol 344 no 1 pp 1ndash11 2011

[52] A K Moorthy O V Savinova J Q Ho V Y-F Wang D VuandGGhosh ldquoThe 20S proteasome processesNF-120581B1 p105 intop50 in a translation-independent mannerrdquo EMBO Journal vol25 no 9 pp 1945ndash1956 2006

[53] L Lin G N DeMartino and W C Greene ldquoCotranslationalbiogenesis of NF-120581B p50 by the 26S proteasomerdquo Cell vol 92no 6 pp 819ndash828 1998

[54] H W L Ziegler-Heitbrock T Sternsdorf J Liese et alldquoPyrrolidine dithiocarbamate inhibits NF-120581B mobilization andTNF production in humanmonocytesrdquo Journal of Immunologyvol 151 no 12 pp 6986ndash6993 1993

[55] C E Carvalho-Sousa S S Cruz-Machado E K Tamura P AC M Fernandes L Pinato et al ldquoMolecular basis for definingthe pineal gland and pinealocytes as targets for tumor necrosisfactorrdquo Frontiers in Endocrinology vol 2 pp 1ndash11 2011

[56] M PMattson ldquoNF-120581B in the survival and plasticity of neuronsrdquoNeurochemical Research vol 30 no 6-7 pp 883ndash893 2005

[57] P A CM Fernandes E Cecon R PMarkus and Z S FerreiraldquoEffect of TNF-120572 on the melatonin synthetic pathway in the ratpineal gland basis for a rsquofeedbackrsquo of the immune response oncircadian timingrdquo Journal of Pineal Research vol 41 no 4 pp344ndash350 2006

[58] S-Y Tsai T E OrsquoBrien and J A McNulty ldquoMicroglia play arole in mediating the effects of cytokines on the structure andfunction of the rat pineal glandrdquo Cell and Tissue Research vol303 no 3 pp 423ndash431 2001

[59] J S Bains and S H R Oliet ldquoGlia they make your memoriesstickrdquo Trends in Neurosciences vol 30 no 8 pp 417ndash424 2007

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2 BioMed Research International

modulators that inhibit melatonin synthesis by decreasingthe gene expression and activity profile of both AANAT andhydroxyindole-O-methyltransferase (HIOMT) (EC 2114)Glutamate is stored in synaptic-like microvesicles in pinealo-cytes and is released upon cholinergic stimulation [13 14]The interaction of glutamate with AMPA-type ionotropicreceptors in the pinealocytes induces greater glutamate exo-cytosis amplifying the cholinergic action [15 16] Recentlyan investigation demonstrated that the glutamate vesiculartransporter could be responsible for pinealocyte depolariza-tion and glutamate release [17] The final effect of glutamateresults from its binding to mGluR3 a class II metabotropicglutamate receptor which is coupled to the inhibitory G-protein (Gi) leading to reductions in the amount of cAMPand the synthesis of melatonin [12]

Studies have shown the presence of many types ofglutamate receptors in the pineal gland In addition to theknown AMPA and mGluR3 (40 42) expression in the pinealgland Yatsushiro and colleagues showed that rat pinealocytesexpress mGluR5 [18] a class I metabotropic receptor relatedto inositol triphosphate (IP

3) formation and intracellular

calcium mobilization whose role on pineal metabolism hasnot yet been defined The presence of glutamate receptors inpineal astrocytes has also been demonstrated NMDA andAMPA ionotropic receptors were characterized in the ratpineal gland [19] while mGluR2 mGluR3 andmGluR5 wereidentified in the gerbil pineal gland [20] These data mayindicate that glutamatergic signaling in the pineal gland ismore complex than previously thought and involves bothastrocytes and pinealocytes similar to the pattern observedin the central nervous system (CNS)

Indeed in the CNS astrocytes are important partners atthe synapses Over the past decade an increasing number ofobservations have progressively challenged the classical viewthat astrocytes are passive participants in synaptic functionIn fact there is a dynamic two-way communication betweenglia and neurons called tripartite synapse inwhich astrocyticprocesses are associated with the presynaptic and postsy-naptic neuronal elements [21] Neurotransmitters released bypresynaptic neurons evoke a calcium increase in adjacentastrocytes which in turn can release several transmitterscalled gliotransmitters including glutamate D-serine ATPand large molecules like atrial natriuretic factor and tumornecrosis factor-120572 (TNF-120572) [21ndash24] These gliotransmittersfeed back onto the presynaptic neuron either to enhance orto depress further release of the neurotransmitters [25] orthey can act on postsynaptic neurons causing excitatory orinhibitory responses [26]

Glutamate signaling in the CNS is classically relatedto the activation of nuclear factor 120581B (NF-120581B) Thus glu-tamate induces the transcription of genes such as nitricoxide synthase cyclooxygenase 2 Mn-superoxide dismu-tase calbindin glutamate receptors Bcl-2 and brain-derivedneurotrophic factor [27ndash29] Additionally recent studieshave shown that astrocytes use NF-120581B as a mediator oftranscriptional responses to a variety of stimuli includingtissue injury and diseases [30] NF-120581B can induce or repressgene expression by binding to DNA sequences known as

120581B elements [31 32] Activation of NF-120581B typically involvesthe phosphorylation of proteins that inhibit NF-120581B (I120581Bs)by the I120581B kinase (IKK) complex which results in I120581Bdegradation [33] NF-120581B is then released from the inhibitorycomplex and translocated to the nucleus In mammaliancells there are five NF-120581B family membersmdashRel A (p65) RelB c-Rel p50p105 (NF-120581B1) and p52p100 (NF-120581B2)mdashanddifferent NF-120581B complexes are formed by diverse homo- andheterodimerization processes [34 35] In the pineal glandtwo independent studies showed that NF-120581B is involved inthe inflammatory response and exhibits a circadian rhythm[36 37]

Based on these data we aimed to investigate the inhib-itory action of glutamate on melatonin synthesis analyzingthe crosstalk between astrocytes and pinealocytes NF-120581Bactivation glutamate receptor expression and intracellularcalcium responses were analyzed in both cell types In thepresent work we demonstrated that the paracrine interac-tions between pinealocytes and astrocytes are fundamentalfor the glutamate inhibitory effect on melatonin synthesisand that it involves the activation of astrocytic NF-120581B tran-scription factor and the release of a soluble factor possiblyTNF-120572

2 Materials and Methods

21 Animals Male Wistar rats (200ndash220 g) were kept undera 12 12 h light-dark cycle (lights on at 06 00 h) in atemperature-controlled room (21plusmn 2∘C) with water and foodad libitum The animals were euthanized by decapitationbetween 09 00 and 10 00 h All experimentswere performedin accordance with the guidelines of the Brazilian Collegefor Animal Experimentation (COBEA) and approved bythe Committee of Ethics in Animal Experimentation of theInstitute of Biomedical Sciences (CEUAmdashPermit number091) University of Sao Paulo (Sao Paulo Brazil)

22 Pineal Gland Culture Pineal glands were cultured aspreviously described [38] Briefly after decapitation the ratpineal glands were removed and immediately placed in ice-cold BGJb medium (Fitton-Jackson Modification GibcoGrand Island NY USA) with phenol red modified by theaddition of bovine serum albumin (1mgmL) 2mM glu-tamine 01mgmL ascorbic acid and penicillin (100UmL)-streptomycin (100120583gmL) Pineal glands were cultivated(37∘C 95 O

2 5 CO

2) in the same BGJb medium in

24-well plates (2 glandswell 200120583Lwell) for 48 h (themedium was changed after the first 24 h) After this periodall glands were placed in fresh medium for 1 h beforetreatment For melatonin analysis the glands were incubatedwith a combination of the following drugs norepinephrine(NE) (1120583M) glutamate (100 and 600 120583M) and pyrrolidinedithiocarbamate (PDTC) (300 120583M) After 5 h the glandswere frozen on dry ice and kept at minus80∘C until they wereassayed All culture media components and drugs used werepurchased from Sigma (St Louis MO USA)

BioMed Research International 3

23 Isolated Pinealocyte Culture Pinealocytes were obtainedby papain digestion (Papain Dissociation System Wor-thington Biochemical Corporation Freehold NJ USA) Theglands were removed and immediately placed in ice-coldDulbeccorsquos Modified Eaglersquos Medium (DMEM) (glucose1000mgL HEPES 59 g and sodium bicarbonate 37 g)(Sigma St Louis MO USA) The tissue was incubatedat 37∘C for 45min in papain (001) and DNase (001)solution After removal of papain and its blockage withovomucoid (2mgmL) the cells were mechanically dispersedand resuspended in DMEM supplemented with 10 fetal calfserum and 1 penicillin-streptomycin to a final concentra-tion of 2 times 105 cellsmL Fifteen milliliters of cells in culturemedium was cultivated in 75 cm2 culture flasks at 37∘C in 5CO295 air for 24 h After 24 h the cells in suspension were

predominantly pinealocytes which were separated after theremoval of all the culture medium content The cells werecentrifuged and resuspended in DMEM at a concentrationof 2 times 105 cellsmL transferred to a 24-well plate (1mL perwell) and kept at 37∘C in 5 CO

295 air for 1 h prior to the

pharmacological treatments For melatonin determination inthe culturemedium the cells were stimulated withNE (1 120583M)alone or in associationwith glutamate (100 or 600120583M) for 5 h

24 Isolated Astrocyte Culture After the removal of thepinealocytes in suspension the astrocytes remained attachedto the culture flasks and were kept in culture for one weekAfter this period the cells were washed twice with Hankssolution and 6mL of trypsin 025 was added for 5minTrypsin action was blocked with DMEM supplemented with10 fetal calf serum The cells were centrifuged at 300 g for5min at 20∘C The pellet was resuspended according to thedifferent assays

25 Pinealocyte and Astrocyte Coculture After the dissocia-tion of the pineal gland cells by papain digestion pinealocytesand astrocytes were cocultivated in the same well (24-wellplates) in DMEM medium with 10 fetal calf serum at 37∘Cin 5 CO

295 air at a concentration of 4 times 105 cellsmL

(1mL per well) for 60 h After that the medium was replacedby fresh medium without fetal calf serum and the cellswere kept for 1 h at 37∘C in 5 CO

295 air prior to the

pharmacological treatments For melatonin determination inthe culturemedium the cells were stimulated withNE (1 120583M)alone or in associationwith glutamate (100 or 600120583M) for 5 hWhenBB1101 (10120583M) [39] the inhibitor of TNF-120572 convertingenzyme (TACE ADAM17) was used it was added to themedium 30min before the treatments described previously

The cells in co-culture were also physically separated byinserts (transwell with 04120583mpore Corning NY USA) withastrocytes in the bottom of the wells and pinealocytes withinthe inserts The incubation procedures described previouslywere reproduced

26 Cell Culture Characterization by Immunocytochem-istry Suspensions of pineal gland cultured cells (astrocytespinealocytes or co-culture) were washed twice in phosphatebuffer (PB 01M pH 74) for medium removal and fixed

with 2 paraformaldehyde in PB for 15min After twoadditional washes in PB cells were spread upon gelatin-and chromoalumen-coated slides and allowed to dry andadhere using a hot plate (37∘C) Cells were then incubatedwith mouse monoclonal antibodies either against the glialfibrillary acidic protein (GFAP Sigma USA) or CR3 com-plement receptor (OX-42 BD Biosciences USA) diluted1 1000ndash1 2000 in PB containing 001 Triton X-100 in awet chamber for 14ndash18 h at room temperature Following 3 times10min washes in PB cells were then incubated with a wheatgerm agglutinin-rhodamine label (WGA Vector Laborato-ries USA) mixed with a donkey anti-mouse IgG conjugatedto fluorescein isothiocyanate (Jackson Labs USA) diluted1 200 and 1 100 respectively for 2 h Slideswere thenwashed3 times 10min and coverslipped with glycerol-carbonate bufferThe material was analyzed using a fluorescence microscopeequipped with standard filters and digital images werecollected and mounted using Adobe Photoshop software(Adobe Systems Inc USA) Controls for the experimentsconsisted of the omission of primary antibodies or agglutininfrom the procedure No staining was observed in these cases

27 Intracellular Calcium Measurement Isolated astrocytesand pinealocytes were obtained as described previously Thecells were placed on a thin glass coverslip and incubated inDMEMwith 16 nMof Fluo-4 (Molecular Probes Inc EugeneOR USA) kept at 37∘C for 1 h and then washed three timeswith the same medium Isolated astrocytes and pinealocyteswere treated with glutamate (600 120583M) or glutamatergicagonists (N-methyl-D-aspartic acidmdashNMDA 100 120583M120572-amino-3-hydroxyl-5-methyl-4-isoxazole-propionatemdashAMPA 50 120583M (S)-35-dihydroxyphenylglycinemdashDHPG50120583M) (Tocris Bioscience Ellisville MO USA) A laserscanning microscope (LSM 510 Carl Zeiss Jena Germany)equipped with the Attofluor ratio imaging system (AttoInstruments Rockville MD USA) was used Measurementswere performed using an excitation filter of 505 nm and theemission was monitored at 530 nm

28 Determination of Melatonin Content Melatonin con-tent in pineal glands and culture medium was determinedby high performance liquid chromatography (HPLC) withelectrochemical detection using Empower software (WatersSystem Milford MA USA) [38] Melatonin was separatedon a resolve C18 column (5 120583m 150 times 39mm) The chro-matographic system was isocratically operated with thefollowing mobile phase 01M sodium acetate 01M citricacid and 015mM EDTA 30 methanol pH 37 at a flowrate of 1mLmin The electrochemical detector potential wasadjusted to +900mV The elution time for melatonin wasapproximately 6min Each gland was sonicated (MicrosonXL 2005 Heat System Inc Farmingdale NY USA) in asolution of 01M perchloric acid containing 002 EDTAand 002 sodium bisulfate Culture medium was dilutedin the same solution of perchloric acid (1 1 volvol) Aftercentrifugation (2min 13000 g) 40120583L of the supernatantwas injected into the chromatographic system (InjectorMod7125 20120583L loop Rheodyne Inc San Francisco CA USA)

4 BioMed Research International

All the salts and reagents used were obtained from Merck(Frankfurter Darmstadt Germany)

29 Electrophoretic Mobility Shift Assay

291 Pineal Gland Nuclear Protein Extracts Nuclear extractswere obtained from pineal gland homogenates as previouslydescribed [37] after incubation for 30min with or withoutglutamate (600 120583M) Briefly the glands were homogenizedin lysis buffer (10mM HEPES 10mM KCl 01mM EDTA10 glycerol and 01mM PMSF) and after 15min nonylphenoxypolyethoxylethanol (NP)-40 (10) was added Thesamples were vigorously mixed centrifuged (12000 g 1min4∘C) twice and resuspended in extraction buffer (10mMHEPES 05M KCl 1mM EDTA 10 glycerol and 01mMPMSF) After incubation on ice for 15min and centrifugation(20000 g 5min 4∘C) the supernatant containing the nuclearproteins was collected and stored at minus80∘C Protein concen-tration was determined using the BioRad protein reagent(BioRad Hercules CA USA)

292 Astrocytes and Pinealocytes Nuclear Protein ExtractsNuclear extracts were obtained from isolated astrocytes andpinealocytes homogenates as previously described [40] afterincubation for 30min with or without glutamate (600120583M)Briefly the cells were homogenized in lysis buffer (10mMHEPES 15mM MgCl

2 10mM KCl 05mM PMSF 01mM

EDTA and 3mMorthovanadate) After addingNP-40 (10)sampleswere vigorouslymixed centrifuged (11000 g 20min4∘C) twice and resuspended in extraction buffer (20mMHEPES 15mMMgCl

2 300mMNaCl 025mM EDTA 25

glycerol 05mM PMSF 3mM orthovanadate 2 120583gmL leu-peptin and 2 120583gmL antipain) After a 20min incubation onice and centrifugation (11000 g 20min 4∘C) the supernatantcontaining the nuclear proteins was collected and stored atminus80∘C The protein concentration was determined using theBioRad protein reagent

293 Gel Shift Assay An electrophoretic mobility shift assayfor NF-120581B was performed utilizing the gel shift assay kitfrom Promega (Madison WI USA) as described previ-ously [41] A 32P-NF-120581B double-stranded consensus oligonu-cleotide probe (51015840-AGTTGAGGGGACTTTCCCAGGC-31015840)and nuclear extracts were used DNA-protein complexeswere separated by electrophoresis through a 6 non-denaturing acrylamidebisacrylamide (375 1) gel in 053Tris-borate-EDTA (TBE) for 2 h at 150V Gels were vac-uum dried and analyzed by autoradiography For compe-tition experiments NF-120581B and transcription initiation fac-tor II (TFIID 51015840-CAGAGCATATAAGGTGAGGTAGGA-31015840)unlabeled double-stranded consensus oligonucleotide wereincluded in ten-fold excess of the molar concentration of 32P-NF-120581B probe to detect specific and nonspecific DNA-proteininteractions respectively Unlabeled oligonucleotides wereadded to the reaction mixture 20min before the addition ofthe radioactive probeThe composition of the complexes wasdetermined by supershift assays adding antibodies (SantaCruzBiotechnologies SantaCruz CAUSA) against different

NF-120581B subunits (p50 p52 c-Rel and p65 1 20 dilution)either before or after the incubation of nuclear extracts withthe labeled oligonucleotide

210 RNA Extraction and RT-PCR Total RNA was extractedfrom isolated astrocytes and pinealocytes usingTRIzol (Invit-rogen Carlsbad CA USA) according to the manufacturerrsquosinstructions cDNA synthesis was performed using Super-Script III Reverse Transcriptase (RT) (Invitrogen CarlsbadCA USA) from 1120583g of total RNA After the RT reaction 2-120583L aliquots were used as a cDNA template for polymerasechain reaction (PCR) amplifications using GoTaq DNAPolymerase (Promega Madison WI USA) The cyclingparameters used were 30 s at 95∘C 30 s at each primerrsquosannealing temperature and 30 s at 72∘C using a MyGeneSeries Gradient Thermocycler (LongGene China) PCRproducts were separated on 12 ethidium bromide-agarosegels and the bands were visualized by digital scanning Theprimers used in this assay are presented in supplementaryTable 1 (see Table 1 in Supplementary Material available athttpdxdoiorg1011552013618432)

211 Flow Cytometry Pineal glands in culture were stimu-lated with NE 1 120583M in the absence or presence of glutamate100 120583M or 600120583M After 5 h of incubation the cells weredissociated via proteolytic digestion with papain In an assayof membrane integrity 50120583L of a propidium iodide solution(20120583g permL in saline buffer)was added to the cells (500120583L)Propidium iodide is a highly water-soluble fluorescent com-pound that cannot pass through intact membranes as it isunable to enter viable cells After membrane disruption itbinds to DNA by intercalating between the bases with littleor no sequence preference After 5min of incubation at roomtemperature the cells were evaluated in FACScalibur flowcytometer equipment (BectonDickinson San Jose CAUSA)using Cell Quest software (Becton Dickinson San Jose CAUSA) Fluorescence was measured using the FL2 channel(orange-red fluorescencemdash58542 nm) DNA fragmentationwas analyzed after DNA staining with propidium iodideaccording to the previously described method [42] Cellswere resuspended in a solution with detergents (300120583Lhypotonic solution containing 20120583gmL propidium iodide01 sodium citrate and 01TritonX-100) that permeabilizethe cells which promptly incorporate the dye into DNAThe pellet was gently resuspended and the cells were thenincubated for 2 h at room temperature Fluorescence wasmeasured and analyzed by flow cytometry as describedpreviously

212 Statistical Analysis Data were always presented asthe mean plusmn SEM Statistical analyses (GraphPad Prism 50software San Diego CA USA) were performed using one-way ANOVA followed by a Bonferroni post hoc test Whenappropriate Studentrsquos 119905-test was applied The graphs relatedto confocal microscopy were prepared utilizing Origin 50software (MicroCal Software Inc Northampton MA USA)Autoradiographs of NF-120581Bwere quantified using ScionImagesoftware (Scion Corp Frederick MD USA)

BioMed Research International 5

GFAP

(a)

WGA

(b)

GFAP

(c)

WGA

50120583M

(d)

Figure 1 Cell cultures immunocytochemistry Digital images of isolated cultures of astrocytes (a) and pinealocytes (b) showing specific celltype staining using a mouse monoclonal antibody against the glial fibrillary acidic protein (GFAP) and a wheat germ agglutinin-rhodaminelabel (WGA) to identify pinealocytes The pictures at the bottom illustrate the distribution of glial cells (c) and pinealocytes (d) in a mixedcoculture The insets show photomicrographies of the cell cultures in contrast phase microscopy astrocytes (a) pinealocytes (b) and co-culture (c and d) In isolated pinealocytes and in co-culture many cells are clustered Magnification 200x (astrocytes and pinealocytes) and100x (co-culture)

3 Results

The cultures used in this study were characterized and val-idated by immunostaining with GFAP a specific marker forastroglial cells and OX-42 a marker for microglia (specificfor activated microglia as the antibody reacts with the CR3complement) This OX-42 antibody is suitable for pinealgland staining because the predominant type of microglia inthis tissue expresses complement type 3 receptors [43] WGAwas used as a pinealocyte marker because it labels photore-ceptors [44 45] and pinealocytes aremodified photoreceptorcells [46 47]This technique proved to be efficient in labelingthese pineal cells (Figure 1)

Cultured isolated astrocytes showed fluorescence forGFAP (Figure 1(a)) but not for OX-42 and WGA (data notshown) Cultured pinealocytes lacked immunostaining forthe two glial markers GFAP and OX-42 (data not shown)and reacted withWGA demonstrating the absolute predom-inance of pinealocytes (Figure 1(b)) Figures 1(c) and 1(d)show the distribution of pinealocytes and astrocytes in themixed culture (co-culture) The round appearance of thelabeled cells in Figure 1 different from that in the insets is dueto the fixation process with paraformaldehyde In Figure 1(d)pinealocytes appear smaller than those in Figure 1(b) most

likely due to the use of trypsin to obtain the cells furtherdissociating the cell clusters

In intact cultured pineal glands the known effect ofglutamate in reducing NE-stimulated melatonin synthesis(119875 lt 005) was observed (Figure 2(a)) On the other handwhen isolated cultured pinealocytes were incubated withglutamate (100 and 600 120583M) in the presence of NE (1 120583M)this effect was abrogated (Figure 2(b)) Glutamatersquos inhibitoryeffect on melatonin synthesis as observed in intact culturedpineal glands was only reproduced when pinealocytes werecultivated in the presence of astrocytes in co-culture (119875 lt001) (Figure 2(c))

To investigate if the contact between astrocytes andpinealocytes is necessary for the inhibitory effect of gluta-mate the two cell types were cocultivated in the same wellbut separated by inserts It was observed that glutamatersquosinhibitory effect on NE-stimulated melatonin synthesis wasexpressed in the same way as when the cells were cultivatedwith physical contact indicating that a diffusible factorreleased by astrocytes acted on the pinealocytes (119875 lt 001)(Figure 2(d))

The possibility of TNF-120572 as a gliotransmitter that medi-ates the glutamate effect was tested using BB1101 a preferredinhibitor of metalloproteinases with sheddase activity that

6 BioMed Research International

18

15

12

9

6

3

0

Mela

toni

n (n

ggl

and)

lowast

NE 1120583MGlu 100120583MGlu 600120583M

+ +

+

+

+

minus minus

minus minus

(a)

0

Mela

toni

n (n

gm

L)

NE 1120583MGlu 100120583MGlu 600120583M

+ +

+

+

+

minus minus

minus minus

120

80

40

(b)

Mela

toni

n (n

gm

L)

NE 1120583MGlu 100120583MGlu 600120583M

+ +

+

+

+

minus minus

minus minus

50

40

30

20

10

0

lowastlowast lowastlowast

(c)

NE 1120583MGlu 600120583M

Mela

toni

n (n

gm

L)

+ +

+minus

lowastlowast

100

80

60

40

20

0

(d)

Figure 2 Glutamate effects on melatonin synthesis (a) Pineal gland culture stimulated with norepinephrine (NE 1120583M) in the absence orpresence of glutamate (Glu 100 and Glu 600120583M) Melatonin content is expressed in nggland as the mean plusmn SEM of 3 experiments One-wayANOVA Bonferroni multiple comparison test lowast119875 lt 005 versus NE 1 120583M 119899 = 12 for each group (b) Isolated rat pinealocyte culture underNE 1120583M stimulation in the absence or presence of Glu 100 or Glu 600120583M 119899 = 6 for each group (c) Pinealocyte and astrocyte co-culturestimulated with NE 1 120583M in the absence or presence of Glu 100 or Glu 600 120583M One-way ANOVA Bonferroni multiple comparison testlowastlowast119875 lt 001 versus NE 1 120583M 119899 = 8 for each group (d) Astrocytes and pinealocytes were cultured together but with physical separation by

inserts Astrocytes were cultivated in the bottom of the plate and pinealocytes were cultivated within the inserts and stimulated with NE1 120583M associated or not associated with Glu 600 120583M 119879 test lowastlowast119875 lt 001 119899 = 4 for each group For the cell cultures melatonin was evaluatedin the culture medium and expressed in ngmL as the mean plusmn SEM of 3 experiments

includes the TACE It was verified that when the cells inco-culture were incubated with BB1101 (10 120583M) prior to thetreatments with glutamate (600 120583M) and NE (1 120583M) it wasable to completely revert the inhibitory effect of glutamate onmelatonin synthesis (119875 lt 005) (Figure 3)

RT-PCR analyses showed mRNA expression of classIII metabotropic glutamate receptors (mGluR1mGluR5 andmGluR2mGluR3 resp) as well as ionotropic NMDA andAMPA-type receptors (GluN1 GluA1 GluA2 and GluA3subunits) in astrocytes and pinealocytes (Figures 4(a) and4(b)) Contrarily mGluR7 mRNA (class III metabotropicreceptors) was not detected in any of the cell types(Figure 4(a)) To determine whether class I mGluRs NMDA

and AMPA ionotropic receptors are functional and incor-porated into the astrocytic and pinealocytic membranes anintracellular calcium analysis by confocal microscopy wasused It was observed that the class I metabotropic receptoragonist DHPG (50120583M) and the ionotropic receptor agonistsNMDA (100120583M) and AMPA (50 120583M) were able to increasethe astrocytes and pinealocytes intracellular calcium contentin a similar way as that observed for glutamate (600 120583M)(Figure 5 Table 1)

Electrophoretic mobility shift assays revealed that gluta-mate (100 and 600120583M) activates NF-120581B in cultured rat pinealglands The result of the competition study showed that theupper complex 1 was displaced by an excess of unlabeled

BioMed Research International 7

60

40

20

0NE 1120583M

Mela

toni

n (n

gm

L)

lowast

+ +

+ +

+

+

+

+minus minus

minus minus

lowastlowast

BB1101 10120583MGlu 600 120583M

Figure 3 (a) Ablation of the inhibitory glutamatergic effect onmelatonin synthesis by the TACE inhibitor BB1101 Pinealocytesand astrocytes were maintained in co-culture and stimulated withnorepinephrine (NE 1 120583M) in association with glutamate (Glu600 120583M) and BB1101 (10120583M) One-way ANOVA Bonferroni mul-tiple comparison test lowastlowast119875 lt 001 versus NE 1 120583M lowast119875 lt 005 versusNE 1 120583M + Glu 600 120583M 119899 = 4 for each group 3 experiments

Table 1 Summary of intracellular [Ca2+] changes in astrocytes andpinealocytes after stimulation

Cell typeagents Number of cellsexamined [Ca2+]

119894response

Isolated astrocytesGlu 600 120583M 68 79AMPA 50 120583M 97 80NMDA 100 120583M 49 90DHPG 50 120583M 80 74

Isolated pinealocytesGlu 600 120583M 66 80AMPA 50 120583M 78 85NMDA 100 120583M 53 90DHPG 50 120583M 60 76

Intracellular [Ca2+] in fluo-4-loaded astrocytes and pinealocytes was mea-sured as described in Section 2 The majority of the stimulated cellsresponded with an increase in intracellular [Ca2+] after the addition of eitherglutamate (600120583M) ionotropic glutamatergic agonists (AMPA 50120583M andNMDA 100120583M) or a class I metabotropic glutamatergic agonist (DHPG50120583M)

NF-120581B but not by a TFIID double-stranded oligonucleotideconsensus sequence demonstrating the specificity of the NF-120581BDNA interactionThe lower complex 2 was less efficientlydisplaced by unlabeledNF-120581B probe (Figure 6(a)) Supershiftanalysis indicated that the antibody against the p50 subunitwas able to shift the DNAprotein interaction present incomplex 1 The presence of antibodies against the p52 p65and c-Rel subunits did not affect DNA-protein complexes(Figures 6(b) and 6(c)) Taken together these results indicatethat at these concentrations glutamate activates the p50p50

homodimer Complex 2 was not displaced by the antibodiesand was not considered to be related to the NF-120581B family

Astrocyte and pinealocyte isolated cultures were used todetermine the cell type in which NF-120581B was being activatedGlutamate (600120583M)was shown to activateNF-120581B exclusivelyin astrocytes and not in pinealocytes (119875 lt 001) (Figure 7)

The involvement of NF-120581B activation in glutamatersquosinhibitory effect on melatonin synthesis was analyzed usinga specific inhibitor of NF-120581B PDTC PDTC (300 120583M) pre-vented the inhibitory effect of glutamate in cultured rat pinealglands stimulated by NE (1 120583M) (119875 lt 001) (Figure 8)

Glutamate cytotoxicity was evaluated because it is knownthat this neurotransmitter in high concentrations couldinduce cell death by cytotoxicity [48] Cell membraneintegrity and DNA fragmentation were assessed by flowcytometric analysis using propidium iodide (PI) Supple-mentary Figure 1 is representative of the results obtainedafter cell incubation with NE (1120583M) in the absence (controlgroup) or presence of glutamate (100 and 600120583M) for 5 hThe histograms show the fluorescence intensity in the FL-2channel which corresponds to the PI emission wavelengthWhen the cell membrane is intact the fluorescence signal islow and is related to autofluorescence (M1) A high signalmeans that PI was able to enter the cell and bind to DNA(M2) Glutamate treatments for 5 h were not able to inducea loss of membrane integrity (Supplementary Figure 1(a))The percentage of cells with ruptured membranes in thecontrol group was 453 whereas it was 502 and 577 inthe 100 and 600 120583M glutamate-treated groups respectively(Supplementary Figure 1(c)) Histograms in SupplementaryFigure 1(b) represent the results of DNA fragmentationanalysis After PI treatment the DNA content in live cellspresented a high fluorescence signal (M2) DNA fragmenta-tion induces the formation of small DNA pieces that exhibitlow fluorescence intensity (M1) The percentages of cellswith DNA fragmentation after glutamate stimulation 100and 600120583M were 286 and 335 respectively which weresimilar to the value of control cells (366) (SupplementaryFigure 1(d))

4 Discussion

Isolated pinealocytes isolated astrocytes and pineal-ocyteastrocyte co-culture were used in this study Theimmunocytochemistry characterization of the cell typespresent in these different cultures demonstrated the pre-dominance of pinealocytes in isolated pinealocyte culturewith the absence of glial cells In the same way astrocytecultures do not contain a significant number of microglialcells or pinealocytes In co-culture pinealocytes are thepredominant cells as astrocytes are present in minorquantities reproducing the proportion present in intactpineal glands (51) Thus the cultures were considered to beappropriate for this study

Data from the literature have shown that glutamate acts asan inhibitory neurotransmitter on melatonin synthesis [12]According to Yamada et al [13 14 16] and Yatsushiro et al[16] glutamate is stored in microvesicles and released from

8 BioMed Research International

mGluR1 mGluR2 mGluR3 mGluR5 mGluR7

M H2O As Pi Hi As Pi Hi As Pi Hi As Pi Hi As Pi Hi

200 bp100 bp

(a)

M H2O As Pi Hi As Pi Hi As Pi Hi As Pi Hi

200 bp100 bp

GluA1 GluA2 GluA3 GluN1

(b)

Figure 4 RT-PCR detection of metabotropic and ionotropic glutamate receptors in isolated astrocytes and pinealocytes (a) RT-PCRdetection of transcripts for mGluR1 (79 bp) mGluR2 (198 bp) mGluR3 (196 bp) mGluR5 (165 bp) and mGluR7 (70 bp) in astrocytes (As)pinealocytes (Pi) and hippocampus (Hi) Molecular weight marker (M) and water (H

2O) are presented in lanes 1 and 2 respectively (b)

RT-PCR detection of transcripts for GluA1 (203 bp) GluA2 (155 bp) GluA3 (193 bp) and GluN1 (201 bp) in astrocytes (As) pinealocytes(Pi) and hippocampus (Hi) The molecular weight marker (M) and PCR product generated with water (H

2O) are presented in lanes 1 and 2

respectively

pinealocytes upon cholinergic stimulation It interacts withionotropic and metabotropic glutamate receptors presentin pinealocyte membranes releasing additional glutamateand inhibiting melatonin synthesis respectively Recently amechanism involving the glutamate transporter GLT-1 wasproposed to be responsible for glutamate release [17] Inthese studies involving pinealocytes an autocrineparacrineprocess was suggested for the influence of glutamate onmelatonin synthesis In the presentworkwe demonstrated animportant role for astrocytes in glutamatersquos modulatory effecton melatonin synthesis that includes a paracrine interactionwith pinealocytes

Whereas in isolated pinealocytes glutamate does notexhibit the typical inhibitory effect on melatonin synthesisas observed in intact pineal glands its association withastrocytes restores this effect These results demonstrate thatastrocytes act as partners that collaborate to produce theglutamate effect In addition the fact that the effect occurredeven when inserts were used preventing the physical contactbetween the cells in co-culture suggests that a solublefactor is a mediator of this process The hypothesis that gapjunctions mediated the interactions between astrocytes andpinealocytes as occurs in the pineal gland [49] was excludedfrom this observationThe cytokine TNF-120572 a gliotransmitterthat is released by astrocytes could be the soluble factorthat is involved in the interactions between astrocytes andpinealocytes In fact the absence of a soluble form of TNF-120572 interferes with the glutamate response

The possibility that the glutamate effects were due to celldeath was investigated by flow cytometry As the cell viabilityand DNA fragmentation levels were similar between controland glutamate-stimulated groups showing no damage topineal cells even when treated with 600120583M of glutamate itis possible to exclude a glutamate cytotoxic effect as a causeof melatonin reduction

Although glutamate receptor expression had been firstdescribed in pinealocytes its expression was also laterdemonstrated in astrocytes mGluR3 and AMPA (GluA1subunit) were shown to be present in pinealocytes [12 16]

and NMDA and AMPA (GluA23) receptors were present inrat pineal gland astrocytes [19] Ionotropic receptor mRNAwas similarly observed in astrocytes in the present studyin addition to the novel observation of metabotropic recep-tor (mGluR1 mGluR2 mGluR3 mGluR5) expression Inpinealocytes the expression of mGluR1R2R3R5 AMPAand NMDA receptors was shownThis evidence supports thepossibility of astrocyte participation in rat pineal glutamater-gic signaling

Confocal microscopy assays using NMDA AMPA andDHPG agonists in isolated astrocytes and pinealocytesrevealed increased intracellular calcium content in both celltypes for all the agonists used PCR and confocal microscopyanalyses suggest both gene and protein expression in classI mGluR AMPA and NMDA ionotropic receptors reveal-ing the importance of the glutamate receptors as signaltransducers in rat pineal gland astrocytes and pinealocytesThus glutamate seems to interact with its receptors inboth cell types but its effect on melatonin synthesis nec-essarily involves astrocyte glutamate receptors The possibleparacrine interactions between pinealocytes and astrocytesseem to be the key in understanding the role of astrocytes inthe rat pineal gland

Transcription factors exert a great influence on pinealphysiology CREB and ICER are two transcription factorsthat intimately control the transcription of AANAT [50]Additionally there are other transcription factors in the ratpineal gland whose roles in pineal melatonin synthesis arenot known [51] NF-120581B is present in the rat pineal glandshowing a circadian rhythm and involvement in inflamma-tory responses [36 37] NF-120581B is a classic possible glutamatemediator and its activation in glial cells was demonstrated toplay a role in inflammatory processes [30]The present resultsdemonstrated NF-120581B activation by glutamate in culturedisolated astrocytes supporting its role as a mediator ofglutamate effects Supershift assay in cultured pineal glandsstimulated with glutamate showed the displacement of thep50 subunit emphasizing the importance of p50p50 homod-imer formationThis is an astrocyte-dependent event because

BioMed Research International 9

0 50 100 150 200 250 300 350

10

20

30

40

50

60

Time (s)

Isolated astrocytes

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

Glu 600120583M

(a)

0 30 60 90 120 150

155

160

165

170

175

180

185

Time (s)

Isolated pinealocytes

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

Glu 600120583M

(b)

0 100 200 300 400 500

10

20

30

Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

AMPA 50120583M

(c)

0 30 60 90 120 150 180

102

104

106

108

110

112

114

Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

AMPA 50120583M

(d)

0 50 100 150 200 250 300

400

600

800

1000

1200

1400

Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

NMDA 100120583M

(e)

0 50 100 150 200 250

900

1200

1500

Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

NMDA 100120583M

(f)

100 150 200 250

126

128

130

132

134

136

Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity) DHPG 50120583M

(g)

0 100 200 300 400

80

90

100

110

120

130

Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

DHPG 50120583M

(h)

Figure 5 Analysis of intracellular calcium in isolated astrocyte and pinealocyte cultures Traces of Fluo-4 fluorescence indicate theintracellular calcium transients from single cells Additions of exogenous glutamate or glutamatergic agonists were performed as indicatedby the arrows

10 BioMed Research International

ControlGlu 100120583MGlu 600120583MUnlabeled NF-120581BUnlabeled TFIID

1

(NS) 2

Free probe

+

minus

minus

minus

minus +

minus

minus

minus

minus

+

minus

minus

minus

minus +

+

minus

minus

minus +

+

minus

minus

minus+

+

minus

minus

minus

+

+

minus

minus

minus

(a)

(NS) 2

Free probe

(p50p50) 1

(NS) 3

+

+

minus

minus

minus

minus

+

minus

minus

minus

minus

minus

+

minus

minus

minus

minus

minus

+

+

minus

minus

minus

minus

+

+

minus

minus

minus

minus

+

+

minus

minus

minus

minus

ControlGlu 100120583MAnti p50Anti p52Anti p65Anti c-Rel

(b)

Free probe

Anti c-Rel

(NS) 2

(p50p50) 1

(NS) 3

ControlGlu 600120583MAnti p50Anti p52Anti p65

+minus minus minus minus minus

+

+ +

+

+ + +

+

+minus minus

minus

minus

minus

minus minus minus minus minus

minus

minus minus minus

minus minus

minus minus minus

minus minus

(c)

Figure 6 Electrophoretic mobility shift assays for NF120581B in the pineal gland (a) Competition studies were performed using nuclear extract(5120583g) from cultured pineal glands treated with glutamate (Glu) 100 and 600 120583M in the absence or presence of unlabeled specific (NF-120581Bconsensus sequence 10-fold molar excess) or nonspecific oligonucleotide (TFIID consensus sequence 10-fold molar excess) as indicated(b c) Supershift assays were performed with the same nuclear extract (5120583g) incubated in the absence or presence of antibodies against thesubunits p50 (1 10 dilution) p65 (1 20 dilution) p52 and c-Rel (1 10 dilution) as indicated Antibodies were added 20min prior to theaddition of the radiolabeled NF-120581B consensus oligonucleotide The position of specific NF-120581BDNA binding complexes p50p50 (band 1 bc) is indicated NS represents no specific binding (bands 2 and 3 a b c) The localization of the free probe is also indicated

BioMed Research International 11

ControlGlu 600120583M

minusminus

minus +

NF-120581B

(a)

20000

15000

10000

0Control Glu 600120583M

5000

lowastlowast

(au

)

(b)

ControlGlu 600120583M

minusminus

minus +

NF-120581B

(c)

(au

)

Control Glu 600120583M

16000

12000

8000

4000

0

(d)

Figure 7 Electrophoretic mobility shift assays for NF120581B in isolated astrocytes and pinealocytes (a) Nuclear proteins (5 120583g) were extractedfrom astrocytes treated with glutamate (Glu) 600120583M (b)Densitometric analysis (arbitrary units) of theNF-120581B band is presented on panel (a)One-way ANOVA Studentrsquos test lowastlowast119875 lt 001 versus control group mean plusmn SEM (c) Nuclear proteins (5120583g) were extracted from pinealocytestreated with Glu 600 120583M (d) Densitometric analysis (arbitrary units) of the NF-120581B band is presented in (c) The position of the specificNF-120581BDNA binding complex is indicated 119899 = 5 for each group 3 experiments

its activation was not observed in pinealocytes Astrocyteand pinealocyte supershift assays were not performed due totechnical limitations related to the amount of nuclear protein

The p50 subunit is synthesized from its precursor p105The processing of p105 and the resulting generation ofmature p50 subunits seem to be a constitutive event as p50homodimers are not regulated by I120581B proteins [34] The p50subunit formation seems to be regulated by cotranslationaland posttranslational mechanisms In the posttranslationalmechanism the p50 subunit is synthesized from its precursorp105 by phosphorylation at its 927 and 932 serine residueswhich allows the ubiquitinization and proteolytic degrada-tion of p105 by the 26S proteasome [52] In the cotranslationalmechanism p50 and p105 can be generated from a singlemRNA [53] There is also evidence that the 20S protea-some constitutively processes p105 to p50 in a ubiquitin-independent manner [52] The p50p50 homodimer wasoriginally considered to be a transcriptional repressor butnow there is substantial evidence that it can also inducegene transcription after a complex formation with the BCL3nuclear protein an atypical member of the I120581B family ofproteins that is considered to be a coactivator of p50p50homodimers [34]

The link between NF-120581B activation by glutamate and thereduction of melatonin synthesis was evidenced with the useof PDTC a NF-120581B translocation inhibitor [54] PDTC pre-vented glutamatersquos inhibitory effect on melatonin synthesisTaken together the crucial presence of astrocytes for theinhibitory effect of glutamate the exclusive activation of NF-120581B by glutamate in the same cell type and the demonstrationof NF-120581Brsquos role inmelatonin synthesis indicate that astrocytesunder glutamate stimulation possibly release a factor thatpromotes a decrease in pinealocyte melatonin synthesisTNF-120572may be a good candidate Indeed TNF-R1 the TNF-120572 receptor that interacts with soluble TNF is present in thepineal gland reinforcing the importance of this signalingpathway [55] Acting as an inhibitor of TACE to prevent TNF-120572 release BB1101 was thus adequate in demonstrating the roleplayed byTNF-120572 In addition the hypothesis that the p50p50homodimer may induce TNF-120572 gene expression and releaseneeds to be clarified

In the CNS astrocytes are known to release gliotransmit-ters such as glutamate D-serine and ATP [21] Moreoverastrocytes can also synthesize and release proinflammatorycytokines in response to NF-120581B activation such as TNF-120572 and interleukin-1 and induce gene expression of iNOS

12 BioMed Research International

NE 1120583MGlu 100120583MGlu 600120583MPDTC 300120583M

Mela

toni

n (n

ggl

and)

20

15

10

5

0

lowastlowast

lowastlowast

+ +

+ + +

++

+

+

+

+

+ +

minus minus minusminus

minus minus minusminus

minus minus minus

Figure 8 Effect of PDTC an inhibitor of NF-120581B on melatoninsynthesis Pineal glands in culture were incubated with PDTC(300120583M) and stimulated by norepinephrine (NE 1120583M) in thepresence of glutamate (100 and 600120583M) andmelatonin content wasevaluated One-way ANOVA Bonferroni multiple comparison test119875 lt 001 versus NE 1 120583M lowastlowast119875 lt 001 versus NE + Glu 100 120583Mand NE + Glu 600 120583M plotted as the mean plusmn SEM 119899 = 12 for eachgroup 3 experiments

PinealocyteAstrocyte

NF-120581Bactivation

AMPANMDAmGluR15

Release of solublefactor

TNF-120572

Glu

TNF-120572 alone (recruiting AMPA)or in association with Glu

mGluR3TNF-R1 AMPA[iCa+2]

Melatoninsynthesis

Figure 9 Diagram illustrating the putative paracrine interactionbetween pinealocytes and astrocytes in the modulation of pinealmelatonin synthesis by glutamate Glutamate (Glu) induces intra-cellular Ca2+ increase in astrocytes with the subsequent activationof NF120581B and release of a soluble factor possibly TNF-120572 This factoralone or in association with Glu promotes melatonin synthesisreduction in pinealocytes

resulting in nitric oxide formation [56] In this work wesuggested that TNF-120572 is the factor that is released byastrocytes under glutamate stimulation because the TACEinhibitor antagonized glutamatersquos effects Reinforcing thisidea recent preliminary data obtained in our laboratorywith the L929 tumor cell lineage which is sensitive to TNF-120572 showed that astrocytes release TNF-120572 when stimulatedby glutamate (data not shown) The literature states that

TNF-120572 decreases the serotonin content and AANAT mRNAexpression resulting in the reduction of N-acetylserotonin[57 58] the immediate precursor of melatonin synthesis Itis not clear whether TNF-120572 acts by itself or in combinationwith glutamate because there are glutamate receptors in thepinealocyte membrane [12 15 16 18] Recent studies havereported the control of hippocampal synapses by TNF-120572 dueto the increased insertion of AMPA receptors in the surfaceof the neuronal soma [59]The same effect could take place inthe pineal gland in which TNF-120572 released by the astrocytesin response to NF-120581B activation could increase the numberof AMPA receptors in the pinealocyte membrane resultingin the inhibition of melatonin synthesis

A model of the putative paracrine interactions betweenastrocytes and pinealocytes in the modulation of pinealmelatonin synthesis by glutamate is shown in Figure 9

5 Conclusions

In summary according to our results we propose thatglutamate acts on glutamate receptors (AMPA NMDAandor mGluR15) in astrocytes increasing the intracellularcalcium concentration promoting NF-120581B activation anddriving the release of a soluble factor possibly TNF-120572 whichacts independently or with glutamate in the pinealocytesresulting in inhibition of melatonin synthesis

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

This work was supported by a grant from FAPESP (0404328-1) Darine Villela was supported by a master fellowshipfrom FAPESP (0556943-4) Anderson Augusto Moutinhoand Victoria Fairbanks Atherino were supported by CNPq(PIBIC)

References

[1] J Calvo J Boya A Borregon and J E Garcia-MaurinoldquoPresence of glial cells in the rat pineal gland a light andelectronmicroscopic immunohistochemical studyrdquoAnatomicalRecord vol 220 no 4 pp 424ndash428 1988

[2] L Vollrath The Pineal Organ Springer New York NY USA1981

[3] R Y Moore J C Speh and J P Card ldquoThe retinohypothalamictract originates from a distinct subset of retinal ganglion cellsrdquoJournal of Comparative Neurology vol 352 no 3 pp 351ndash3661995

[4] D Sugden ldquoMelatonin biosynthesis in the mammalian pinealglandrdquo Experientia vol 45 no 10 pp 922ndash932 1989

[5] A F Afeche D CMVillelaM V Abrahao R Peres J Cipolla-Neto et al ldquoMelatonin and pineal glandrdquo in New Researchon Neurosecretory Systems E Romano Ed pp 151ndash177 NovaScience Publishers Hauppauge NY USA 2008

[6] J Arendt Melatonin and the Mammalian Pineal Gland Chap-man amp Hall London UK 1995

BioMed Research International 13

[7] M Ehret P Pevet and M Maitre ldquoTryptophan hydroxylasesynthesis is induced by 3101584051015840-cyclic adenosine monophosphateduring circadian rhythm in the rat pineal glandrdquo Journal ofNeurochemistry vol 57 no 5 pp 1516ndash1521 1991

[8] D C Klein G R Berg and J Weller ldquoMelatonin synthesisadenosine 3101584051015840-monophosphate and norepinephrine stimulateN-acetyltransferaserdquo Science vol 168 no 3934 pp 979ndash9801970

[9] D C Klein N L Schaad M A A Namboordiri L Yu and JL Weller ldquoControl of N-acetyltransferaserdquo Biochemical SocietyTransactions vol 20 no 2 pp 299ndash304 1992

[10] V Simonneaux and C Ribelayga ldquoGeneration of the melatoninendocrine message in mammals a review of the complexregulation of melatonin synthesis by norepinephrine peptidesand other pineal transmittersrdquoPharmacological Reviews vol 55no 2 pp 325ndash395 2003

[11] S Ishio H Yamada C M Craft and Y MoriyamaldquoHydroxyindole-O-methyltransferase is another target forL-glutamate-evoked inhibition of melatonin synthesis in ratpinealocytesrdquo Brain Research vol 850 no 1-2 pp 73ndash78 1999

[12] H Yamada S Yatsushiro S Ishio et al ldquoMetabotropic gluta-mate receptors negatively regulate melatonin synthesis in ratpinealocytesrdquo Journal of Neuroscience vol 18 no 6 pp 2056ndash2062 1998

[13] H Yamada A Yamamoto M Takahashi H Michibata HKumon and Y Moriyama ldquoThe L-type Ca2+ channel isinvolved in microvesicle-mediated glutamate exocytosis fromrat pinealocytesrdquo Journal of Pineal Research vol 21 no 3 pp165ndash174 1996

[14] H Yamada A Yamamoto S Yodozawa et al ldquoMicrovesicle-mediated exocytosis of glutamate is a novel paracrine-likechemical transduction mechanism and inhibits melatoninsecretion in rat pinealocytesrdquo Journal of Pineal Research vol 21no 3 pp 175ndash191 1996

[15] H Yamada A Ogura S Koizumi A Yamaguchi and YMoriyama ldquoAcetylcholine triggers L-glutamate exocytosis vianicotinic receptors and inhibits melatonin synthesis in ratpinealocytesrdquo Journal of Neuroscience vol 18 no 13 pp 4946ndash4952 1998

[16] S Yatsushiro H Yamada M Hayashi A Yamamoto andY Moriyama ldquoIonotropic glutamate receptors trigger micro-vesicle-mediated exocytosis of L-glutamate in rat pinealocytesrdquoJournal of Neurochemistry vol 75 no 1 pp 288ndash297 2000

[17] M-HKim SUehara AMuroyama BHille YMoriyama andD-S Koh ldquoGlutamate transporter-mediated glutamate secre-tion in the mammalian pineal glandrdquo Journal of Neurosciencevol 28 no 43 pp 10852ndash10863 2008

[18] S Yatsushiro H Yamada M Hayashi S Tsuboi and YMoriyama ldquoFunctional expression of metabotropic glutamatereceptor type 5 in rat pinealocytesrdquo NeuroReport vol 10 no 7pp 1599ndash1603 1999

[19] C Kaur V Sivakumar and E A Ling ldquoExpression ofN-methyl-D-aspartate (NMDA) and 120572-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) GluR23 receptors inthe developing rat pineal glandrdquo Journal of Pineal Research vol39 no 3 pp 294ndash301 2005

[20] H Pabst and P Redecker ldquoInterstitial glial cells of the gerbilpineal gland display immunoreactivity for the metabotropicglutamate receptors mGluR23 and mGluR5rdquo Brain Researchvol 838 no 1-2 pp 60ndash68 1999

[21] M M Halassa T Fellin and P G Haydon ldquoThe tripartitesynapse roles for gliotransmission in health and diseaserdquoTrends in Molecular Medicine vol 13 no 2 pp 54ndash63 2007

[22] S M Finkbeiner ldquoGlial calciumrdquo Glia vol 9 no 2 pp 83ndash1041993

[23] E A Newman ldquoNew roles for astrocytes regulation of synaptictransmissionrdquo Trends in Neurosciences vol 26 no 10 pp 536ndash542 2003

[24] J T Porter and K D McCarthy ldquoAstrocytic neurotransmitterreceptors in situ and in vivordquo Progress in Neurobiology vol 51no 4 pp 439ndash455 1997

[25] V Parpura T A Basarsky F Liu K Jeftinija S Jeftinija and PG Haydon ldquoGlutamate-mediated astrocyte-neuron signallingrdquoNature vol 369 no 6483 pp 744ndash747 1994

[26] T D Hassinger P B Atkinson G J Strecker et al ldquoEvidencefor glutamate-mediated activation of hippocampal neurons byglial calcium wavesrdquo Journal of Neurobiology vol 28 no 2 pp159ndash170 1995

[27] R H Lipsky K Xu D Zhu et al ldquoNuclear factor 120581B is a criticaldeterminant in N-methyl-D-aspartate receptor-mediated neu-roprotectionrdquo Journal of Neurochemistry vol 78 no 2 pp 254ndash264 2001

[28] M P Mattson C Culmsee Z Yu and S Camandola ldquoRoles ofnuclear factor 120581B in neuronal survival and plasticityrdquo Journal ofNeurochemistry vol 74 no 2 pp 443ndash456 2000

[29] C DMunhoz B Garcıa-Bueno J L MMadrigal L B LepschC Scavone and J C Leza ldquoStress-induced neuroinflammationmechanisms and new pharmacological targetsrdquo Brazilian Jour-nal of Medical and Biological Research vol 41 no 12 pp 1037ndash1046 2008

[30] S Camandola andM PMattson ldquoNF-ΚB as a therapeutic targetin neurodegenerative diseasesrdquo Expert Opinion on TherapeuticTargets vol 11 no 2 pp 123ndash132 2007

[31] B Kaltschmidt D Widera and C Kaltschmidt ldquoSignaling viaNF-120581B in the nervous systemrdquo Biochimica et Biophysica Actavol 1745 no 3 pp 287ndash299 2005

[32] P T Massa H Aleyasin D S Park X Mao and S W BargerldquoNF120581B in neurons the uncertainty principle in neurobiologyrdquoJournal of Neurochemistry vol 97 no 3 pp 607ndash618 2006

[33] H Hacker and M Karin ldquoRegulation and function of IKK andIKK-related kinasesrdquo Sciencersquos STKE vol 2006 no 357 p re132006

[34] S Beinke and S C Ley ldquoFunctions of NF-120581B1 and NF-120581B2 inimmune cell biologyrdquo Biochemical Journal vol 382 no 2 pp393ndash409 2004

[35] N D Perkins ldquoIntegrating cell-signalling pathways with NF-120581Band IKK functionrdquo Nature Reviews Molecular Cell Biology vol8 pp 49ndash62 2007

[36] E Cecon P A Fernandes L Pinato Z S Ferreira and R PMarkus ldquoDaily variation of constitutively activated nuclear fac-tor 120581B (NFKB) in rat pineal glandrdquoChronobiology Internationalvol 27 no 1 pp 52ndash67 2010

[37] Z S Ferreira P A C M Fernandes D Duma J Assreuy MC W Avellar and R P Markus ldquoCorticosterone modulatesnoradrenaline-induced melatonin synthesis through inhibitionof nuclear factor 120581Brdquo Journal of Pineal Research vol 38 no 3pp 182ndash188 2005

[38] S C Afeche R Barbosa J H Scialfa I M Terra A CCassola and J Cipolla-Neto ldquoEffects of the blockade of highvoltage-activated calcium channels on in vitro pineal melatoninsynthesisrdquo Cell Biochemistry and Function vol 24 no 6 pp499ndash505 2006

14 BioMed Research International

[39] B Barlaam T G Bird C Lambert-Van Der Brempt DCampbell S J Foster and R Maciewicz ldquoNew 120572-substitutedsuccinate-based hydroxamic acids as TNF120572 convertaseinhibitorsrdquo Journal of Medicinal Chemistry vol 42 no 23 pp4890ndash4908 1999

[40] I Glezer C D Munhoz E M Kawamoto T MarcourakisM C Werneck Avellar and C Scavone ldquoMK-801 and 7-Ni attenuate the activation of brain NF-120581B induced by LPSrdquoNeuropharmacology vol 45 no 8 pp 1120ndash1129 2003

[41] Y Rong and M Baudry ldquoSeizure activity results in a rapidinduction of nuclear factor-120581B in adult but not juvenile ratlimbic structuresrdquo Journal of Neurochemistry vol 67 no 2 pp662ndash668 1996

[42] I Nicoletti G Migliorati M C Pagliacci F Grignani and CRiccardi ldquoA rapid and simple method for measuring thymocyteapoptosis by propidium iodide staining and flow cytometryrdquoJournal of Immunological Methods vol 139 no 2 pp 271ndash2791991

[43] Y F Jiang-Shieh C H Wu M L Chang J Y Shiehand C Y Wen ldquoRegional heterogeneity in immunoreactivemacrophagesmicroglia in the rat pineal glandrdquo Journal ofPineal Research vol 35 no 1 pp 45ndash53 2003

[44] K-O Soderstrom ldquoLectin binding to the human retinardquoAnatomical Record vol 220 no 2 pp 219ndash223 1988

[45] F Uehara M Sameshima T Muramatsu and N OhbaldquoLocalization of fluorescence-labeled lectin binding sites onphotoreceptor cells of the monkey retinardquo Experimental EyeResearch vol 36 no 1 pp 113ndash123 1983

[46] H-W Korf ldquoThe pineal organ as a component of the biologicalclock phylogenetic and ontogenetic considerationsrdquo Annals ofthe New York Academy of Sciences vol 719 pp 13ndash42 1994

[47] E Maronde and J H Stehle ldquoThe mammalian pineal glandknown facts unknown facetsrdquo Trends in Endocrinology andMetabolism vol 18 no 4 pp 142ndash149 2007

[48] X-M Zhang and J Zhu ldquoKainic acid-induced neurotoxicitytargeting glial responses and glia-derived cytokinesrdquo CurrentNeuropharmacology vol 9 no 2 pp 388ndash398 2011

[49] D Stellwagen and R CMalenka ldquoSynaptic scalingmediated byglial TNF-120572rdquo Nature vol 440 no 7087 pp 1054ndash1059 2006

[50] E Maronde M Pfeffer J Olcese et al ldquoTranscription factorsin neuroendocrine regulation rhythmic changes in pCREB andICER levels framemelatonin synthesisrdquo Journal of Neurosciencevol 19 no 9 pp 3326ndash3336 1999

[51] D M Bustos M J Bailey D Sugden et al ldquoGlobal dailydynamics of the pineal transcriptomerdquoCell and Tissue Researchvol 344 no 1 pp 1ndash11 2011

[52] A K Moorthy O V Savinova J Q Ho V Y-F Wang D VuandGGhosh ldquoThe 20S proteasome processesNF-120581B1 p105 intop50 in a translation-independent mannerrdquo EMBO Journal vol25 no 9 pp 1945ndash1956 2006

[53] L Lin G N DeMartino and W C Greene ldquoCotranslationalbiogenesis of NF-120581B p50 by the 26S proteasomerdquo Cell vol 92no 6 pp 819ndash828 1998

[54] H W L Ziegler-Heitbrock T Sternsdorf J Liese et alldquoPyrrolidine dithiocarbamate inhibits NF-120581B mobilization andTNF production in humanmonocytesrdquo Journal of Immunologyvol 151 no 12 pp 6986ndash6993 1993

[55] C E Carvalho-Sousa S S Cruz-Machado E K Tamura P AC M Fernandes L Pinato et al ldquoMolecular basis for definingthe pineal gland and pinealocytes as targets for tumor necrosisfactorrdquo Frontiers in Endocrinology vol 2 pp 1ndash11 2011

[56] M PMattson ldquoNF-120581B in the survival and plasticity of neuronsrdquoNeurochemical Research vol 30 no 6-7 pp 883ndash893 2005

[57] P A CM Fernandes E Cecon R PMarkus and Z S FerreiraldquoEffect of TNF-120572 on the melatonin synthetic pathway in the ratpineal gland basis for a rsquofeedbackrsquo of the immune response oncircadian timingrdquo Journal of Pineal Research vol 41 no 4 pp344ndash350 2006

[58] S-Y Tsai T E OrsquoBrien and J A McNulty ldquoMicroglia play arole in mediating the effects of cytokines on the structure andfunction of the rat pineal glandrdquo Cell and Tissue Research vol303 no 3 pp 423ndash431 2001

[59] J S Bains and S H R Oliet ldquoGlia they make your memoriesstickrdquo Trends in Neurosciences vol 30 no 8 pp 417ndash424 2007

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BioMed Research International 3

23 Isolated Pinealocyte Culture Pinealocytes were obtainedby papain digestion (Papain Dissociation System Wor-thington Biochemical Corporation Freehold NJ USA) Theglands were removed and immediately placed in ice-coldDulbeccorsquos Modified Eaglersquos Medium (DMEM) (glucose1000mgL HEPES 59 g and sodium bicarbonate 37 g)(Sigma St Louis MO USA) The tissue was incubatedat 37∘C for 45min in papain (001) and DNase (001)solution After removal of papain and its blockage withovomucoid (2mgmL) the cells were mechanically dispersedand resuspended in DMEM supplemented with 10 fetal calfserum and 1 penicillin-streptomycin to a final concentra-tion of 2 times 105 cellsmL Fifteen milliliters of cells in culturemedium was cultivated in 75 cm2 culture flasks at 37∘C in 5CO295 air for 24 h After 24 h the cells in suspension were

predominantly pinealocytes which were separated after theremoval of all the culture medium content The cells werecentrifuged and resuspended in DMEM at a concentrationof 2 times 105 cellsmL transferred to a 24-well plate (1mL perwell) and kept at 37∘C in 5 CO

295 air for 1 h prior to the

pharmacological treatments For melatonin determination inthe culturemedium the cells were stimulated withNE (1 120583M)alone or in associationwith glutamate (100 or 600120583M) for 5 h

24 Isolated Astrocyte Culture After the removal of thepinealocytes in suspension the astrocytes remained attachedto the culture flasks and were kept in culture for one weekAfter this period the cells were washed twice with Hankssolution and 6mL of trypsin 025 was added for 5minTrypsin action was blocked with DMEM supplemented with10 fetal calf serum The cells were centrifuged at 300 g for5min at 20∘C The pellet was resuspended according to thedifferent assays

25 Pinealocyte and Astrocyte Coculture After the dissocia-tion of the pineal gland cells by papain digestion pinealocytesand astrocytes were cocultivated in the same well (24-wellplates) in DMEM medium with 10 fetal calf serum at 37∘Cin 5 CO

295 air at a concentration of 4 times 105 cellsmL

(1mL per well) for 60 h After that the medium was replacedby fresh medium without fetal calf serum and the cellswere kept for 1 h at 37∘C in 5 CO

295 air prior to the

pharmacological treatments For melatonin determination inthe culturemedium the cells were stimulated withNE (1 120583M)alone or in associationwith glutamate (100 or 600120583M) for 5 hWhenBB1101 (10120583M) [39] the inhibitor of TNF-120572 convertingenzyme (TACE ADAM17) was used it was added to themedium 30min before the treatments described previously

The cells in co-culture were also physically separated byinserts (transwell with 04120583mpore Corning NY USA) withastrocytes in the bottom of the wells and pinealocytes withinthe inserts The incubation procedures described previouslywere reproduced

26 Cell Culture Characterization by Immunocytochem-istry Suspensions of pineal gland cultured cells (astrocytespinealocytes or co-culture) were washed twice in phosphatebuffer (PB 01M pH 74) for medium removal and fixed

with 2 paraformaldehyde in PB for 15min After twoadditional washes in PB cells were spread upon gelatin-and chromoalumen-coated slides and allowed to dry andadhere using a hot plate (37∘C) Cells were then incubatedwith mouse monoclonal antibodies either against the glialfibrillary acidic protein (GFAP Sigma USA) or CR3 com-plement receptor (OX-42 BD Biosciences USA) diluted1 1000ndash1 2000 in PB containing 001 Triton X-100 in awet chamber for 14ndash18 h at room temperature Following 3 times10min washes in PB cells were then incubated with a wheatgerm agglutinin-rhodamine label (WGA Vector Laborato-ries USA) mixed with a donkey anti-mouse IgG conjugatedto fluorescein isothiocyanate (Jackson Labs USA) diluted1 200 and 1 100 respectively for 2 h Slideswere thenwashed3 times 10min and coverslipped with glycerol-carbonate bufferThe material was analyzed using a fluorescence microscopeequipped with standard filters and digital images werecollected and mounted using Adobe Photoshop software(Adobe Systems Inc USA) Controls for the experimentsconsisted of the omission of primary antibodies or agglutininfrom the procedure No staining was observed in these cases

27 Intracellular Calcium Measurement Isolated astrocytesand pinealocytes were obtained as described previously Thecells were placed on a thin glass coverslip and incubated inDMEMwith 16 nMof Fluo-4 (Molecular Probes Inc EugeneOR USA) kept at 37∘C for 1 h and then washed three timeswith the same medium Isolated astrocytes and pinealocyteswere treated with glutamate (600 120583M) or glutamatergicagonists (N-methyl-D-aspartic acidmdashNMDA 100 120583M120572-amino-3-hydroxyl-5-methyl-4-isoxazole-propionatemdashAMPA 50 120583M (S)-35-dihydroxyphenylglycinemdashDHPG50120583M) (Tocris Bioscience Ellisville MO USA) A laserscanning microscope (LSM 510 Carl Zeiss Jena Germany)equipped with the Attofluor ratio imaging system (AttoInstruments Rockville MD USA) was used Measurementswere performed using an excitation filter of 505 nm and theemission was monitored at 530 nm

28 Determination of Melatonin Content Melatonin con-tent in pineal glands and culture medium was determinedby high performance liquid chromatography (HPLC) withelectrochemical detection using Empower software (WatersSystem Milford MA USA) [38] Melatonin was separatedon a resolve C18 column (5 120583m 150 times 39mm) The chro-matographic system was isocratically operated with thefollowing mobile phase 01M sodium acetate 01M citricacid and 015mM EDTA 30 methanol pH 37 at a flowrate of 1mLmin The electrochemical detector potential wasadjusted to +900mV The elution time for melatonin wasapproximately 6min Each gland was sonicated (MicrosonXL 2005 Heat System Inc Farmingdale NY USA) in asolution of 01M perchloric acid containing 002 EDTAand 002 sodium bisulfate Culture medium was dilutedin the same solution of perchloric acid (1 1 volvol) Aftercentrifugation (2min 13000 g) 40120583L of the supernatantwas injected into the chromatographic system (InjectorMod7125 20120583L loop Rheodyne Inc San Francisco CA USA)

4 BioMed Research International

All the salts and reagents used were obtained from Merck(Frankfurter Darmstadt Germany)

29 Electrophoretic Mobility Shift Assay

291 Pineal Gland Nuclear Protein Extracts Nuclear extractswere obtained from pineal gland homogenates as previouslydescribed [37] after incubation for 30min with or withoutglutamate (600 120583M) Briefly the glands were homogenizedin lysis buffer (10mM HEPES 10mM KCl 01mM EDTA10 glycerol and 01mM PMSF) and after 15min nonylphenoxypolyethoxylethanol (NP)-40 (10) was added Thesamples were vigorously mixed centrifuged (12000 g 1min4∘C) twice and resuspended in extraction buffer (10mMHEPES 05M KCl 1mM EDTA 10 glycerol and 01mMPMSF) After incubation on ice for 15min and centrifugation(20000 g 5min 4∘C) the supernatant containing the nuclearproteins was collected and stored at minus80∘C Protein concen-tration was determined using the BioRad protein reagent(BioRad Hercules CA USA)

292 Astrocytes and Pinealocytes Nuclear Protein ExtractsNuclear extracts were obtained from isolated astrocytes andpinealocytes homogenates as previously described [40] afterincubation for 30min with or without glutamate (600120583M)Briefly the cells were homogenized in lysis buffer (10mMHEPES 15mM MgCl

2 10mM KCl 05mM PMSF 01mM

EDTA and 3mMorthovanadate) After addingNP-40 (10)sampleswere vigorouslymixed centrifuged (11000 g 20min4∘C) twice and resuspended in extraction buffer (20mMHEPES 15mMMgCl

2 300mMNaCl 025mM EDTA 25

glycerol 05mM PMSF 3mM orthovanadate 2 120583gmL leu-peptin and 2 120583gmL antipain) After a 20min incubation onice and centrifugation (11000 g 20min 4∘C) the supernatantcontaining the nuclear proteins was collected and stored atminus80∘C The protein concentration was determined using theBioRad protein reagent

293 Gel Shift Assay An electrophoretic mobility shift assayfor NF-120581B was performed utilizing the gel shift assay kitfrom Promega (Madison WI USA) as described previ-ously [41] A 32P-NF-120581B double-stranded consensus oligonu-cleotide probe (51015840-AGTTGAGGGGACTTTCCCAGGC-31015840)and nuclear extracts were used DNA-protein complexeswere separated by electrophoresis through a 6 non-denaturing acrylamidebisacrylamide (375 1) gel in 053Tris-borate-EDTA (TBE) for 2 h at 150V Gels were vac-uum dried and analyzed by autoradiography For compe-tition experiments NF-120581B and transcription initiation fac-tor II (TFIID 51015840-CAGAGCATATAAGGTGAGGTAGGA-31015840)unlabeled double-stranded consensus oligonucleotide wereincluded in ten-fold excess of the molar concentration of 32P-NF-120581B probe to detect specific and nonspecific DNA-proteininteractions respectively Unlabeled oligonucleotides wereadded to the reaction mixture 20min before the addition ofthe radioactive probeThe composition of the complexes wasdetermined by supershift assays adding antibodies (SantaCruzBiotechnologies SantaCruz CAUSA) against different

NF-120581B subunits (p50 p52 c-Rel and p65 1 20 dilution)either before or after the incubation of nuclear extracts withthe labeled oligonucleotide

210 RNA Extraction and RT-PCR Total RNA was extractedfrom isolated astrocytes and pinealocytes usingTRIzol (Invit-rogen Carlsbad CA USA) according to the manufacturerrsquosinstructions cDNA synthesis was performed using Super-Script III Reverse Transcriptase (RT) (Invitrogen CarlsbadCA USA) from 1120583g of total RNA After the RT reaction 2-120583L aliquots were used as a cDNA template for polymerasechain reaction (PCR) amplifications using GoTaq DNAPolymerase (Promega Madison WI USA) The cyclingparameters used were 30 s at 95∘C 30 s at each primerrsquosannealing temperature and 30 s at 72∘C using a MyGeneSeries Gradient Thermocycler (LongGene China) PCRproducts were separated on 12 ethidium bromide-agarosegels and the bands were visualized by digital scanning Theprimers used in this assay are presented in supplementaryTable 1 (see Table 1 in Supplementary Material available athttpdxdoiorg1011552013618432)

211 Flow Cytometry Pineal glands in culture were stimu-lated with NE 1 120583M in the absence or presence of glutamate100 120583M or 600120583M After 5 h of incubation the cells weredissociated via proteolytic digestion with papain In an assayof membrane integrity 50120583L of a propidium iodide solution(20120583g permL in saline buffer)was added to the cells (500120583L)Propidium iodide is a highly water-soluble fluorescent com-pound that cannot pass through intact membranes as it isunable to enter viable cells After membrane disruption itbinds to DNA by intercalating between the bases with littleor no sequence preference After 5min of incubation at roomtemperature the cells were evaluated in FACScalibur flowcytometer equipment (BectonDickinson San Jose CAUSA)using Cell Quest software (Becton Dickinson San Jose CAUSA) Fluorescence was measured using the FL2 channel(orange-red fluorescencemdash58542 nm) DNA fragmentationwas analyzed after DNA staining with propidium iodideaccording to the previously described method [42] Cellswere resuspended in a solution with detergents (300120583Lhypotonic solution containing 20120583gmL propidium iodide01 sodium citrate and 01TritonX-100) that permeabilizethe cells which promptly incorporate the dye into DNAThe pellet was gently resuspended and the cells were thenincubated for 2 h at room temperature Fluorescence wasmeasured and analyzed by flow cytometry as describedpreviously

212 Statistical Analysis Data were always presented asthe mean plusmn SEM Statistical analyses (GraphPad Prism 50software San Diego CA USA) were performed using one-way ANOVA followed by a Bonferroni post hoc test Whenappropriate Studentrsquos 119905-test was applied The graphs relatedto confocal microscopy were prepared utilizing Origin 50software (MicroCal Software Inc Northampton MA USA)Autoradiographs of NF-120581Bwere quantified using ScionImagesoftware (Scion Corp Frederick MD USA)

BioMed Research International 5

GFAP

(a)

WGA

(b)

GFAP

(c)

WGA

50120583M

(d)

Figure 1 Cell cultures immunocytochemistry Digital images of isolated cultures of astrocytes (a) and pinealocytes (b) showing specific celltype staining using a mouse monoclonal antibody against the glial fibrillary acidic protein (GFAP) and a wheat germ agglutinin-rhodaminelabel (WGA) to identify pinealocytes The pictures at the bottom illustrate the distribution of glial cells (c) and pinealocytes (d) in a mixedcoculture The insets show photomicrographies of the cell cultures in contrast phase microscopy astrocytes (a) pinealocytes (b) and co-culture (c and d) In isolated pinealocytes and in co-culture many cells are clustered Magnification 200x (astrocytes and pinealocytes) and100x (co-culture)

3 Results

The cultures used in this study were characterized and val-idated by immunostaining with GFAP a specific marker forastroglial cells and OX-42 a marker for microglia (specificfor activated microglia as the antibody reacts with the CR3complement) This OX-42 antibody is suitable for pinealgland staining because the predominant type of microglia inthis tissue expresses complement type 3 receptors [43] WGAwas used as a pinealocyte marker because it labels photore-ceptors [44 45] and pinealocytes aremodified photoreceptorcells [46 47]This technique proved to be efficient in labelingthese pineal cells (Figure 1)

Cultured isolated astrocytes showed fluorescence forGFAP (Figure 1(a)) but not for OX-42 and WGA (data notshown) Cultured pinealocytes lacked immunostaining forthe two glial markers GFAP and OX-42 (data not shown)and reacted withWGA demonstrating the absolute predom-inance of pinealocytes (Figure 1(b)) Figures 1(c) and 1(d)show the distribution of pinealocytes and astrocytes in themixed culture (co-culture) The round appearance of thelabeled cells in Figure 1 different from that in the insets is dueto the fixation process with paraformaldehyde In Figure 1(d)pinealocytes appear smaller than those in Figure 1(b) most

likely due to the use of trypsin to obtain the cells furtherdissociating the cell clusters

In intact cultured pineal glands the known effect ofglutamate in reducing NE-stimulated melatonin synthesis(119875 lt 005) was observed (Figure 2(a)) On the other handwhen isolated cultured pinealocytes were incubated withglutamate (100 and 600 120583M) in the presence of NE (1 120583M)this effect was abrogated (Figure 2(b)) Glutamatersquos inhibitoryeffect on melatonin synthesis as observed in intact culturedpineal glands was only reproduced when pinealocytes werecultivated in the presence of astrocytes in co-culture (119875 lt001) (Figure 2(c))

To investigate if the contact between astrocytes andpinealocytes is necessary for the inhibitory effect of gluta-mate the two cell types were cocultivated in the same wellbut separated by inserts It was observed that glutamatersquosinhibitory effect on NE-stimulated melatonin synthesis wasexpressed in the same way as when the cells were cultivatedwith physical contact indicating that a diffusible factorreleased by astrocytes acted on the pinealocytes (119875 lt 001)(Figure 2(d))

The possibility of TNF-120572 as a gliotransmitter that medi-ates the glutamate effect was tested using BB1101 a preferredinhibitor of metalloproteinases with sheddase activity that

6 BioMed Research International

18

15

12

9

6

3

0

Mela

toni

n (n

ggl

and)

lowast

NE 1120583MGlu 100120583MGlu 600120583M

+ +

+

+

+

minus minus

minus minus

(a)

0

Mela

toni

n (n

gm

L)

NE 1120583MGlu 100120583MGlu 600120583M

+ +

+

+

+

minus minus

minus minus

120

80

40

(b)

Mela

toni

n (n

gm

L)

NE 1120583MGlu 100120583MGlu 600120583M

+ +

+

+

+

minus minus

minus minus

50

40

30

20

10

0

lowastlowast lowastlowast

(c)

NE 1120583MGlu 600120583M

Mela

toni

n (n

gm

L)

+ +

+minus

lowastlowast

100

80

60

40

20

0

(d)

Figure 2 Glutamate effects on melatonin synthesis (a) Pineal gland culture stimulated with norepinephrine (NE 1120583M) in the absence orpresence of glutamate (Glu 100 and Glu 600120583M) Melatonin content is expressed in nggland as the mean plusmn SEM of 3 experiments One-wayANOVA Bonferroni multiple comparison test lowast119875 lt 005 versus NE 1 120583M 119899 = 12 for each group (b) Isolated rat pinealocyte culture underNE 1120583M stimulation in the absence or presence of Glu 100 or Glu 600120583M 119899 = 6 for each group (c) Pinealocyte and astrocyte co-culturestimulated with NE 1 120583M in the absence or presence of Glu 100 or Glu 600 120583M One-way ANOVA Bonferroni multiple comparison testlowastlowast119875 lt 001 versus NE 1 120583M 119899 = 8 for each group (d) Astrocytes and pinealocytes were cultured together but with physical separation by

inserts Astrocytes were cultivated in the bottom of the plate and pinealocytes were cultivated within the inserts and stimulated with NE1 120583M associated or not associated with Glu 600 120583M 119879 test lowastlowast119875 lt 001 119899 = 4 for each group For the cell cultures melatonin was evaluatedin the culture medium and expressed in ngmL as the mean plusmn SEM of 3 experiments

includes the TACE It was verified that when the cells inco-culture were incubated with BB1101 (10 120583M) prior to thetreatments with glutamate (600 120583M) and NE (1 120583M) it wasable to completely revert the inhibitory effect of glutamate onmelatonin synthesis (119875 lt 005) (Figure 3)

RT-PCR analyses showed mRNA expression of classIII metabotropic glutamate receptors (mGluR1mGluR5 andmGluR2mGluR3 resp) as well as ionotropic NMDA andAMPA-type receptors (GluN1 GluA1 GluA2 and GluA3subunits) in astrocytes and pinealocytes (Figures 4(a) and4(b)) Contrarily mGluR7 mRNA (class III metabotropicreceptors) was not detected in any of the cell types(Figure 4(a)) To determine whether class I mGluRs NMDA

and AMPA ionotropic receptors are functional and incor-porated into the astrocytic and pinealocytic membranes anintracellular calcium analysis by confocal microscopy wasused It was observed that the class I metabotropic receptoragonist DHPG (50120583M) and the ionotropic receptor agonistsNMDA (100120583M) and AMPA (50 120583M) were able to increasethe astrocytes and pinealocytes intracellular calcium contentin a similar way as that observed for glutamate (600 120583M)(Figure 5 Table 1)

Electrophoretic mobility shift assays revealed that gluta-mate (100 and 600120583M) activates NF-120581B in cultured rat pinealglands The result of the competition study showed that theupper complex 1 was displaced by an excess of unlabeled

BioMed Research International 7

60

40

20

0NE 1120583M

Mela

toni

n (n

gm

L)

lowast

+ +

+ +

+

+

+

+minus minus

minus minus

lowastlowast

BB1101 10120583MGlu 600 120583M

Figure 3 (a) Ablation of the inhibitory glutamatergic effect onmelatonin synthesis by the TACE inhibitor BB1101 Pinealocytesand astrocytes were maintained in co-culture and stimulated withnorepinephrine (NE 1 120583M) in association with glutamate (Glu600 120583M) and BB1101 (10120583M) One-way ANOVA Bonferroni mul-tiple comparison test lowastlowast119875 lt 001 versus NE 1 120583M lowast119875 lt 005 versusNE 1 120583M + Glu 600 120583M 119899 = 4 for each group 3 experiments

Table 1 Summary of intracellular [Ca2+] changes in astrocytes andpinealocytes after stimulation

Cell typeagents Number of cellsexamined [Ca2+]

119894response

Isolated astrocytesGlu 600 120583M 68 79AMPA 50 120583M 97 80NMDA 100 120583M 49 90DHPG 50 120583M 80 74

Isolated pinealocytesGlu 600 120583M 66 80AMPA 50 120583M 78 85NMDA 100 120583M 53 90DHPG 50 120583M 60 76

Intracellular [Ca2+] in fluo-4-loaded astrocytes and pinealocytes was mea-sured as described in Section 2 The majority of the stimulated cellsresponded with an increase in intracellular [Ca2+] after the addition of eitherglutamate (600120583M) ionotropic glutamatergic agonists (AMPA 50120583M andNMDA 100120583M) or a class I metabotropic glutamatergic agonist (DHPG50120583M)

NF-120581B but not by a TFIID double-stranded oligonucleotideconsensus sequence demonstrating the specificity of the NF-120581BDNA interactionThe lower complex 2 was less efficientlydisplaced by unlabeledNF-120581B probe (Figure 6(a)) Supershiftanalysis indicated that the antibody against the p50 subunitwas able to shift the DNAprotein interaction present incomplex 1 The presence of antibodies against the p52 p65and c-Rel subunits did not affect DNA-protein complexes(Figures 6(b) and 6(c)) Taken together these results indicatethat at these concentrations glutamate activates the p50p50

homodimer Complex 2 was not displaced by the antibodiesand was not considered to be related to the NF-120581B family

Astrocyte and pinealocyte isolated cultures were used todetermine the cell type in which NF-120581B was being activatedGlutamate (600120583M)was shown to activateNF-120581B exclusivelyin astrocytes and not in pinealocytes (119875 lt 001) (Figure 7)

The involvement of NF-120581B activation in glutamatersquosinhibitory effect on melatonin synthesis was analyzed usinga specific inhibitor of NF-120581B PDTC PDTC (300 120583M) pre-vented the inhibitory effect of glutamate in cultured rat pinealglands stimulated by NE (1 120583M) (119875 lt 001) (Figure 8)

Glutamate cytotoxicity was evaluated because it is knownthat this neurotransmitter in high concentrations couldinduce cell death by cytotoxicity [48] Cell membraneintegrity and DNA fragmentation were assessed by flowcytometric analysis using propidium iodide (PI) Supple-mentary Figure 1 is representative of the results obtainedafter cell incubation with NE (1120583M) in the absence (controlgroup) or presence of glutamate (100 and 600120583M) for 5 hThe histograms show the fluorescence intensity in the FL-2channel which corresponds to the PI emission wavelengthWhen the cell membrane is intact the fluorescence signal islow and is related to autofluorescence (M1) A high signalmeans that PI was able to enter the cell and bind to DNA(M2) Glutamate treatments for 5 h were not able to inducea loss of membrane integrity (Supplementary Figure 1(a))The percentage of cells with ruptured membranes in thecontrol group was 453 whereas it was 502 and 577 inthe 100 and 600 120583M glutamate-treated groups respectively(Supplementary Figure 1(c)) Histograms in SupplementaryFigure 1(b) represent the results of DNA fragmentationanalysis After PI treatment the DNA content in live cellspresented a high fluorescence signal (M2) DNA fragmenta-tion induces the formation of small DNA pieces that exhibitlow fluorescence intensity (M1) The percentages of cellswith DNA fragmentation after glutamate stimulation 100and 600120583M were 286 and 335 respectively which weresimilar to the value of control cells (366) (SupplementaryFigure 1(d))

4 Discussion

Isolated pinealocytes isolated astrocytes and pineal-ocyteastrocyte co-culture were used in this study Theimmunocytochemistry characterization of the cell typespresent in these different cultures demonstrated the pre-dominance of pinealocytes in isolated pinealocyte culturewith the absence of glial cells In the same way astrocytecultures do not contain a significant number of microglialcells or pinealocytes In co-culture pinealocytes are thepredominant cells as astrocytes are present in minorquantities reproducing the proportion present in intactpineal glands (51) Thus the cultures were considered to beappropriate for this study

Data from the literature have shown that glutamate acts asan inhibitory neurotransmitter on melatonin synthesis [12]According to Yamada et al [13 14 16] and Yatsushiro et al[16] glutamate is stored in microvesicles and released from

8 BioMed Research International

mGluR1 mGluR2 mGluR3 mGluR5 mGluR7

M H2O As Pi Hi As Pi Hi As Pi Hi As Pi Hi As Pi Hi

200 bp100 bp

(a)

M H2O As Pi Hi As Pi Hi As Pi Hi As Pi Hi

200 bp100 bp

GluA1 GluA2 GluA3 GluN1

(b)

Figure 4 RT-PCR detection of metabotropic and ionotropic glutamate receptors in isolated astrocytes and pinealocytes (a) RT-PCRdetection of transcripts for mGluR1 (79 bp) mGluR2 (198 bp) mGluR3 (196 bp) mGluR5 (165 bp) and mGluR7 (70 bp) in astrocytes (As)pinealocytes (Pi) and hippocampus (Hi) Molecular weight marker (M) and water (H

2O) are presented in lanes 1 and 2 respectively (b)

RT-PCR detection of transcripts for GluA1 (203 bp) GluA2 (155 bp) GluA3 (193 bp) and GluN1 (201 bp) in astrocytes (As) pinealocytes(Pi) and hippocampus (Hi) The molecular weight marker (M) and PCR product generated with water (H

2O) are presented in lanes 1 and 2

respectively

pinealocytes upon cholinergic stimulation It interacts withionotropic and metabotropic glutamate receptors presentin pinealocyte membranes releasing additional glutamateand inhibiting melatonin synthesis respectively Recently amechanism involving the glutamate transporter GLT-1 wasproposed to be responsible for glutamate release [17] Inthese studies involving pinealocytes an autocrineparacrineprocess was suggested for the influence of glutamate onmelatonin synthesis In the presentworkwe demonstrated animportant role for astrocytes in glutamatersquos modulatory effecton melatonin synthesis that includes a paracrine interactionwith pinealocytes

Whereas in isolated pinealocytes glutamate does notexhibit the typical inhibitory effect on melatonin synthesisas observed in intact pineal glands its association withastrocytes restores this effect These results demonstrate thatastrocytes act as partners that collaborate to produce theglutamate effect In addition the fact that the effect occurredeven when inserts were used preventing the physical contactbetween the cells in co-culture suggests that a solublefactor is a mediator of this process The hypothesis that gapjunctions mediated the interactions between astrocytes andpinealocytes as occurs in the pineal gland [49] was excludedfrom this observationThe cytokine TNF-120572 a gliotransmitterthat is released by astrocytes could be the soluble factorthat is involved in the interactions between astrocytes andpinealocytes In fact the absence of a soluble form of TNF-120572 interferes with the glutamate response

The possibility that the glutamate effects were due to celldeath was investigated by flow cytometry As the cell viabilityand DNA fragmentation levels were similar between controland glutamate-stimulated groups showing no damage topineal cells even when treated with 600120583M of glutamate itis possible to exclude a glutamate cytotoxic effect as a causeof melatonin reduction

Although glutamate receptor expression had been firstdescribed in pinealocytes its expression was also laterdemonstrated in astrocytes mGluR3 and AMPA (GluA1subunit) were shown to be present in pinealocytes [12 16]

and NMDA and AMPA (GluA23) receptors were present inrat pineal gland astrocytes [19] Ionotropic receptor mRNAwas similarly observed in astrocytes in the present studyin addition to the novel observation of metabotropic recep-tor (mGluR1 mGluR2 mGluR3 mGluR5) expression Inpinealocytes the expression of mGluR1R2R3R5 AMPAand NMDA receptors was shownThis evidence supports thepossibility of astrocyte participation in rat pineal glutamater-gic signaling

Confocal microscopy assays using NMDA AMPA andDHPG agonists in isolated astrocytes and pinealocytesrevealed increased intracellular calcium content in both celltypes for all the agonists used PCR and confocal microscopyanalyses suggest both gene and protein expression in classI mGluR AMPA and NMDA ionotropic receptors reveal-ing the importance of the glutamate receptors as signaltransducers in rat pineal gland astrocytes and pinealocytesThus glutamate seems to interact with its receptors inboth cell types but its effect on melatonin synthesis nec-essarily involves astrocyte glutamate receptors The possibleparacrine interactions between pinealocytes and astrocytesseem to be the key in understanding the role of astrocytes inthe rat pineal gland

Transcription factors exert a great influence on pinealphysiology CREB and ICER are two transcription factorsthat intimately control the transcription of AANAT [50]Additionally there are other transcription factors in the ratpineal gland whose roles in pineal melatonin synthesis arenot known [51] NF-120581B is present in the rat pineal glandshowing a circadian rhythm and involvement in inflamma-tory responses [36 37] NF-120581B is a classic possible glutamatemediator and its activation in glial cells was demonstrated toplay a role in inflammatory processes [30]The present resultsdemonstrated NF-120581B activation by glutamate in culturedisolated astrocytes supporting its role as a mediator ofglutamate effects Supershift assay in cultured pineal glandsstimulated with glutamate showed the displacement of thep50 subunit emphasizing the importance of p50p50 homod-imer formationThis is an astrocyte-dependent event because

BioMed Research International 9

0 50 100 150 200 250 300 350

10

20

30

40

50

60

Time (s)

Isolated astrocytes

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

Glu 600120583M

(a)

0 30 60 90 120 150

155

160

165

170

175

180

185

Time (s)

Isolated pinealocytes

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

Glu 600120583M

(b)

0 100 200 300 400 500

10

20

30

Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

AMPA 50120583M

(c)

0 30 60 90 120 150 180

102

104

106

108

110

112

114

Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

AMPA 50120583M

(d)

0 50 100 150 200 250 300

400

600

800

1000

1200

1400

Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

NMDA 100120583M

(e)

0 50 100 150 200 250

900

1200

1500

Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

NMDA 100120583M

(f)

100 150 200 250

126

128

130

132

134

136

Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity) DHPG 50120583M

(g)

0 100 200 300 400

80

90

100

110

120

130

Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

DHPG 50120583M

(h)

Figure 5 Analysis of intracellular calcium in isolated astrocyte and pinealocyte cultures Traces of Fluo-4 fluorescence indicate theintracellular calcium transients from single cells Additions of exogenous glutamate or glutamatergic agonists were performed as indicatedby the arrows

10 BioMed Research International

ControlGlu 100120583MGlu 600120583MUnlabeled NF-120581BUnlabeled TFIID

1

(NS) 2

Free probe

+

minus

minus

minus

minus +

minus

minus

minus

minus

+

minus

minus

minus

minus +

+

minus

minus

minus +

+

minus

minus

minus+

+

minus

minus

minus

+

+

minus

minus

minus

(a)

(NS) 2

Free probe

(p50p50) 1

(NS) 3

+

+

minus

minus

minus

minus

+

minus

minus

minus

minus

minus

+

minus

minus

minus

minus

minus

+

+

minus

minus

minus

minus

+

+

minus

minus

minus

minus

+

+

minus

minus

minus

minus

ControlGlu 100120583MAnti p50Anti p52Anti p65Anti c-Rel

(b)

Free probe

Anti c-Rel

(NS) 2

(p50p50) 1

(NS) 3

ControlGlu 600120583MAnti p50Anti p52Anti p65

+minus minus minus minus minus

+

+ +

+

+ + +

+

+minus minus

minus

minus

minus

minus minus minus minus minus

minus

minus minus minus

minus minus

minus minus minus

minus minus

(c)

Figure 6 Electrophoretic mobility shift assays for NF120581B in the pineal gland (a) Competition studies were performed using nuclear extract(5120583g) from cultured pineal glands treated with glutamate (Glu) 100 and 600 120583M in the absence or presence of unlabeled specific (NF-120581Bconsensus sequence 10-fold molar excess) or nonspecific oligonucleotide (TFIID consensus sequence 10-fold molar excess) as indicated(b c) Supershift assays were performed with the same nuclear extract (5120583g) incubated in the absence or presence of antibodies against thesubunits p50 (1 10 dilution) p65 (1 20 dilution) p52 and c-Rel (1 10 dilution) as indicated Antibodies were added 20min prior to theaddition of the radiolabeled NF-120581B consensus oligonucleotide The position of specific NF-120581BDNA binding complexes p50p50 (band 1 bc) is indicated NS represents no specific binding (bands 2 and 3 a b c) The localization of the free probe is also indicated

BioMed Research International 11

ControlGlu 600120583M

minusminus

minus +

NF-120581B

(a)

20000

15000

10000

0Control Glu 600120583M

5000

lowastlowast

(au

)

(b)

ControlGlu 600120583M

minusminus

minus +

NF-120581B

(c)

(au

)

Control Glu 600120583M

16000

12000

8000

4000

0

(d)

Figure 7 Electrophoretic mobility shift assays for NF120581B in isolated astrocytes and pinealocytes (a) Nuclear proteins (5 120583g) were extractedfrom astrocytes treated with glutamate (Glu) 600120583M (b)Densitometric analysis (arbitrary units) of theNF-120581B band is presented on panel (a)One-way ANOVA Studentrsquos test lowastlowast119875 lt 001 versus control group mean plusmn SEM (c) Nuclear proteins (5120583g) were extracted from pinealocytestreated with Glu 600 120583M (d) Densitometric analysis (arbitrary units) of the NF-120581B band is presented in (c) The position of the specificNF-120581BDNA binding complex is indicated 119899 = 5 for each group 3 experiments

its activation was not observed in pinealocytes Astrocyteand pinealocyte supershift assays were not performed due totechnical limitations related to the amount of nuclear protein

The p50 subunit is synthesized from its precursor p105The processing of p105 and the resulting generation ofmature p50 subunits seem to be a constitutive event as p50homodimers are not regulated by I120581B proteins [34] The p50subunit formation seems to be regulated by cotranslationaland posttranslational mechanisms In the posttranslationalmechanism the p50 subunit is synthesized from its precursorp105 by phosphorylation at its 927 and 932 serine residueswhich allows the ubiquitinization and proteolytic degrada-tion of p105 by the 26S proteasome [52] In the cotranslationalmechanism p50 and p105 can be generated from a singlemRNA [53] There is also evidence that the 20S protea-some constitutively processes p105 to p50 in a ubiquitin-independent manner [52] The p50p50 homodimer wasoriginally considered to be a transcriptional repressor butnow there is substantial evidence that it can also inducegene transcription after a complex formation with the BCL3nuclear protein an atypical member of the I120581B family ofproteins that is considered to be a coactivator of p50p50homodimers [34]

The link between NF-120581B activation by glutamate and thereduction of melatonin synthesis was evidenced with the useof PDTC a NF-120581B translocation inhibitor [54] PDTC pre-vented glutamatersquos inhibitory effect on melatonin synthesisTaken together the crucial presence of astrocytes for theinhibitory effect of glutamate the exclusive activation of NF-120581B by glutamate in the same cell type and the demonstrationof NF-120581Brsquos role inmelatonin synthesis indicate that astrocytesunder glutamate stimulation possibly release a factor thatpromotes a decrease in pinealocyte melatonin synthesisTNF-120572may be a good candidate Indeed TNF-R1 the TNF-120572 receptor that interacts with soluble TNF is present in thepineal gland reinforcing the importance of this signalingpathway [55] Acting as an inhibitor of TACE to prevent TNF-120572 release BB1101 was thus adequate in demonstrating the roleplayed byTNF-120572 In addition the hypothesis that the p50p50homodimer may induce TNF-120572 gene expression and releaseneeds to be clarified

In the CNS astrocytes are known to release gliotransmit-ters such as glutamate D-serine and ATP [21] Moreoverastrocytes can also synthesize and release proinflammatorycytokines in response to NF-120581B activation such as TNF-120572 and interleukin-1 and induce gene expression of iNOS

12 BioMed Research International

NE 1120583MGlu 100120583MGlu 600120583MPDTC 300120583M

Mela

toni

n (n

ggl

and)

20

15

10

5

0

lowastlowast

lowastlowast

+ +

+ + +

++

+

+

+

+

+ +

minus minus minusminus

minus minus minusminus

minus minus minus

Figure 8 Effect of PDTC an inhibitor of NF-120581B on melatoninsynthesis Pineal glands in culture were incubated with PDTC(300120583M) and stimulated by norepinephrine (NE 1120583M) in thepresence of glutamate (100 and 600120583M) andmelatonin content wasevaluated One-way ANOVA Bonferroni multiple comparison test119875 lt 001 versus NE 1 120583M lowastlowast119875 lt 001 versus NE + Glu 100 120583Mand NE + Glu 600 120583M plotted as the mean plusmn SEM 119899 = 12 for eachgroup 3 experiments

PinealocyteAstrocyte

NF-120581Bactivation

AMPANMDAmGluR15

Release of solublefactor

TNF-120572

Glu

TNF-120572 alone (recruiting AMPA)or in association with Glu

mGluR3TNF-R1 AMPA[iCa+2]

Melatoninsynthesis

Figure 9 Diagram illustrating the putative paracrine interactionbetween pinealocytes and astrocytes in the modulation of pinealmelatonin synthesis by glutamate Glutamate (Glu) induces intra-cellular Ca2+ increase in astrocytes with the subsequent activationof NF120581B and release of a soluble factor possibly TNF-120572 This factoralone or in association with Glu promotes melatonin synthesisreduction in pinealocytes

resulting in nitric oxide formation [56] In this work wesuggested that TNF-120572 is the factor that is released byastrocytes under glutamate stimulation because the TACEinhibitor antagonized glutamatersquos effects Reinforcing thisidea recent preliminary data obtained in our laboratorywith the L929 tumor cell lineage which is sensitive to TNF-120572 showed that astrocytes release TNF-120572 when stimulatedby glutamate (data not shown) The literature states that

TNF-120572 decreases the serotonin content and AANAT mRNAexpression resulting in the reduction of N-acetylserotonin[57 58] the immediate precursor of melatonin synthesis Itis not clear whether TNF-120572 acts by itself or in combinationwith glutamate because there are glutamate receptors in thepinealocyte membrane [12 15 16 18] Recent studies havereported the control of hippocampal synapses by TNF-120572 dueto the increased insertion of AMPA receptors in the surfaceof the neuronal soma [59]The same effect could take place inthe pineal gland in which TNF-120572 released by the astrocytesin response to NF-120581B activation could increase the numberof AMPA receptors in the pinealocyte membrane resultingin the inhibition of melatonin synthesis

A model of the putative paracrine interactions betweenastrocytes and pinealocytes in the modulation of pinealmelatonin synthesis by glutamate is shown in Figure 9

5 Conclusions

In summary according to our results we propose thatglutamate acts on glutamate receptors (AMPA NMDAandor mGluR15) in astrocytes increasing the intracellularcalcium concentration promoting NF-120581B activation anddriving the release of a soluble factor possibly TNF-120572 whichacts independently or with glutamate in the pinealocytesresulting in inhibition of melatonin synthesis

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

This work was supported by a grant from FAPESP (0404328-1) Darine Villela was supported by a master fellowshipfrom FAPESP (0556943-4) Anderson Augusto Moutinhoand Victoria Fairbanks Atherino were supported by CNPq(PIBIC)

References

[1] J Calvo J Boya A Borregon and J E Garcia-MaurinoldquoPresence of glial cells in the rat pineal gland a light andelectronmicroscopic immunohistochemical studyrdquoAnatomicalRecord vol 220 no 4 pp 424ndash428 1988

[2] L Vollrath The Pineal Organ Springer New York NY USA1981

[3] R Y Moore J C Speh and J P Card ldquoThe retinohypothalamictract originates from a distinct subset of retinal ganglion cellsrdquoJournal of Comparative Neurology vol 352 no 3 pp 351ndash3661995

[4] D Sugden ldquoMelatonin biosynthesis in the mammalian pinealglandrdquo Experientia vol 45 no 10 pp 922ndash932 1989

[5] A F Afeche D CMVillelaM V Abrahao R Peres J Cipolla-Neto et al ldquoMelatonin and pineal glandrdquo in New Researchon Neurosecretory Systems E Romano Ed pp 151ndash177 NovaScience Publishers Hauppauge NY USA 2008

[6] J Arendt Melatonin and the Mammalian Pineal Gland Chap-man amp Hall London UK 1995

BioMed Research International 13

[7] M Ehret P Pevet and M Maitre ldquoTryptophan hydroxylasesynthesis is induced by 3101584051015840-cyclic adenosine monophosphateduring circadian rhythm in the rat pineal glandrdquo Journal ofNeurochemistry vol 57 no 5 pp 1516ndash1521 1991

[8] D C Klein G R Berg and J Weller ldquoMelatonin synthesisadenosine 3101584051015840-monophosphate and norepinephrine stimulateN-acetyltransferaserdquo Science vol 168 no 3934 pp 979ndash9801970

[9] D C Klein N L Schaad M A A Namboordiri L Yu and JL Weller ldquoControl of N-acetyltransferaserdquo Biochemical SocietyTransactions vol 20 no 2 pp 299ndash304 1992

[10] V Simonneaux and C Ribelayga ldquoGeneration of the melatoninendocrine message in mammals a review of the complexregulation of melatonin synthesis by norepinephrine peptidesand other pineal transmittersrdquoPharmacological Reviews vol 55no 2 pp 325ndash395 2003

[11] S Ishio H Yamada C M Craft and Y MoriyamaldquoHydroxyindole-O-methyltransferase is another target forL-glutamate-evoked inhibition of melatonin synthesis in ratpinealocytesrdquo Brain Research vol 850 no 1-2 pp 73ndash78 1999

[12] H Yamada S Yatsushiro S Ishio et al ldquoMetabotropic gluta-mate receptors negatively regulate melatonin synthesis in ratpinealocytesrdquo Journal of Neuroscience vol 18 no 6 pp 2056ndash2062 1998

[13] H Yamada A Yamamoto M Takahashi H Michibata HKumon and Y Moriyama ldquoThe L-type Ca2+ channel isinvolved in microvesicle-mediated glutamate exocytosis fromrat pinealocytesrdquo Journal of Pineal Research vol 21 no 3 pp165ndash174 1996

[14] H Yamada A Yamamoto S Yodozawa et al ldquoMicrovesicle-mediated exocytosis of glutamate is a novel paracrine-likechemical transduction mechanism and inhibits melatoninsecretion in rat pinealocytesrdquo Journal of Pineal Research vol 21no 3 pp 175ndash191 1996

[15] H Yamada A Ogura S Koizumi A Yamaguchi and YMoriyama ldquoAcetylcholine triggers L-glutamate exocytosis vianicotinic receptors and inhibits melatonin synthesis in ratpinealocytesrdquo Journal of Neuroscience vol 18 no 13 pp 4946ndash4952 1998

[16] S Yatsushiro H Yamada M Hayashi A Yamamoto andY Moriyama ldquoIonotropic glutamate receptors trigger micro-vesicle-mediated exocytosis of L-glutamate in rat pinealocytesrdquoJournal of Neurochemistry vol 75 no 1 pp 288ndash297 2000

[17] M-HKim SUehara AMuroyama BHille YMoriyama andD-S Koh ldquoGlutamate transporter-mediated glutamate secre-tion in the mammalian pineal glandrdquo Journal of Neurosciencevol 28 no 43 pp 10852ndash10863 2008

[18] S Yatsushiro H Yamada M Hayashi S Tsuboi and YMoriyama ldquoFunctional expression of metabotropic glutamatereceptor type 5 in rat pinealocytesrdquo NeuroReport vol 10 no 7pp 1599ndash1603 1999

[19] C Kaur V Sivakumar and E A Ling ldquoExpression ofN-methyl-D-aspartate (NMDA) and 120572-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) GluR23 receptors inthe developing rat pineal glandrdquo Journal of Pineal Research vol39 no 3 pp 294ndash301 2005

[20] H Pabst and P Redecker ldquoInterstitial glial cells of the gerbilpineal gland display immunoreactivity for the metabotropicglutamate receptors mGluR23 and mGluR5rdquo Brain Researchvol 838 no 1-2 pp 60ndash68 1999

[21] M M Halassa T Fellin and P G Haydon ldquoThe tripartitesynapse roles for gliotransmission in health and diseaserdquoTrends in Molecular Medicine vol 13 no 2 pp 54ndash63 2007

[22] S M Finkbeiner ldquoGlial calciumrdquo Glia vol 9 no 2 pp 83ndash1041993

[23] E A Newman ldquoNew roles for astrocytes regulation of synaptictransmissionrdquo Trends in Neurosciences vol 26 no 10 pp 536ndash542 2003

[24] J T Porter and K D McCarthy ldquoAstrocytic neurotransmitterreceptors in situ and in vivordquo Progress in Neurobiology vol 51no 4 pp 439ndash455 1997

[25] V Parpura T A Basarsky F Liu K Jeftinija S Jeftinija and PG Haydon ldquoGlutamate-mediated astrocyte-neuron signallingrdquoNature vol 369 no 6483 pp 744ndash747 1994

[26] T D Hassinger P B Atkinson G J Strecker et al ldquoEvidencefor glutamate-mediated activation of hippocampal neurons byglial calcium wavesrdquo Journal of Neurobiology vol 28 no 2 pp159ndash170 1995

[27] R H Lipsky K Xu D Zhu et al ldquoNuclear factor 120581B is a criticaldeterminant in N-methyl-D-aspartate receptor-mediated neu-roprotectionrdquo Journal of Neurochemistry vol 78 no 2 pp 254ndash264 2001

[28] M P Mattson C Culmsee Z Yu and S Camandola ldquoRoles ofnuclear factor 120581B in neuronal survival and plasticityrdquo Journal ofNeurochemistry vol 74 no 2 pp 443ndash456 2000

[29] C DMunhoz B Garcıa-Bueno J L MMadrigal L B LepschC Scavone and J C Leza ldquoStress-induced neuroinflammationmechanisms and new pharmacological targetsrdquo Brazilian Jour-nal of Medical and Biological Research vol 41 no 12 pp 1037ndash1046 2008

[30] S Camandola andM PMattson ldquoNF-ΚB as a therapeutic targetin neurodegenerative diseasesrdquo Expert Opinion on TherapeuticTargets vol 11 no 2 pp 123ndash132 2007

[31] B Kaltschmidt D Widera and C Kaltschmidt ldquoSignaling viaNF-120581B in the nervous systemrdquo Biochimica et Biophysica Actavol 1745 no 3 pp 287ndash299 2005

[32] P T Massa H Aleyasin D S Park X Mao and S W BargerldquoNF120581B in neurons the uncertainty principle in neurobiologyrdquoJournal of Neurochemistry vol 97 no 3 pp 607ndash618 2006

[33] H Hacker and M Karin ldquoRegulation and function of IKK andIKK-related kinasesrdquo Sciencersquos STKE vol 2006 no 357 p re132006

[34] S Beinke and S C Ley ldquoFunctions of NF-120581B1 and NF-120581B2 inimmune cell biologyrdquo Biochemical Journal vol 382 no 2 pp393ndash409 2004

[35] N D Perkins ldquoIntegrating cell-signalling pathways with NF-120581Band IKK functionrdquo Nature Reviews Molecular Cell Biology vol8 pp 49ndash62 2007

[36] E Cecon P A Fernandes L Pinato Z S Ferreira and R PMarkus ldquoDaily variation of constitutively activated nuclear fac-tor 120581B (NFKB) in rat pineal glandrdquoChronobiology Internationalvol 27 no 1 pp 52ndash67 2010

[37] Z S Ferreira P A C M Fernandes D Duma J Assreuy MC W Avellar and R P Markus ldquoCorticosterone modulatesnoradrenaline-induced melatonin synthesis through inhibitionof nuclear factor 120581Brdquo Journal of Pineal Research vol 38 no 3pp 182ndash188 2005

[38] S C Afeche R Barbosa J H Scialfa I M Terra A CCassola and J Cipolla-Neto ldquoEffects of the blockade of highvoltage-activated calcium channels on in vitro pineal melatoninsynthesisrdquo Cell Biochemistry and Function vol 24 no 6 pp499ndash505 2006

14 BioMed Research International

[39] B Barlaam T G Bird C Lambert-Van Der Brempt DCampbell S J Foster and R Maciewicz ldquoNew 120572-substitutedsuccinate-based hydroxamic acids as TNF120572 convertaseinhibitorsrdquo Journal of Medicinal Chemistry vol 42 no 23 pp4890ndash4908 1999

[40] I Glezer C D Munhoz E M Kawamoto T MarcourakisM C Werneck Avellar and C Scavone ldquoMK-801 and 7-Ni attenuate the activation of brain NF-120581B induced by LPSrdquoNeuropharmacology vol 45 no 8 pp 1120ndash1129 2003

[41] Y Rong and M Baudry ldquoSeizure activity results in a rapidinduction of nuclear factor-120581B in adult but not juvenile ratlimbic structuresrdquo Journal of Neurochemistry vol 67 no 2 pp662ndash668 1996

[42] I Nicoletti G Migliorati M C Pagliacci F Grignani and CRiccardi ldquoA rapid and simple method for measuring thymocyteapoptosis by propidium iodide staining and flow cytometryrdquoJournal of Immunological Methods vol 139 no 2 pp 271ndash2791991

[43] Y F Jiang-Shieh C H Wu M L Chang J Y Shiehand C Y Wen ldquoRegional heterogeneity in immunoreactivemacrophagesmicroglia in the rat pineal glandrdquo Journal ofPineal Research vol 35 no 1 pp 45ndash53 2003

[44] K-O Soderstrom ldquoLectin binding to the human retinardquoAnatomical Record vol 220 no 2 pp 219ndash223 1988

[45] F Uehara M Sameshima T Muramatsu and N OhbaldquoLocalization of fluorescence-labeled lectin binding sites onphotoreceptor cells of the monkey retinardquo Experimental EyeResearch vol 36 no 1 pp 113ndash123 1983

[46] H-W Korf ldquoThe pineal organ as a component of the biologicalclock phylogenetic and ontogenetic considerationsrdquo Annals ofthe New York Academy of Sciences vol 719 pp 13ndash42 1994

[47] E Maronde and J H Stehle ldquoThe mammalian pineal glandknown facts unknown facetsrdquo Trends in Endocrinology andMetabolism vol 18 no 4 pp 142ndash149 2007

[48] X-M Zhang and J Zhu ldquoKainic acid-induced neurotoxicitytargeting glial responses and glia-derived cytokinesrdquo CurrentNeuropharmacology vol 9 no 2 pp 388ndash398 2011

[49] D Stellwagen and R CMalenka ldquoSynaptic scalingmediated byglial TNF-120572rdquo Nature vol 440 no 7087 pp 1054ndash1059 2006

[50] E Maronde M Pfeffer J Olcese et al ldquoTranscription factorsin neuroendocrine regulation rhythmic changes in pCREB andICER levels framemelatonin synthesisrdquo Journal of Neurosciencevol 19 no 9 pp 3326ndash3336 1999

[51] D M Bustos M J Bailey D Sugden et al ldquoGlobal dailydynamics of the pineal transcriptomerdquoCell and Tissue Researchvol 344 no 1 pp 1ndash11 2011

[52] A K Moorthy O V Savinova J Q Ho V Y-F Wang D VuandGGhosh ldquoThe 20S proteasome processesNF-120581B1 p105 intop50 in a translation-independent mannerrdquo EMBO Journal vol25 no 9 pp 1945ndash1956 2006

[53] L Lin G N DeMartino and W C Greene ldquoCotranslationalbiogenesis of NF-120581B p50 by the 26S proteasomerdquo Cell vol 92no 6 pp 819ndash828 1998

[54] H W L Ziegler-Heitbrock T Sternsdorf J Liese et alldquoPyrrolidine dithiocarbamate inhibits NF-120581B mobilization andTNF production in humanmonocytesrdquo Journal of Immunologyvol 151 no 12 pp 6986ndash6993 1993

[55] C E Carvalho-Sousa S S Cruz-Machado E K Tamura P AC M Fernandes L Pinato et al ldquoMolecular basis for definingthe pineal gland and pinealocytes as targets for tumor necrosisfactorrdquo Frontiers in Endocrinology vol 2 pp 1ndash11 2011

[56] M PMattson ldquoNF-120581B in the survival and plasticity of neuronsrdquoNeurochemical Research vol 30 no 6-7 pp 883ndash893 2005

[57] P A CM Fernandes E Cecon R PMarkus and Z S FerreiraldquoEffect of TNF-120572 on the melatonin synthetic pathway in the ratpineal gland basis for a rsquofeedbackrsquo of the immune response oncircadian timingrdquo Journal of Pineal Research vol 41 no 4 pp344ndash350 2006

[58] S-Y Tsai T E OrsquoBrien and J A McNulty ldquoMicroglia play arole in mediating the effects of cytokines on the structure andfunction of the rat pineal glandrdquo Cell and Tissue Research vol303 no 3 pp 423ndash431 2001

[59] J S Bains and S H R Oliet ldquoGlia they make your memoriesstickrdquo Trends in Neurosciences vol 30 no 8 pp 417ndash424 2007

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

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Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Research and TreatmentAIDS

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Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 BioMed Research International

All the salts and reagents used were obtained from Merck(Frankfurter Darmstadt Germany)

29 Electrophoretic Mobility Shift Assay

291 Pineal Gland Nuclear Protein Extracts Nuclear extractswere obtained from pineal gland homogenates as previouslydescribed [37] after incubation for 30min with or withoutglutamate (600 120583M) Briefly the glands were homogenizedin lysis buffer (10mM HEPES 10mM KCl 01mM EDTA10 glycerol and 01mM PMSF) and after 15min nonylphenoxypolyethoxylethanol (NP)-40 (10) was added Thesamples were vigorously mixed centrifuged (12000 g 1min4∘C) twice and resuspended in extraction buffer (10mMHEPES 05M KCl 1mM EDTA 10 glycerol and 01mMPMSF) After incubation on ice for 15min and centrifugation(20000 g 5min 4∘C) the supernatant containing the nuclearproteins was collected and stored at minus80∘C Protein concen-tration was determined using the BioRad protein reagent(BioRad Hercules CA USA)

292 Astrocytes and Pinealocytes Nuclear Protein ExtractsNuclear extracts were obtained from isolated astrocytes andpinealocytes homogenates as previously described [40] afterincubation for 30min with or without glutamate (600120583M)Briefly the cells were homogenized in lysis buffer (10mMHEPES 15mM MgCl

2 10mM KCl 05mM PMSF 01mM

EDTA and 3mMorthovanadate) After addingNP-40 (10)sampleswere vigorouslymixed centrifuged (11000 g 20min4∘C) twice and resuspended in extraction buffer (20mMHEPES 15mMMgCl

2 300mMNaCl 025mM EDTA 25

glycerol 05mM PMSF 3mM orthovanadate 2 120583gmL leu-peptin and 2 120583gmL antipain) After a 20min incubation onice and centrifugation (11000 g 20min 4∘C) the supernatantcontaining the nuclear proteins was collected and stored atminus80∘C The protein concentration was determined using theBioRad protein reagent

293 Gel Shift Assay An electrophoretic mobility shift assayfor NF-120581B was performed utilizing the gel shift assay kitfrom Promega (Madison WI USA) as described previ-ously [41] A 32P-NF-120581B double-stranded consensus oligonu-cleotide probe (51015840-AGTTGAGGGGACTTTCCCAGGC-31015840)and nuclear extracts were used DNA-protein complexeswere separated by electrophoresis through a 6 non-denaturing acrylamidebisacrylamide (375 1) gel in 053Tris-borate-EDTA (TBE) for 2 h at 150V Gels were vac-uum dried and analyzed by autoradiography For compe-tition experiments NF-120581B and transcription initiation fac-tor II (TFIID 51015840-CAGAGCATATAAGGTGAGGTAGGA-31015840)unlabeled double-stranded consensus oligonucleotide wereincluded in ten-fold excess of the molar concentration of 32P-NF-120581B probe to detect specific and nonspecific DNA-proteininteractions respectively Unlabeled oligonucleotides wereadded to the reaction mixture 20min before the addition ofthe radioactive probeThe composition of the complexes wasdetermined by supershift assays adding antibodies (SantaCruzBiotechnologies SantaCruz CAUSA) against different

NF-120581B subunits (p50 p52 c-Rel and p65 1 20 dilution)either before or after the incubation of nuclear extracts withthe labeled oligonucleotide

210 RNA Extraction and RT-PCR Total RNA was extractedfrom isolated astrocytes and pinealocytes usingTRIzol (Invit-rogen Carlsbad CA USA) according to the manufacturerrsquosinstructions cDNA synthesis was performed using Super-Script III Reverse Transcriptase (RT) (Invitrogen CarlsbadCA USA) from 1120583g of total RNA After the RT reaction 2-120583L aliquots were used as a cDNA template for polymerasechain reaction (PCR) amplifications using GoTaq DNAPolymerase (Promega Madison WI USA) The cyclingparameters used were 30 s at 95∘C 30 s at each primerrsquosannealing temperature and 30 s at 72∘C using a MyGeneSeries Gradient Thermocycler (LongGene China) PCRproducts were separated on 12 ethidium bromide-agarosegels and the bands were visualized by digital scanning Theprimers used in this assay are presented in supplementaryTable 1 (see Table 1 in Supplementary Material available athttpdxdoiorg1011552013618432)

211 Flow Cytometry Pineal glands in culture were stimu-lated with NE 1 120583M in the absence or presence of glutamate100 120583M or 600120583M After 5 h of incubation the cells weredissociated via proteolytic digestion with papain In an assayof membrane integrity 50120583L of a propidium iodide solution(20120583g permL in saline buffer)was added to the cells (500120583L)Propidium iodide is a highly water-soluble fluorescent com-pound that cannot pass through intact membranes as it isunable to enter viable cells After membrane disruption itbinds to DNA by intercalating between the bases with littleor no sequence preference After 5min of incubation at roomtemperature the cells were evaluated in FACScalibur flowcytometer equipment (BectonDickinson San Jose CAUSA)using Cell Quest software (Becton Dickinson San Jose CAUSA) Fluorescence was measured using the FL2 channel(orange-red fluorescencemdash58542 nm) DNA fragmentationwas analyzed after DNA staining with propidium iodideaccording to the previously described method [42] Cellswere resuspended in a solution with detergents (300120583Lhypotonic solution containing 20120583gmL propidium iodide01 sodium citrate and 01TritonX-100) that permeabilizethe cells which promptly incorporate the dye into DNAThe pellet was gently resuspended and the cells were thenincubated for 2 h at room temperature Fluorescence wasmeasured and analyzed by flow cytometry as describedpreviously

212 Statistical Analysis Data were always presented asthe mean plusmn SEM Statistical analyses (GraphPad Prism 50software San Diego CA USA) were performed using one-way ANOVA followed by a Bonferroni post hoc test Whenappropriate Studentrsquos 119905-test was applied The graphs relatedto confocal microscopy were prepared utilizing Origin 50software (MicroCal Software Inc Northampton MA USA)Autoradiographs of NF-120581Bwere quantified using ScionImagesoftware (Scion Corp Frederick MD USA)

BioMed Research International 5

GFAP

(a)

WGA

(b)

GFAP

(c)

WGA

50120583M

(d)

Figure 1 Cell cultures immunocytochemistry Digital images of isolated cultures of astrocytes (a) and pinealocytes (b) showing specific celltype staining using a mouse monoclonal antibody against the glial fibrillary acidic protein (GFAP) and a wheat germ agglutinin-rhodaminelabel (WGA) to identify pinealocytes The pictures at the bottom illustrate the distribution of glial cells (c) and pinealocytes (d) in a mixedcoculture The insets show photomicrographies of the cell cultures in contrast phase microscopy astrocytes (a) pinealocytes (b) and co-culture (c and d) In isolated pinealocytes and in co-culture many cells are clustered Magnification 200x (astrocytes and pinealocytes) and100x (co-culture)

3 Results

The cultures used in this study were characterized and val-idated by immunostaining with GFAP a specific marker forastroglial cells and OX-42 a marker for microglia (specificfor activated microglia as the antibody reacts with the CR3complement) This OX-42 antibody is suitable for pinealgland staining because the predominant type of microglia inthis tissue expresses complement type 3 receptors [43] WGAwas used as a pinealocyte marker because it labels photore-ceptors [44 45] and pinealocytes aremodified photoreceptorcells [46 47]This technique proved to be efficient in labelingthese pineal cells (Figure 1)

Cultured isolated astrocytes showed fluorescence forGFAP (Figure 1(a)) but not for OX-42 and WGA (data notshown) Cultured pinealocytes lacked immunostaining forthe two glial markers GFAP and OX-42 (data not shown)and reacted withWGA demonstrating the absolute predom-inance of pinealocytes (Figure 1(b)) Figures 1(c) and 1(d)show the distribution of pinealocytes and astrocytes in themixed culture (co-culture) The round appearance of thelabeled cells in Figure 1 different from that in the insets is dueto the fixation process with paraformaldehyde In Figure 1(d)pinealocytes appear smaller than those in Figure 1(b) most

likely due to the use of trypsin to obtain the cells furtherdissociating the cell clusters

In intact cultured pineal glands the known effect ofglutamate in reducing NE-stimulated melatonin synthesis(119875 lt 005) was observed (Figure 2(a)) On the other handwhen isolated cultured pinealocytes were incubated withglutamate (100 and 600 120583M) in the presence of NE (1 120583M)this effect was abrogated (Figure 2(b)) Glutamatersquos inhibitoryeffect on melatonin synthesis as observed in intact culturedpineal glands was only reproduced when pinealocytes werecultivated in the presence of astrocytes in co-culture (119875 lt001) (Figure 2(c))

To investigate if the contact between astrocytes andpinealocytes is necessary for the inhibitory effect of gluta-mate the two cell types were cocultivated in the same wellbut separated by inserts It was observed that glutamatersquosinhibitory effect on NE-stimulated melatonin synthesis wasexpressed in the same way as when the cells were cultivatedwith physical contact indicating that a diffusible factorreleased by astrocytes acted on the pinealocytes (119875 lt 001)(Figure 2(d))

The possibility of TNF-120572 as a gliotransmitter that medi-ates the glutamate effect was tested using BB1101 a preferredinhibitor of metalloproteinases with sheddase activity that

6 BioMed Research International

18

15

12

9

6

3

0

Mela

toni

n (n

ggl

and)

lowast

NE 1120583MGlu 100120583MGlu 600120583M

+ +

+

+

+

minus minus

minus minus

(a)

0

Mela

toni

n (n

gm

L)

NE 1120583MGlu 100120583MGlu 600120583M

+ +

+

+

+

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minus minus

120

80

40

(b)

Mela

toni

n (n

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NE 1120583MGlu 100120583MGlu 600120583M

+ +

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40

30

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toni

n (n

gm

L)

+ +

+minus

lowastlowast

100

80

60

40

20

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(d)

Figure 2 Glutamate effects on melatonin synthesis (a) Pineal gland culture stimulated with norepinephrine (NE 1120583M) in the absence orpresence of glutamate (Glu 100 and Glu 600120583M) Melatonin content is expressed in nggland as the mean plusmn SEM of 3 experiments One-wayANOVA Bonferroni multiple comparison test lowast119875 lt 005 versus NE 1 120583M 119899 = 12 for each group (b) Isolated rat pinealocyte culture underNE 1120583M stimulation in the absence or presence of Glu 100 or Glu 600120583M 119899 = 6 for each group (c) Pinealocyte and astrocyte co-culturestimulated with NE 1 120583M in the absence or presence of Glu 100 or Glu 600 120583M One-way ANOVA Bonferroni multiple comparison testlowastlowast119875 lt 001 versus NE 1 120583M 119899 = 8 for each group (d) Astrocytes and pinealocytes were cultured together but with physical separation by

inserts Astrocytes were cultivated in the bottom of the plate and pinealocytes were cultivated within the inserts and stimulated with NE1 120583M associated or not associated with Glu 600 120583M 119879 test lowastlowast119875 lt 001 119899 = 4 for each group For the cell cultures melatonin was evaluatedin the culture medium and expressed in ngmL as the mean plusmn SEM of 3 experiments

includes the TACE It was verified that when the cells inco-culture were incubated with BB1101 (10 120583M) prior to thetreatments with glutamate (600 120583M) and NE (1 120583M) it wasable to completely revert the inhibitory effect of glutamate onmelatonin synthesis (119875 lt 005) (Figure 3)

RT-PCR analyses showed mRNA expression of classIII metabotropic glutamate receptors (mGluR1mGluR5 andmGluR2mGluR3 resp) as well as ionotropic NMDA andAMPA-type receptors (GluN1 GluA1 GluA2 and GluA3subunits) in astrocytes and pinealocytes (Figures 4(a) and4(b)) Contrarily mGluR7 mRNA (class III metabotropicreceptors) was not detected in any of the cell types(Figure 4(a)) To determine whether class I mGluRs NMDA

and AMPA ionotropic receptors are functional and incor-porated into the astrocytic and pinealocytic membranes anintracellular calcium analysis by confocal microscopy wasused It was observed that the class I metabotropic receptoragonist DHPG (50120583M) and the ionotropic receptor agonistsNMDA (100120583M) and AMPA (50 120583M) were able to increasethe astrocytes and pinealocytes intracellular calcium contentin a similar way as that observed for glutamate (600 120583M)(Figure 5 Table 1)

Electrophoretic mobility shift assays revealed that gluta-mate (100 and 600120583M) activates NF-120581B in cultured rat pinealglands The result of the competition study showed that theupper complex 1 was displaced by an excess of unlabeled

BioMed Research International 7

60

40

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0NE 1120583M

Mela

toni

n (n

gm

L)

lowast

+ +

+ +

+

+

+

+minus minus

minus minus

lowastlowast

BB1101 10120583MGlu 600 120583M

Figure 3 (a) Ablation of the inhibitory glutamatergic effect onmelatonin synthesis by the TACE inhibitor BB1101 Pinealocytesand astrocytes were maintained in co-culture and stimulated withnorepinephrine (NE 1 120583M) in association with glutamate (Glu600 120583M) and BB1101 (10120583M) One-way ANOVA Bonferroni mul-tiple comparison test lowastlowast119875 lt 001 versus NE 1 120583M lowast119875 lt 005 versusNE 1 120583M + Glu 600 120583M 119899 = 4 for each group 3 experiments

Table 1 Summary of intracellular [Ca2+] changes in astrocytes andpinealocytes after stimulation

Cell typeagents Number of cellsexamined [Ca2+]

119894response

Isolated astrocytesGlu 600 120583M 68 79AMPA 50 120583M 97 80NMDA 100 120583M 49 90DHPG 50 120583M 80 74

Isolated pinealocytesGlu 600 120583M 66 80AMPA 50 120583M 78 85NMDA 100 120583M 53 90DHPG 50 120583M 60 76

Intracellular [Ca2+] in fluo-4-loaded astrocytes and pinealocytes was mea-sured as described in Section 2 The majority of the stimulated cellsresponded with an increase in intracellular [Ca2+] after the addition of eitherglutamate (600120583M) ionotropic glutamatergic agonists (AMPA 50120583M andNMDA 100120583M) or a class I metabotropic glutamatergic agonist (DHPG50120583M)

NF-120581B but not by a TFIID double-stranded oligonucleotideconsensus sequence demonstrating the specificity of the NF-120581BDNA interactionThe lower complex 2 was less efficientlydisplaced by unlabeledNF-120581B probe (Figure 6(a)) Supershiftanalysis indicated that the antibody against the p50 subunitwas able to shift the DNAprotein interaction present incomplex 1 The presence of antibodies against the p52 p65and c-Rel subunits did not affect DNA-protein complexes(Figures 6(b) and 6(c)) Taken together these results indicatethat at these concentrations glutamate activates the p50p50

homodimer Complex 2 was not displaced by the antibodiesand was not considered to be related to the NF-120581B family

Astrocyte and pinealocyte isolated cultures were used todetermine the cell type in which NF-120581B was being activatedGlutamate (600120583M)was shown to activateNF-120581B exclusivelyin astrocytes and not in pinealocytes (119875 lt 001) (Figure 7)

The involvement of NF-120581B activation in glutamatersquosinhibitory effect on melatonin synthesis was analyzed usinga specific inhibitor of NF-120581B PDTC PDTC (300 120583M) pre-vented the inhibitory effect of glutamate in cultured rat pinealglands stimulated by NE (1 120583M) (119875 lt 001) (Figure 8)

Glutamate cytotoxicity was evaluated because it is knownthat this neurotransmitter in high concentrations couldinduce cell death by cytotoxicity [48] Cell membraneintegrity and DNA fragmentation were assessed by flowcytometric analysis using propidium iodide (PI) Supple-mentary Figure 1 is representative of the results obtainedafter cell incubation with NE (1120583M) in the absence (controlgroup) or presence of glutamate (100 and 600120583M) for 5 hThe histograms show the fluorescence intensity in the FL-2channel which corresponds to the PI emission wavelengthWhen the cell membrane is intact the fluorescence signal islow and is related to autofluorescence (M1) A high signalmeans that PI was able to enter the cell and bind to DNA(M2) Glutamate treatments for 5 h were not able to inducea loss of membrane integrity (Supplementary Figure 1(a))The percentage of cells with ruptured membranes in thecontrol group was 453 whereas it was 502 and 577 inthe 100 and 600 120583M glutamate-treated groups respectively(Supplementary Figure 1(c)) Histograms in SupplementaryFigure 1(b) represent the results of DNA fragmentationanalysis After PI treatment the DNA content in live cellspresented a high fluorescence signal (M2) DNA fragmenta-tion induces the formation of small DNA pieces that exhibitlow fluorescence intensity (M1) The percentages of cellswith DNA fragmentation after glutamate stimulation 100and 600120583M were 286 and 335 respectively which weresimilar to the value of control cells (366) (SupplementaryFigure 1(d))

4 Discussion

Isolated pinealocytes isolated astrocytes and pineal-ocyteastrocyte co-culture were used in this study Theimmunocytochemistry characterization of the cell typespresent in these different cultures demonstrated the pre-dominance of pinealocytes in isolated pinealocyte culturewith the absence of glial cells In the same way astrocytecultures do not contain a significant number of microglialcells or pinealocytes In co-culture pinealocytes are thepredominant cells as astrocytes are present in minorquantities reproducing the proportion present in intactpineal glands (51) Thus the cultures were considered to beappropriate for this study

Data from the literature have shown that glutamate acts asan inhibitory neurotransmitter on melatonin synthesis [12]According to Yamada et al [13 14 16] and Yatsushiro et al[16] glutamate is stored in microvesicles and released from

8 BioMed Research International

mGluR1 mGluR2 mGluR3 mGluR5 mGluR7

M H2O As Pi Hi As Pi Hi As Pi Hi As Pi Hi As Pi Hi

200 bp100 bp

(a)

M H2O As Pi Hi As Pi Hi As Pi Hi As Pi Hi

200 bp100 bp

GluA1 GluA2 GluA3 GluN1

(b)

Figure 4 RT-PCR detection of metabotropic and ionotropic glutamate receptors in isolated astrocytes and pinealocytes (a) RT-PCRdetection of transcripts for mGluR1 (79 bp) mGluR2 (198 bp) mGluR3 (196 bp) mGluR5 (165 bp) and mGluR7 (70 bp) in astrocytes (As)pinealocytes (Pi) and hippocampus (Hi) Molecular weight marker (M) and water (H

2O) are presented in lanes 1 and 2 respectively (b)

RT-PCR detection of transcripts for GluA1 (203 bp) GluA2 (155 bp) GluA3 (193 bp) and GluN1 (201 bp) in astrocytes (As) pinealocytes(Pi) and hippocampus (Hi) The molecular weight marker (M) and PCR product generated with water (H

2O) are presented in lanes 1 and 2

respectively

pinealocytes upon cholinergic stimulation It interacts withionotropic and metabotropic glutamate receptors presentin pinealocyte membranes releasing additional glutamateand inhibiting melatonin synthesis respectively Recently amechanism involving the glutamate transporter GLT-1 wasproposed to be responsible for glutamate release [17] Inthese studies involving pinealocytes an autocrineparacrineprocess was suggested for the influence of glutamate onmelatonin synthesis In the presentworkwe demonstrated animportant role for astrocytes in glutamatersquos modulatory effecton melatonin synthesis that includes a paracrine interactionwith pinealocytes

Whereas in isolated pinealocytes glutamate does notexhibit the typical inhibitory effect on melatonin synthesisas observed in intact pineal glands its association withastrocytes restores this effect These results demonstrate thatastrocytes act as partners that collaborate to produce theglutamate effect In addition the fact that the effect occurredeven when inserts were used preventing the physical contactbetween the cells in co-culture suggests that a solublefactor is a mediator of this process The hypothesis that gapjunctions mediated the interactions between astrocytes andpinealocytes as occurs in the pineal gland [49] was excludedfrom this observationThe cytokine TNF-120572 a gliotransmitterthat is released by astrocytes could be the soluble factorthat is involved in the interactions between astrocytes andpinealocytes In fact the absence of a soluble form of TNF-120572 interferes with the glutamate response

The possibility that the glutamate effects were due to celldeath was investigated by flow cytometry As the cell viabilityand DNA fragmentation levels were similar between controland glutamate-stimulated groups showing no damage topineal cells even when treated with 600120583M of glutamate itis possible to exclude a glutamate cytotoxic effect as a causeof melatonin reduction

Although glutamate receptor expression had been firstdescribed in pinealocytes its expression was also laterdemonstrated in astrocytes mGluR3 and AMPA (GluA1subunit) were shown to be present in pinealocytes [12 16]

and NMDA and AMPA (GluA23) receptors were present inrat pineal gland astrocytes [19] Ionotropic receptor mRNAwas similarly observed in astrocytes in the present studyin addition to the novel observation of metabotropic recep-tor (mGluR1 mGluR2 mGluR3 mGluR5) expression Inpinealocytes the expression of mGluR1R2R3R5 AMPAand NMDA receptors was shownThis evidence supports thepossibility of astrocyte participation in rat pineal glutamater-gic signaling

Confocal microscopy assays using NMDA AMPA andDHPG agonists in isolated astrocytes and pinealocytesrevealed increased intracellular calcium content in both celltypes for all the agonists used PCR and confocal microscopyanalyses suggest both gene and protein expression in classI mGluR AMPA and NMDA ionotropic receptors reveal-ing the importance of the glutamate receptors as signaltransducers in rat pineal gland astrocytes and pinealocytesThus glutamate seems to interact with its receptors inboth cell types but its effect on melatonin synthesis nec-essarily involves astrocyte glutamate receptors The possibleparacrine interactions between pinealocytes and astrocytesseem to be the key in understanding the role of astrocytes inthe rat pineal gland

Transcription factors exert a great influence on pinealphysiology CREB and ICER are two transcription factorsthat intimately control the transcription of AANAT [50]Additionally there are other transcription factors in the ratpineal gland whose roles in pineal melatonin synthesis arenot known [51] NF-120581B is present in the rat pineal glandshowing a circadian rhythm and involvement in inflamma-tory responses [36 37] NF-120581B is a classic possible glutamatemediator and its activation in glial cells was demonstrated toplay a role in inflammatory processes [30]The present resultsdemonstrated NF-120581B activation by glutamate in culturedisolated astrocytes supporting its role as a mediator ofglutamate effects Supershift assay in cultured pineal glandsstimulated with glutamate showed the displacement of thep50 subunit emphasizing the importance of p50p50 homod-imer formationThis is an astrocyte-dependent event because

BioMed Research International 9

0 50 100 150 200 250 300 350

10

20

30

40

50

60

Time (s)

Isolated astrocytes

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

Glu 600120583M

(a)

0 30 60 90 120 150

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Time (s)

Isolated pinealocytes

Fluo

-4 d

ye ch

ange

s(fl

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scen

cein

tens

ity)

Glu 600120583M

(b)

0 100 200 300 400 500

10

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Time (s)

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cein

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AMPA 50120583M

(c)

0 30 60 90 120 150 180

102

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s(fl

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cein

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AMPA 50120583M

(d)

0 50 100 150 200 250 300

400

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-4 d

ye ch

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s(fl

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cein

tens

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NMDA 100120583M

(e)

0 50 100 150 200 250

900

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Time (s)

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-4 d

ye ch

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s(fl

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cein

tens

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NMDA 100120583M

(f)

100 150 200 250

126

128

130

132

134

136

Time (s)

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-4 d

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s(fl

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tens

ity) DHPG 50120583M

(g)

0 100 200 300 400

80

90

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Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

DHPG 50120583M

(h)

Figure 5 Analysis of intracellular calcium in isolated astrocyte and pinealocyte cultures Traces of Fluo-4 fluorescence indicate theintracellular calcium transients from single cells Additions of exogenous glutamate or glutamatergic agonists were performed as indicatedby the arrows

10 BioMed Research International

ControlGlu 100120583MGlu 600120583MUnlabeled NF-120581BUnlabeled TFIID

1

(NS) 2

Free probe

+

minus

minus

minus

minus +

minus

minus

minus

minus

+

minus

minus

minus

minus +

+

minus

minus

minus +

+

minus

minus

minus+

+

minus

minus

minus

+

+

minus

minus

minus

(a)

(NS) 2

Free probe

(p50p50) 1

(NS) 3

+

+

minus

minus

minus

minus

+

minus

minus

minus

minus

minus

+

minus

minus

minus

minus

minus

+

+

minus

minus

minus

minus

+

+

minus

minus

minus

minus

+

+

minus

minus

minus

minus

ControlGlu 100120583MAnti p50Anti p52Anti p65Anti c-Rel

(b)

Free probe

Anti c-Rel

(NS) 2

(p50p50) 1

(NS) 3

ControlGlu 600120583MAnti p50Anti p52Anti p65

+minus minus minus minus minus

+

+ +

+

+ + +

+

+minus minus

minus

minus

minus

minus minus minus minus minus

minus

minus minus minus

minus minus

minus minus minus

minus minus

(c)

Figure 6 Electrophoretic mobility shift assays for NF120581B in the pineal gland (a) Competition studies were performed using nuclear extract(5120583g) from cultured pineal glands treated with glutamate (Glu) 100 and 600 120583M in the absence or presence of unlabeled specific (NF-120581Bconsensus sequence 10-fold molar excess) or nonspecific oligonucleotide (TFIID consensus sequence 10-fold molar excess) as indicated(b c) Supershift assays were performed with the same nuclear extract (5120583g) incubated in the absence or presence of antibodies against thesubunits p50 (1 10 dilution) p65 (1 20 dilution) p52 and c-Rel (1 10 dilution) as indicated Antibodies were added 20min prior to theaddition of the radiolabeled NF-120581B consensus oligonucleotide The position of specific NF-120581BDNA binding complexes p50p50 (band 1 bc) is indicated NS represents no specific binding (bands 2 and 3 a b c) The localization of the free probe is also indicated

BioMed Research International 11

ControlGlu 600120583M

minusminus

minus +

NF-120581B

(a)

20000

15000

10000

0Control Glu 600120583M

5000

lowastlowast

(au

)

(b)

ControlGlu 600120583M

minusminus

minus +

NF-120581B

(c)

(au

)

Control Glu 600120583M

16000

12000

8000

4000

0

(d)

Figure 7 Electrophoretic mobility shift assays for NF120581B in isolated astrocytes and pinealocytes (a) Nuclear proteins (5 120583g) were extractedfrom astrocytes treated with glutamate (Glu) 600120583M (b)Densitometric analysis (arbitrary units) of theNF-120581B band is presented on panel (a)One-way ANOVA Studentrsquos test lowastlowast119875 lt 001 versus control group mean plusmn SEM (c) Nuclear proteins (5120583g) were extracted from pinealocytestreated with Glu 600 120583M (d) Densitometric analysis (arbitrary units) of the NF-120581B band is presented in (c) The position of the specificNF-120581BDNA binding complex is indicated 119899 = 5 for each group 3 experiments

its activation was not observed in pinealocytes Astrocyteand pinealocyte supershift assays were not performed due totechnical limitations related to the amount of nuclear protein

The p50 subunit is synthesized from its precursor p105The processing of p105 and the resulting generation ofmature p50 subunits seem to be a constitutive event as p50homodimers are not regulated by I120581B proteins [34] The p50subunit formation seems to be regulated by cotranslationaland posttranslational mechanisms In the posttranslationalmechanism the p50 subunit is synthesized from its precursorp105 by phosphorylation at its 927 and 932 serine residueswhich allows the ubiquitinization and proteolytic degrada-tion of p105 by the 26S proteasome [52] In the cotranslationalmechanism p50 and p105 can be generated from a singlemRNA [53] There is also evidence that the 20S protea-some constitutively processes p105 to p50 in a ubiquitin-independent manner [52] The p50p50 homodimer wasoriginally considered to be a transcriptional repressor butnow there is substantial evidence that it can also inducegene transcription after a complex formation with the BCL3nuclear protein an atypical member of the I120581B family ofproteins that is considered to be a coactivator of p50p50homodimers [34]

The link between NF-120581B activation by glutamate and thereduction of melatonin synthesis was evidenced with the useof PDTC a NF-120581B translocation inhibitor [54] PDTC pre-vented glutamatersquos inhibitory effect on melatonin synthesisTaken together the crucial presence of astrocytes for theinhibitory effect of glutamate the exclusive activation of NF-120581B by glutamate in the same cell type and the demonstrationof NF-120581Brsquos role inmelatonin synthesis indicate that astrocytesunder glutamate stimulation possibly release a factor thatpromotes a decrease in pinealocyte melatonin synthesisTNF-120572may be a good candidate Indeed TNF-R1 the TNF-120572 receptor that interacts with soluble TNF is present in thepineal gland reinforcing the importance of this signalingpathway [55] Acting as an inhibitor of TACE to prevent TNF-120572 release BB1101 was thus adequate in demonstrating the roleplayed byTNF-120572 In addition the hypothesis that the p50p50homodimer may induce TNF-120572 gene expression and releaseneeds to be clarified

In the CNS astrocytes are known to release gliotransmit-ters such as glutamate D-serine and ATP [21] Moreoverastrocytes can also synthesize and release proinflammatorycytokines in response to NF-120581B activation such as TNF-120572 and interleukin-1 and induce gene expression of iNOS

12 BioMed Research International

NE 1120583MGlu 100120583MGlu 600120583MPDTC 300120583M

Mela

toni

n (n

ggl

and)

20

15

10

5

0

lowastlowast

lowastlowast

+ +

+ + +

++

+

+

+

+

+ +

minus minus minusminus

minus minus minusminus

minus minus minus

Figure 8 Effect of PDTC an inhibitor of NF-120581B on melatoninsynthesis Pineal glands in culture were incubated with PDTC(300120583M) and stimulated by norepinephrine (NE 1120583M) in thepresence of glutamate (100 and 600120583M) andmelatonin content wasevaluated One-way ANOVA Bonferroni multiple comparison test119875 lt 001 versus NE 1 120583M lowastlowast119875 lt 001 versus NE + Glu 100 120583Mand NE + Glu 600 120583M plotted as the mean plusmn SEM 119899 = 12 for eachgroup 3 experiments

PinealocyteAstrocyte

NF-120581Bactivation

AMPANMDAmGluR15

Release of solublefactor

TNF-120572

Glu

TNF-120572 alone (recruiting AMPA)or in association with Glu

mGluR3TNF-R1 AMPA[iCa+2]

Melatoninsynthesis

Figure 9 Diagram illustrating the putative paracrine interactionbetween pinealocytes and astrocytes in the modulation of pinealmelatonin synthesis by glutamate Glutamate (Glu) induces intra-cellular Ca2+ increase in astrocytes with the subsequent activationof NF120581B and release of a soluble factor possibly TNF-120572 This factoralone or in association with Glu promotes melatonin synthesisreduction in pinealocytes

resulting in nitric oxide formation [56] In this work wesuggested that TNF-120572 is the factor that is released byastrocytes under glutamate stimulation because the TACEinhibitor antagonized glutamatersquos effects Reinforcing thisidea recent preliminary data obtained in our laboratorywith the L929 tumor cell lineage which is sensitive to TNF-120572 showed that astrocytes release TNF-120572 when stimulatedby glutamate (data not shown) The literature states that

TNF-120572 decreases the serotonin content and AANAT mRNAexpression resulting in the reduction of N-acetylserotonin[57 58] the immediate precursor of melatonin synthesis Itis not clear whether TNF-120572 acts by itself or in combinationwith glutamate because there are glutamate receptors in thepinealocyte membrane [12 15 16 18] Recent studies havereported the control of hippocampal synapses by TNF-120572 dueto the increased insertion of AMPA receptors in the surfaceof the neuronal soma [59]The same effect could take place inthe pineal gland in which TNF-120572 released by the astrocytesin response to NF-120581B activation could increase the numberof AMPA receptors in the pinealocyte membrane resultingin the inhibition of melatonin synthesis

A model of the putative paracrine interactions betweenastrocytes and pinealocytes in the modulation of pinealmelatonin synthesis by glutamate is shown in Figure 9

5 Conclusions

In summary according to our results we propose thatglutamate acts on glutamate receptors (AMPA NMDAandor mGluR15) in astrocytes increasing the intracellularcalcium concentration promoting NF-120581B activation anddriving the release of a soluble factor possibly TNF-120572 whichacts independently or with glutamate in the pinealocytesresulting in inhibition of melatonin synthesis

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

This work was supported by a grant from FAPESP (0404328-1) Darine Villela was supported by a master fellowshipfrom FAPESP (0556943-4) Anderson Augusto Moutinhoand Victoria Fairbanks Atherino were supported by CNPq(PIBIC)

References

[1] J Calvo J Boya A Borregon and J E Garcia-MaurinoldquoPresence of glial cells in the rat pineal gland a light andelectronmicroscopic immunohistochemical studyrdquoAnatomicalRecord vol 220 no 4 pp 424ndash428 1988

[2] L Vollrath The Pineal Organ Springer New York NY USA1981

[3] R Y Moore J C Speh and J P Card ldquoThe retinohypothalamictract originates from a distinct subset of retinal ganglion cellsrdquoJournal of Comparative Neurology vol 352 no 3 pp 351ndash3661995

[4] D Sugden ldquoMelatonin biosynthesis in the mammalian pinealglandrdquo Experientia vol 45 no 10 pp 922ndash932 1989

[5] A F Afeche D CMVillelaM V Abrahao R Peres J Cipolla-Neto et al ldquoMelatonin and pineal glandrdquo in New Researchon Neurosecretory Systems E Romano Ed pp 151ndash177 NovaScience Publishers Hauppauge NY USA 2008

[6] J Arendt Melatonin and the Mammalian Pineal Gland Chap-man amp Hall London UK 1995

BioMed Research International 13

[7] M Ehret P Pevet and M Maitre ldquoTryptophan hydroxylasesynthesis is induced by 3101584051015840-cyclic adenosine monophosphateduring circadian rhythm in the rat pineal glandrdquo Journal ofNeurochemistry vol 57 no 5 pp 1516ndash1521 1991

[8] D C Klein G R Berg and J Weller ldquoMelatonin synthesisadenosine 3101584051015840-monophosphate and norepinephrine stimulateN-acetyltransferaserdquo Science vol 168 no 3934 pp 979ndash9801970

[9] D C Klein N L Schaad M A A Namboordiri L Yu and JL Weller ldquoControl of N-acetyltransferaserdquo Biochemical SocietyTransactions vol 20 no 2 pp 299ndash304 1992

[10] V Simonneaux and C Ribelayga ldquoGeneration of the melatoninendocrine message in mammals a review of the complexregulation of melatonin synthesis by norepinephrine peptidesand other pineal transmittersrdquoPharmacological Reviews vol 55no 2 pp 325ndash395 2003

[11] S Ishio H Yamada C M Craft and Y MoriyamaldquoHydroxyindole-O-methyltransferase is another target forL-glutamate-evoked inhibition of melatonin synthesis in ratpinealocytesrdquo Brain Research vol 850 no 1-2 pp 73ndash78 1999

[12] H Yamada S Yatsushiro S Ishio et al ldquoMetabotropic gluta-mate receptors negatively regulate melatonin synthesis in ratpinealocytesrdquo Journal of Neuroscience vol 18 no 6 pp 2056ndash2062 1998

[13] H Yamada A Yamamoto M Takahashi H Michibata HKumon and Y Moriyama ldquoThe L-type Ca2+ channel isinvolved in microvesicle-mediated glutamate exocytosis fromrat pinealocytesrdquo Journal of Pineal Research vol 21 no 3 pp165ndash174 1996

[14] H Yamada A Yamamoto S Yodozawa et al ldquoMicrovesicle-mediated exocytosis of glutamate is a novel paracrine-likechemical transduction mechanism and inhibits melatoninsecretion in rat pinealocytesrdquo Journal of Pineal Research vol 21no 3 pp 175ndash191 1996

[15] H Yamada A Ogura S Koizumi A Yamaguchi and YMoriyama ldquoAcetylcholine triggers L-glutamate exocytosis vianicotinic receptors and inhibits melatonin synthesis in ratpinealocytesrdquo Journal of Neuroscience vol 18 no 13 pp 4946ndash4952 1998

[16] S Yatsushiro H Yamada M Hayashi A Yamamoto andY Moriyama ldquoIonotropic glutamate receptors trigger micro-vesicle-mediated exocytosis of L-glutamate in rat pinealocytesrdquoJournal of Neurochemistry vol 75 no 1 pp 288ndash297 2000

[17] M-HKim SUehara AMuroyama BHille YMoriyama andD-S Koh ldquoGlutamate transporter-mediated glutamate secre-tion in the mammalian pineal glandrdquo Journal of Neurosciencevol 28 no 43 pp 10852ndash10863 2008

[18] S Yatsushiro H Yamada M Hayashi S Tsuboi and YMoriyama ldquoFunctional expression of metabotropic glutamatereceptor type 5 in rat pinealocytesrdquo NeuroReport vol 10 no 7pp 1599ndash1603 1999

[19] C Kaur V Sivakumar and E A Ling ldquoExpression ofN-methyl-D-aspartate (NMDA) and 120572-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) GluR23 receptors inthe developing rat pineal glandrdquo Journal of Pineal Research vol39 no 3 pp 294ndash301 2005

[20] H Pabst and P Redecker ldquoInterstitial glial cells of the gerbilpineal gland display immunoreactivity for the metabotropicglutamate receptors mGluR23 and mGluR5rdquo Brain Researchvol 838 no 1-2 pp 60ndash68 1999

[21] M M Halassa T Fellin and P G Haydon ldquoThe tripartitesynapse roles for gliotransmission in health and diseaserdquoTrends in Molecular Medicine vol 13 no 2 pp 54ndash63 2007

[22] S M Finkbeiner ldquoGlial calciumrdquo Glia vol 9 no 2 pp 83ndash1041993

[23] E A Newman ldquoNew roles for astrocytes regulation of synaptictransmissionrdquo Trends in Neurosciences vol 26 no 10 pp 536ndash542 2003

[24] J T Porter and K D McCarthy ldquoAstrocytic neurotransmitterreceptors in situ and in vivordquo Progress in Neurobiology vol 51no 4 pp 439ndash455 1997

[25] V Parpura T A Basarsky F Liu K Jeftinija S Jeftinija and PG Haydon ldquoGlutamate-mediated astrocyte-neuron signallingrdquoNature vol 369 no 6483 pp 744ndash747 1994

[26] T D Hassinger P B Atkinson G J Strecker et al ldquoEvidencefor glutamate-mediated activation of hippocampal neurons byglial calcium wavesrdquo Journal of Neurobiology vol 28 no 2 pp159ndash170 1995

[27] R H Lipsky K Xu D Zhu et al ldquoNuclear factor 120581B is a criticaldeterminant in N-methyl-D-aspartate receptor-mediated neu-roprotectionrdquo Journal of Neurochemistry vol 78 no 2 pp 254ndash264 2001

[28] M P Mattson C Culmsee Z Yu and S Camandola ldquoRoles ofnuclear factor 120581B in neuronal survival and plasticityrdquo Journal ofNeurochemistry vol 74 no 2 pp 443ndash456 2000

[29] C DMunhoz B Garcıa-Bueno J L MMadrigal L B LepschC Scavone and J C Leza ldquoStress-induced neuroinflammationmechanisms and new pharmacological targetsrdquo Brazilian Jour-nal of Medical and Biological Research vol 41 no 12 pp 1037ndash1046 2008

[30] S Camandola andM PMattson ldquoNF-ΚB as a therapeutic targetin neurodegenerative diseasesrdquo Expert Opinion on TherapeuticTargets vol 11 no 2 pp 123ndash132 2007

[31] B Kaltschmidt D Widera and C Kaltschmidt ldquoSignaling viaNF-120581B in the nervous systemrdquo Biochimica et Biophysica Actavol 1745 no 3 pp 287ndash299 2005

[32] P T Massa H Aleyasin D S Park X Mao and S W BargerldquoNF120581B in neurons the uncertainty principle in neurobiologyrdquoJournal of Neurochemistry vol 97 no 3 pp 607ndash618 2006

[33] H Hacker and M Karin ldquoRegulation and function of IKK andIKK-related kinasesrdquo Sciencersquos STKE vol 2006 no 357 p re132006

[34] S Beinke and S C Ley ldquoFunctions of NF-120581B1 and NF-120581B2 inimmune cell biologyrdquo Biochemical Journal vol 382 no 2 pp393ndash409 2004

[35] N D Perkins ldquoIntegrating cell-signalling pathways with NF-120581Band IKK functionrdquo Nature Reviews Molecular Cell Biology vol8 pp 49ndash62 2007

[36] E Cecon P A Fernandes L Pinato Z S Ferreira and R PMarkus ldquoDaily variation of constitutively activated nuclear fac-tor 120581B (NFKB) in rat pineal glandrdquoChronobiology Internationalvol 27 no 1 pp 52ndash67 2010

[37] Z S Ferreira P A C M Fernandes D Duma J Assreuy MC W Avellar and R P Markus ldquoCorticosterone modulatesnoradrenaline-induced melatonin synthesis through inhibitionof nuclear factor 120581Brdquo Journal of Pineal Research vol 38 no 3pp 182ndash188 2005

[38] S C Afeche R Barbosa J H Scialfa I M Terra A CCassola and J Cipolla-Neto ldquoEffects of the blockade of highvoltage-activated calcium channels on in vitro pineal melatoninsynthesisrdquo Cell Biochemistry and Function vol 24 no 6 pp499ndash505 2006

14 BioMed Research International

[39] B Barlaam T G Bird C Lambert-Van Der Brempt DCampbell S J Foster and R Maciewicz ldquoNew 120572-substitutedsuccinate-based hydroxamic acids as TNF120572 convertaseinhibitorsrdquo Journal of Medicinal Chemistry vol 42 no 23 pp4890ndash4908 1999

[40] I Glezer C D Munhoz E M Kawamoto T MarcourakisM C Werneck Avellar and C Scavone ldquoMK-801 and 7-Ni attenuate the activation of brain NF-120581B induced by LPSrdquoNeuropharmacology vol 45 no 8 pp 1120ndash1129 2003

[41] Y Rong and M Baudry ldquoSeizure activity results in a rapidinduction of nuclear factor-120581B in adult but not juvenile ratlimbic structuresrdquo Journal of Neurochemistry vol 67 no 2 pp662ndash668 1996

[42] I Nicoletti G Migliorati M C Pagliacci F Grignani and CRiccardi ldquoA rapid and simple method for measuring thymocyteapoptosis by propidium iodide staining and flow cytometryrdquoJournal of Immunological Methods vol 139 no 2 pp 271ndash2791991

[43] Y F Jiang-Shieh C H Wu M L Chang J Y Shiehand C Y Wen ldquoRegional heterogeneity in immunoreactivemacrophagesmicroglia in the rat pineal glandrdquo Journal ofPineal Research vol 35 no 1 pp 45ndash53 2003

[44] K-O Soderstrom ldquoLectin binding to the human retinardquoAnatomical Record vol 220 no 2 pp 219ndash223 1988

[45] F Uehara M Sameshima T Muramatsu and N OhbaldquoLocalization of fluorescence-labeled lectin binding sites onphotoreceptor cells of the monkey retinardquo Experimental EyeResearch vol 36 no 1 pp 113ndash123 1983

[46] H-W Korf ldquoThe pineal organ as a component of the biologicalclock phylogenetic and ontogenetic considerationsrdquo Annals ofthe New York Academy of Sciences vol 719 pp 13ndash42 1994

[47] E Maronde and J H Stehle ldquoThe mammalian pineal glandknown facts unknown facetsrdquo Trends in Endocrinology andMetabolism vol 18 no 4 pp 142ndash149 2007

[48] X-M Zhang and J Zhu ldquoKainic acid-induced neurotoxicitytargeting glial responses and glia-derived cytokinesrdquo CurrentNeuropharmacology vol 9 no 2 pp 388ndash398 2011

[49] D Stellwagen and R CMalenka ldquoSynaptic scalingmediated byglial TNF-120572rdquo Nature vol 440 no 7087 pp 1054ndash1059 2006

[50] E Maronde M Pfeffer J Olcese et al ldquoTranscription factorsin neuroendocrine regulation rhythmic changes in pCREB andICER levels framemelatonin synthesisrdquo Journal of Neurosciencevol 19 no 9 pp 3326ndash3336 1999

[51] D M Bustos M J Bailey D Sugden et al ldquoGlobal dailydynamics of the pineal transcriptomerdquoCell and Tissue Researchvol 344 no 1 pp 1ndash11 2011

[52] A K Moorthy O V Savinova J Q Ho V Y-F Wang D VuandGGhosh ldquoThe 20S proteasome processesNF-120581B1 p105 intop50 in a translation-independent mannerrdquo EMBO Journal vol25 no 9 pp 1945ndash1956 2006

[53] L Lin G N DeMartino and W C Greene ldquoCotranslationalbiogenesis of NF-120581B p50 by the 26S proteasomerdquo Cell vol 92no 6 pp 819ndash828 1998

[54] H W L Ziegler-Heitbrock T Sternsdorf J Liese et alldquoPyrrolidine dithiocarbamate inhibits NF-120581B mobilization andTNF production in humanmonocytesrdquo Journal of Immunologyvol 151 no 12 pp 6986ndash6993 1993

[55] C E Carvalho-Sousa S S Cruz-Machado E K Tamura P AC M Fernandes L Pinato et al ldquoMolecular basis for definingthe pineal gland and pinealocytes as targets for tumor necrosisfactorrdquo Frontiers in Endocrinology vol 2 pp 1ndash11 2011

[56] M PMattson ldquoNF-120581B in the survival and plasticity of neuronsrdquoNeurochemical Research vol 30 no 6-7 pp 883ndash893 2005

[57] P A CM Fernandes E Cecon R PMarkus and Z S FerreiraldquoEffect of TNF-120572 on the melatonin synthetic pathway in the ratpineal gland basis for a rsquofeedbackrsquo of the immune response oncircadian timingrdquo Journal of Pineal Research vol 41 no 4 pp344ndash350 2006

[58] S-Y Tsai T E OrsquoBrien and J A McNulty ldquoMicroglia play arole in mediating the effects of cytokines on the structure andfunction of the rat pineal glandrdquo Cell and Tissue Research vol303 no 3 pp 423ndash431 2001

[59] J S Bains and S H R Oliet ldquoGlia they make your memoriesstickrdquo Trends in Neurosciences vol 30 no 8 pp 417ndash424 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

BioMed Research International 5

GFAP

(a)

WGA

(b)

GFAP

(c)

WGA

50120583M

(d)

Figure 1 Cell cultures immunocytochemistry Digital images of isolated cultures of astrocytes (a) and pinealocytes (b) showing specific celltype staining using a mouse monoclonal antibody against the glial fibrillary acidic protein (GFAP) and a wheat germ agglutinin-rhodaminelabel (WGA) to identify pinealocytes The pictures at the bottom illustrate the distribution of glial cells (c) and pinealocytes (d) in a mixedcoculture The insets show photomicrographies of the cell cultures in contrast phase microscopy astrocytes (a) pinealocytes (b) and co-culture (c and d) In isolated pinealocytes and in co-culture many cells are clustered Magnification 200x (astrocytes and pinealocytes) and100x (co-culture)

3 Results

The cultures used in this study were characterized and val-idated by immunostaining with GFAP a specific marker forastroglial cells and OX-42 a marker for microglia (specificfor activated microglia as the antibody reacts with the CR3complement) This OX-42 antibody is suitable for pinealgland staining because the predominant type of microglia inthis tissue expresses complement type 3 receptors [43] WGAwas used as a pinealocyte marker because it labels photore-ceptors [44 45] and pinealocytes aremodified photoreceptorcells [46 47]This technique proved to be efficient in labelingthese pineal cells (Figure 1)

Cultured isolated astrocytes showed fluorescence forGFAP (Figure 1(a)) but not for OX-42 and WGA (data notshown) Cultured pinealocytes lacked immunostaining forthe two glial markers GFAP and OX-42 (data not shown)and reacted withWGA demonstrating the absolute predom-inance of pinealocytes (Figure 1(b)) Figures 1(c) and 1(d)show the distribution of pinealocytes and astrocytes in themixed culture (co-culture) The round appearance of thelabeled cells in Figure 1 different from that in the insets is dueto the fixation process with paraformaldehyde In Figure 1(d)pinealocytes appear smaller than those in Figure 1(b) most

likely due to the use of trypsin to obtain the cells furtherdissociating the cell clusters

In intact cultured pineal glands the known effect ofglutamate in reducing NE-stimulated melatonin synthesis(119875 lt 005) was observed (Figure 2(a)) On the other handwhen isolated cultured pinealocytes were incubated withglutamate (100 and 600 120583M) in the presence of NE (1 120583M)this effect was abrogated (Figure 2(b)) Glutamatersquos inhibitoryeffect on melatonin synthesis as observed in intact culturedpineal glands was only reproduced when pinealocytes werecultivated in the presence of astrocytes in co-culture (119875 lt001) (Figure 2(c))

To investigate if the contact between astrocytes andpinealocytes is necessary for the inhibitory effect of gluta-mate the two cell types were cocultivated in the same wellbut separated by inserts It was observed that glutamatersquosinhibitory effect on NE-stimulated melatonin synthesis wasexpressed in the same way as when the cells were cultivatedwith physical contact indicating that a diffusible factorreleased by astrocytes acted on the pinealocytes (119875 lt 001)(Figure 2(d))

The possibility of TNF-120572 as a gliotransmitter that medi-ates the glutamate effect was tested using BB1101 a preferredinhibitor of metalloproteinases with sheddase activity that

6 BioMed Research International

18

15

12

9

6

3

0

Mela

toni

n (n

ggl

and)

lowast

NE 1120583MGlu 100120583MGlu 600120583M

+ +

+

+

+

minus minus

minus minus

(a)

0

Mela

toni

n (n

gm

L)

NE 1120583MGlu 100120583MGlu 600120583M

+ +

+

+

+

minus minus

minus minus

120

80

40

(b)

Mela

toni

n (n

gm

L)

NE 1120583MGlu 100120583MGlu 600120583M

+ +

+

+

+

minus minus

minus minus

50

40

30

20

10

0

lowastlowast lowastlowast

(c)

NE 1120583MGlu 600120583M

Mela

toni

n (n

gm

L)

+ +

+minus

lowastlowast

100

80

60

40

20

0

(d)

Figure 2 Glutamate effects on melatonin synthesis (a) Pineal gland culture stimulated with norepinephrine (NE 1120583M) in the absence orpresence of glutamate (Glu 100 and Glu 600120583M) Melatonin content is expressed in nggland as the mean plusmn SEM of 3 experiments One-wayANOVA Bonferroni multiple comparison test lowast119875 lt 005 versus NE 1 120583M 119899 = 12 for each group (b) Isolated rat pinealocyte culture underNE 1120583M stimulation in the absence or presence of Glu 100 or Glu 600120583M 119899 = 6 for each group (c) Pinealocyte and astrocyte co-culturestimulated with NE 1 120583M in the absence or presence of Glu 100 or Glu 600 120583M One-way ANOVA Bonferroni multiple comparison testlowastlowast119875 lt 001 versus NE 1 120583M 119899 = 8 for each group (d) Astrocytes and pinealocytes were cultured together but with physical separation by

inserts Astrocytes were cultivated in the bottom of the plate and pinealocytes were cultivated within the inserts and stimulated with NE1 120583M associated or not associated with Glu 600 120583M 119879 test lowastlowast119875 lt 001 119899 = 4 for each group For the cell cultures melatonin was evaluatedin the culture medium and expressed in ngmL as the mean plusmn SEM of 3 experiments

includes the TACE It was verified that when the cells inco-culture were incubated with BB1101 (10 120583M) prior to thetreatments with glutamate (600 120583M) and NE (1 120583M) it wasable to completely revert the inhibitory effect of glutamate onmelatonin synthesis (119875 lt 005) (Figure 3)

RT-PCR analyses showed mRNA expression of classIII metabotropic glutamate receptors (mGluR1mGluR5 andmGluR2mGluR3 resp) as well as ionotropic NMDA andAMPA-type receptors (GluN1 GluA1 GluA2 and GluA3subunits) in astrocytes and pinealocytes (Figures 4(a) and4(b)) Contrarily mGluR7 mRNA (class III metabotropicreceptors) was not detected in any of the cell types(Figure 4(a)) To determine whether class I mGluRs NMDA

and AMPA ionotropic receptors are functional and incor-porated into the astrocytic and pinealocytic membranes anintracellular calcium analysis by confocal microscopy wasused It was observed that the class I metabotropic receptoragonist DHPG (50120583M) and the ionotropic receptor agonistsNMDA (100120583M) and AMPA (50 120583M) were able to increasethe astrocytes and pinealocytes intracellular calcium contentin a similar way as that observed for glutamate (600 120583M)(Figure 5 Table 1)

Electrophoretic mobility shift assays revealed that gluta-mate (100 and 600120583M) activates NF-120581B in cultured rat pinealglands The result of the competition study showed that theupper complex 1 was displaced by an excess of unlabeled

BioMed Research International 7

60

40

20

0NE 1120583M

Mela

toni

n (n

gm

L)

lowast

+ +

+ +

+

+

+

+minus minus

minus minus

lowastlowast

BB1101 10120583MGlu 600 120583M

Figure 3 (a) Ablation of the inhibitory glutamatergic effect onmelatonin synthesis by the TACE inhibitor BB1101 Pinealocytesand astrocytes were maintained in co-culture and stimulated withnorepinephrine (NE 1 120583M) in association with glutamate (Glu600 120583M) and BB1101 (10120583M) One-way ANOVA Bonferroni mul-tiple comparison test lowastlowast119875 lt 001 versus NE 1 120583M lowast119875 lt 005 versusNE 1 120583M + Glu 600 120583M 119899 = 4 for each group 3 experiments

Table 1 Summary of intracellular [Ca2+] changes in astrocytes andpinealocytes after stimulation

Cell typeagents Number of cellsexamined [Ca2+]

119894response

Isolated astrocytesGlu 600 120583M 68 79AMPA 50 120583M 97 80NMDA 100 120583M 49 90DHPG 50 120583M 80 74

Isolated pinealocytesGlu 600 120583M 66 80AMPA 50 120583M 78 85NMDA 100 120583M 53 90DHPG 50 120583M 60 76

Intracellular [Ca2+] in fluo-4-loaded astrocytes and pinealocytes was mea-sured as described in Section 2 The majority of the stimulated cellsresponded with an increase in intracellular [Ca2+] after the addition of eitherglutamate (600120583M) ionotropic glutamatergic agonists (AMPA 50120583M andNMDA 100120583M) or a class I metabotropic glutamatergic agonist (DHPG50120583M)

NF-120581B but not by a TFIID double-stranded oligonucleotideconsensus sequence demonstrating the specificity of the NF-120581BDNA interactionThe lower complex 2 was less efficientlydisplaced by unlabeledNF-120581B probe (Figure 6(a)) Supershiftanalysis indicated that the antibody against the p50 subunitwas able to shift the DNAprotein interaction present incomplex 1 The presence of antibodies against the p52 p65and c-Rel subunits did not affect DNA-protein complexes(Figures 6(b) and 6(c)) Taken together these results indicatethat at these concentrations glutamate activates the p50p50

homodimer Complex 2 was not displaced by the antibodiesand was not considered to be related to the NF-120581B family

Astrocyte and pinealocyte isolated cultures were used todetermine the cell type in which NF-120581B was being activatedGlutamate (600120583M)was shown to activateNF-120581B exclusivelyin astrocytes and not in pinealocytes (119875 lt 001) (Figure 7)

The involvement of NF-120581B activation in glutamatersquosinhibitory effect on melatonin synthesis was analyzed usinga specific inhibitor of NF-120581B PDTC PDTC (300 120583M) pre-vented the inhibitory effect of glutamate in cultured rat pinealglands stimulated by NE (1 120583M) (119875 lt 001) (Figure 8)

Glutamate cytotoxicity was evaluated because it is knownthat this neurotransmitter in high concentrations couldinduce cell death by cytotoxicity [48] Cell membraneintegrity and DNA fragmentation were assessed by flowcytometric analysis using propidium iodide (PI) Supple-mentary Figure 1 is representative of the results obtainedafter cell incubation with NE (1120583M) in the absence (controlgroup) or presence of glutamate (100 and 600120583M) for 5 hThe histograms show the fluorescence intensity in the FL-2channel which corresponds to the PI emission wavelengthWhen the cell membrane is intact the fluorescence signal islow and is related to autofluorescence (M1) A high signalmeans that PI was able to enter the cell and bind to DNA(M2) Glutamate treatments for 5 h were not able to inducea loss of membrane integrity (Supplementary Figure 1(a))The percentage of cells with ruptured membranes in thecontrol group was 453 whereas it was 502 and 577 inthe 100 and 600 120583M glutamate-treated groups respectively(Supplementary Figure 1(c)) Histograms in SupplementaryFigure 1(b) represent the results of DNA fragmentationanalysis After PI treatment the DNA content in live cellspresented a high fluorescence signal (M2) DNA fragmenta-tion induces the formation of small DNA pieces that exhibitlow fluorescence intensity (M1) The percentages of cellswith DNA fragmentation after glutamate stimulation 100and 600120583M were 286 and 335 respectively which weresimilar to the value of control cells (366) (SupplementaryFigure 1(d))

4 Discussion

Isolated pinealocytes isolated astrocytes and pineal-ocyteastrocyte co-culture were used in this study Theimmunocytochemistry characterization of the cell typespresent in these different cultures demonstrated the pre-dominance of pinealocytes in isolated pinealocyte culturewith the absence of glial cells In the same way astrocytecultures do not contain a significant number of microglialcells or pinealocytes In co-culture pinealocytes are thepredominant cells as astrocytes are present in minorquantities reproducing the proportion present in intactpineal glands (51) Thus the cultures were considered to beappropriate for this study

Data from the literature have shown that glutamate acts asan inhibitory neurotransmitter on melatonin synthesis [12]According to Yamada et al [13 14 16] and Yatsushiro et al[16] glutamate is stored in microvesicles and released from

8 BioMed Research International

mGluR1 mGluR2 mGluR3 mGluR5 mGluR7

M H2O As Pi Hi As Pi Hi As Pi Hi As Pi Hi As Pi Hi

200 bp100 bp

(a)

M H2O As Pi Hi As Pi Hi As Pi Hi As Pi Hi

200 bp100 bp

GluA1 GluA2 GluA3 GluN1

(b)

Figure 4 RT-PCR detection of metabotropic and ionotropic glutamate receptors in isolated astrocytes and pinealocytes (a) RT-PCRdetection of transcripts for mGluR1 (79 bp) mGluR2 (198 bp) mGluR3 (196 bp) mGluR5 (165 bp) and mGluR7 (70 bp) in astrocytes (As)pinealocytes (Pi) and hippocampus (Hi) Molecular weight marker (M) and water (H

2O) are presented in lanes 1 and 2 respectively (b)

RT-PCR detection of transcripts for GluA1 (203 bp) GluA2 (155 bp) GluA3 (193 bp) and GluN1 (201 bp) in astrocytes (As) pinealocytes(Pi) and hippocampus (Hi) The molecular weight marker (M) and PCR product generated with water (H

2O) are presented in lanes 1 and 2

respectively

pinealocytes upon cholinergic stimulation It interacts withionotropic and metabotropic glutamate receptors presentin pinealocyte membranes releasing additional glutamateand inhibiting melatonin synthesis respectively Recently amechanism involving the glutamate transporter GLT-1 wasproposed to be responsible for glutamate release [17] Inthese studies involving pinealocytes an autocrineparacrineprocess was suggested for the influence of glutamate onmelatonin synthesis In the presentworkwe demonstrated animportant role for astrocytes in glutamatersquos modulatory effecton melatonin synthesis that includes a paracrine interactionwith pinealocytes

Whereas in isolated pinealocytes glutamate does notexhibit the typical inhibitory effect on melatonin synthesisas observed in intact pineal glands its association withastrocytes restores this effect These results demonstrate thatastrocytes act as partners that collaborate to produce theglutamate effect In addition the fact that the effect occurredeven when inserts were used preventing the physical contactbetween the cells in co-culture suggests that a solublefactor is a mediator of this process The hypothesis that gapjunctions mediated the interactions between astrocytes andpinealocytes as occurs in the pineal gland [49] was excludedfrom this observationThe cytokine TNF-120572 a gliotransmitterthat is released by astrocytes could be the soluble factorthat is involved in the interactions between astrocytes andpinealocytes In fact the absence of a soluble form of TNF-120572 interferes with the glutamate response

The possibility that the glutamate effects were due to celldeath was investigated by flow cytometry As the cell viabilityand DNA fragmentation levels were similar between controland glutamate-stimulated groups showing no damage topineal cells even when treated with 600120583M of glutamate itis possible to exclude a glutamate cytotoxic effect as a causeof melatonin reduction

Although glutamate receptor expression had been firstdescribed in pinealocytes its expression was also laterdemonstrated in astrocytes mGluR3 and AMPA (GluA1subunit) were shown to be present in pinealocytes [12 16]

and NMDA and AMPA (GluA23) receptors were present inrat pineal gland astrocytes [19] Ionotropic receptor mRNAwas similarly observed in astrocytes in the present studyin addition to the novel observation of metabotropic recep-tor (mGluR1 mGluR2 mGluR3 mGluR5) expression Inpinealocytes the expression of mGluR1R2R3R5 AMPAand NMDA receptors was shownThis evidence supports thepossibility of astrocyte participation in rat pineal glutamater-gic signaling

Confocal microscopy assays using NMDA AMPA andDHPG agonists in isolated astrocytes and pinealocytesrevealed increased intracellular calcium content in both celltypes for all the agonists used PCR and confocal microscopyanalyses suggest both gene and protein expression in classI mGluR AMPA and NMDA ionotropic receptors reveal-ing the importance of the glutamate receptors as signaltransducers in rat pineal gland astrocytes and pinealocytesThus glutamate seems to interact with its receptors inboth cell types but its effect on melatonin synthesis nec-essarily involves astrocyte glutamate receptors The possibleparacrine interactions between pinealocytes and astrocytesseem to be the key in understanding the role of astrocytes inthe rat pineal gland

Transcription factors exert a great influence on pinealphysiology CREB and ICER are two transcription factorsthat intimately control the transcription of AANAT [50]Additionally there are other transcription factors in the ratpineal gland whose roles in pineal melatonin synthesis arenot known [51] NF-120581B is present in the rat pineal glandshowing a circadian rhythm and involvement in inflamma-tory responses [36 37] NF-120581B is a classic possible glutamatemediator and its activation in glial cells was demonstrated toplay a role in inflammatory processes [30]The present resultsdemonstrated NF-120581B activation by glutamate in culturedisolated astrocytes supporting its role as a mediator ofglutamate effects Supershift assay in cultured pineal glandsstimulated with glutamate showed the displacement of thep50 subunit emphasizing the importance of p50p50 homod-imer formationThis is an astrocyte-dependent event because

BioMed Research International 9

0 50 100 150 200 250 300 350

10

20

30

40

50

60

Time (s)

Isolated astrocytes

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

Glu 600120583M

(a)

0 30 60 90 120 150

155

160

165

170

175

180

185

Time (s)

Isolated pinealocytes

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

Glu 600120583M

(b)

0 100 200 300 400 500

10

20

30

Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

AMPA 50120583M

(c)

0 30 60 90 120 150 180

102

104

106

108

110

112

114

Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

AMPA 50120583M

(d)

0 50 100 150 200 250 300

400

600

800

1000

1200

1400

Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

NMDA 100120583M

(e)

0 50 100 150 200 250

900

1200

1500

Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

NMDA 100120583M

(f)

100 150 200 250

126

128

130

132

134

136

Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity) DHPG 50120583M

(g)

0 100 200 300 400

80

90

100

110

120

130

Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

DHPG 50120583M

(h)

Figure 5 Analysis of intracellular calcium in isolated astrocyte and pinealocyte cultures Traces of Fluo-4 fluorescence indicate theintracellular calcium transients from single cells Additions of exogenous glutamate or glutamatergic agonists were performed as indicatedby the arrows

10 BioMed Research International

ControlGlu 100120583MGlu 600120583MUnlabeled NF-120581BUnlabeled TFIID

1

(NS) 2

Free probe

+

minus

minus

minus

minus +

minus

minus

minus

minus

+

minus

minus

minus

minus +

+

minus

minus

minus +

+

minus

minus

minus+

+

minus

minus

minus

+

+

minus

minus

minus

(a)

(NS) 2

Free probe

(p50p50) 1

(NS) 3

+

+

minus

minus

minus

minus

+

minus

minus

minus

minus

minus

+

minus

minus

minus

minus

minus

+

+

minus

minus

minus

minus

+

+

minus

minus

minus

minus

+

+

minus

minus

minus

minus

ControlGlu 100120583MAnti p50Anti p52Anti p65Anti c-Rel

(b)

Free probe

Anti c-Rel

(NS) 2

(p50p50) 1

(NS) 3

ControlGlu 600120583MAnti p50Anti p52Anti p65

+minus minus minus minus minus

+

+ +

+

+ + +

+

+minus minus

minus

minus

minus

minus minus minus minus minus

minus

minus minus minus

minus minus

minus minus minus

minus minus

(c)

Figure 6 Electrophoretic mobility shift assays for NF120581B in the pineal gland (a) Competition studies were performed using nuclear extract(5120583g) from cultured pineal glands treated with glutamate (Glu) 100 and 600 120583M in the absence or presence of unlabeled specific (NF-120581Bconsensus sequence 10-fold molar excess) or nonspecific oligonucleotide (TFIID consensus sequence 10-fold molar excess) as indicated(b c) Supershift assays were performed with the same nuclear extract (5120583g) incubated in the absence or presence of antibodies against thesubunits p50 (1 10 dilution) p65 (1 20 dilution) p52 and c-Rel (1 10 dilution) as indicated Antibodies were added 20min prior to theaddition of the radiolabeled NF-120581B consensus oligonucleotide The position of specific NF-120581BDNA binding complexes p50p50 (band 1 bc) is indicated NS represents no specific binding (bands 2 and 3 a b c) The localization of the free probe is also indicated

BioMed Research International 11

ControlGlu 600120583M

minusminus

minus +

NF-120581B

(a)

20000

15000

10000

0Control Glu 600120583M

5000

lowastlowast

(au

)

(b)

ControlGlu 600120583M

minusminus

minus +

NF-120581B

(c)

(au

)

Control Glu 600120583M

16000

12000

8000

4000

0

(d)

Figure 7 Electrophoretic mobility shift assays for NF120581B in isolated astrocytes and pinealocytes (a) Nuclear proteins (5 120583g) were extractedfrom astrocytes treated with glutamate (Glu) 600120583M (b)Densitometric analysis (arbitrary units) of theNF-120581B band is presented on panel (a)One-way ANOVA Studentrsquos test lowastlowast119875 lt 001 versus control group mean plusmn SEM (c) Nuclear proteins (5120583g) were extracted from pinealocytestreated with Glu 600 120583M (d) Densitometric analysis (arbitrary units) of the NF-120581B band is presented in (c) The position of the specificNF-120581BDNA binding complex is indicated 119899 = 5 for each group 3 experiments

its activation was not observed in pinealocytes Astrocyteand pinealocyte supershift assays were not performed due totechnical limitations related to the amount of nuclear protein

The p50 subunit is synthesized from its precursor p105The processing of p105 and the resulting generation ofmature p50 subunits seem to be a constitutive event as p50homodimers are not regulated by I120581B proteins [34] The p50subunit formation seems to be regulated by cotranslationaland posttranslational mechanisms In the posttranslationalmechanism the p50 subunit is synthesized from its precursorp105 by phosphorylation at its 927 and 932 serine residueswhich allows the ubiquitinization and proteolytic degrada-tion of p105 by the 26S proteasome [52] In the cotranslationalmechanism p50 and p105 can be generated from a singlemRNA [53] There is also evidence that the 20S protea-some constitutively processes p105 to p50 in a ubiquitin-independent manner [52] The p50p50 homodimer wasoriginally considered to be a transcriptional repressor butnow there is substantial evidence that it can also inducegene transcription after a complex formation with the BCL3nuclear protein an atypical member of the I120581B family ofproteins that is considered to be a coactivator of p50p50homodimers [34]

The link between NF-120581B activation by glutamate and thereduction of melatonin synthesis was evidenced with the useof PDTC a NF-120581B translocation inhibitor [54] PDTC pre-vented glutamatersquos inhibitory effect on melatonin synthesisTaken together the crucial presence of astrocytes for theinhibitory effect of glutamate the exclusive activation of NF-120581B by glutamate in the same cell type and the demonstrationof NF-120581Brsquos role inmelatonin synthesis indicate that astrocytesunder glutamate stimulation possibly release a factor thatpromotes a decrease in pinealocyte melatonin synthesisTNF-120572may be a good candidate Indeed TNF-R1 the TNF-120572 receptor that interacts with soluble TNF is present in thepineal gland reinforcing the importance of this signalingpathway [55] Acting as an inhibitor of TACE to prevent TNF-120572 release BB1101 was thus adequate in demonstrating the roleplayed byTNF-120572 In addition the hypothesis that the p50p50homodimer may induce TNF-120572 gene expression and releaseneeds to be clarified

In the CNS astrocytes are known to release gliotransmit-ters such as glutamate D-serine and ATP [21] Moreoverastrocytes can also synthesize and release proinflammatorycytokines in response to NF-120581B activation such as TNF-120572 and interleukin-1 and induce gene expression of iNOS

12 BioMed Research International

NE 1120583MGlu 100120583MGlu 600120583MPDTC 300120583M

Mela

toni

n (n

ggl

and)

20

15

10

5

0

lowastlowast

lowastlowast

+ +

+ + +

++

+

+

+

+

+ +

minus minus minusminus

minus minus minusminus

minus minus minus

Figure 8 Effect of PDTC an inhibitor of NF-120581B on melatoninsynthesis Pineal glands in culture were incubated with PDTC(300120583M) and stimulated by norepinephrine (NE 1120583M) in thepresence of glutamate (100 and 600120583M) andmelatonin content wasevaluated One-way ANOVA Bonferroni multiple comparison test119875 lt 001 versus NE 1 120583M lowastlowast119875 lt 001 versus NE + Glu 100 120583Mand NE + Glu 600 120583M plotted as the mean plusmn SEM 119899 = 12 for eachgroup 3 experiments

PinealocyteAstrocyte

NF-120581Bactivation

AMPANMDAmGluR15

Release of solublefactor

TNF-120572

Glu

TNF-120572 alone (recruiting AMPA)or in association with Glu

mGluR3TNF-R1 AMPA[iCa+2]

Melatoninsynthesis

Figure 9 Diagram illustrating the putative paracrine interactionbetween pinealocytes and astrocytes in the modulation of pinealmelatonin synthesis by glutamate Glutamate (Glu) induces intra-cellular Ca2+ increase in astrocytes with the subsequent activationof NF120581B and release of a soluble factor possibly TNF-120572 This factoralone or in association with Glu promotes melatonin synthesisreduction in pinealocytes

resulting in nitric oxide formation [56] In this work wesuggested that TNF-120572 is the factor that is released byastrocytes under glutamate stimulation because the TACEinhibitor antagonized glutamatersquos effects Reinforcing thisidea recent preliminary data obtained in our laboratorywith the L929 tumor cell lineage which is sensitive to TNF-120572 showed that astrocytes release TNF-120572 when stimulatedby glutamate (data not shown) The literature states that

TNF-120572 decreases the serotonin content and AANAT mRNAexpression resulting in the reduction of N-acetylserotonin[57 58] the immediate precursor of melatonin synthesis Itis not clear whether TNF-120572 acts by itself or in combinationwith glutamate because there are glutamate receptors in thepinealocyte membrane [12 15 16 18] Recent studies havereported the control of hippocampal synapses by TNF-120572 dueto the increased insertion of AMPA receptors in the surfaceof the neuronal soma [59]The same effect could take place inthe pineal gland in which TNF-120572 released by the astrocytesin response to NF-120581B activation could increase the numberof AMPA receptors in the pinealocyte membrane resultingin the inhibition of melatonin synthesis

A model of the putative paracrine interactions betweenastrocytes and pinealocytes in the modulation of pinealmelatonin synthesis by glutamate is shown in Figure 9

5 Conclusions

In summary according to our results we propose thatglutamate acts on glutamate receptors (AMPA NMDAandor mGluR15) in astrocytes increasing the intracellularcalcium concentration promoting NF-120581B activation anddriving the release of a soluble factor possibly TNF-120572 whichacts independently or with glutamate in the pinealocytesresulting in inhibition of melatonin synthesis

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

This work was supported by a grant from FAPESP (0404328-1) Darine Villela was supported by a master fellowshipfrom FAPESP (0556943-4) Anderson Augusto Moutinhoand Victoria Fairbanks Atherino were supported by CNPq(PIBIC)

References

[1] J Calvo J Boya A Borregon and J E Garcia-MaurinoldquoPresence of glial cells in the rat pineal gland a light andelectronmicroscopic immunohistochemical studyrdquoAnatomicalRecord vol 220 no 4 pp 424ndash428 1988

[2] L Vollrath The Pineal Organ Springer New York NY USA1981

[3] R Y Moore J C Speh and J P Card ldquoThe retinohypothalamictract originates from a distinct subset of retinal ganglion cellsrdquoJournal of Comparative Neurology vol 352 no 3 pp 351ndash3661995

[4] D Sugden ldquoMelatonin biosynthesis in the mammalian pinealglandrdquo Experientia vol 45 no 10 pp 922ndash932 1989

[5] A F Afeche D CMVillelaM V Abrahao R Peres J Cipolla-Neto et al ldquoMelatonin and pineal glandrdquo in New Researchon Neurosecretory Systems E Romano Ed pp 151ndash177 NovaScience Publishers Hauppauge NY USA 2008

[6] J Arendt Melatonin and the Mammalian Pineal Gland Chap-man amp Hall London UK 1995

BioMed Research International 13

[7] M Ehret P Pevet and M Maitre ldquoTryptophan hydroxylasesynthesis is induced by 3101584051015840-cyclic adenosine monophosphateduring circadian rhythm in the rat pineal glandrdquo Journal ofNeurochemistry vol 57 no 5 pp 1516ndash1521 1991

[8] D C Klein G R Berg and J Weller ldquoMelatonin synthesisadenosine 3101584051015840-monophosphate and norepinephrine stimulateN-acetyltransferaserdquo Science vol 168 no 3934 pp 979ndash9801970

[9] D C Klein N L Schaad M A A Namboordiri L Yu and JL Weller ldquoControl of N-acetyltransferaserdquo Biochemical SocietyTransactions vol 20 no 2 pp 299ndash304 1992

[10] V Simonneaux and C Ribelayga ldquoGeneration of the melatoninendocrine message in mammals a review of the complexregulation of melatonin synthesis by norepinephrine peptidesand other pineal transmittersrdquoPharmacological Reviews vol 55no 2 pp 325ndash395 2003

[11] S Ishio H Yamada C M Craft and Y MoriyamaldquoHydroxyindole-O-methyltransferase is another target forL-glutamate-evoked inhibition of melatonin synthesis in ratpinealocytesrdquo Brain Research vol 850 no 1-2 pp 73ndash78 1999

[12] H Yamada S Yatsushiro S Ishio et al ldquoMetabotropic gluta-mate receptors negatively regulate melatonin synthesis in ratpinealocytesrdquo Journal of Neuroscience vol 18 no 6 pp 2056ndash2062 1998

[13] H Yamada A Yamamoto M Takahashi H Michibata HKumon and Y Moriyama ldquoThe L-type Ca2+ channel isinvolved in microvesicle-mediated glutamate exocytosis fromrat pinealocytesrdquo Journal of Pineal Research vol 21 no 3 pp165ndash174 1996

[14] H Yamada A Yamamoto S Yodozawa et al ldquoMicrovesicle-mediated exocytosis of glutamate is a novel paracrine-likechemical transduction mechanism and inhibits melatoninsecretion in rat pinealocytesrdquo Journal of Pineal Research vol 21no 3 pp 175ndash191 1996

[15] H Yamada A Ogura S Koizumi A Yamaguchi and YMoriyama ldquoAcetylcholine triggers L-glutamate exocytosis vianicotinic receptors and inhibits melatonin synthesis in ratpinealocytesrdquo Journal of Neuroscience vol 18 no 13 pp 4946ndash4952 1998

[16] S Yatsushiro H Yamada M Hayashi A Yamamoto andY Moriyama ldquoIonotropic glutamate receptors trigger micro-vesicle-mediated exocytosis of L-glutamate in rat pinealocytesrdquoJournal of Neurochemistry vol 75 no 1 pp 288ndash297 2000

[17] M-HKim SUehara AMuroyama BHille YMoriyama andD-S Koh ldquoGlutamate transporter-mediated glutamate secre-tion in the mammalian pineal glandrdquo Journal of Neurosciencevol 28 no 43 pp 10852ndash10863 2008

[18] S Yatsushiro H Yamada M Hayashi S Tsuboi and YMoriyama ldquoFunctional expression of metabotropic glutamatereceptor type 5 in rat pinealocytesrdquo NeuroReport vol 10 no 7pp 1599ndash1603 1999

[19] C Kaur V Sivakumar and E A Ling ldquoExpression ofN-methyl-D-aspartate (NMDA) and 120572-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) GluR23 receptors inthe developing rat pineal glandrdquo Journal of Pineal Research vol39 no 3 pp 294ndash301 2005

[20] H Pabst and P Redecker ldquoInterstitial glial cells of the gerbilpineal gland display immunoreactivity for the metabotropicglutamate receptors mGluR23 and mGluR5rdquo Brain Researchvol 838 no 1-2 pp 60ndash68 1999

[21] M M Halassa T Fellin and P G Haydon ldquoThe tripartitesynapse roles for gliotransmission in health and diseaserdquoTrends in Molecular Medicine vol 13 no 2 pp 54ndash63 2007

[22] S M Finkbeiner ldquoGlial calciumrdquo Glia vol 9 no 2 pp 83ndash1041993

[23] E A Newman ldquoNew roles for astrocytes regulation of synaptictransmissionrdquo Trends in Neurosciences vol 26 no 10 pp 536ndash542 2003

[24] J T Porter and K D McCarthy ldquoAstrocytic neurotransmitterreceptors in situ and in vivordquo Progress in Neurobiology vol 51no 4 pp 439ndash455 1997

[25] V Parpura T A Basarsky F Liu K Jeftinija S Jeftinija and PG Haydon ldquoGlutamate-mediated astrocyte-neuron signallingrdquoNature vol 369 no 6483 pp 744ndash747 1994

[26] T D Hassinger P B Atkinson G J Strecker et al ldquoEvidencefor glutamate-mediated activation of hippocampal neurons byglial calcium wavesrdquo Journal of Neurobiology vol 28 no 2 pp159ndash170 1995

[27] R H Lipsky K Xu D Zhu et al ldquoNuclear factor 120581B is a criticaldeterminant in N-methyl-D-aspartate receptor-mediated neu-roprotectionrdquo Journal of Neurochemistry vol 78 no 2 pp 254ndash264 2001

[28] M P Mattson C Culmsee Z Yu and S Camandola ldquoRoles ofnuclear factor 120581B in neuronal survival and plasticityrdquo Journal ofNeurochemistry vol 74 no 2 pp 443ndash456 2000

[29] C DMunhoz B Garcıa-Bueno J L MMadrigal L B LepschC Scavone and J C Leza ldquoStress-induced neuroinflammationmechanisms and new pharmacological targetsrdquo Brazilian Jour-nal of Medical and Biological Research vol 41 no 12 pp 1037ndash1046 2008

[30] S Camandola andM PMattson ldquoNF-ΚB as a therapeutic targetin neurodegenerative diseasesrdquo Expert Opinion on TherapeuticTargets vol 11 no 2 pp 123ndash132 2007

[31] B Kaltschmidt D Widera and C Kaltschmidt ldquoSignaling viaNF-120581B in the nervous systemrdquo Biochimica et Biophysica Actavol 1745 no 3 pp 287ndash299 2005

[32] P T Massa H Aleyasin D S Park X Mao and S W BargerldquoNF120581B in neurons the uncertainty principle in neurobiologyrdquoJournal of Neurochemistry vol 97 no 3 pp 607ndash618 2006

[33] H Hacker and M Karin ldquoRegulation and function of IKK andIKK-related kinasesrdquo Sciencersquos STKE vol 2006 no 357 p re132006

[34] S Beinke and S C Ley ldquoFunctions of NF-120581B1 and NF-120581B2 inimmune cell biologyrdquo Biochemical Journal vol 382 no 2 pp393ndash409 2004

[35] N D Perkins ldquoIntegrating cell-signalling pathways with NF-120581Band IKK functionrdquo Nature Reviews Molecular Cell Biology vol8 pp 49ndash62 2007

[36] E Cecon P A Fernandes L Pinato Z S Ferreira and R PMarkus ldquoDaily variation of constitutively activated nuclear fac-tor 120581B (NFKB) in rat pineal glandrdquoChronobiology Internationalvol 27 no 1 pp 52ndash67 2010

[37] Z S Ferreira P A C M Fernandes D Duma J Assreuy MC W Avellar and R P Markus ldquoCorticosterone modulatesnoradrenaline-induced melatonin synthesis through inhibitionof nuclear factor 120581Brdquo Journal of Pineal Research vol 38 no 3pp 182ndash188 2005

[38] S C Afeche R Barbosa J H Scialfa I M Terra A CCassola and J Cipolla-Neto ldquoEffects of the blockade of highvoltage-activated calcium channels on in vitro pineal melatoninsynthesisrdquo Cell Biochemistry and Function vol 24 no 6 pp499ndash505 2006

14 BioMed Research International

[39] B Barlaam T G Bird C Lambert-Van Der Brempt DCampbell S J Foster and R Maciewicz ldquoNew 120572-substitutedsuccinate-based hydroxamic acids as TNF120572 convertaseinhibitorsrdquo Journal of Medicinal Chemistry vol 42 no 23 pp4890ndash4908 1999

[40] I Glezer C D Munhoz E M Kawamoto T MarcourakisM C Werneck Avellar and C Scavone ldquoMK-801 and 7-Ni attenuate the activation of brain NF-120581B induced by LPSrdquoNeuropharmacology vol 45 no 8 pp 1120ndash1129 2003

[41] Y Rong and M Baudry ldquoSeizure activity results in a rapidinduction of nuclear factor-120581B in adult but not juvenile ratlimbic structuresrdquo Journal of Neurochemistry vol 67 no 2 pp662ndash668 1996

[42] I Nicoletti G Migliorati M C Pagliacci F Grignani and CRiccardi ldquoA rapid and simple method for measuring thymocyteapoptosis by propidium iodide staining and flow cytometryrdquoJournal of Immunological Methods vol 139 no 2 pp 271ndash2791991

[43] Y F Jiang-Shieh C H Wu M L Chang J Y Shiehand C Y Wen ldquoRegional heterogeneity in immunoreactivemacrophagesmicroglia in the rat pineal glandrdquo Journal ofPineal Research vol 35 no 1 pp 45ndash53 2003

[44] K-O Soderstrom ldquoLectin binding to the human retinardquoAnatomical Record vol 220 no 2 pp 219ndash223 1988

[45] F Uehara M Sameshima T Muramatsu and N OhbaldquoLocalization of fluorescence-labeled lectin binding sites onphotoreceptor cells of the monkey retinardquo Experimental EyeResearch vol 36 no 1 pp 113ndash123 1983

[46] H-W Korf ldquoThe pineal organ as a component of the biologicalclock phylogenetic and ontogenetic considerationsrdquo Annals ofthe New York Academy of Sciences vol 719 pp 13ndash42 1994

[47] E Maronde and J H Stehle ldquoThe mammalian pineal glandknown facts unknown facetsrdquo Trends in Endocrinology andMetabolism vol 18 no 4 pp 142ndash149 2007

[48] X-M Zhang and J Zhu ldquoKainic acid-induced neurotoxicitytargeting glial responses and glia-derived cytokinesrdquo CurrentNeuropharmacology vol 9 no 2 pp 388ndash398 2011

[49] D Stellwagen and R CMalenka ldquoSynaptic scalingmediated byglial TNF-120572rdquo Nature vol 440 no 7087 pp 1054ndash1059 2006

[50] E Maronde M Pfeffer J Olcese et al ldquoTranscription factorsin neuroendocrine regulation rhythmic changes in pCREB andICER levels framemelatonin synthesisrdquo Journal of Neurosciencevol 19 no 9 pp 3326ndash3336 1999

[51] D M Bustos M J Bailey D Sugden et al ldquoGlobal dailydynamics of the pineal transcriptomerdquoCell and Tissue Researchvol 344 no 1 pp 1ndash11 2011

[52] A K Moorthy O V Savinova J Q Ho V Y-F Wang D VuandGGhosh ldquoThe 20S proteasome processesNF-120581B1 p105 intop50 in a translation-independent mannerrdquo EMBO Journal vol25 no 9 pp 1945ndash1956 2006

[53] L Lin G N DeMartino and W C Greene ldquoCotranslationalbiogenesis of NF-120581B p50 by the 26S proteasomerdquo Cell vol 92no 6 pp 819ndash828 1998

[54] H W L Ziegler-Heitbrock T Sternsdorf J Liese et alldquoPyrrolidine dithiocarbamate inhibits NF-120581B mobilization andTNF production in humanmonocytesrdquo Journal of Immunologyvol 151 no 12 pp 6986ndash6993 1993

[55] C E Carvalho-Sousa S S Cruz-Machado E K Tamura P AC M Fernandes L Pinato et al ldquoMolecular basis for definingthe pineal gland and pinealocytes as targets for tumor necrosisfactorrdquo Frontiers in Endocrinology vol 2 pp 1ndash11 2011

[56] M PMattson ldquoNF-120581B in the survival and plasticity of neuronsrdquoNeurochemical Research vol 30 no 6-7 pp 883ndash893 2005

[57] P A CM Fernandes E Cecon R PMarkus and Z S FerreiraldquoEffect of TNF-120572 on the melatonin synthetic pathway in the ratpineal gland basis for a rsquofeedbackrsquo of the immune response oncircadian timingrdquo Journal of Pineal Research vol 41 no 4 pp344ndash350 2006

[58] S-Y Tsai T E OrsquoBrien and J A McNulty ldquoMicroglia play arole in mediating the effects of cytokines on the structure andfunction of the rat pineal glandrdquo Cell and Tissue Research vol303 no 3 pp 423ndash431 2001

[59] J S Bains and S H R Oliet ldquoGlia they make your memoriesstickrdquo Trends in Neurosciences vol 30 no 8 pp 417ndash424 2007

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

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Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Research and TreatmentAIDS

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 BioMed Research International

18

15

12

9

6

3

0

Mela

toni

n (n

ggl

and)

lowast

NE 1120583MGlu 100120583MGlu 600120583M

+ +

+

+

+

minus minus

minus minus

(a)

0

Mela

toni

n (n

gm

L)

NE 1120583MGlu 100120583MGlu 600120583M

+ +

+

+

+

minus minus

minus minus

120

80

40

(b)

Mela

toni

n (n

gm

L)

NE 1120583MGlu 100120583MGlu 600120583M

+ +

+

+

+

minus minus

minus minus

50

40

30

20

10

0

lowastlowast lowastlowast

(c)

NE 1120583MGlu 600120583M

Mela

toni

n (n

gm

L)

+ +

+minus

lowastlowast

100

80

60

40

20

0

(d)

Figure 2 Glutamate effects on melatonin synthesis (a) Pineal gland culture stimulated with norepinephrine (NE 1120583M) in the absence orpresence of glutamate (Glu 100 and Glu 600120583M) Melatonin content is expressed in nggland as the mean plusmn SEM of 3 experiments One-wayANOVA Bonferroni multiple comparison test lowast119875 lt 005 versus NE 1 120583M 119899 = 12 for each group (b) Isolated rat pinealocyte culture underNE 1120583M stimulation in the absence or presence of Glu 100 or Glu 600120583M 119899 = 6 for each group (c) Pinealocyte and astrocyte co-culturestimulated with NE 1 120583M in the absence or presence of Glu 100 or Glu 600 120583M One-way ANOVA Bonferroni multiple comparison testlowastlowast119875 lt 001 versus NE 1 120583M 119899 = 8 for each group (d) Astrocytes and pinealocytes were cultured together but with physical separation by

inserts Astrocytes were cultivated in the bottom of the plate and pinealocytes were cultivated within the inserts and stimulated with NE1 120583M associated or not associated with Glu 600 120583M 119879 test lowastlowast119875 lt 001 119899 = 4 for each group For the cell cultures melatonin was evaluatedin the culture medium and expressed in ngmL as the mean plusmn SEM of 3 experiments

includes the TACE It was verified that when the cells inco-culture were incubated with BB1101 (10 120583M) prior to thetreatments with glutamate (600 120583M) and NE (1 120583M) it wasable to completely revert the inhibitory effect of glutamate onmelatonin synthesis (119875 lt 005) (Figure 3)

RT-PCR analyses showed mRNA expression of classIII metabotropic glutamate receptors (mGluR1mGluR5 andmGluR2mGluR3 resp) as well as ionotropic NMDA andAMPA-type receptors (GluN1 GluA1 GluA2 and GluA3subunits) in astrocytes and pinealocytes (Figures 4(a) and4(b)) Contrarily mGluR7 mRNA (class III metabotropicreceptors) was not detected in any of the cell types(Figure 4(a)) To determine whether class I mGluRs NMDA

and AMPA ionotropic receptors are functional and incor-porated into the astrocytic and pinealocytic membranes anintracellular calcium analysis by confocal microscopy wasused It was observed that the class I metabotropic receptoragonist DHPG (50120583M) and the ionotropic receptor agonistsNMDA (100120583M) and AMPA (50 120583M) were able to increasethe astrocytes and pinealocytes intracellular calcium contentin a similar way as that observed for glutamate (600 120583M)(Figure 5 Table 1)

Electrophoretic mobility shift assays revealed that gluta-mate (100 and 600120583M) activates NF-120581B in cultured rat pinealglands The result of the competition study showed that theupper complex 1 was displaced by an excess of unlabeled

BioMed Research International 7

60

40

20

0NE 1120583M

Mela

toni

n (n

gm

L)

lowast

+ +

+ +

+

+

+

+minus minus

minus minus

lowastlowast

BB1101 10120583MGlu 600 120583M

Figure 3 (a) Ablation of the inhibitory glutamatergic effect onmelatonin synthesis by the TACE inhibitor BB1101 Pinealocytesand astrocytes were maintained in co-culture and stimulated withnorepinephrine (NE 1 120583M) in association with glutamate (Glu600 120583M) and BB1101 (10120583M) One-way ANOVA Bonferroni mul-tiple comparison test lowastlowast119875 lt 001 versus NE 1 120583M lowast119875 lt 005 versusNE 1 120583M + Glu 600 120583M 119899 = 4 for each group 3 experiments

Table 1 Summary of intracellular [Ca2+] changes in astrocytes andpinealocytes after stimulation

Cell typeagents Number of cellsexamined [Ca2+]

119894response

Isolated astrocytesGlu 600 120583M 68 79AMPA 50 120583M 97 80NMDA 100 120583M 49 90DHPG 50 120583M 80 74

Isolated pinealocytesGlu 600 120583M 66 80AMPA 50 120583M 78 85NMDA 100 120583M 53 90DHPG 50 120583M 60 76

Intracellular [Ca2+] in fluo-4-loaded astrocytes and pinealocytes was mea-sured as described in Section 2 The majority of the stimulated cellsresponded with an increase in intracellular [Ca2+] after the addition of eitherglutamate (600120583M) ionotropic glutamatergic agonists (AMPA 50120583M andNMDA 100120583M) or a class I metabotropic glutamatergic agonist (DHPG50120583M)

NF-120581B but not by a TFIID double-stranded oligonucleotideconsensus sequence demonstrating the specificity of the NF-120581BDNA interactionThe lower complex 2 was less efficientlydisplaced by unlabeledNF-120581B probe (Figure 6(a)) Supershiftanalysis indicated that the antibody against the p50 subunitwas able to shift the DNAprotein interaction present incomplex 1 The presence of antibodies against the p52 p65and c-Rel subunits did not affect DNA-protein complexes(Figures 6(b) and 6(c)) Taken together these results indicatethat at these concentrations glutamate activates the p50p50

homodimer Complex 2 was not displaced by the antibodiesand was not considered to be related to the NF-120581B family

Astrocyte and pinealocyte isolated cultures were used todetermine the cell type in which NF-120581B was being activatedGlutamate (600120583M)was shown to activateNF-120581B exclusivelyin astrocytes and not in pinealocytes (119875 lt 001) (Figure 7)

The involvement of NF-120581B activation in glutamatersquosinhibitory effect on melatonin synthesis was analyzed usinga specific inhibitor of NF-120581B PDTC PDTC (300 120583M) pre-vented the inhibitory effect of glutamate in cultured rat pinealglands stimulated by NE (1 120583M) (119875 lt 001) (Figure 8)

Glutamate cytotoxicity was evaluated because it is knownthat this neurotransmitter in high concentrations couldinduce cell death by cytotoxicity [48] Cell membraneintegrity and DNA fragmentation were assessed by flowcytometric analysis using propidium iodide (PI) Supple-mentary Figure 1 is representative of the results obtainedafter cell incubation with NE (1120583M) in the absence (controlgroup) or presence of glutamate (100 and 600120583M) for 5 hThe histograms show the fluorescence intensity in the FL-2channel which corresponds to the PI emission wavelengthWhen the cell membrane is intact the fluorescence signal islow and is related to autofluorescence (M1) A high signalmeans that PI was able to enter the cell and bind to DNA(M2) Glutamate treatments for 5 h were not able to inducea loss of membrane integrity (Supplementary Figure 1(a))The percentage of cells with ruptured membranes in thecontrol group was 453 whereas it was 502 and 577 inthe 100 and 600 120583M glutamate-treated groups respectively(Supplementary Figure 1(c)) Histograms in SupplementaryFigure 1(b) represent the results of DNA fragmentationanalysis After PI treatment the DNA content in live cellspresented a high fluorescence signal (M2) DNA fragmenta-tion induces the formation of small DNA pieces that exhibitlow fluorescence intensity (M1) The percentages of cellswith DNA fragmentation after glutamate stimulation 100and 600120583M were 286 and 335 respectively which weresimilar to the value of control cells (366) (SupplementaryFigure 1(d))

4 Discussion

Isolated pinealocytes isolated astrocytes and pineal-ocyteastrocyte co-culture were used in this study Theimmunocytochemistry characterization of the cell typespresent in these different cultures demonstrated the pre-dominance of pinealocytes in isolated pinealocyte culturewith the absence of glial cells In the same way astrocytecultures do not contain a significant number of microglialcells or pinealocytes In co-culture pinealocytes are thepredominant cells as astrocytes are present in minorquantities reproducing the proportion present in intactpineal glands (51) Thus the cultures were considered to beappropriate for this study

Data from the literature have shown that glutamate acts asan inhibitory neurotransmitter on melatonin synthesis [12]According to Yamada et al [13 14 16] and Yatsushiro et al[16] glutamate is stored in microvesicles and released from

8 BioMed Research International

mGluR1 mGluR2 mGluR3 mGluR5 mGluR7

M H2O As Pi Hi As Pi Hi As Pi Hi As Pi Hi As Pi Hi

200 bp100 bp

(a)

M H2O As Pi Hi As Pi Hi As Pi Hi As Pi Hi

200 bp100 bp

GluA1 GluA2 GluA3 GluN1

(b)

Figure 4 RT-PCR detection of metabotropic and ionotropic glutamate receptors in isolated astrocytes and pinealocytes (a) RT-PCRdetection of transcripts for mGluR1 (79 bp) mGluR2 (198 bp) mGluR3 (196 bp) mGluR5 (165 bp) and mGluR7 (70 bp) in astrocytes (As)pinealocytes (Pi) and hippocampus (Hi) Molecular weight marker (M) and water (H

2O) are presented in lanes 1 and 2 respectively (b)

RT-PCR detection of transcripts for GluA1 (203 bp) GluA2 (155 bp) GluA3 (193 bp) and GluN1 (201 bp) in astrocytes (As) pinealocytes(Pi) and hippocampus (Hi) The molecular weight marker (M) and PCR product generated with water (H

2O) are presented in lanes 1 and 2

respectively

pinealocytes upon cholinergic stimulation It interacts withionotropic and metabotropic glutamate receptors presentin pinealocyte membranes releasing additional glutamateand inhibiting melatonin synthesis respectively Recently amechanism involving the glutamate transporter GLT-1 wasproposed to be responsible for glutamate release [17] Inthese studies involving pinealocytes an autocrineparacrineprocess was suggested for the influence of glutamate onmelatonin synthesis In the presentworkwe demonstrated animportant role for astrocytes in glutamatersquos modulatory effecton melatonin synthesis that includes a paracrine interactionwith pinealocytes

Whereas in isolated pinealocytes glutamate does notexhibit the typical inhibitory effect on melatonin synthesisas observed in intact pineal glands its association withastrocytes restores this effect These results demonstrate thatastrocytes act as partners that collaborate to produce theglutamate effect In addition the fact that the effect occurredeven when inserts were used preventing the physical contactbetween the cells in co-culture suggests that a solublefactor is a mediator of this process The hypothesis that gapjunctions mediated the interactions between astrocytes andpinealocytes as occurs in the pineal gland [49] was excludedfrom this observationThe cytokine TNF-120572 a gliotransmitterthat is released by astrocytes could be the soluble factorthat is involved in the interactions between astrocytes andpinealocytes In fact the absence of a soluble form of TNF-120572 interferes with the glutamate response

The possibility that the glutamate effects were due to celldeath was investigated by flow cytometry As the cell viabilityand DNA fragmentation levels were similar between controland glutamate-stimulated groups showing no damage topineal cells even when treated with 600120583M of glutamate itis possible to exclude a glutamate cytotoxic effect as a causeof melatonin reduction

Although glutamate receptor expression had been firstdescribed in pinealocytes its expression was also laterdemonstrated in astrocytes mGluR3 and AMPA (GluA1subunit) were shown to be present in pinealocytes [12 16]

and NMDA and AMPA (GluA23) receptors were present inrat pineal gland astrocytes [19] Ionotropic receptor mRNAwas similarly observed in astrocytes in the present studyin addition to the novel observation of metabotropic recep-tor (mGluR1 mGluR2 mGluR3 mGluR5) expression Inpinealocytes the expression of mGluR1R2R3R5 AMPAand NMDA receptors was shownThis evidence supports thepossibility of astrocyte participation in rat pineal glutamater-gic signaling

Confocal microscopy assays using NMDA AMPA andDHPG agonists in isolated astrocytes and pinealocytesrevealed increased intracellular calcium content in both celltypes for all the agonists used PCR and confocal microscopyanalyses suggest both gene and protein expression in classI mGluR AMPA and NMDA ionotropic receptors reveal-ing the importance of the glutamate receptors as signaltransducers in rat pineal gland astrocytes and pinealocytesThus glutamate seems to interact with its receptors inboth cell types but its effect on melatonin synthesis nec-essarily involves astrocyte glutamate receptors The possibleparacrine interactions between pinealocytes and astrocytesseem to be the key in understanding the role of astrocytes inthe rat pineal gland

Transcription factors exert a great influence on pinealphysiology CREB and ICER are two transcription factorsthat intimately control the transcription of AANAT [50]Additionally there are other transcription factors in the ratpineal gland whose roles in pineal melatonin synthesis arenot known [51] NF-120581B is present in the rat pineal glandshowing a circadian rhythm and involvement in inflamma-tory responses [36 37] NF-120581B is a classic possible glutamatemediator and its activation in glial cells was demonstrated toplay a role in inflammatory processes [30]The present resultsdemonstrated NF-120581B activation by glutamate in culturedisolated astrocytes supporting its role as a mediator ofglutamate effects Supershift assay in cultured pineal glandsstimulated with glutamate showed the displacement of thep50 subunit emphasizing the importance of p50p50 homod-imer formationThis is an astrocyte-dependent event because

BioMed Research International 9

0 50 100 150 200 250 300 350

10

20

30

40

50

60

Time (s)

Isolated astrocytes

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

Glu 600120583M

(a)

0 30 60 90 120 150

155

160

165

170

175

180

185

Time (s)

Isolated pinealocytes

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

Glu 600120583M

(b)

0 100 200 300 400 500

10

20

30

Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

AMPA 50120583M

(c)

0 30 60 90 120 150 180

102

104

106

108

110

112

114

Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

AMPA 50120583M

(d)

0 50 100 150 200 250 300

400

600

800

1000

1200

1400

Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

NMDA 100120583M

(e)

0 50 100 150 200 250

900

1200

1500

Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

NMDA 100120583M

(f)

100 150 200 250

126

128

130

132

134

136

Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity) DHPG 50120583M

(g)

0 100 200 300 400

80

90

100

110

120

130

Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

DHPG 50120583M

(h)

Figure 5 Analysis of intracellular calcium in isolated astrocyte and pinealocyte cultures Traces of Fluo-4 fluorescence indicate theintracellular calcium transients from single cells Additions of exogenous glutamate or glutamatergic agonists were performed as indicatedby the arrows

10 BioMed Research International

ControlGlu 100120583MGlu 600120583MUnlabeled NF-120581BUnlabeled TFIID

1

(NS) 2

Free probe

+

minus

minus

minus

minus +

minus

minus

minus

minus

+

minus

minus

minus

minus +

+

minus

minus

minus +

+

minus

minus

minus+

+

minus

minus

minus

+

+

minus

minus

minus

(a)

(NS) 2

Free probe

(p50p50) 1

(NS) 3

+

+

minus

minus

minus

minus

+

minus

minus

minus

minus

minus

+

minus

minus

minus

minus

minus

+

+

minus

minus

minus

minus

+

+

minus

minus

minus

minus

+

+

minus

minus

minus

minus

ControlGlu 100120583MAnti p50Anti p52Anti p65Anti c-Rel

(b)

Free probe

Anti c-Rel

(NS) 2

(p50p50) 1

(NS) 3

ControlGlu 600120583MAnti p50Anti p52Anti p65

+minus minus minus minus minus

+

+ +

+

+ + +

+

+minus minus

minus

minus

minus

minus minus minus minus minus

minus

minus minus minus

minus minus

minus minus minus

minus minus

(c)

Figure 6 Electrophoretic mobility shift assays for NF120581B in the pineal gland (a) Competition studies were performed using nuclear extract(5120583g) from cultured pineal glands treated with glutamate (Glu) 100 and 600 120583M in the absence or presence of unlabeled specific (NF-120581Bconsensus sequence 10-fold molar excess) or nonspecific oligonucleotide (TFIID consensus sequence 10-fold molar excess) as indicated(b c) Supershift assays were performed with the same nuclear extract (5120583g) incubated in the absence or presence of antibodies against thesubunits p50 (1 10 dilution) p65 (1 20 dilution) p52 and c-Rel (1 10 dilution) as indicated Antibodies were added 20min prior to theaddition of the radiolabeled NF-120581B consensus oligonucleotide The position of specific NF-120581BDNA binding complexes p50p50 (band 1 bc) is indicated NS represents no specific binding (bands 2 and 3 a b c) The localization of the free probe is also indicated

BioMed Research International 11

ControlGlu 600120583M

minusminus

minus +

NF-120581B

(a)

20000

15000

10000

0Control Glu 600120583M

5000

lowastlowast

(au

)

(b)

ControlGlu 600120583M

minusminus

minus +

NF-120581B

(c)

(au

)

Control Glu 600120583M

16000

12000

8000

4000

0

(d)

Figure 7 Electrophoretic mobility shift assays for NF120581B in isolated astrocytes and pinealocytes (a) Nuclear proteins (5 120583g) were extractedfrom astrocytes treated with glutamate (Glu) 600120583M (b)Densitometric analysis (arbitrary units) of theNF-120581B band is presented on panel (a)One-way ANOVA Studentrsquos test lowastlowast119875 lt 001 versus control group mean plusmn SEM (c) Nuclear proteins (5120583g) were extracted from pinealocytestreated with Glu 600 120583M (d) Densitometric analysis (arbitrary units) of the NF-120581B band is presented in (c) The position of the specificNF-120581BDNA binding complex is indicated 119899 = 5 for each group 3 experiments

its activation was not observed in pinealocytes Astrocyteand pinealocyte supershift assays were not performed due totechnical limitations related to the amount of nuclear protein

The p50 subunit is synthesized from its precursor p105The processing of p105 and the resulting generation ofmature p50 subunits seem to be a constitutive event as p50homodimers are not regulated by I120581B proteins [34] The p50subunit formation seems to be regulated by cotranslationaland posttranslational mechanisms In the posttranslationalmechanism the p50 subunit is synthesized from its precursorp105 by phosphorylation at its 927 and 932 serine residueswhich allows the ubiquitinization and proteolytic degrada-tion of p105 by the 26S proteasome [52] In the cotranslationalmechanism p50 and p105 can be generated from a singlemRNA [53] There is also evidence that the 20S protea-some constitutively processes p105 to p50 in a ubiquitin-independent manner [52] The p50p50 homodimer wasoriginally considered to be a transcriptional repressor butnow there is substantial evidence that it can also inducegene transcription after a complex formation with the BCL3nuclear protein an atypical member of the I120581B family ofproteins that is considered to be a coactivator of p50p50homodimers [34]

The link between NF-120581B activation by glutamate and thereduction of melatonin synthesis was evidenced with the useof PDTC a NF-120581B translocation inhibitor [54] PDTC pre-vented glutamatersquos inhibitory effect on melatonin synthesisTaken together the crucial presence of astrocytes for theinhibitory effect of glutamate the exclusive activation of NF-120581B by glutamate in the same cell type and the demonstrationof NF-120581Brsquos role inmelatonin synthesis indicate that astrocytesunder glutamate stimulation possibly release a factor thatpromotes a decrease in pinealocyte melatonin synthesisTNF-120572may be a good candidate Indeed TNF-R1 the TNF-120572 receptor that interacts with soluble TNF is present in thepineal gland reinforcing the importance of this signalingpathway [55] Acting as an inhibitor of TACE to prevent TNF-120572 release BB1101 was thus adequate in demonstrating the roleplayed byTNF-120572 In addition the hypothesis that the p50p50homodimer may induce TNF-120572 gene expression and releaseneeds to be clarified

In the CNS astrocytes are known to release gliotransmit-ters such as glutamate D-serine and ATP [21] Moreoverastrocytes can also synthesize and release proinflammatorycytokines in response to NF-120581B activation such as TNF-120572 and interleukin-1 and induce gene expression of iNOS

12 BioMed Research International

NE 1120583MGlu 100120583MGlu 600120583MPDTC 300120583M

Mela

toni

n (n

ggl

and)

20

15

10

5

0

lowastlowast

lowastlowast

+ +

+ + +

++

+

+

+

+

+ +

minus minus minusminus

minus minus minusminus

minus minus minus

Figure 8 Effect of PDTC an inhibitor of NF-120581B on melatoninsynthesis Pineal glands in culture were incubated with PDTC(300120583M) and stimulated by norepinephrine (NE 1120583M) in thepresence of glutamate (100 and 600120583M) andmelatonin content wasevaluated One-way ANOVA Bonferroni multiple comparison test119875 lt 001 versus NE 1 120583M lowastlowast119875 lt 001 versus NE + Glu 100 120583Mand NE + Glu 600 120583M plotted as the mean plusmn SEM 119899 = 12 for eachgroup 3 experiments

PinealocyteAstrocyte

NF-120581Bactivation

AMPANMDAmGluR15

Release of solublefactor

TNF-120572

Glu

TNF-120572 alone (recruiting AMPA)or in association with Glu

mGluR3TNF-R1 AMPA[iCa+2]

Melatoninsynthesis

Figure 9 Diagram illustrating the putative paracrine interactionbetween pinealocytes and astrocytes in the modulation of pinealmelatonin synthesis by glutamate Glutamate (Glu) induces intra-cellular Ca2+ increase in astrocytes with the subsequent activationof NF120581B and release of a soluble factor possibly TNF-120572 This factoralone or in association with Glu promotes melatonin synthesisreduction in pinealocytes

resulting in nitric oxide formation [56] In this work wesuggested that TNF-120572 is the factor that is released byastrocytes under glutamate stimulation because the TACEinhibitor antagonized glutamatersquos effects Reinforcing thisidea recent preliminary data obtained in our laboratorywith the L929 tumor cell lineage which is sensitive to TNF-120572 showed that astrocytes release TNF-120572 when stimulatedby glutamate (data not shown) The literature states that

TNF-120572 decreases the serotonin content and AANAT mRNAexpression resulting in the reduction of N-acetylserotonin[57 58] the immediate precursor of melatonin synthesis Itis not clear whether TNF-120572 acts by itself or in combinationwith glutamate because there are glutamate receptors in thepinealocyte membrane [12 15 16 18] Recent studies havereported the control of hippocampal synapses by TNF-120572 dueto the increased insertion of AMPA receptors in the surfaceof the neuronal soma [59]The same effect could take place inthe pineal gland in which TNF-120572 released by the astrocytesin response to NF-120581B activation could increase the numberof AMPA receptors in the pinealocyte membrane resultingin the inhibition of melatonin synthesis

A model of the putative paracrine interactions betweenastrocytes and pinealocytes in the modulation of pinealmelatonin synthesis by glutamate is shown in Figure 9

5 Conclusions

In summary according to our results we propose thatglutamate acts on glutamate receptors (AMPA NMDAandor mGluR15) in astrocytes increasing the intracellularcalcium concentration promoting NF-120581B activation anddriving the release of a soluble factor possibly TNF-120572 whichacts independently or with glutamate in the pinealocytesresulting in inhibition of melatonin synthesis

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

This work was supported by a grant from FAPESP (0404328-1) Darine Villela was supported by a master fellowshipfrom FAPESP (0556943-4) Anderson Augusto Moutinhoand Victoria Fairbanks Atherino were supported by CNPq(PIBIC)

References

[1] J Calvo J Boya A Borregon and J E Garcia-MaurinoldquoPresence of glial cells in the rat pineal gland a light andelectronmicroscopic immunohistochemical studyrdquoAnatomicalRecord vol 220 no 4 pp 424ndash428 1988

[2] L Vollrath The Pineal Organ Springer New York NY USA1981

[3] R Y Moore J C Speh and J P Card ldquoThe retinohypothalamictract originates from a distinct subset of retinal ganglion cellsrdquoJournal of Comparative Neurology vol 352 no 3 pp 351ndash3661995

[4] D Sugden ldquoMelatonin biosynthesis in the mammalian pinealglandrdquo Experientia vol 45 no 10 pp 922ndash932 1989

[5] A F Afeche D CMVillelaM V Abrahao R Peres J Cipolla-Neto et al ldquoMelatonin and pineal glandrdquo in New Researchon Neurosecretory Systems E Romano Ed pp 151ndash177 NovaScience Publishers Hauppauge NY USA 2008

[6] J Arendt Melatonin and the Mammalian Pineal Gland Chap-man amp Hall London UK 1995

BioMed Research International 13

[7] M Ehret P Pevet and M Maitre ldquoTryptophan hydroxylasesynthesis is induced by 3101584051015840-cyclic adenosine monophosphateduring circadian rhythm in the rat pineal glandrdquo Journal ofNeurochemistry vol 57 no 5 pp 1516ndash1521 1991

[8] D C Klein G R Berg and J Weller ldquoMelatonin synthesisadenosine 3101584051015840-monophosphate and norepinephrine stimulateN-acetyltransferaserdquo Science vol 168 no 3934 pp 979ndash9801970

[9] D C Klein N L Schaad M A A Namboordiri L Yu and JL Weller ldquoControl of N-acetyltransferaserdquo Biochemical SocietyTransactions vol 20 no 2 pp 299ndash304 1992

[10] V Simonneaux and C Ribelayga ldquoGeneration of the melatoninendocrine message in mammals a review of the complexregulation of melatonin synthesis by norepinephrine peptidesand other pineal transmittersrdquoPharmacological Reviews vol 55no 2 pp 325ndash395 2003

[11] S Ishio H Yamada C M Craft and Y MoriyamaldquoHydroxyindole-O-methyltransferase is another target forL-glutamate-evoked inhibition of melatonin synthesis in ratpinealocytesrdquo Brain Research vol 850 no 1-2 pp 73ndash78 1999

[12] H Yamada S Yatsushiro S Ishio et al ldquoMetabotropic gluta-mate receptors negatively regulate melatonin synthesis in ratpinealocytesrdquo Journal of Neuroscience vol 18 no 6 pp 2056ndash2062 1998

[13] H Yamada A Yamamoto M Takahashi H Michibata HKumon and Y Moriyama ldquoThe L-type Ca2+ channel isinvolved in microvesicle-mediated glutamate exocytosis fromrat pinealocytesrdquo Journal of Pineal Research vol 21 no 3 pp165ndash174 1996

[14] H Yamada A Yamamoto S Yodozawa et al ldquoMicrovesicle-mediated exocytosis of glutamate is a novel paracrine-likechemical transduction mechanism and inhibits melatoninsecretion in rat pinealocytesrdquo Journal of Pineal Research vol 21no 3 pp 175ndash191 1996

[15] H Yamada A Ogura S Koizumi A Yamaguchi and YMoriyama ldquoAcetylcholine triggers L-glutamate exocytosis vianicotinic receptors and inhibits melatonin synthesis in ratpinealocytesrdquo Journal of Neuroscience vol 18 no 13 pp 4946ndash4952 1998

[16] S Yatsushiro H Yamada M Hayashi A Yamamoto andY Moriyama ldquoIonotropic glutamate receptors trigger micro-vesicle-mediated exocytosis of L-glutamate in rat pinealocytesrdquoJournal of Neurochemistry vol 75 no 1 pp 288ndash297 2000

[17] M-HKim SUehara AMuroyama BHille YMoriyama andD-S Koh ldquoGlutamate transporter-mediated glutamate secre-tion in the mammalian pineal glandrdquo Journal of Neurosciencevol 28 no 43 pp 10852ndash10863 2008

[18] S Yatsushiro H Yamada M Hayashi S Tsuboi and YMoriyama ldquoFunctional expression of metabotropic glutamatereceptor type 5 in rat pinealocytesrdquo NeuroReport vol 10 no 7pp 1599ndash1603 1999

[19] C Kaur V Sivakumar and E A Ling ldquoExpression ofN-methyl-D-aspartate (NMDA) and 120572-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) GluR23 receptors inthe developing rat pineal glandrdquo Journal of Pineal Research vol39 no 3 pp 294ndash301 2005

[20] H Pabst and P Redecker ldquoInterstitial glial cells of the gerbilpineal gland display immunoreactivity for the metabotropicglutamate receptors mGluR23 and mGluR5rdquo Brain Researchvol 838 no 1-2 pp 60ndash68 1999

[21] M M Halassa T Fellin and P G Haydon ldquoThe tripartitesynapse roles for gliotransmission in health and diseaserdquoTrends in Molecular Medicine vol 13 no 2 pp 54ndash63 2007

[22] S M Finkbeiner ldquoGlial calciumrdquo Glia vol 9 no 2 pp 83ndash1041993

[23] E A Newman ldquoNew roles for astrocytes regulation of synaptictransmissionrdquo Trends in Neurosciences vol 26 no 10 pp 536ndash542 2003

[24] J T Porter and K D McCarthy ldquoAstrocytic neurotransmitterreceptors in situ and in vivordquo Progress in Neurobiology vol 51no 4 pp 439ndash455 1997

[25] V Parpura T A Basarsky F Liu K Jeftinija S Jeftinija and PG Haydon ldquoGlutamate-mediated astrocyte-neuron signallingrdquoNature vol 369 no 6483 pp 744ndash747 1994

[26] T D Hassinger P B Atkinson G J Strecker et al ldquoEvidencefor glutamate-mediated activation of hippocampal neurons byglial calcium wavesrdquo Journal of Neurobiology vol 28 no 2 pp159ndash170 1995

[27] R H Lipsky K Xu D Zhu et al ldquoNuclear factor 120581B is a criticaldeterminant in N-methyl-D-aspartate receptor-mediated neu-roprotectionrdquo Journal of Neurochemistry vol 78 no 2 pp 254ndash264 2001

[28] M P Mattson C Culmsee Z Yu and S Camandola ldquoRoles ofnuclear factor 120581B in neuronal survival and plasticityrdquo Journal ofNeurochemistry vol 74 no 2 pp 443ndash456 2000

[29] C DMunhoz B Garcıa-Bueno J L MMadrigal L B LepschC Scavone and J C Leza ldquoStress-induced neuroinflammationmechanisms and new pharmacological targetsrdquo Brazilian Jour-nal of Medical and Biological Research vol 41 no 12 pp 1037ndash1046 2008

[30] S Camandola andM PMattson ldquoNF-ΚB as a therapeutic targetin neurodegenerative diseasesrdquo Expert Opinion on TherapeuticTargets vol 11 no 2 pp 123ndash132 2007

[31] B Kaltschmidt D Widera and C Kaltschmidt ldquoSignaling viaNF-120581B in the nervous systemrdquo Biochimica et Biophysica Actavol 1745 no 3 pp 287ndash299 2005

[32] P T Massa H Aleyasin D S Park X Mao and S W BargerldquoNF120581B in neurons the uncertainty principle in neurobiologyrdquoJournal of Neurochemistry vol 97 no 3 pp 607ndash618 2006

[33] H Hacker and M Karin ldquoRegulation and function of IKK andIKK-related kinasesrdquo Sciencersquos STKE vol 2006 no 357 p re132006

[34] S Beinke and S C Ley ldquoFunctions of NF-120581B1 and NF-120581B2 inimmune cell biologyrdquo Biochemical Journal vol 382 no 2 pp393ndash409 2004

[35] N D Perkins ldquoIntegrating cell-signalling pathways with NF-120581Band IKK functionrdquo Nature Reviews Molecular Cell Biology vol8 pp 49ndash62 2007

[36] E Cecon P A Fernandes L Pinato Z S Ferreira and R PMarkus ldquoDaily variation of constitutively activated nuclear fac-tor 120581B (NFKB) in rat pineal glandrdquoChronobiology Internationalvol 27 no 1 pp 52ndash67 2010

[37] Z S Ferreira P A C M Fernandes D Duma J Assreuy MC W Avellar and R P Markus ldquoCorticosterone modulatesnoradrenaline-induced melatonin synthesis through inhibitionof nuclear factor 120581Brdquo Journal of Pineal Research vol 38 no 3pp 182ndash188 2005

[38] S C Afeche R Barbosa J H Scialfa I M Terra A CCassola and J Cipolla-Neto ldquoEffects of the blockade of highvoltage-activated calcium channels on in vitro pineal melatoninsynthesisrdquo Cell Biochemistry and Function vol 24 no 6 pp499ndash505 2006

14 BioMed Research International

[39] B Barlaam T G Bird C Lambert-Van Der Brempt DCampbell S J Foster and R Maciewicz ldquoNew 120572-substitutedsuccinate-based hydroxamic acids as TNF120572 convertaseinhibitorsrdquo Journal of Medicinal Chemistry vol 42 no 23 pp4890ndash4908 1999

[40] I Glezer C D Munhoz E M Kawamoto T MarcourakisM C Werneck Avellar and C Scavone ldquoMK-801 and 7-Ni attenuate the activation of brain NF-120581B induced by LPSrdquoNeuropharmacology vol 45 no 8 pp 1120ndash1129 2003

[41] Y Rong and M Baudry ldquoSeizure activity results in a rapidinduction of nuclear factor-120581B in adult but not juvenile ratlimbic structuresrdquo Journal of Neurochemistry vol 67 no 2 pp662ndash668 1996

[42] I Nicoletti G Migliorati M C Pagliacci F Grignani and CRiccardi ldquoA rapid and simple method for measuring thymocyteapoptosis by propidium iodide staining and flow cytometryrdquoJournal of Immunological Methods vol 139 no 2 pp 271ndash2791991

[43] Y F Jiang-Shieh C H Wu M L Chang J Y Shiehand C Y Wen ldquoRegional heterogeneity in immunoreactivemacrophagesmicroglia in the rat pineal glandrdquo Journal ofPineal Research vol 35 no 1 pp 45ndash53 2003

[44] K-O Soderstrom ldquoLectin binding to the human retinardquoAnatomical Record vol 220 no 2 pp 219ndash223 1988

[45] F Uehara M Sameshima T Muramatsu and N OhbaldquoLocalization of fluorescence-labeled lectin binding sites onphotoreceptor cells of the monkey retinardquo Experimental EyeResearch vol 36 no 1 pp 113ndash123 1983

[46] H-W Korf ldquoThe pineal organ as a component of the biologicalclock phylogenetic and ontogenetic considerationsrdquo Annals ofthe New York Academy of Sciences vol 719 pp 13ndash42 1994

[47] E Maronde and J H Stehle ldquoThe mammalian pineal glandknown facts unknown facetsrdquo Trends in Endocrinology andMetabolism vol 18 no 4 pp 142ndash149 2007

[48] X-M Zhang and J Zhu ldquoKainic acid-induced neurotoxicitytargeting glial responses and glia-derived cytokinesrdquo CurrentNeuropharmacology vol 9 no 2 pp 388ndash398 2011

[49] D Stellwagen and R CMalenka ldquoSynaptic scalingmediated byglial TNF-120572rdquo Nature vol 440 no 7087 pp 1054ndash1059 2006

[50] E Maronde M Pfeffer J Olcese et al ldquoTranscription factorsin neuroendocrine regulation rhythmic changes in pCREB andICER levels framemelatonin synthesisrdquo Journal of Neurosciencevol 19 no 9 pp 3326ndash3336 1999

[51] D M Bustos M J Bailey D Sugden et al ldquoGlobal dailydynamics of the pineal transcriptomerdquoCell and Tissue Researchvol 344 no 1 pp 1ndash11 2011

[52] A K Moorthy O V Savinova J Q Ho V Y-F Wang D VuandGGhosh ldquoThe 20S proteasome processesNF-120581B1 p105 intop50 in a translation-independent mannerrdquo EMBO Journal vol25 no 9 pp 1945ndash1956 2006

[53] L Lin G N DeMartino and W C Greene ldquoCotranslationalbiogenesis of NF-120581B p50 by the 26S proteasomerdquo Cell vol 92no 6 pp 819ndash828 1998

[54] H W L Ziegler-Heitbrock T Sternsdorf J Liese et alldquoPyrrolidine dithiocarbamate inhibits NF-120581B mobilization andTNF production in humanmonocytesrdquo Journal of Immunologyvol 151 no 12 pp 6986ndash6993 1993

[55] C E Carvalho-Sousa S S Cruz-Machado E K Tamura P AC M Fernandes L Pinato et al ldquoMolecular basis for definingthe pineal gland and pinealocytes as targets for tumor necrosisfactorrdquo Frontiers in Endocrinology vol 2 pp 1ndash11 2011

[56] M PMattson ldquoNF-120581B in the survival and plasticity of neuronsrdquoNeurochemical Research vol 30 no 6-7 pp 883ndash893 2005

[57] P A CM Fernandes E Cecon R PMarkus and Z S FerreiraldquoEffect of TNF-120572 on the melatonin synthetic pathway in the ratpineal gland basis for a rsquofeedbackrsquo of the immune response oncircadian timingrdquo Journal of Pineal Research vol 41 no 4 pp344ndash350 2006

[58] S-Y Tsai T E OrsquoBrien and J A McNulty ldquoMicroglia play arole in mediating the effects of cytokines on the structure andfunction of the rat pineal glandrdquo Cell and Tissue Research vol303 no 3 pp 423ndash431 2001

[59] J S Bains and S H R Oliet ldquoGlia they make your memoriesstickrdquo Trends in Neurosciences vol 30 no 8 pp 417ndash424 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

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Computational and Mathematical Methods in Medicine

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Research and TreatmentAIDS

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

BioMed Research International 7

60

40

20

0NE 1120583M

Mela

toni

n (n

gm

L)

lowast

+ +

+ +

+

+

+

+minus minus

minus minus

lowastlowast

BB1101 10120583MGlu 600 120583M

Figure 3 (a) Ablation of the inhibitory glutamatergic effect onmelatonin synthesis by the TACE inhibitor BB1101 Pinealocytesand astrocytes were maintained in co-culture and stimulated withnorepinephrine (NE 1 120583M) in association with glutamate (Glu600 120583M) and BB1101 (10120583M) One-way ANOVA Bonferroni mul-tiple comparison test lowastlowast119875 lt 001 versus NE 1 120583M lowast119875 lt 005 versusNE 1 120583M + Glu 600 120583M 119899 = 4 for each group 3 experiments

Table 1 Summary of intracellular [Ca2+] changes in astrocytes andpinealocytes after stimulation

Cell typeagents Number of cellsexamined [Ca2+]

119894response

Isolated astrocytesGlu 600 120583M 68 79AMPA 50 120583M 97 80NMDA 100 120583M 49 90DHPG 50 120583M 80 74

Isolated pinealocytesGlu 600 120583M 66 80AMPA 50 120583M 78 85NMDA 100 120583M 53 90DHPG 50 120583M 60 76

Intracellular [Ca2+] in fluo-4-loaded astrocytes and pinealocytes was mea-sured as described in Section 2 The majority of the stimulated cellsresponded with an increase in intracellular [Ca2+] after the addition of eitherglutamate (600120583M) ionotropic glutamatergic agonists (AMPA 50120583M andNMDA 100120583M) or a class I metabotropic glutamatergic agonist (DHPG50120583M)

NF-120581B but not by a TFIID double-stranded oligonucleotideconsensus sequence demonstrating the specificity of the NF-120581BDNA interactionThe lower complex 2 was less efficientlydisplaced by unlabeledNF-120581B probe (Figure 6(a)) Supershiftanalysis indicated that the antibody against the p50 subunitwas able to shift the DNAprotein interaction present incomplex 1 The presence of antibodies against the p52 p65and c-Rel subunits did not affect DNA-protein complexes(Figures 6(b) and 6(c)) Taken together these results indicatethat at these concentrations glutamate activates the p50p50

homodimer Complex 2 was not displaced by the antibodiesand was not considered to be related to the NF-120581B family

Astrocyte and pinealocyte isolated cultures were used todetermine the cell type in which NF-120581B was being activatedGlutamate (600120583M)was shown to activateNF-120581B exclusivelyin astrocytes and not in pinealocytes (119875 lt 001) (Figure 7)

The involvement of NF-120581B activation in glutamatersquosinhibitory effect on melatonin synthesis was analyzed usinga specific inhibitor of NF-120581B PDTC PDTC (300 120583M) pre-vented the inhibitory effect of glutamate in cultured rat pinealglands stimulated by NE (1 120583M) (119875 lt 001) (Figure 8)

Glutamate cytotoxicity was evaluated because it is knownthat this neurotransmitter in high concentrations couldinduce cell death by cytotoxicity [48] Cell membraneintegrity and DNA fragmentation were assessed by flowcytometric analysis using propidium iodide (PI) Supple-mentary Figure 1 is representative of the results obtainedafter cell incubation with NE (1120583M) in the absence (controlgroup) or presence of glutamate (100 and 600120583M) for 5 hThe histograms show the fluorescence intensity in the FL-2channel which corresponds to the PI emission wavelengthWhen the cell membrane is intact the fluorescence signal islow and is related to autofluorescence (M1) A high signalmeans that PI was able to enter the cell and bind to DNA(M2) Glutamate treatments for 5 h were not able to inducea loss of membrane integrity (Supplementary Figure 1(a))The percentage of cells with ruptured membranes in thecontrol group was 453 whereas it was 502 and 577 inthe 100 and 600 120583M glutamate-treated groups respectively(Supplementary Figure 1(c)) Histograms in SupplementaryFigure 1(b) represent the results of DNA fragmentationanalysis After PI treatment the DNA content in live cellspresented a high fluorescence signal (M2) DNA fragmenta-tion induces the formation of small DNA pieces that exhibitlow fluorescence intensity (M1) The percentages of cellswith DNA fragmentation after glutamate stimulation 100and 600120583M were 286 and 335 respectively which weresimilar to the value of control cells (366) (SupplementaryFigure 1(d))

4 Discussion

Isolated pinealocytes isolated astrocytes and pineal-ocyteastrocyte co-culture were used in this study Theimmunocytochemistry characterization of the cell typespresent in these different cultures demonstrated the pre-dominance of pinealocytes in isolated pinealocyte culturewith the absence of glial cells In the same way astrocytecultures do not contain a significant number of microglialcells or pinealocytes In co-culture pinealocytes are thepredominant cells as astrocytes are present in minorquantities reproducing the proportion present in intactpineal glands (51) Thus the cultures were considered to beappropriate for this study

Data from the literature have shown that glutamate acts asan inhibitory neurotransmitter on melatonin synthesis [12]According to Yamada et al [13 14 16] and Yatsushiro et al[16] glutamate is stored in microvesicles and released from

8 BioMed Research International

mGluR1 mGluR2 mGluR3 mGluR5 mGluR7

M H2O As Pi Hi As Pi Hi As Pi Hi As Pi Hi As Pi Hi

200 bp100 bp

(a)

M H2O As Pi Hi As Pi Hi As Pi Hi As Pi Hi

200 bp100 bp

GluA1 GluA2 GluA3 GluN1

(b)

Figure 4 RT-PCR detection of metabotropic and ionotropic glutamate receptors in isolated astrocytes and pinealocytes (a) RT-PCRdetection of transcripts for mGluR1 (79 bp) mGluR2 (198 bp) mGluR3 (196 bp) mGluR5 (165 bp) and mGluR7 (70 bp) in astrocytes (As)pinealocytes (Pi) and hippocampus (Hi) Molecular weight marker (M) and water (H

2O) are presented in lanes 1 and 2 respectively (b)

RT-PCR detection of transcripts for GluA1 (203 bp) GluA2 (155 bp) GluA3 (193 bp) and GluN1 (201 bp) in astrocytes (As) pinealocytes(Pi) and hippocampus (Hi) The molecular weight marker (M) and PCR product generated with water (H

2O) are presented in lanes 1 and 2

respectively

pinealocytes upon cholinergic stimulation It interacts withionotropic and metabotropic glutamate receptors presentin pinealocyte membranes releasing additional glutamateand inhibiting melatonin synthesis respectively Recently amechanism involving the glutamate transporter GLT-1 wasproposed to be responsible for glutamate release [17] Inthese studies involving pinealocytes an autocrineparacrineprocess was suggested for the influence of glutamate onmelatonin synthesis In the presentworkwe demonstrated animportant role for astrocytes in glutamatersquos modulatory effecton melatonin synthesis that includes a paracrine interactionwith pinealocytes

Whereas in isolated pinealocytes glutamate does notexhibit the typical inhibitory effect on melatonin synthesisas observed in intact pineal glands its association withastrocytes restores this effect These results demonstrate thatastrocytes act as partners that collaborate to produce theglutamate effect In addition the fact that the effect occurredeven when inserts were used preventing the physical contactbetween the cells in co-culture suggests that a solublefactor is a mediator of this process The hypothesis that gapjunctions mediated the interactions between astrocytes andpinealocytes as occurs in the pineal gland [49] was excludedfrom this observationThe cytokine TNF-120572 a gliotransmitterthat is released by astrocytes could be the soluble factorthat is involved in the interactions between astrocytes andpinealocytes In fact the absence of a soluble form of TNF-120572 interferes with the glutamate response

The possibility that the glutamate effects were due to celldeath was investigated by flow cytometry As the cell viabilityand DNA fragmentation levels were similar between controland glutamate-stimulated groups showing no damage topineal cells even when treated with 600120583M of glutamate itis possible to exclude a glutamate cytotoxic effect as a causeof melatonin reduction

Although glutamate receptor expression had been firstdescribed in pinealocytes its expression was also laterdemonstrated in astrocytes mGluR3 and AMPA (GluA1subunit) were shown to be present in pinealocytes [12 16]

and NMDA and AMPA (GluA23) receptors were present inrat pineal gland astrocytes [19] Ionotropic receptor mRNAwas similarly observed in astrocytes in the present studyin addition to the novel observation of metabotropic recep-tor (mGluR1 mGluR2 mGluR3 mGluR5) expression Inpinealocytes the expression of mGluR1R2R3R5 AMPAand NMDA receptors was shownThis evidence supports thepossibility of astrocyte participation in rat pineal glutamater-gic signaling

Confocal microscopy assays using NMDA AMPA andDHPG agonists in isolated astrocytes and pinealocytesrevealed increased intracellular calcium content in both celltypes for all the agonists used PCR and confocal microscopyanalyses suggest both gene and protein expression in classI mGluR AMPA and NMDA ionotropic receptors reveal-ing the importance of the glutamate receptors as signaltransducers in rat pineal gland astrocytes and pinealocytesThus glutamate seems to interact with its receptors inboth cell types but its effect on melatonin synthesis nec-essarily involves astrocyte glutamate receptors The possibleparacrine interactions between pinealocytes and astrocytesseem to be the key in understanding the role of astrocytes inthe rat pineal gland

Transcription factors exert a great influence on pinealphysiology CREB and ICER are two transcription factorsthat intimately control the transcription of AANAT [50]Additionally there are other transcription factors in the ratpineal gland whose roles in pineal melatonin synthesis arenot known [51] NF-120581B is present in the rat pineal glandshowing a circadian rhythm and involvement in inflamma-tory responses [36 37] NF-120581B is a classic possible glutamatemediator and its activation in glial cells was demonstrated toplay a role in inflammatory processes [30]The present resultsdemonstrated NF-120581B activation by glutamate in culturedisolated astrocytes supporting its role as a mediator ofglutamate effects Supershift assay in cultured pineal glandsstimulated with glutamate showed the displacement of thep50 subunit emphasizing the importance of p50p50 homod-imer formationThis is an astrocyte-dependent event because

BioMed Research International 9

0 50 100 150 200 250 300 350

10

20

30

40

50

60

Time (s)

Isolated astrocytes

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

Glu 600120583M

(a)

0 30 60 90 120 150

155

160

165

170

175

180

185

Time (s)

Isolated pinealocytes

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

Glu 600120583M

(b)

0 100 200 300 400 500

10

20

30

Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

AMPA 50120583M

(c)

0 30 60 90 120 150 180

102

104

106

108

110

112

114

Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

AMPA 50120583M

(d)

0 50 100 150 200 250 300

400

600

800

1000

1200

1400

Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

NMDA 100120583M

(e)

0 50 100 150 200 250

900

1200

1500

Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

NMDA 100120583M

(f)

100 150 200 250

126

128

130

132

134

136

Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity) DHPG 50120583M

(g)

0 100 200 300 400

80

90

100

110

120

130

Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

DHPG 50120583M

(h)

Figure 5 Analysis of intracellular calcium in isolated astrocyte and pinealocyte cultures Traces of Fluo-4 fluorescence indicate theintracellular calcium transients from single cells Additions of exogenous glutamate or glutamatergic agonists were performed as indicatedby the arrows

10 BioMed Research International

ControlGlu 100120583MGlu 600120583MUnlabeled NF-120581BUnlabeled TFIID

1

(NS) 2

Free probe

+

minus

minus

minus

minus +

minus

minus

minus

minus

+

minus

minus

minus

minus +

+

minus

minus

minus +

+

minus

minus

minus+

+

minus

minus

minus

+

+

minus

minus

minus

(a)

(NS) 2

Free probe

(p50p50) 1

(NS) 3

+

+

minus

minus

minus

minus

+

minus

minus

minus

minus

minus

+

minus

minus

minus

minus

minus

+

+

minus

minus

minus

minus

+

+

minus

minus

minus

minus

+

+

minus

minus

minus

minus

ControlGlu 100120583MAnti p50Anti p52Anti p65Anti c-Rel

(b)

Free probe

Anti c-Rel

(NS) 2

(p50p50) 1

(NS) 3

ControlGlu 600120583MAnti p50Anti p52Anti p65

+minus minus minus minus minus

+

+ +

+

+ + +

+

+minus minus

minus

minus

minus

minus minus minus minus minus

minus

minus minus minus

minus minus

minus minus minus

minus minus

(c)

Figure 6 Electrophoretic mobility shift assays for NF120581B in the pineal gland (a) Competition studies were performed using nuclear extract(5120583g) from cultured pineal glands treated with glutamate (Glu) 100 and 600 120583M in the absence or presence of unlabeled specific (NF-120581Bconsensus sequence 10-fold molar excess) or nonspecific oligonucleotide (TFIID consensus sequence 10-fold molar excess) as indicated(b c) Supershift assays were performed with the same nuclear extract (5120583g) incubated in the absence or presence of antibodies against thesubunits p50 (1 10 dilution) p65 (1 20 dilution) p52 and c-Rel (1 10 dilution) as indicated Antibodies were added 20min prior to theaddition of the radiolabeled NF-120581B consensus oligonucleotide The position of specific NF-120581BDNA binding complexes p50p50 (band 1 bc) is indicated NS represents no specific binding (bands 2 and 3 a b c) The localization of the free probe is also indicated

BioMed Research International 11

ControlGlu 600120583M

minusminus

minus +

NF-120581B

(a)

20000

15000

10000

0Control Glu 600120583M

5000

lowastlowast

(au

)

(b)

ControlGlu 600120583M

minusminus

minus +

NF-120581B

(c)

(au

)

Control Glu 600120583M

16000

12000

8000

4000

0

(d)

Figure 7 Electrophoretic mobility shift assays for NF120581B in isolated astrocytes and pinealocytes (a) Nuclear proteins (5 120583g) were extractedfrom astrocytes treated with glutamate (Glu) 600120583M (b)Densitometric analysis (arbitrary units) of theNF-120581B band is presented on panel (a)One-way ANOVA Studentrsquos test lowastlowast119875 lt 001 versus control group mean plusmn SEM (c) Nuclear proteins (5120583g) were extracted from pinealocytestreated with Glu 600 120583M (d) Densitometric analysis (arbitrary units) of the NF-120581B band is presented in (c) The position of the specificNF-120581BDNA binding complex is indicated 119899 = 5 for each group 3 experiments

its activation was not observed in pinealocytes Astrocyteand pinealocyte supershift assays were not performed due totechnical limitations related to the amount of nuclear protein

The p50 subunit is synthesized from its precursor p105The processing of p105 and the resulting generation ofmature p50 subunits seem to be a constitutive event as p50homodimers are not regulated by I120581B proteins [34] The p50subunit formation seems to be regulated by cotranslationaland posttranslational mechanisms In the posttranslationalmechanism the p50 subunit is synthesized from its precursorp105 by phosphorylation at its 927 and 932 serine residueswhich allows the ubiquitinization and proteolytic degrada-tion of p105 by the 26S proteasome [52] In the cotranslationalmechanism p50 and p105 can be generated from a singlemRNA [53] There is also evidence that the 20S protea-some constitutively processes p105 to p50 in a ubiquitin-independent manner [52] The p50p50 homodimer wasoriginally considered to be a transcriptional repressor butnow there is substantial evidence that it can also inducegene transcription after a complex formation with the BCL3nuclear protein an atypical member of the I120581B family ofproteins that is considered to be a coactivator of p50p50homodimers [34]

The link between NF-120581B activation by glutamate and thereduction of melatonin synthesis was evidenced with the useof PDTC a NF-120581B translocation inhibitor [54] PDTC pre-vented glutamatersquos inhibitory effect on melatonin synthesisTaken together the crucial presence of astrocytes for theinhibitory effect of glutamate the exclusive activation of NF-120581B by glutamate in the same cell type and the demonstrationof NF-120581Brsquos role inmelatonin synthesis indicate that astrocytesunder glutamate stimulation possibly release a factor thatpromotes a decrease in pinealocyte melatonin synthesisTNF-120572may be a good candidate Indeed TNF-R1 the TNF-120572 receptor that interacts with soluble TNF is present in thepineal gland reinforcing the importance of this signalingpathway [55] Acting as an inhibitor of TACE to prevent TNF-120572 release BB1101 was thus adequate in demonstrating the roleplayed byTNF-120572 In addition the hypothesis that the p50p50homodimer may induce TNF-120572 gene expression and releaseneeds to be clarified

In the CNS astrocytes are known to release gliotransmit-ters such as glutamate D-serine and ATP [21] Moreoverastrocytes can also synthesize and release proinflammatorycytokines in response to NF-120581B activation such as TNF-120572 and interleukin-1 and induce gene expression of iNOS

12 BioMed Research International

NE 1120583MGlu 100120583MGlu 600120583MPDTC 300120583M

Mela

toni

n (n

ggl

and)

20

15

10

5

0

lowastlowast

lowastlowast

+ +

+ + +

++

+

+

+

+

+ +

minus minus minusminus

minus minus minusminus

minus minus minus

Figure 8 Effect of PDTC an inhibitor of NF-120581B on melatoninsynthesis Pineal glands in culture were incubated with PDTC(300120583M) and stimulated by norepinephrine (NE 1120583M) in thepresence of glutamate (100 and 600120583M) andmelatonin content wasevaluated One-way ANOVA Bonferroni multiple comparison test119875 lt 001 versus NE 1 120583M lowastlowast119875 lt 001 versus NE + Glu 100 120583Mand NE + Glu 600 120583M plotted as the mean plusmn SEM 119899 = 12 for eachgroup 3 experiments

PinealocyteAstrocyte

NF-120581Bactivation

AMPANMDAmGluR15

Release of solublefactor

TNF-120572

Glu

TNF-120572 alone (recruiting AMPA)or in association with Glu

mGluR3TNF-R1 AMPA[iCa+2]

Melatoninsynthesis

Figure 9 Diagram illustrating the putative paracrine interactionbetween pinealocytes and astrocytes in the modulation of pinealmelatonin synthesis by glutamate Glutamate (Glu) induces intra-cellular Ca2+ increase in astrocytes with the subsequent activationof NF120581B and release of a soluble factor possibly TNF-120572 This factoralone or in association with Glu promotes melatonin synthesisreduction in pinealocytes

resulting in nitric oxide formation [56] In this work wesuggested that TNF-120572 is the factor that is released byastrocytes under glutamate stimulation because the TACEinhibitor antagonized glutamatersquos effects Reinforcing thisidea recent preliminary data obtained in our laboratorywith the L929 tumor cell lineage which is sensitive to TNF-120572 showed that astrocytes release TNF-120572 when stimulatedby glutamate (data not shown) The literature states that

TNF-120572 decreases the serotonin content and AANAT mRNAexpression resulting in the reduction of N-acetylserotonin[57 58] the immediate precursor of melatonin synthesis Itis not clear whether TNF-120572 acts by itself or in combinationwith glutamate because there are glutamate receptors in thepinealocyte membrane [12 15 16 18] Recent studies havereported the control of hippocampal synapses by TNF-120572 dueto the increased insertion of AMPA receptors in the surfaceof the neuronal soma [59]The same effect could take place inthe pineal gland in which TNF-120572 released by the astrocytesin response to NF-120581B activation could increase the numberof AMPA receptors in the pinealocyte membrane resultingin the inhibition of melatonin synthesis

A model of the putative paracrine interactions betweenastrocytes and pinealocytes in the modulation of pinealmelatonin synthesis by glutamate is shown in Figure 9

5 Conclusions

In summary according to our results we propose thatglutamate acts on glutamate receptors (AMPA NMDAandor mGluR15) in astrocytes increasing the intracellularcalcium concentration promoting NF-120581B activation anddriving the release of a soluble factor possibly TNF-120572 whichacts independently or with glutamate in the pinealocytesresulting in inhibition of melatonin synthesis

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

This work was supported by a grant from FAPESP (0404328-1) Darine Villela was supported by a master fellowshipfrom FAPESP (0556943-4) Anderson Augusto Moutinhoand Victoria Fairbanks Atherino were supported by CNPq(PIBIC)

References

[1] J Calvo J Boya A Borregon and J E Garcia-MaurinoldquoPresence of glial cells in the rat pineal gland a light andelectronmicroscopic immunohistochemical studyrdquoAnatomicalRecord vol 220 no 4 pp 424ndash428 1988

[2] L Vollrath The Pineal Organ Springer New York NY USA1981

[3] R Y Moore J C Speh and J P Card ldquoThe retinohypothalamictract originates from a distinct subset of retinal ganglion cellsrdquoJournal of Comparative Neurology vol 352 no 3 pp 351ndash3661995

[4] D Sugden ldquoMelatonin biosynthesis in the mammalian pinealglandrdquo Experientia vol 45 no 10 pp 922ndash932 1989

[5] A F Afeche D CMVillelaM V Abrahao R Peres J Cipolla-Neto et al ldquoMelatonin and pineal glandrdquo in New Researchon Neurosecretory Systems E Romano Ed pp 151ndash177 NovaScience Publishers Hauppauge NY USA 2008

[6] J Arendt Melatonin and the Mammalian Pineal Gland Chap-man amp Hall London UK 1995

BioMed Research International 13

[7] M Ehret P Pevet and M Maitre ldquoTryptophan hydroxylasesynthesis is induced by 3101584051015840-cyclic adenosine monophosphateduring circadian rhythm in the rat pineal glandrdquo Journal ofNeurochemistry vol 57 no 5 pp 1516ndash1521 1991

[8] D C Klein G R Berg and J Weller ldquoMelatonin synthesisadenosine 3101584051015840-monophosphate and norepinephrine stimulateN-acetyltransferaserdquo Science vol 168 no 3934 pp 979ndash9801970

[9] D C Klein N L Schaad M A A Namboordiri L Yu and JL Weller ldquoControl of N-acetyltransferaserdquo Biochemical SocietyTransactions vol 20 no 2 pp 299ndash304 1992

[10] V Simonneaux and C Ribelayga ldquoGeneration of the melatoninendocrine message in mammals a review of the complexregulation of melatonin synthesis by norepinephrine peptidesand other pineal transmittersrdquoPharmacological Reviews vol 55no 2 pp 325ndash395 2003

[11] S Ishio H Yamada C M Craft and Y MoriyamaldquoHydroxyindole-O-methyltransferase is another target forL-glutamate-evoked inhibition of melatonin synthesis in ratpinealocytesrdquo Brain Research vol 850 no 1-2 pp 73ndash78 1999

[12] H Yamada S Yatsushiro S Ishio et al ldquoMetabotropic gluta-mate receptors negatively regulate melatonin synthesis in ratpinealocytesrdquo Journal of Neuroscience vol 18 no 6 pp 2056ndash2062 1998

[13] H Yamada A Yamamoto M Takahashi H Michibata HKumon and Y Moriyama ldquoThe L-type Ca2+ channel isinvolved in microvesicle-mediated glutamate exocytosis fromrat pinealocytesrdquo Journal of Pineal Research vol 21 no 3 pp165ndash174 1996

[14] H Yamada A Yamamoto S Yodozawa et al ldquoMicrovesicle-mediated exocytosis of glutamate is a novel paracrine-likechemical transduction mechanism and inhibits melatoninsecretion in rat pinealocytesrdquo Journal of Pineal Research vol 21no 3 pp 175ndash191 1996

[15] H Yamada A Ogura S Koizumi A Yamaguchi and YMoriyama ldquoAcetylcholine triggers L-glutamate exocytosis vianicotinic receptors and inhibits melatonin synthesis in ratpinealocytesrdquo Journal of Neuroscience vol 18 no 13 pp 4946ndash4952 1998

[16] S Yatsushiro H Yamada M Hayashi A Yamamoto andY Moriyama ldquoIonotropic glutamate receptors trigger micro-vesicle-mediated exocytosis of L-glutamate in rat pinealocytesrdquoJournal of Neurochemistry vol 75 no 1 pp 288ndash297 2000

[17] M-HKim SUehara AMuroyama BHille YMoriyama andD-S Koh ldquoGlutamate transporter-mediated glutamate secre-tion in the mammalian pineal glandrdquo Journal of Neurosciencevol 28 no 43 pp 10852ndash10863 2008

[18] S Yatsushiro H Yamada M Hayashi S Tsuboi and YMoriyama ldquoFunctional expression of metabotropic glutamatereceptor type 5 in rat pinealocytesrdquo NeuroReport vol 10 no 7pp 1599ndash1603 1999

[19] C Kaur V Sivakumar and E A Ling ldquoExpression ofN-methyl-D-aspartate (NMDA) and 120572-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) GluR23 receptors inthe developing rat pineal glandrdquo Journal of Pineal Research vol39 no 3 pp 294ndash301 2005

[20] H Pabst and P Redecker ldquoInterstitial glial cells of the gerbilpineal gland display immunoreactivity for the metabotropicglutamate receptors mGluR23 and mGluR5rdquo Brain Researchvol 838 no 1-2 pp 60ndash68 1999

[21] M M Halassa T Fellin and P G Haydon ldquoThe tripartitesynapse roles for gliotransmission in health and diseaserdquoTrends in Molecular Medicine vol 13 no 2 pp 54ndash63 2007

[22] S M Finkbeiner ldquoGlial calciumrdquo Glia vol 9 no 2 pp 83ndash1041993

[23] E A Newman ldquoNew roles for astrocytes regulation of synaptictransmissionrdquo Trends in Neurosciences vol 26 no 10 pp 536ndash542 2003

[24] J T Porter and K D McCarthy ldquoAstrocytic neurotransmitterreceptors in situ and in vivordquo Progress in Neurobiology vol 51no 4 pp 439ndash455 1997

[25] V Parpura T A Basarsky F Liu K Jeftinija S Jeftinija and PG Haydon ldquoGlutamate-mediated astrocyte-neuron signallingrdquoNature vol 369 no 6483 pp 744ndash747 1994

[26] T D Hassinger P B Atkinson G J Strecker et al ldquoEvidencefor glutamate-mediated activation of hippocampal neurons byglial calcium wavesrdquo Journal of Neurobiology vol 28 no 2 pp159ndash170 1995

[27] R H Lipsky K Xu D Zhu et al ldquoNuclear factor 120581B is a criticaldeterminant in N-methyl-D-aspartate receptor-mediated neu-roprotectionrdquo Journal of Neurochemistry vol 78 no 2 pp 254ndash264 2001

[28] M P Mattson C Culmsee Z Yu and S Camandola ldquoRoles ofnuclear factor 120581B in neuronal survival and plasticityrdquo Journal ofNeurochemistry vol 74 no 2 pp 443ndash456 2000

[29] C DMunhoz B Garcıa-Bueno J L MMadrigal L B LepschC Scavone and J C Leza ldquoStress-induced neuroinflammationmechanisms and new pharmacological targetsrdquo Brazilian Jour-nal of Medical and Biological Research vol 41 no 12 pp 1037ndash1046 2008

[30] S Camandola andM PMattson ldquoNF-ΚB as a therapeutic targetin neurodegenerative diseasesrdquo Expert Opinion on TherapeuticTargets vol 11 no 2 pp 123ndash132 2007

[31] B Kaltschmidt D Widera and C Kaltschmidt ldquoSignaling viaNF-120581B in the nervous systemrdquo Biochimica et Biophysica Actavol 1745 no 3 pp 287ndash299 2005

[32] P T Massa H Aleyasin D S Park X Mao and S W BargerldquoNF120581B in neurons the uncertainty principle in neurobiologyrdquoJournal of Neurochemistry vol 97 no 3 pp 607ndash618 2006

[33] H Hacker and M Karin ldquoRegulation and function of IKK andIKK-related kinasesrdquo Sciencersquos STKE vol 2006 no 357 p re132006

[34] S Beinke and S C Ley ldquoFunctions of NF-120581B1 and NF-120581B2 inimmune cell biologyrdquo Biochemical Journal vol 382 no 2 pp393ndash409 2004

[35] N D Perkins ldquoIntegrating cell-signalling pathways with NF-120581Band IKK functionrdquo Nature Reviews Molecular Cell Biology vol8 pp 49ndash62 2007

[36] E Cecon P A Fernandes L Pinato Z S Ferreira and R PMarkus ldquoDaily variation of constitutively activated nuclear fac-tor 120581B (NFKB) in rat pineal glandrdquoChronobiology Internationalvol 27 no 1 pp 52ndash67 2010

[37] Z S Ferreira P A C M Fernandes D Duma J Assreuy MC W Avellar and R P Markus ldquoCorticosterone modulatesnoradrenaline-induced melatonin synthesis through inhibitionof nuclear factor 120581Brdquo Journal of Pineal Research vol 38 no 3pp 182ndash188 2005

[38] S C Afeche R Barbosa J H Scialfa I M Terra A CCassola and J Cipolla-Neto ldquoEffects of the blockade of highvoltage-activated calcium channels on in vitro pineal melatoninsynthesisrdquo Cell Biochemistry and Function vol 24 no 6 pp499ndash505 2006

14 BioMed Research International

[39] B Barlaam T G Bird C Lambert-Van Der Brempt DCampbell S J Foster and R Maciewicz ldquoNew 120572-substitutedsuccinate-based hydroxamic acids as TNF120572 convertaseinhibitorsrdquo Journal of Medicinal Chemistry vol 42 no 23 pp4890ndash4908 1999

[40] I Glezer C D Munhoz E M Kawamoto T MarcourakisM C Werneck Avellar and C Scavone ldquoMK-801 and 7-Ni attenuate the activation of brain NF-120581B induced by LPSrdquoNeuropharmacology vol 45 no 8 pp 1120ndash1129 2003

[41] Y Rong and M Baudry ldquoSeizure activity results in a rapidinduction of nuclear factor-120581B in adult but not juvenile ratlimbic structuresrdquo Journal of Neurochemistry vol 67 no 2 pp662ndash668 1996

[42] I Nicoletti G Migliorati M C Pagliacci F Grignani and CRiccardi ldquoA rapid and simple method for measuring thymocyteapoptosis by propidium iodide staining and flow cytometryrdquoJournal of Immunological Methods vol 139 no 2 pp 271ndash2791991

[43] Y F Jiang-Shieh C H Wu M L Chang J Y Shiehand C Y Wen ldquoRegional heterogeneity in immunoreactivemacrophagesmicroglia in the rat pineal glandrdquo Journal ofPineal Research vol 35 no 1 pp 45ndash53 2003

[44] K-O Soderstrom ldquoLectin binding to the human retinardquoAnatomical Record vol 220 no 2 pp 219ndash223 1988

[45] F Uehara M Sameshima T Muramatsu and N OhbaldquoLocalization of fluorescence-labeled lectin binding sites onphotoreceptor cells of the monkey retinardquo Experimental EyeResearch vol 36 no 1 pp 113ndash123 1983

[46] H-W Korf ldquoThe pineal organ as a component of the biologicalclock phylogenetic and ontogenetic considerationsrdquo Annals ofthe New York Academy of Sciences vol 719 pp 13ndash42 1994

[47] E Maronde and J H Stehle ldquoThe mammalian pineal glandknown facts unknown facetsrdquo Trends in Endocrinology andMetabolism vol 18 no 4 pp 142ndash149 2007

[48] X-M Zhang and J Zhu ldquoKainic acid-induced neurotoxicitytargeting glial responses and glia-derived cytokinesrdquo CurrentNeuropharmacology vol 9 no 2 pp 388ndash398 2011

[49] D Stellwagen and R CMalenka ldquoSynaptic scalingmediated byglial TNF-120572rdquo Nature vol 440 no 7087 pp 1054ndash1059 2006

[50] E Maronde M Pfeffer J Olcese et al ldquoTranscription factorsin neuroendocrine regulation rhythmic changes in pCREB andICER levels framemelatonin synthesisrdquo Journal of Neurosciencevol 19 no 9 pp 3326ndash3336 1999

[51] D M Bustos M J Bailey D Sugden et al ldquoGlobal dailydynamics of the pineal transcriptomerdquoCell and Tissue Researchvol 344 no 1 pp 1ndash11 2011

[52] A K Moorthy O V Savinova J Q Ho V Y-F Wang D VuandGGhosh ldquoThe 20S proteasome processesNF-120581B1 p105 intop50 in a translation-independent mannerrdquo EMBO Journal vol25 no 9 pp 1945ndash1956 2006

[53] L Lin G N DeMartino and W C Greene ldquoCotranslationalbiogenesis of NF-120581B p50 by the 26S proteasomerdquo Cell vol 92no 6 pp 819ndash828 1998

[54] H W L Ziegler-Heitbrock T Sternsdorf J Liese et alldquoPyrrolidine dithiocarbamate inhibits NF-120581B mobilization andTNF production in humanmonocytesrdquo Journal of Immunologyvol 151 no 12 pp 6986ndash6993 1993

[55] C E Carvalho-Sousa S S Cruz-Machado E K Tamura P AC M Fernandes L Pinato et al ldquoMolecular basis for definingthe pineal gland and pinealocytes as targets for tumor necrosisfactorrdquo Frontiers in Endocrinology vol 2 pp 1ndash11 2011

[56] M PMattson ldquoNF-120581B in the survival and plasticity of neuronsrdquoNeurochemical Research vol 30 no 6-7 pp 883ndash893 2005

[57] P A CM Fernandes E Cecon R PMarkus and Z S FerreiraldquoEffect of TNF-120572 on the melatonin synthetic pathway in the ratpineal gland basis for a rsquofeedbackrsquo of the immune response oncircadian timingrdquo Journal of Pineal Research vol 41 no 4 pp344ndash350 2006

[58] S-Y Tsai T E OrsquoBrien and J A McNulty ldquoMicroglia play arole in mediating the effects of cytokines on the structure andfunction of the rat pineal glandrdquo Cell and Tissue Research vol303 no 3 pp 423ndash431 2001

[59] J S Bains and S H R Oliet ldquoGlia they make your memoriesstickrdquo Trends in Neurosciences vol 30 no 8 pp 417ndash424 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

8 BioMed Research International

mGluR1 mGluR2 mGluR3 mGluR5 mGluR7

M H2O As Pi Hi As Pi Hi As Pi Hi As Pi Hi As Pi Hi

200 bp100 bp

(a)

M H2O As Pi Hi As Pi Hi As Pi Hi As Pi Hi

200 bp100 bp

GluA1 GluA2 GluA3 GluN1

(b)

Figure 4 RT-PCR detection of metabotropic and ionotropic glutamate receptors in isolated astrocytes and pinealocytes (a) RT-PCRdetection of transcripts for mGluR1 (79 bp) mGluR2 (198 bp) mGluR3 (196 bp) mGluR5 (165 bp) and mGluR7 (70 bp) in astrocytes (As)pinealocytes (Pi) and hippocampus (Hi) Molecular weight marker (M) and water (H

2O) are presented in lanes 1 and 2 respectively (b)

RT-PCR detection of transcripts for GluA1 (203 bp) GluA2 (155 bp) GluA3 (193 bp) and GluN1 (201 bp) in astrocytes (As) pinealocytes(Pi) and hippocampus (Hi) The molecular weight marker (M) and PCR product generated with water (H

2O) are presented in lanes 1 and 2

respectively

pinealocytes upon cholinergic stimulation It interacts withionotropic and metabotropic glutamate receptors presentin pinealocyte membranes releasing additional glutamateand inhibiting melatonin synthesis respectively Recently amechanism involving the glutamate transporter GLT-1 wasproposed to be responsible for glutamate release [17] Inthese studies involving pinealocytes an autocrineparacrineprocess was suggested for the influence of glutamate onmelatonin synthesis In the presentworkwe demonstrated animportant role for astrocytes in glutamatersquos modulatory effecton melatonin synthesis that includes a paracrine interactionwith pinealocytes

Whereas in isolated pinealocytes glutamate does notexhibit the typical inhibitory effect on melatonin synthesisas observed in intact pineal glands its association withastrocytes restores this effect These results demonstrate thatastrocytes act as partners that collaborate to produce theglutamate effect In addition the fact that the effect occurredeven when inserts were used preventing the physical contactbetween the cells in co-culture suggests that a solublefactor is a mediator of this process The hypothesis that gapjunctions mediated the interactions between astrocytes andpinealocytes as occurs in the pineal gland [49] was excludedfrom this observationThe cytokine TNF-120572 a gliotransmitterthat is released by astrocytes could be the soluble factorthat is involved in the interactions between astrocytes andpinealocytes In fact the absence of a soluble form of TNF-120572 interferes with the glutamate response

The possibility that the glutamate effects were due to celldeath was investigated by flow cytometry As the cell viabilityand DNA fragmentation levels were similar between controland glutamate-stimulated groups showing no damage topineal cells even when treated with 600120583M of glutamate itis possible to exclude a glutamate cytotoxic effect as a causeof melatonin reduction

Although glutamate receptor expression had been firstdescribed in pinealocytes its expression was also laterdemonstrated in astrocytes mGluR3 and AMPA (GluA1subunit) were shown to be present in pinealocytes [12 16]

and NMDA and AMPA (GluA23) receptors were present inrat pineal gland astrocytes [19] Ionotropic receptor mRNAwas similarly observed in astrocytes in the present studyin addition to the novel observation of metabotropic recep-tor (mGluR1 mGluR2 mGluR3 mGluR5) expression Inpinealocytes the expression of mGluR1R2R3R5 AMPAand NMDA receptors was shownThis evidence supports thepossibility of astrocyte participation in rat pineal glutamater-gic signaling

Confocal microscopy assays using NMDA AMPA andDHPG agonists in isolated astrocytes and pinealocytesrevealed increased intracellular calcium content in both celltypes for all the agonists used PCR and confocal microscopyanalyses suggest both gene and protein expression in classI mGluR AMPA and NMDA ionotropic receptors reveal-ing the importance of the glutamate receptors as signaltransducers in rat pineal gland astrocytes and pinealocytesThus glutamate seems to interact with its receptors inboth cell types but its effect on melatonin synthesis nec-essarily involves astrocyte glutamate receptors The possibleparacrine interactions between pinealocytes and astrocytesseem to be the key in understanding the role of astrocytes inthe rat pineal gland

Transcription factors exert a great influence on pinealphysiology CREB and ICER are two transcription factorsthat intimately control the transcription of AANAT [50]Additionally there are other transcription factors in the ratpineal gland whose roles in pineal melatonin synthesis arenot known [51] NF-120581B is present in the rat pineal glandshowing a circadian rhythm and involvement in inflamma-tory responses [36 37] NF-120581B is a classic possible glutamatemediator and its activation in glial cells was demonstrated toplay a role in inflammatory processes [30]The present resultsdemonstrated NF-120581B activation by glutamate in culturedisolated astrocytes supporting its role as a mediator ofglutamate effects Supershift assay in cultured pineal glandsstimulated with glutamate showed the displacement of thep50 subunit emphasizing the importance of p50p50 homod-imer formationThis is an astrocyte-dependent event because

BioMed Research International 9

0 50 100 150 200 250 300 350

10

20

30

40

50

60

Time (s)

Isolated astrocytes

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

Glu 600120583M

(a)

0 30 60 90 120 150

155

160

165

170

175

180

185

Time (s)

Isolated pinealocytes

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

Glu 600120583M

(b)

0 100 200 300 400 500

10

20

30

Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

AMPA 50120583M

(c)

0 30 60 90 120 150 180

102

104

106

108

110

112

114

Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

AMPA 50120583M

(d)

0 50 100 150 200 250 300

400

600

800

1000

1200

1400

Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

NMDA 100120583M

(e)

0 50 100 150 200 250

900

1200

1500

Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

NMDA 100120583M

(f)

100 150 200 250

126

128

130

132

134

136

Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity) DHPG 50120583M

(g)

0 100 200 300 400

80

90

100

110

120

130

Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

DHPG 50120583M

(h)

Figure 5 Analysis of intracellular calcium in isolated astrocyte and pinealocyte cultures Traces of Fluo-4 fluorescence indicate theintracellular calcium transients from single cells Additions of exogenous glutamate or glutamatergic agonists were performed as indicatedby the arrows

10 BioMed Research International

ControlGlu 100120583MGlu 600120583MUnlabeled NF-120581BUnlabeled TFIID

1

(NS) 2

Free probe

+

minus

minus

minus

minus +

minus

minus

minus

minus

+

minus

minus

minus

minus +

+

minus

minus

minus +

+

minus

minus

minus+

+

minus

minus

minus

+

+

minus

minus

minus

(a)

(NS) 2

Free probe

(p50p50) 1

(NS) 3

+

+

minus

minus

minus

minus

+

minus

minus

minus

minus

minus

+

minus

minus

minus

minus

minus

+

+

minus

minus

minus

minus

+

+

minus

minus

minus

minus

+

+

minus

minus

minus

minus

ControlGlu 100120583MAnti p50Anti p52Anti p65Anti c-Rel

(b)

Free probe

Anti c-Rel

(NS) 2

(p50p50) 1

(NS) 3

ControlGlu 600120583MAnti p50Anti p52Anti p65

+minus minus minus minus minus

+

+ +

+

+ + +

+

+minus minus

minus

minus

minus

minus minus minus minus minus

minus

minus minus minus

minus minus

minus minus minus

minus minus

(c)

Figure 6 Electrophoretic mobility shift assays for NF120581B in the pineal gland (a) Competition studies were performed using nuclear extract(5120583g) from cultured pineal glands treated with glutamate (Glu) 100 and 600 120583M in the absence or presence of unlabeled specific (NF-120581Bconsensus sequence 10-fold molar excess) or nonspecific oligonucleotide (TFIID consensus sequence 10-fold molar excess) as indicated(b c) Supershift assays were performed with the same nuclear extract (5120583g) incubated in the absence or presence of antibodies against thesubunits p50 (1 10 dilution) p65 (1 20 dilution) p52 and c-Rel (1 10 dilution) as indicated Antibodies were added 20min prior to theaddition of the radiolabeled NF-120581B consensus oligonucleotide The position of specific NF-120581BDNA binding complexes p50p50 (band 1 bc) is indicated NS represents no specific binding (bands 2 and 3 a b c) The localization of the free probe is also indicated

BioMed Research International 11

ControlGlu 600120583M

minusminus

minus +

NF-120581B

(a)

20000

15000

10000

0Control Glu 600120583M

5000

lowastlowast

(au

)

(b)

ControlGlu 600120583M

minusminus

minus +

NF-120581B

(c)

(au

)

Control Glu 600120583M

16000

12000

8000

4000

0

(d)

Figure 7 Electrophoretic mobility shift assays for NF120581B in isolated astrocytes and pinealocytes (a) Nuclear proteins (5 120583g) were extractedfrom astrocytes treated with glutamate (Glu) 600120583M (b)Densitometric analysis (arbitrary units) of theNF-120581B band is presented on panel (a)One-way ANOVA Studentrsquos test lowastlowast119875 lt 001 versus control group mean plusmn SEM (c) Nuclear proteins (5120583g) were extracted from pinealocytestreated with Glu 600 120583M (d) Densitometric analysis (arbitrary units) of the NF-120581B band is presented in (c) The position of the specificNF-120581BDNA binding complex is indicated 119899 = 5 for each group 3 experiments

its activation was not observed in pinealocytes Astrocyteand pinealocyte supershift assays were not performed due totechnical limitations related to the amount of nuclear protein

The p50 subunit is synthesized from its precursor p105The processing of p105 and the resulting generation ofmature p50 subunits seem to be a constitutive event as p50homodimers are not regulated by I120581B proteins [34] The p50subunit formation seems to be regulated by cotranslationaland posttranslational mechanisms In the posttranslationalmechanism the p50 subunit is synthesized from its precursorp105 by phosphorylation at its 927 and 932 serine residueswhich allows the ubiquitinization and proteolytic degrada-tion of p105 by the 26S proteasome [52] In the cotranslationalmechanism p50 and p105 can be generated from a singlemRNA [53] There is also evidence that the 20S protea-some constitutively processes p105 to p50 in a ubiquitin-independent manner [52] The p50p50 homodimer wasoriginally considered to be a transcriptional repressor butnow there is substantial evidence that it can also inducegene transcription after a complex formation with the BCL3nuclear protein an atypical member of the I120581B family ofproteins that is considered to be a coactivator of p50p50homodimers [34]

The link between NF-120581B activation by glutamate and thereduction of melatonin synthesis was evidenced with the useof PDTC a NF-120581B translocation inhibitor [54] PDTC pre-vented glutamatersquos inhibitory effect on melatonin synthesisTaken together the crucial presence of astrocytes for theinhibitory effect of glutamate the exclusive activation of NF-120581B by glutamate in the same cell type and the demonstrationof NF-120581Brsquos role inmelatonin synthesis indicate that astrocytesunder glutamate stimulation possibly release a factor thatpromotes a decrease in pinealocyte melatonin synthesisTNF-120572may be a good candidate Indeed TNF-R1 the TNF-120572 receptor that interacts with soluble TNF is present in thepineal gland reinforcing the importance of this signalingpathway [55] Acting as an inhibitor of TACE to prevent TNF-120572 release BB1101 was thus adequate in demonstrating the roleplayed byTNF-120572 In addition the hypothesis that the p50p50homodimer may induce TNF-120572 gene expression and releaseneeds to be clarified

In the CNS astrocytes are known to release gliotransmit-ters such as glutamate D-serine and ATP [21] Moreoverastrocytes can also synthesize and release proinflammatorycytokines in response to NF-120581B activation such as TNF-120572 and interleukin-1 and induce gene expression of iNOS

12 BioMed Research International

NE 1120583MGlu 100120583MGlu 600120583MPDTC 300120583M

Mela

toni

n (n

ggl

and)

20

15

10

5

0

lowastlowast

lowastlowast

+ +

+ + +

++

+

+

+

+

+ +

minus minus minusminus

minus minus minusminus

minus minus minus

Figure 8 Effect of PDTC an inhibitor of NF-120581B on melatoninsynthesis Pineal glands in culture were incubated with PDTC(300120583M) and stimulated by norepinephrine (NE 1120583M) in thepresence of glutamate (100 and 600120583M) andmelatonin content wasevaluated One-way ANOVA Bonferroni multiple comparison test119875 lt 001 versus NE 1 120583M lowastlowast119875 lt 001 versus NE + Glu 100 120583Mand NE + Glu 600 120583M plotted as the mean plusmn SEM 119899 = 12 for eachgroup 3 experiments

PinealocyteAstrocyte

NF-120581Bactivation

AMPANMDAmGluR15

Release of solublefactor

TNF-120572

Glu

TNF-120572 alone (recruiting AMPA)or in association with Glu

mGluR3TNF-R1 AMPA[iCa+2]

Melatoninsynthesis

Figure 9 Diagram illustrating the putative paracrine interactionbetween pinealocytes and astrocytes in the modulation of pinealmelatonin synthesis by glutamate Glutamate (Glu) induces intra-cellular Ca2+ increase in astrocytes with the subsequent activationof NF120581B and release of a soluble factor possibly TNF-120572 This factoralone or in association with Glu promotes melatonin synthesisreduction in pinealocytes

resulting in nitric oxide formation [56] In this work wesuggested that TNF-120572 is the factor that is released byastrocytes under glutamate stimulation because the TACEinhibitor antagonized glutamatersquos effects Reinforcing thisidea recent preliminary data obtained in our laboratorywith the L929 tumor cell lineage which is sensitive to TNF-120572 showed that astrocytes release TNF-120572 when stimulatedby glutamate (data not shown) The literature states that

TNF-120572 decreases the serotonin content and AANAT mRNAexpression resulting in the reduction of N-acetylserotonin[57 58] the immediate precursor of melatonin synthesis Itis not clear whether TNF-120572 acts by itself or in combinationwith glutamate because there are glutamate receptors in thepinealocyte membrane [12 15 16 18] Recent studies havereported the control of hippocampal synapses by TNF-120572 dueto the increased insertion of AMPA receptors in the surfaceof the neuronal soma [59]The same effect could take place inthe pineal gland in which TNF-120572 released by the astrocytesin response to NF-120581B activation could increase the numberof AMPA receptors in the pinealocyte membrane resultingin the inhibition of melatonin synthesis

A model of the putative paracrine interactions betweenastrocytes and pinealocytes in the modulation of pinealmelatonin synthesis by glutamate is shown in Figure 9

5 Conclusions

In summary according to our results we propose thatglutamate acts on glutamate receptors (AMPA NMDAandor mGluR15) in astrocytes increasing the intracellularcalcium concentration promoting NF-120581B activation anddriving the release of a soluble factor possibly TNF-120572 whichacts independently or with glutamate in the pinealocytesresulting in inhibition of melatonin synthesis

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

This work was supported by a grant from FAPESP (0404328-1) Darine Villela was supported by a master fellowshipfrom FAPESP (0556943-4) Anderson Augusto Moutinhoand Victoria Fairbanks Atherino were supported by CNPq(PIBIC)

References

[1] J Calvo J Boya A Borregon and J E Garcia-MaurinoldquoPresence of glial cells in the rat pineal gland a light andelectronmicroscopic immunohistochemical studyrdquoAnatomicalRecord vol 220 no 4 pp 424ndash428 1988

[2] L Vollrath The Pineal Organ Springer New York NY USA1981

[3] R Y Moore J C Speh and J P Card ldquoThe retinohypothalamictract originates from a distinct subset of retinal ganglion cellsrdquoJournal of Comparative Neurology vol 352 no 3 pp 351ndash3661995

[4] D Sugden ldquoMelatonin biosynthesis in the mammalian pinealglandrdquo Experientia vol 45 no 10 pp 922ndash932 1989

[5] A F Afeche D CMVillelaM V Abrahao R Peres J Cipolla-Neto et al ldquoMelatonin and pineal glandrdquo in New Researchon Neurosecretory Systems E Romano Ed pp 151ndash177 NovaScience Publishers Hauppauge NY USA 2008

[6] J Arendt Melatonin and the Mammalian Pineal Gland Chap-man amp Hall London UK 1995

BioMed Research International 13

[7] M Ehret P Pevet and M Maitre ldquoTryptophan hydroxylasesynthesis is induced by 3101584051015840-cyclic adenosine monophosphateduring circadian rhythm in the rat pineal glandrdquo Journal ofNeurochemistry vol 57 no 5 pp 1516ndash1521 1991

[8] D C Klein G R Berg and J Weller ldquoMelatonin synthesisadenosine 3101584051015840-monophosphate and norepinephrine stimulateN-acetyltransferaserdquo Science vol 168 no 3934 pp 979ndash9801970

[9] D C Klein N L Schaad M A A Namboordiri L Yu and JL Weller ldquoControl of N-acetyltransferaserdquo Biochemical SocietyTransactions vol 20 no 2 pp 299ndash304 1992

[10] V Simonneaux and C Ribelayga ldquoGeneration of the melatoninendocrine message in mammals a review of the complexregulation of melatonin synthesis by norepinephrine peptidesand other pineal transmittersrdquoPharmacological Reviews vol 55no 2 pp 325ndash395 2003

[11] S Ishio H Yamada C M Craft and Y MoriyamaldquoHydroxyindole-O-methyltransferase is another target forL-glutamate-evoked inhibition of melatonin synthesis in ratpinealocytesrdquo Brain Research vol 850 no 1-2 pp 73ndash78 1999

[12] H Yamada S Yatsushiro S Ishio et al ldquoMetabotropic gluta-mate receptors negatively regulate melatonin synthesis in ratpinealocytesrdquo Journal of Neuroscience vol 18 no 6 pp 2056ndash2062 1998

[13] H Yamada A Yamamoto M Takahashi H Michibata HKumon and Y Moriyama ldquoThe L-type Ca2+ channel isinvolved in microvesicle-mediated glutamate exocytosis fromrat pinealocytesrdquo Journal of Pineal Research vol 21 no 3 pp165ndash174 1996

[14] H Yamada A Yamamoto S Yodozawa et al ldquoMicrovesicle-mediated exocytosis of glutamate is a novel paracrine-likechemical transduction mechanism and inhibits melatoninsecretion in rat pinealocytesrdquo Journal of Pineal Research vol 21no 3 pp 175ndash191 1996

[15] H Yamada A Ogura S Koizumi A Yamaguchi and YMoriyama ldquoAcetylcholine triggers L-glutamate exocytosis vianicotinic receptors and inhibits melatonin synthesis in ratpinealocytesrdquo Journal of Neuroscience vol 18 no 13 pp 4946ndash4952 1998

[16] S Yatsushiro H Yamada M Hayashi A Yamamoto andY Moriyama ldquoIonotropic glutamate receptors trigger micro-vesicle-mediated exocytosis of L-glutamate in rat pinealocytesrdquoJournal of Neurochemistry vol 75 no 1 pp 288ndash297 2000

[17] M-HKim SUehara AMuroyama BHille YMoriyama andD-S Koh ldquoGlutamate transporter-mediated glutamate secre-tion in the mammalian pineal glandrdquo Journal of Neurosciencevol 28 no 43 pp 10852ndash10863 2008

[18] S Yatsushiro H Yamada M Hayashi S Tsuboi and YMoriyama ldquoFunctional expression of metabotropic glutamatereceptor type 5 in rat pinealocytesrdquo NeuroReport vol 10 no 7pp 1599ndash1603 1999

[19] C Kaur V Sivakumar and E A Ling ldquoExpression ofN-methyl-D-aspartate (NMDA) and 120572-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) GluR23 receptors inthe developing rat pineal glandrdquo Journal of Pineal Research vol39 no 3 pp 294ndash301 2005

[20] H Pabst and P Redecker ldquoInterstitial glial cells of the gerbilpineal gland display immunoreactivity for the metabotropicglutamate receptors mGluR23 and mGluR5rdquo Brain Researchvol 838 no 1-2 pp 60ndash68 1999

[21] M M Halassa T Fellin and P G Haydon ldquoThe tripartitesynapse roles for gliotransmission in health and diseaserdquoTrends in Molecular Medicine vol 13 no 2 pp 54ndash63 2007

[22] S M Finkbeiner ldquoGlial calciumrdquo Glia vol 9 no 2 pp 83ndash1041993

[23] E A Newman ldquoNew roles for astrocytes regulation of synaptictransmissionrdquo Trends in Neurosciences vol 26 no 10 pp 536ndash542 2003

[24] J T Porter and K D McCarthy ldquoAstrocytic neurotransmitterreceptors in situ and in vivordquo Progress in Neurobiology vol 51no 4 pp 439ndash455 1997

[25] V Parpura T A Basarsky F Liu K Jeftinija S Jeftinija and PG Haydon ldquoGlutamate-mediated astrocyte-neuron signallingrdquoNature vol 369 no 6483 pp 744ndash747 1994

[26] T D Hassinger P B Atkinson G J Strecker et al ldquoEvidencefor glutamate-mediated activation of hippocampal neurons byglial calcium wavesrdquo Journal of Neurobiology vol 28 no 2 pp159ndash170 1995

[27] R H Lipsky K Xu D Zhu et al ldquoNuclear factor 120581B is a criticaldeterminant in N-methyl-D-aspartate receptor-mediated neu-roprotectionrdquo Journal of Neurochemistry vol 78 no 2 pp 254ndash264 2001

[28] M P Mattson C Culmsee Z Yu and S Camandola ldquoRoles ofnuclear factor 120581B in neuronal survival and plasticityrdquo Journal ofNeurochemistry vol 74 no 2 pp 443ndash456 2000

[29] C DMunhoz B Garcıa-Bueno J L MMadrigal L B LepschC Scavone and J C Leza ldquoStress-induced neuroinflammationmechanisms and new pharmacological targetsrdquo Brazilian Jour-nal of Medical and Biological Research vol 41 no 12 pp 1037ndash1046 2008

[30] S Camandola andM PMattson ldquoNF-ΚB as a therapeutic targetin neurodegenerative diseasesrdquo Expert Opinion on TherapeuticTargets vol 11 no 2 pp 123ndash132 2007

[31] B Kaltschmidt D Widera and C Kaltschmidt ldquoSignaling viaNF-120581B in the nervous systemrdquo Biochimica et Biophysica Actavol 1745 no 3 pp 287ndash299 2005

[32] P T Massa H Aleyasin D S Park X Mao and S W BargerldquoNF120581B in neurons the uncertainty principle in neurobiologyrdquoJournal of Neurochemistry vol 97 no 3 pp 607ndash618 2006

[33] H Hacker and M Karin ldquoRegulation and function of IKK andIKK-related kinasesrdquo Sciencersquos STKE vol 2006 no 357 p re132006

[34] S Beinke and S C Ley ldquoFunctions of NF-120581B1 and NF-120581B2 inimmune cell biologyrdquo Biochemical Journal vol 382 no 2 pp393ndash409 2004

[35] N D Perkins ldquoIntegrating cell-signalling pathways with NF-120581Band IKK functionrdquo Nature Reviews Molecular Cell Biology vol8 pp 49ndash62 2007

[36] E Cecon P A Fernandes L Pinato Z S Ferreira and R PMarkus ldquoDaily variation of constitutively activated nuclear fac-tor 120581B (NFKB) in rat pineal glandrdquoChronobiology Internationalvol 27 no 1 pp 52ndash67 2010

[37] Z S Ferreira P A C M Fernandes D Duma J Assreuy MC W Avellar and R P Markus ldquoCorticosterone modulatesnoradrenaline-induced melatonin synthesis through inhibitionof nuclear factor 120581Brdquo Journal of Pineal Research vol 38 no 3pp 182ndash188 2005

[38] S C Afeche R Barbosa J H Scialfa I M Terra A CCassola and J Cipolla-Neto ldquoEffects of the blockade of highvoltage-activated calcium channels on in vitro pineal melatoninsynthesisrdquo Cell Biochemistry and Function vol 24 no 6 pp499ndash505 2006

14 BioMed Research International

[39] B Barlaam T G Bird C Lambert-Van Der Brempt DCampbell S J Foster and R Maciewicz ldquoNew 120572-substitutedsuccinate-based hydroxamic acids as TNF120572 convertaseinhibitorsrdquo Journal of Medicinal Chemistry vol 42 no 23 pp4890ndash4908 1999

[40] I Glezer C D Munhoz E M Kawamoto T MarcourakisM C Werneck Avellar and C Scavone ldquoMK-801 and 7-Ni attenuate the activation of brain NF-120581B induced by LPSrdquoNeuropharmacology vol 45 no 8 pp 1120ndash1129 2003

[41] Y Rong and M Baudry ldquoSeizure activity results in a rapidinduction of nuclear factor-120581B in adult but not juvenile ratlimbic structuresrdquo Journal of Neurochemistry vol 67 no 2 pp662ndash668 1996

[42] I Nicoletti G Migliorati M C Pagliacci F Grignani and CRiccardi ldquoA rapid and simple method for measuring thymocyteapoptosis by propidium iodide staining and flow cytometryrdquoJournal of Immunological Methods vol 139 no 2 pp 271ndash2791991

[43] Y F Jiang-Shieh C H Wu M L Chang J Y Shiehand C Y Wen ldquoRegional heterogeneity in immunoreactivemacrophagesmicroglia in the rat pineal glandrdquo Journal ofPineal Research vol 35 no 1 pp 45ndash53 2003

[44] K-O Soderstrom ldquoLectin binding to the human retinardquoAnatomical Record vol 220 no 2 pp 219ndash223 1988

[45] F Uehara M Sameshima T Muramatsu and N OhbaldquoLocalization of fluorescence-labeled lectin binding sites onphotoreceptor cells of the monkey retinardquo Experimental EyeResearch vol 36 no 1 pp 113ndash123 1983

[46] H-W Korf ldquoThe pineal organ as a component of the biologicalclock phylogenetic and ontogenetic considerationsrdquo Annals ofthe New York Academy of Sciences vol 719 pp 13ndash42 1994

[47] E Maronde and J H Stehle ldquoThe mammalian pineal glandknown facts unknown facetsrdquo Trends in Endocrinology andMetabolism vol 18 no 4 pp 142ndash149 2007

[48] X-M Zhang and J Zhu ldquoKainic acid-induced neurotoxicitytargeting glial responses and glia-derived cytokinesrdquo CurrentNeuropharmacology vol 9 no 2 pp 388ndash398 2011

[49] D Stellwagen and R CMalenka ldquoSynaptic scalingmediated byglial TNF-120572rdquo Nature vol 440 no 7087 pp 1054ndash1059 2006

[50] E Maronde M Pfeffer J Olcese et al ldquoTranscription factorsin neuroendocrine regulation rhythmic changes in pCREB andICER levels framemelatonin synthesisrdquo Journal of Neurosciencevol 19 no 9 pp 3326ndash3336 1999

[51] D M Bustos M J Bailey D Sugden et al ldquoGlobal dailydynamics of the pineal transcriptomerdquoCell and Tissue Researchvol 344 no 1 pp 1ndash11 2011

[52] A K Moorthy O V Savinova J Q Ho V Y-F Wang D VuandGGhosh ldquoThe 20S proteasome processesNF-120581B1 p105 intop50 in a translation-independent mannerrdquo EMBO Journal vol25 no 9 pp 1945ndash1956 2006

[53] L Lin G N DeMartino and W C Greene ldquoCotranslationalbiogenesis of NF-120581B p50 by the 26S proteasomerdquo Cell vol 92no 6 pp 819ndash828 1998

[54] H W L Ziegler-Heitbrock T Sternsdorf J Liese et alldquoPyrrolidine dithiocarbamate inhibits NF-120581B mobilization andTNF production in humanmonocytesrdquo Journal of Immunologyvol 151 no 12 pp 6986ndash6993 1993

[55] C E Carvalho-Sousa S S Cruz-Machado E K Tamura P AC M Fernandes L Pinato et al ldquoMolecular basis for definingthe pineal gland and pinealocytes as targets for tumor necrosisfactorrdquo Frontiers in Endocrinology vol 2 pp 1ndash11 2011

[56] M PMattson ldquoNF-120581B in the survival and plasticity of neuronsrdquoNeurochemical Research vol 30 no 6-7 pp 883ndash893 2005

[57] P A CM Fernandes E Cecon R PMarkus and Z S FerreiraldquoEffect of TNF-120572 on the melatonin synthetic pathway in the ratpineal gland basis for a rsquofeedbackrsquo of the immune response oncircadian timingrdquo Journal of Pineal Research vol 41 no 4 pp344ndash350 2006

[58] S-Y Tsai T E OrsquoBrien and J A McNulty ldquoMicroglia play arole in mediating the effects of cytokines on the structure andfunction of the rat pineal glandrdquo Cell and Tissue Research vol303 no 3 pp 423ndash431 2001

[59] J S Bains and S H R Oliet ldquoGlia they make your memoriesstickrdquo Trends in Neurosciences vol 30 no 8 pp 417ndash424 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

BioMed Research International 9

0 50 100 150 200 250 300 350

10

20

30

40

50

60

Time (s)

Isolated astrocytes

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

Glu 600120583M

(a)

0 30 60 90 120 150

155

160

165

170

175

180

185

Time (s)

Isolated pinealocytes

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

Glu 600120583M

(b)

0 100 200 300 400 500

10

20

30

Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

AMPA 50120583M

(c)

0 30 60 90 120 150 180

102

104

106

108

110

112

114

Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

AMPA 50120583M

(d)

0 50 100 150 200 250 300

400

600

800

1000

1200

1400

Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

NMDA 100120583M

(e)

0 50 100 150 200 250

900

1200

1500

Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

NMDA 100120583M

(f)

100 150 200 250

126

128

130

132

134

136

Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity) DHPG 50120583M

(g)

0 100 200 300 400

80

90

100

110

120

130

Time (s)

Fluo

-4 d

ye ch

ange

s(fl

uore

scen

cein

tens

ity)

DHPG 50120583M

(h)

Figure 5 Analysis of intracellular calcium in isolated astrocyte and pinealocyte cultures Traces of Fluo-4 fluorescence indicate theintracellular calcium transients from single cells Additions of exogenous glutamate or glutamatergic agonists were performed as indicatedby the arrows

10 BioMed Research International

ControlGlu 100120583MGlu 600120583MUnlabeled NF-120581BUnlabeled TFIID

1

(NS) 2

Free probe

+

minus

minus

minus

minus +

minus

minus

minus

minus

+

minus

minus

minus

minus +

+

minus

minus

minus +

+

minus

minus

minus+

+

minus

minus

minus

+

+

minus

minus

minus

(a)

(NS) 2

Free probe

(p50p50) 1

(NS) 3

+

+

minus

minus

minus

minus

+

minus

minus

minus

minus

minus

+

minus

minus

minus

minus

minus

+

+

minus

minus

minus

minus

+

+

minus

minus

minus

minus

+

+

minus

minus

minus

minus

ControlGlu 100120583MAnti p50Anti p52Anti p65Anti c-Rel

(b)

Free probe

Anti c-Rel

(NS) 2

(p50p50) 1

(NS) 3

ControlGlu 600120583MAnti p50Anti p52Anti p65

+minus minus minus minus minus

+

+ +

+

+ + +

+

+minus minus

minus

minus

minus

minus minus minus minus minus

minus

minus minus minus

minus minus

minus minus minus

minus minus

(c)

Figure 6 Electrophoretic mobility shift assays for NF120581B in the pineal gland (a) Competition studies were performed using nuclear extract(5120583g) from cultured pineal glands treated with glutamate (Glu) 100 and 600 120583M in the absence or presence of unlabeled specific (NF-120581Bconsensus sequence 10-fold molar excess) or nonspecific oligonucleotide (TFIID consensus sequence 10-fold molar excess) as indicated(b c) Supershift assays were performed with the same nuclear extract (5120583g) incubated in the absence or presence of antibodies against thesubunits p50 (1 10 dilution) p65 (1 20 dilution) p52 and c-Rel (1 10 dilution) as indicated Antibodies were added 20min prior to theaddition of the radiolabeled NF-120581B consensus oligonucleotide The position of specific NF-120581BDNA binding complexes p50p50 (band 1 bc) is indicated NS represents no specific binding (bands 2 and 3 a b c) The localization of the free probe is also indicated

BioMed Research International 11

ControlGlu 600120583M

minusminus

minus +

NF-120581B

(a)

20000

15000

10000

0Control Glu 600120583M

5000

lowastlowast

(au

)

(b)

ControlGlu 600120583M

minusminus

minus +

NF-120581B

(c)

(au

)

Control Glu 600120583M

16000

12000

8000

4000

0

(d)

Figure 7 Electrophoretic mobility shift assays for NF120581B in isolated astrocytes and pinealocytes (a) Nuclear proteins (5 120583g) were extractedfrom astrocytes treated with glutamate (Glu) 600120583M (b)Densitometric analysis (arbitrary units) of theNF-120581B band is presented on panel (a)One-way ANOVA Studentrsquos test lowastlowast119875 lt 001 versus control group mean plusmn SEM (c) Nuclear proteins (5120583g) were extracted from pinealocytestreated with Glu 600 120583M (d) Densitometric analysis (arbitrary units) of the NF-120581B band is presented in (c) The position of the specificNF-120581BDNA binding complex is indicated 119899 = 5 for each group 3 experiments

its activation was not observed in pinealocytes Astrocyteand pinealocyte supershift assays were not performed due totechnical limitations related to the amount of nuclear protein

The p50 subunit is synthesized from its precursor p105The processing of p105 and the resulting generation ofmature p50 subunits seem to be a constitutive event as p50homodimers are not regulated by I120581B proteins [34] The p50subunit formation seems to be regulated by cotranslationaland posttranslational mechanisms In the posttranslationalmechanism the p50 subunit is synthesized from its precursorp105 by phosphorylation at its 927 and 932 serine residueswhich allows the ubiquitinization and proteolytic degrada-tion of p105 by the 26S proteasome [52] In the cotranslationalmechanism p50 and p105 can be generated from a singlemRNA [53] There is also evidence that the 20S protea-some constitutively processes p105 to p50 in a ubiquitin-independent manner [52] The p50p50 homodimer wasoriginally considered to be a transcriptional repressor butnow there is substantial evidence that it can also inducegene transcription after a complex formation with the BCL3nuclear protein an atypical member of the I120581B family ofproteins that is considered to be a coactivator of p50p50homodimers [34]

The link between NF-120581B activation by glutamate and thereduction of melatonin synthesis was evidenced with the useof PDTC a NF-120581B translocation inhibitor [54] PDTC pre-vented glutamatersquos inhibitory effect on melatonin synthesisTaken together the crucial presence of astrocytes for theinhibitory effect of glutamate the exclusive activation of NF-120581B by glutamate in the same cell type and the demonstrationof NF-120581Brsquos role inmelatonin synthesis indicate that astrocytesunder glutamate stimulation possibly release a factor thatpromotes a decrease in pinealocyte melatonin synthesisTNF-120572may be a good candidate Indeed TNF-R1 the TNF-120572 receptor that interacts with soluble TNF is present in thepineal gland reinforcing the importance of this signalingpathway [55] Acting as an inhibitor of TACE to prevent TNF-120572 release BB1101 was thus adequate in demonstrating the roleplayed byTNF-120572 In addition the hypothesis that the p50p50homodimer may induce TNF-120572 gene expression and releaseneeds to be clarified

In the CNS astrocytes are known to release gliotransmit-ters such as glutamate D-serine and ATP [21] Moreoverastrocytes can also synthesize and release proinflammatorycytokines in response to NF-120581B activation such as TNF-120572 and interleukin-1 and induce gene expression of iNOS

12 BioMed Research International

NE 1120583MGlu 100120583MGlu 600120583MPDTC 300120583M

Mela

toni

n (n

ggl

and)

20

15

10

5

0

lowastlowast

lowastlowast

+ +

+ + +

++

+

+

+

+

+ +

minus minus minusminus

minus minus minusminus

minus minus minus

Figure 8 Effect of PDTC an inhibitor of NF-120581B on melatoninsynthesis Pineal glands in culture were incubated with PDTC(300120583M) and stimulated by norepinephrine (NE 1120583M) in thepresence of glutamate (100 and 600120583M) andmelatonin content wasevaluated One-way ANOVA Bonferroni multiple comparison test119875 lt 001 versus NE 1 120583M lowastlowast119875 lt 001 versus NE + Glu 100 120583Mand NE + Glu 600 120583M plotted as the mean plusmn SEM 119899 = 12 for eachgroup 3 experiments

PinealocyteAstrocyte

NF-120581Bactivation

AMPANMDAmGluR15

Release of solublefactor

TNF-120572

Glu

TNF-120572 alone (recruiting AMPA)or in association with Glu

mGluR3TNF-R1 AMPA[iCa+2]

Melatoninsynthesis

Figure 9 Diagram illustrating the putative paracrine interactionbetween pinealocytes and astrocytes in the modulation of pinealmelatonin synthesis by glutamate Glutamate (Glu) induces intra-cellular Ca2+ increase in astrocytes with the subsequent activationof NF120581B and release of a soluble factor possibly TNF-120572 This factoralone or in association with Glu promotes melatonin synthesisreduction in pinealocytes

resulting in nitric oxide formation [56] In this work wesuggested that TNF-120572 is the factor that is released byastrocytes under glutamate stimulation because the TACEinhibitor antagonized glutamatersquos effects Reinforcing thisidea recent preliminary data obtained in our laboratorywith the L929 tumor cell lineage which is sensitive to TNF-120572 showed that astrocytes release TNF-120572 when stimulatedby glutamate (data not shown) The literature states that

TNF-120572 decreases the serotonin content and AANAT mRNAexpression resulting in the reduction of N-acetylserotonin[57 58] the immediate precursor of melatonin synthesis Itis not clear whether TNF-120572 acts by itself or in combinationwith glutamate because there are glutamate receptors in thepinealocyte membrane [12 15 16 18] Recent studies havereported the control of hippocampal synapses by TNF-120572 dueto the increased insertion of AMPA receptors in the surfaceof the neuronal soma [59]The same effect could take place inthe pineal gland in which TNF-120572 released by the astrocytesin response to NF-120581B activation could increase the numberof AMPA receptors in the pinealocyte membrane resultingin the inhibition of melatonin synthesis

A model of the putative paracrine interactions betweenastrocytes and pinealocytes in the modulation of pinealmelatonin synthesis by glutamate is shown in Figure 9

5 Conclusions

In summary according to our results we propose thatglutamate acts on glutamate receptors (AMPA NMDAandor mGluR15) in astrocytes increasing the intracellularcalcium concentration promoting NF-120581B activation anddriving the release of a soluble factor possibly TNF-120572 whichacts independently or with glutamate in the pinealocytesresulting in inhibition of melatonin synthesis

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

This work was supported by a grant from FAPESP (0404328-1) Darine Villela was supported by a master fellowshipfrom FAPESP (0556943-4) Anderson Augusto Moutinhoand Victoria Fairbanks Atherino were supported by CNPq(PIBIC)

References

[1] J Calvo J Boya A Borregon and J E Garcia-MaurinoldquoPresence of glial cells in the rat pineal gland a light andelectronmicroscopic immunohistochemical studyrdquoAnatomicalRecord vol 220 no 4 pp 424ndash428 1988

[2] L Vollrath The Pineal Organ Springer New York NY USA1981

[3] R Y Moore J C Speh and J P Card ldquoThe retinohypothalamictract originates from a distinct subset of retinal ganglion cellsrdquoJournal of Comparative Neurology vol 352 no 3 pp 351ndash3661995

[4] D Sugden ldquoMelatonin biosynthesis in the mammalian pinealglandrdquo Experientia vol 45 no 10 pp 922ndash932 1989

[5] A F Afeche D CMVillelaM V Abrahao R Peres J Cipolla-Neto et al ldquoMelatonin and pineal glandrdquo in New Researchon Neurosecretory Systems E Romano Ed pp 151ndash177 NovaScience Publishers Hauppauge NY USA 2008

[6] J Arendt Melatonin and the Mammalian Pineal Gland Chap-man amp Hall London UK 1995

BioMed Research International 13

[7] M Ehret P Pevet and M Maitre ldquoTryptophan hydroxylasesynthesis is induced by 3101584051015840-cyclic adenosine monophosphateduring circadian rhythm in the rat pineal glandrdquo Journal ofNeurochemistry vol 57 no 5 pp 1516ndash1521 1991

[8] D C Klein G R Berg and J Weller ldquoMelatonin synthesisadenosine 3101584051015840-monophosphate and norepinephrine stimulateN-acetyltransferaserdquo Science vol 168 no 3934 pp 979ndash9801970

[9] D C Klein N L Schaad M A A Namboordiri L Yu and JL Weller ldquoControl of N-acetyltransferaserdquo Biochemical SocietyTransactions vol 20 no 2 pp 299ndash304 1992

[10] V Simonneaux and C Ribelayga ldquoGeneration of the melatoninendocrine message in mammals a review of the complexregulation of melatonin synthesis by norepinephrine peptidesand other pineal transmittersrdquoPharmacological Reviews vol 55no 2 pp 325ndash395 2003

[11] S Ishio H Yamada C M Craft and Y MoriyamaldquoHydroxyindole-O-methyltransferase is another target forL-glutamate-evoked inhibition of melatonin synthesis in ratpinealocytesrdquo Brain Research vol 850 no 1-2 pp 73ndash78 1999

[12] H Yamada S Yatsushiro S Ishio et al ldquoMetabotropic gluta-mate receptors negatively regulate melatonin synthesis in ratpinealocytesrdquo Journal of Neuroscience vol 18 no 6 pp 2056ndash2062 1998

[13] H Yamada A Yamamoto M Takahashi H Michibata HKumon and Y Moriyama ldquoThe L-type Ca2+ channel isinvolved in microvesicle-mediated glutamate exocytosis fromrat pinealocytesrdquo Journal of Pineal Research vol 21 no 3 pp165ndash174 1996

[14] H Yamada A Yamamoto S Yodozawa et al ldquoMicrovesicle-mediated exocytosis of glutamate is a novel paracrine-likechemical transduction mechanism and inhibits melatoninsecretion in rat pinealocytesrdquo Journal of Pineal Research vol 21no 3 pp 175ndash191 1996

[15] H Yamada A Ogura S Koizumi A Yamaguchi and YMoriyama ldquoAcetylcholine triggers L-glutamate exocytosis vianicotinic receptors and inhibits melatonin synthesis in ratpinealocytesrdquo Journal of Neuroscience vol 18 no 13 pp 4946ndash4952 1998

[16] S Yatsushiro H Yamada M Hayashi A Yamamoto andY Moriyama ldquoIonotropic glutamate receptors trigger micro-vesicle-mediated exocytosis of L-glutamate in rat pinealocytesrdquoJournal of Neurochemistry vol 75 no 1 pp 288ndash297 2000

[17] M-HKim SUehara AMuroyama BHille YMoriyama andD-S Koh ldquoGlutamate transporter-mediated glutamate secre-tion in the mammalian pineal glandrdquo Journal of Neurosciencevol 28 no 43 pp 10852ndash10863 2008

[18] S Yatsushiro H Yamada M Hayashi S Tsuboi and YMoriyama ldquoFunctional expression of metabotropic glutamatereceptor type 5 in rat pinealocytesrdquo NeuroReport vol 10 no 7pp 1599ndash1603 1999

[19] C Kaur V Sivakumar and E A Ling ldquoExpression ofN-methyl-D-aspartate (NMDA) and 120572-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) GluR23 receptors inthe developing rat pineal glandrdquo Journal of Pineal Research vol39 no 3 pp 294ndash301 2005

[20] H Pabst and P Redecker ldquoInterstitial glial cells of the gerbilpineal gland display immunoreactivity for the metabotropicglutamate receptors mGluR23 and mGluR5rdquo Brain Researchvol 838 no 1-2 pp 60ndash68 1999

[21] M M Halassa T Fellin and P G Haydon ldquoThe tripartitesynapse roles for gliotransmission in health and diseaserdquoTrends in Molecular Medicine vol 13 no 2 pp 54ndash63 2007

[22] S M Finkbeiner ldquoGlial calciumrdquo Glia vol 9 no 2 pp 83ndash1041993

[23] E A Newman ldquoNew roles for astrocytes regulation of synaptictransmissionrdquo Trends in Neurosciences vol 26 no 10 pp 536ndash542 2003

[24] J T Porter and K D McCarthy ldquoAstrocytic neurotransmitterreceptors in situ and in vivordquo Progress in Neurobiology vol 51no 4 pp 439ndash455 1997

[25] V Parpura T A Basarsky F Liu K Jeftinija S Jeftinija and PG Haydon ldquoGlutamate-mediated astrocyte-neuron signallingrdquoNature vol 369 no 6483 pp 744ndash747 1994

[26] T D Hassinger P B Atkinson G J Strecker et al ldquoEvidencefor glutamate-mediated activation of hippocampal neurons byglial calcium wavesrdquo Journal of Neurobiology vol 28 no 2 pp159ndash170 1995

[27] R H Lipsky K Xu D Zhu et al ldquoNuclear factor 120581B is a criticaldeterminant in N-methyl-D-aspartate receptor-mediated neu-roprotectionrdquo Journal of Neurochemistry vol 78 no 2 pp 254ndash264 2001

[28] M P Mattson C Culmsee Z Yu and S Camandola ldquoRoles ofnuclear factor 120581B in neuronal survival and plasticityrdquo Journal ofNeurochemistry vol 74 no 2 pp 443ndash456 2000

[29] C DMunhoz B Garcıa-Bueno J L MMadrigal L B LepschC Scavone and J C Leza ldquoStress-induced neuroinflammationmechanisms and new pharmacological targetsrdquo Brazilian Jour-nal of Medical and Biological Research vol 41 no 12 pp 1037ndash1046 2008

[30] S Camandola andM PMattson ldquoNF-ΚB as a therapeutic targetin neurodegenerative diseasesrdquo Expert Opinion on TherapeuticTargets vol 11 no 2 pp 123ndash132 2007

[31] B Kaltschmidt D Widera and C Kaltschmidt ldquoSignaling viaNF-120581B in the nervous systemrdquo Biochimica et Biophysica Actavol 1745 no 3 pp 287ndash299 2005

[32] P T Massa H Aleyasin D S Park X Mao and S W BargerldquoNF120581B in neurons the uncertainty principle in neurobiologyrdquoJournal of Neurochemistry vol 97 no 3 pp 607ndash618 2006

[33] H Hacker and M Karin ldquoRegulation and function of IKK andIKK-related kinasesrdquo Sciencersquos STKE vol 2006 no 357 p re132006

[34] S Beinke and S C Ley ldquoFunctions of NF-120581B1 and NF-120581B2 inimmune cell biologyrdquo Biochemical Journal vol 382 no 2 pp393ndash409 2004

[35] N D Perkins ldquoIntegrating cell-signalling pathways with NF-120581Band IKK functionrdquo Nature Reviews Molecular Cell Biology vol8 pp 49ndash62 2007

[36] E Cecon P A Fernandes L Pinato Z S Ferreira and R PMarkus ldquoDaily variation of constitutively activated nuclear fac-tor 120581B (NFKB) in rat pineal glandrdquoChronobiology Internationalvol 27 no 1 pp 52ndash67 2010

[37] Z S Ferreira P A C M Fernandes D Duma J Assreuy MC W Avellar and R P Markus ldquoCorticosterone modulatesnoradrenaline-induced melatonin synthesis through inhibitionof nuclear factor 120581Brdquo Journal of Pineal Research vol 38 no 3pp 182ndash188 2005

[38] S C Afeche R Barbosa J H Scialfa I M Terra A CCassola and J Cipolla-Neto ldquoEffects of the blockade of highvoltage-activated calcium channels on in vitro pineal melatoninsynthesisrdquo Cell Biochemistry and Function vol 24 no 6 pp499ndash505 2006

14 BioMed Research International

[39] B Barlaam T G Bird C Lambert-Van Der Brempt DCampbell S J Foster and R Maciewicz ldquoNew 120572-substitutedsuccinate-based hydroxamic acids as TNF120572 convertaseinhibitorsrdquo Journal of Medicinal Chemistry vol 42 no 23 pp4890ndash4908 1999

[40] I Glezer C D Munhoz E M Kawamoto T MarcourakisM C Werneck Avellar and C Scavone ldquoMK-801 and 7-Ni attenuate the activation of brain NF-120581B induced by LPSrdquoNeuropharmacology vol 45 no 8 pp 1120ndash1129 2003

[41] Y Rong and M Baudry ldquoSeizure activity results in a rapidinduction of nuclear factor-120581B in adult but not juvenile ratlimbic structuresrdquo Journal of Neurochemistry vol 67 no 2 pp662ndash668 1996

[42] I Nicoletti G Migliorati M C Pagliacci F Grignani and CRiccardi ldquoA rapid and simple method for measuring thymocyteapoptosis by propidium iodide staining and flow cytometryrdquoJournal of Immunological Methods vol 139 no 2 pp 271ndash2791991

[43] Y F Jiang-Shieh C H Wu M L Chang J Y Shiehand C Y Wen ldquoRegional heterogeneity in immunoreactivemacrophagesmicroglia in the rat pineal glandrdquo Journal ofPineal Research vol 35 no 1 pp 45ndash53 2003

[44] K-O Soderstrom ldquoLectin binding to the human retinardquoAnatomical Record vol 220 no 2 pp 219ndash223 1988

[45] F Uehara M Sameshima T Muramatsu and N OhbaldquoLocalization of fluorescence-labeled lectin binding sites onphotoreceptor cells of the monkey retinardquo Experimental EyeResearch vol 36 no 1 pp 113ndash123 1983

[46] H-W Korf ldquoThe pineal organ as a component of the biologicalclock phylogenetic and ontogenetic considerationsrdquo Annals ofthe New York Academy of Sciences vol 719 pp 13ndash42 1994

[47] E Maronde and J H Stehle ldquoThe mammalian pineal glandknown facts unknown facetsrdquo Trends in Endocrinology andMetabolism vol 18 no 4 pp 142ndash149 2007

[48] X-M Zhang and J Zhu ldquoKainic acid-induced neurotoxicitytargeting glial responses and glia-derived cytokinesrdquo CurrentNeuropharmacology vol 9 no 2 pp 388ndash398 2011

[49] D Stellwagen and R CMalenka ldquoSynaptic scalingmediated byglial TNF-120572rdquo Nature vol 440 no 7087 pp 1054ndash1059 2006

[50] E Maronde M Pfeffer J Olcese et al ldquoTranscription factorsin neuroendocrine regulation rhythmic changes in pCREB andICER levels framemelatonin synthesisrdquo Journal of Neurosciencevol 19 no 9 pp 3326ndash3336 1999

[51] D M Bustos M J Bailey D Sugden et al ldquoGlobal dailydynamics of the pineal transcriptomerdquoCell and Tissue Researchvol 344 no 1 pp 1ndash11 2011

[52] A K Moorthy O V Savinova J Q Ho V Y-F Wang D VuandGGhosh ldquoThe 20S proteasome processesNF-120581B1 p105 intop50 in a translation-independent mannerrdquo EMBO Journal vol25 no 9 pp 1945ndash1956 2006

[53] L Lin G N DeMartino and W C Greene ldquoCotranslationalbiogenesis of NF-120581B p50 by the 26S proteasomerdquo Cell vol 92no 6 pp 819ndash828 1998

[54] H W L Ziegler-Heitbrock T Sternsdorf J Liese et alldquoPyrrolidine dithiocarbamate inhibits NF-120581B mobilization andTNF production in humanmonocytesrdquo Journal of Immunologyvol 151 no 12 pp 6986ndash6993 1993

[55] C E Carvalho-Sousa S S Cruz-Machado E K Tamura P AC M Fernandes L Pinato et al ldquoMolecular basis for definingthe pineal gland and pinealocytes as targets for tumor necrosisfactorrdquo Frontiers in Endocrinology vol 2 pp 1ndash11 2011

[56] M PMattson ldquoNF-120581B in the survival and plasticity of neuronsrdquoNeurochemical Research vol 30 no 6-7 pp 883ndash893 2005

[57] P A CM Fernandes E Cecon R PMarkus and Z S FerreiraldquoEffect of TNF-120572 on the melatonin synthetic pathway in the ratpineal gland basis for a rsquofeedbackrsquo of the immune response oncircadian timingrdquo Journal of Pineal Research vol 41 no 4 pp344ndash350 2006

[58] S-Y Tsai T E OrsquoBrien and J A McNulty ldquoMicroglia play arole in mediating the effects of cytokines on the structure andfunction of the rat pineal glandrdquo Cell and Tissue Research vol303 no 3 pp 423ndash431 2001

[59] J S Bains and S H R Oliet ldquoGlia they make your memoriesstickrdquo Trends in Neurosciences vol 30 no 8 pp 417ndash424 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

10 BioMed Research International

ControlGlu 100120583MGlu 600120583MUnlabeled NF-120581BUnlabeled TFIID

1

(NS) 2

Free probe

+

minus

minus

minus

minus +

minus

minus

minus

minus

+

minus

minus

minus

minus +

+

minus

minus

minus +

+

minus

minus

minus+

+

minus

minus

minus

+

+

minus

minus

minus

(a)

(NS) 2

Free probe

(p50p50) 1

(NS) 3

+

+

minus

minus

minus

minus

+

minus

minus

minus

minus

minus

+

minus

minus

minus

minus

minus

+

+

minus

minus

minus

minus

+

+

minus

minus

minus

minus

+

+

minus

minus

minus

minus

ControlGlu 100120583MAnti p50Anti p52Anti p65Anti c-Rel

(b)

Free probe

Anti c-Rel

(NS) 2

(p50p50) 1

(NS) 3

ControlGlu 600120583MAnti p50Anti p52Anti p65

+minus minus minus minus minus

+

+ +

+

+ + +

+

+minus minus

minus

minus

minus

minus minus minus minus minus

minus

minus minus minus

minus minus

minus minus minus

minus minus

(c)

Figure 6 Electrophoretic mobility shift assays for NF120581B in the pineal gland (a) Competition studies were performed using nuclear extract(5120583g) from cultured pineal glands treated with glutamate (Glu) 100 and 600 120583M in the absence or presence of unlabeled specific (NF-120581Bconsensus sequence 10-fold molar excess) or nonspecific oligonucleotide (TFIID consensus sequence 10-fold molar excess) as indicated(b c) Supershift assays were performed with the same nuclear extract (5120583g) incubated in the absence or presence of antibodies against thesubunits p50 (1 10 dilution) p65 (1 20 dilution) p52 and c-Rel (1 10 dilution) as indicated Antibodies were added 20min prior to theaddition of the radiolabeled NF-120581B consensus oligonucleotide The position of specific NF-120581BDNA binding complexes p50p50 (band 1 bc) is indicated NS represents no specific binding (bands 2 and 3 a b c) The localization of the free probe is also indicated

BioMed Research International 11

ControlGlu 600120583M

minusminus

minus +

NF-120581B

(a)

20000

15000

10000

0Control Glu 600120583M

5000

lowastlowast

(au

)

(b)

ControlGlu 600120583M

minusminus

minus +

NF-120581B

(c)

(au

)

Control Glu 600120583M

16000

12000

8000

4000

0

(d)

Figure 7 Electrophoretic mobility shift assays for NF120581B in isolated astrocytes and pinealocytes (a) Nuclear proteins (5 120583g) were extractedfrom astrocytes treated with glutamate (Glu) 600120583M (b)Densitometric analysis (arbitrary units) of theNF-120581B band is presented on panel (a)One-way ANOVA Studentrsquos test lowastlowast119875 lt 001 versus control group mean plusmn SEM (c) Nuclear proteins (5120583g) were extracted from pinealocytestreated with Glu 600 120583M (d) Densitometric analysis (arbitrary units) of the NF-120581B band is presented in (c) The position of the specificNF-120581BDNA binding complex is indicated 119899 = 5 for each group 3 experiments

its activation was not observed in pinealocytes Astrocyteand pinealocyte supershift assays were not performed due totechnical limitations related to the amount of nuclear protein

The p50 subunit is synthesized from its precursor p105The processing of p105 and the resulting generation ofmature p50 subunits seem to be a constitutive event as p50homodimers are not regulated by I120581B proteins [34] The p50subunit formation seems to be regulated by cotranslationaland posttranslational mechanisms In the posttranslationalmechanism the p50 subunit is synthesized from its precursorp105 by phosphorylation at its 927 and 932 serine residueswhich allows the ubiquitinization and proteolytic degrada-tion of p105 by the 26S proteasome [52] In the cotranslationalmechanism p50 and p105 can be generated from a singlemRNA [53] There is also evidence that the 20S protea-some constitutively processes p105 to p50 in a ubiquitin-independent manner [52] The p50p50 homodimer wasoriginally considered to be a transcriptional repressor butnow there is substantial evidence that it can also inducegene transcription after a complex formation with the BCL3nuclear protein an atypical member of the I120581B family ofproteins that is considered to be a coactivator of p50p50homodimers [34]

The link between NF-120581B activation by glutamate and thereduction of melatonin synthesis was evidenced with the useof PDTC a NF-120581B translocation inhibitor [54] PDTC pre-vented glutamatersquos inhibitory effect on melatonin synthesisTaken together the crucial presence of astrocytes for theinhibitory effect of glutamate the exclusive activation of NF-120581B by glutamate in the same cell type and the demonstrationof NF-120581Brsquos role inmelatonin synthesis indicate that astrocytesunder glutamate stimulation possibly release a factor thatpromotes a decrease in pinealocyte melatonin synthesisTNF-120572may be a good candidate Indeed TNF-R1 the TNF-120572 receptor that interacts with soluble TNF is present in thepineal gland reinforcing the importance of this signalingpathway [55] Acting as an inhibitor of TACE to prevent TNF-120572 release BB1101 was thus adequate in demonstrating the roleplayed byTNF-120572 In addition the hypothesis that the p50p50homodimer may induce TNF-120572 gene expression and releaseneeds to be clarified

In the CNS astrocytes are known to release gliotransmit-ters such as glutamate D-serine and ATP [21] Moreoverastrocytes can also synthesize and release proinflammatorycytokines in response to NF-120581B activation such as TNF-120572 and interleukin-1 and induce gene expression of iNOS

12 BioMed Research International

NE 1120583MGlu 100120583MGlu 600120583MPDTC 300120583M

Mela

toni

n (n

ggl

and)

20

15

10

5

0

lowastlowast

lowastlowast

+ +

+ + +

++

+

+

+

+

+ +

minus minus minusminus

minus minus minusminus

minus minus minus

Figure 8 Effect of PDTC an inhibitor of NF-120581B on melatoninsynthesis Pineal glands in culture were incubated with PDTC(300120583M) and stimulated by norepinephrine (NE 1120583M) in thepresence of glutamate (100 and 600120583M) andmelatonin content wasevaluated One-way ANOVA Bonferroni multiple comparison test119875 lt 001 versus NE 1 120583M lowastlowast119875 lt 001 versus NE + Glu 100 120583Mand NE + Glu 600 120583M plotted as the mean plusmn SEM 119899 = 12 for eachgroup 3 experiments

PinealocyteAstrocyte

NF-120581Bactivation

AMPANMDAmGluR15

Release of solublefactor

TNF-120572

Glu

TNF-120572 alone (recruiting AMPA)or in association with Glu

mGluR3TNF-R1 AMPA[iCa+2]

Melatoninsynthesis

Figure 9 Diagram illustrating the putative paracrine interactionbetween pinealocytes and astrocytes in the modulation of pinealmelatonin synthesis by glutamate Glutamate (Glu) induces intra-cellular Ca2+ increase in astrocytes with the subsequent activationof NF120581B and release of a soluble factor possibly TNF-120572 This factoralone or in association with Glu promotes melatonin synthesisreduction in pinealocytes

resulting in nitric oxide formation [56] In this work wesuggested that TNF-120572 is the factor that is released byastrocytes under glutamate stimulation because the TACEinhibitor antagonized glutamatersquos effects Reinforcing thisidea recent preliminary data obtained in our laboratorywith the L929 tumor cell lineage which is sensitive to TNF-120572 showed that astrocytes release TNF-120572 when stimulatedby glutamate (data not shown) The literature states that

TNF-120572 decreases the serotonin content and AANAT mRNAexpression resulting in the reduction of N-acetylserotonin[57 58] the immediate precursor of melatonin synthesis Itis not clear whether TNF-120572 acts by itself or in combinationwith glutamate because there are glutamate receptors in thepinealocyte membrane [12 15 16 18] Recent studies havereported the control of hippocampal synapses by TNF-120572 dueto the increased insertion of AMPA receptors in the surfaceof the neuronal soma [59]The same effect could take place inthe pineal gland in which TNF-120572 released by the astrocytesin response to NF-120581B activation could increase the numberof AMPA receptors in the pinealocyte membrane resultingin the inhibition of melatonin synthesis

A model of the putative paracrine interactions betweenastrocytes and pinealocytes in the modulation of pinealmelatonin synthesis by glutamate is shown in Figure 9

5 Conclusions

In summary according to our results we propose thatglutamate acts on glutamate receptors (AMPA NMDAandor mGluR15) in astrocytes increasing the intracellularcalcium concentration promoting NF-120581B activation anddriving the release of a soluble factor possibly TNF-120572 whichacts independently or with glutamate in the pinealocytesresulting in inhibition of melatonin synthesis

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

This work was supported by a grant from FAPESP (0404328-1) Darine Villela was supported by a master fellowshipfrom FAPESP (0556943-4) Anderson Augusto Moutinhoand Victoria Fairbanks Atherino were supported by CNPq(PIBIC)

References

[1] J Calvo J Boya A Borregon and J E Garcia-MaurinoldquoPresence of glial cells in the rat pineal gland a light andelectronmicroscopic immunohistochemical studyrdquoAnatomicalRecord vol 220 no 4 pp 424ndash428 1988

[2] L Vollrath The Pineal Organ Springer New York NY USA1981

[3] R Y Moore J C Speh and J P Card ldquoThe retinohypothalamictract originates from a distinct subset of retinal ganglion cellsrdquoJournal of Comparative Neurology vol 352 no 3 pp 351ndash3661995

[4] D Sugden ldquoMelatonin biosynthesis in the mammalian pinealglandrdquo Experientia vol 45 no 10 pp 922ndash932 1989

[5] A F Afeche D CMVillelaM V Abrahao R Peres J Cipolla-Neto et al ldquoMelatonin and pineal glandrdquo in New Researchon Neurosecretory Systems E Romano Ed pp 151ndash177 NovaScience Publishers Hauppauge NY USA 2008

[6] J Arendt Melatonin and the Mammalian Pineal Gland Chap-man amp Hall London UK 1995

BioMed Research International 13

[7] M Ehret P Pevet and M Maitre ldquoTryptophan hydroxylasesynthesis is induced by 3101584051015840-cyclic adenosine monophosphateduring circadian rhythm in the rat pineal glandrdquo Journal ofNeurochemistry vol 57 no 5 pp 1516ndash1521 1991

[8] D C Klein G R Berg and J Weller ldquoMelatonin synthesisadenosine 3101584051015840-monophosphate and norepinephrine stimulateN-acetyltransferaserdquo Science vol 168 no 3934 pp 979ndash9801970

[9] D C Klein N L Schaad M A A Namboordiri L Yu and JL Weller ldquoControl of N-acetyltransferaserdquo Biochemical SocietyTransactions vol 20 no 2 pp 299ndash304 1992

[10] V Simonneaux and C Ribelayga ldquoGeneration of the melatoninendocrine message in mammals a review of the complexregulation of melatonin synthesis by norepinephrine peptidesand other pineal transmittersrdquoPharmacological Reviews vol 55no 2 pp 325ndash395 2003

[11] S Ishio H Yamada C M Craft and Y MoriyamaldquoHydroxyindole-O-methyltransferase is another target forL-glutamate-evoked inhibition of melatonin synthesis in ratpinealocytesrdquo Brain Research vol 850 no 1-2 pp 73ndash78 1999

[12] H Yamada S Yatsushiro S Ishio et al ldquoMetabotropic gluta-mate receptors negatively regulate melatonin synthesis in ratpinealocytesrdquo Journal of Neuroscience vol 18 no 6 pp 2056ndash2062 1998

[13] H Yamada A Yamamoto M Takahashi H Michibata HKumon and Y Moriyama ldquoThe L-type Ca2+ channel isinvolved in microvesicle-mediated glutamate exocytosis fromrat pinealocytesrdquo Journal of Pineal Research vol 21 no 3 pp165ndash174 1996

[14] H Yamada A Yamamoto S Yodozawa et al ldquoMicrovesicle-mediated exocytosis of glutamate is a novel paracrine-likechemical transduction mechanism and inhibits melatoninsecretion in rat pinealocytesrdquo Journal of Pineal Research vol 21no 3 pp 175ndash191 1996

[15] H Yamada A Ogura S Koizumi A Yamaguchi and YMoriyama ldquoAcetylcholine triggers L-glutamate exocytosis vianicotinic receptors and inhibits melatonin synthesis in ratpinealocytesrdquo Journal of Neuroscience vol 18 no 13 pp 4946ndash4952 1998

[16] S Yatsushiro H Yamada M Hayashi A Yamamoto andY Moriyama ldquoIonotropic glutamate receptors trigger micro-vesicle-mediated exocytosis of L-glutamate in rat pinealocytesrdquoJournal of Neurochemistry vol 75 no 1 pp 288ndash297 2000

[17] M-HKim SUehara AMuroyama BHille YMoriyama andD-S Koh ldquoGlutamate transporter-mediated glutamate secre-tion in the mammalian pineal glandrdquo Journal of Neurosciencevol 28 no 43 pp 10852ndash10863 2008

[18] S Yatsushiro H Yamada M Hayashi S Tsuboi and YMoriyama ldquoFunctional expression of metabotropic glutamatereceptor type 5 in rat pinealocytesrdquo NeuroReport vol 10 no 7pp 1599ndash1603 1999

[19] C Kaur V Sivakumar and E A Ling ldquoExpression ofN-methyl-D-aspartate (NMDA) and 120572-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) GluR23 receptors inthe developing rat pineal glandrdquo Journal of Pineal Research vol39 no 3 pp 294ndash301 2005

[20] H Pabst and P Redecker ldquoInterstitial glial cells of the gerbilpineal gland display immunoreactivity for the metabotropicglutamate receptors mGluR23 and mGluR5rdquo Brain Researchvol 838 no 1-2 pp 60ndash68 1999

[21] M M Halassa T Fellin and P G Haydon ldquoThe tripartitesynapse roles for gliotransmission in health and diseaserdquoTrends in Molecular Medicine vol 13 no 2 pp 54ndash63 2007

[22] S M Finkbeiner ldquoGlial calciumrdquo Glia vol 9 no 2 pp 83ndash1041993

[23] E A Newman ldquoNew roles for astrocytes regulation of synaptictransmissionrdquo Trends in Neurosciences vol 26 no 10 pp 536ndash542 2003

[24] J T Porter and K D McCarthy ldquoAstrocytic neurotransmitterreceptors in situ and in vivordquo Progress in Neurobiology vol 51no 4 pp 439ndash455 1997

[25] V Parpura T A Basarsky F Liu K Jeftinija S Jeftinija and PG Haydon ldquoGlutamate-mediated astrocyte-neuron signallingrdquoNature vol 369 no 6483 pp 744ndash747 1994

[26] T D Hassinger P B Atkinson G J Strecker et al ldquoEvidencefor glutamate-mediated activation of hippocampal neurons byglial calcium wavesrdquo Journal of Neurobiology vol 28 no 2 pp159ndash170 1995

[27] R H Lipsky K Xu D Zhu et al ldquoNuclear factor 120581B is a criticaldeterminant in N-methyl-D-aspartate receptor-mediated neu-roprotectionrdquo Journal of Neurochemistry vol 78 no 2 pp 254ndash264 2001

[28] M P Mattson C Culmsee Z Yu and S Camandola ldquoRoles ofnuclear factor 120581B in neuronal survival and plasticityrdquo Journal ofNeurochemistry vol 74 no 2 pp 443ndash456 2000

[29] C DMunhoz B Garcıa-Bueno J L MMadrigal L B LepschC Scavone and J C Leza ldquoStress-induced neuroinflammationmechanisms and new pharmacological targetsrdquo Brazilian Jour-nal of Medical and Biological Research vol 41 no 12 pp 1037ndash1046 2008

[30] S Camandola andM PMattson ldquoNF-ΚB as a therapeutic targetin neurodegenerative diseasesrdquo Expert Opinion on TherapeuticTargets vol 11 no 2 pp 123ndash132 2007

[31] B Kaltschmidt D Widera and C Kaltschmidt ldquoSignaling viaNF-120581B in the nervous systemrdquo Biochimica et Biophysica Actavol 1745 no 3 pp 287ndash299 2005

[32] P T Massa H Aleyasin D S Park X Mao and S W BargerldquoNF120581B in neurons the uncertainty principle in neurobiologyrdquoJournal of Neurochemistry vol 97 no 3 pp 607ndash618 2006

[33] H Hacker and M Karin ldquoRegulation and function of IKK andIKK-related kinasesrdquo Sciencersquos STKE vol 2006 no 357 p re132006

[34] S Beinke and S C Ley ldquoFunctions of NF-120581B1 and NF-120581B2 inimmune cell biologyrdquo Biochemical Journal vol 382 no 2 pp393ndash409 2004

[35] N D Perkins ldquoIntegrating cell-signalling pathways with NF-120581Band IKK functionrdquo Nature Reviews Molecular Cell Biology vol8 pp 49ndash62 2007

[36] E Cecon P A Fernandes L Pinato Z S Ferreira and R PMarkus ldquoDaily variation of constitutively activated nuclear fac-tor 120581B (NFKB) in rat pineal glandrdquoChronobiology Internationalvol 27 no 1 pp 52ndash67 2010

[37] Z S Ferreira P A C M Fernandes D Duma J Assreuy MC W Avellar and R P Markus ldquoCorticosterone modulatesnoradrenaline-induced melatonin synthesis through inhibitionof nuclear factor 120581Brdquo Journal of Pineal Research vol 38 no 3pp 182ndash188 2005

[38] S C Afeche R Barbosa J H Scialfa I M Terra A CCassola and J Cipolla-Neto ldquoEffects of the blockade of highvoltage-activated calcium channels on in vitro pineal melatoninsynthesisrdquo Cell Biochemistry and Function vol 24 no 6 pp499ndash505 2006

14 BioMed Research International

[39] B Barlaam T G Bird C Lambert-Van Der Brempt DCampbell S J Foster and R Maciewicz ldquoNew 120572-substitutedsuccinate-based hydroxamic acids as TNF120572 convertaseinhibitorsrdquo Journal of Medicinal Chemistry vol 42 no 23 pp4890ndash4908 1999

[40] I Glezer C D Munhoz E M Kawamoto T MarcourakisM C Werneck Avellar and C Scavone ldquoMK-801 and 7-Ni attenuate the activation of brain NF-120581B induced by LPSrdquoNeuropharmacology vol 45 no 8 pp 1120ndash1129 2003

[41] Y Rong and M Baudry ldquoSeizure activity results in a rapidinduction of nuclear factor-120581B in adult but not juvenile ratlimbic structuresrdquo Journal of Neurochemistry vol 67 no 2 pp662ndash668 1996

[42] I Nicoletti G Migliorati M C Pagliacci F Grignani and CRiccardi ldquoA rapid and simple method for measuring thymocyteapoptosis by propidium iodide staining and flow cytometryrdquoJournal of Immunological Methods vol 139 no 2 pp 271ndash2791991

[43] Y F Jiang-Shieh C H Wu M L Chang J Y Shiehand C Y Wen ldquoRegional heterogeneity in immunoreactivemacrophagesmicroglia in the rat pineal glandrdquo Journal ofPineal Research vol 35 no 1 pp 45ndash53 2003

[44] K-O Soderstrom ldquoLectin binding to the human retinardquoAnatomical Record vol 220 no 2 pp 219ndash223 1988

[45] F Uehara M Sameshima T Muramatsu and N OhbaldquoLocalization of fluorescence-labeled lectin binding sites onphotoreceptor cells of the monkey retinardquo Experimental EyeResearch vol 36 no 1 pp 113ndash123 1983

[46] H-W Korf ldquoThe pineal organ as a component of the biologicalclock phylogenetic and ontogenetic considerationsrdquo Annals ofthe New York Academy of Sciences vol 719 pp 13ndash42 1994

[47] E Maronde and J H Stehle ldquoThe mammalian pineal glandknown facts unknown facetsrdquo Trends in Endocrinology andMetabolism vol 18 no 4 pp 142ndash149 2007

[48] X-M Zhang and J Zhu ldquoKainic acid-induced neurotoxicitytargeting glial responses and glia-derived cytokinesrdquo CurrentNeuropharmacology vol 9 no 2 pp 388ndash398 2011

[49] D Stellwagen and R CMalenka ldquoSynaptic scalingmediated byglial TNF-120572rdquo Nature vol 440 no 7087 pp 1054ndash1059 2006

[50] E Maronde M Pfeffer J Olcese et al ldquoTranscription factorsin neuroendocrine regulation rhythmic changes in pCREB andICER levels framemelatonin synthesisrdquo Journal of Neurosciencevol 19 no 9 pp 3326ndash3336 1999

[51] D M Bustos M J Bailey D Sugden et al ldquoGlobal dailydynamics of the pineal transcriptomerdquoCell and Tissue Researchvol 344 no 1 pp 1ndash11 2011

[52] A K Moorthy O V Savinova J Q Ho V Y-F Wang D VuandGGhosh ldquoThe 20S proteasome processesNF-120581B1 p105 intop50 in a translation-independent mannerrdquo EMBO Journal vol25 no 9 pp 1945ndash1956 2006

[53] L Lin G N DeMartino and W C Greene ldquoCotranslationalbiogenesis of NF-120581B p50 by the 26S proteasomerdquo Cell vol 92no 6 pp 819ndash828 1998

[54] H W L Ziegler-Heitbrock T Sternsdorf J Liese et alldquoPyrrolidine dithiocarbamate inhibits NF-120581B mobilization andTNF production in humanmonocytesrdquo Journal of Immunologyvol 151 no 12 pp 6986ndash6993 1993

[55] C E Carvalho-Sousa S S Cruz-Machado E K Tamura P AC M Fernandes L Pinato et al ldquoMolecular basis for definingthe pineal gland and pinealocytes as targets for tumor necrosisfactorrdquo Frontiers in Endocrinology vol 2 pp 1ndash11 2011

[56] M PMattson ldquoNF-120581B in the survival and plasticity of neuronsrdquoNeurochemical Research vol 30 no 6-7 pp 883ndash893 2005

[57] P A CM Fernandes E Cecon R PMarkus and Z S FerreiraldquoEffect of TNF-120572 on the melatonin synthetic pathway in the ratpineal gland basis for a rsquofeedbackrsquo of the immune response oncircadian timingrdquo Journal of Pineal Research vol 41 no 4 pp344ndash350 2006

[58] S-Y Tsai T E OrsquoBrien and J A McNulty ldquoMicroglia play arole in mediating the effects of cytokines on the structure andfunction of the rat pineal glandrdquo Cell and Tissue Research vol303 no 3 pp 423ndash431 2001

[59] J S Bains and S H R Oliet ldquoGlia they make your memoriesstickrdquo Trends in Neurosciences vol 30 no 8 pp 417ndash424 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

BioMed Research International 11

ControlGlu 600120583M

minusminus

minus +

NF-120581B

(a)

20000

15000

10000

0Control Glu 600120583M

5000

lowastlowast

(au

)

(b)

ControlGlu 600120583M

minusminus

minus +

NF-120581B

(c)

(au

)

Control Glu 600120583M

16000

12000

8000

4000

0

(d)

Figure 7 Electrophoretic mobility shift assays for NF120581B in isolated astrocytes and pinealocytes (a) Nuclear proteins (5 120583g) were extractedfrom astrocytes treated with glutamate (Glu) 600120583M (b)Densitometric analysis (arbitrary units) of theNF-120581B band is presented on panel (a)One-way ANOVA Studentrsquos test lowastlowast119875 lt 001 versus control group mean plusmn SEM (c) Nuclear proteins (5120583g) were extracted from pinealocytestreated with Glu 600 120583M (d) Densitometric analysis (arbitrary units) of the NF-120581B band is presented in (c) The position of the specificNF-120581BDNA binding complex is indicated 119899 = 5 for each group 3 experiments

its activation was not observed in pinealocytes Astrocyteand pinealocyte supershift assays were not performed due totechnical limitations related to the amount of nuclear protein

The p50 subunit is synthesized from its precursor p105The processing of p105 and the resulting generation ofmature p50 subunits seem to be a constitutive event as p50homodimers are not regulated by I120581B proteins [34] The p50subunit formation seems to be regulated by cotranslationaland posttranslational mechanisms In the posttranslationalmechanism the p50 subunit is synthesized from its precursorp105 by phosphorylation at its 927 and 932 serine residueswhich allows the ubiquitinization and proteolytic degrada-tion of p105 by the 26S proteasome [52] In the cotranslationalmechanism p50 and p105 can be generated from a singlemRNA [53] There is also evidence that the 20S protea-some constitutively processes p105 to p50 in a ubiquitin-independent manner [52] The p50p50 homodimer wasoriginally considered to be a transcriptional repressor butnow there is substantial evidence that it can also inducegene transcription after a complex formation with the BCL3nuclear protein an atypical member of the I120581B family ofproteins that is considered to be a coactivator of p50p50homodimers [34]

The link between NF-120581B activation by glutamate and thereduction of melatonin synthesis was evidenced with the useof PDTC a NF-120581B translocation inhibitor [54] PDTC pre-vented glutamatersquos inhibitory effect on melatonin synthesisTaken together the crucial presence of astrocytes for theinhibitory effect of glutamate the exclusive activation of NF-120581B by glutamate in the same cell type and the demonstrationof NF-120581Brsquos role inmelatonin synthesis indicate that astrocytesunder glutamate stimulation possibly release a factor thatpromotes a decrease in pinealocyte melatonin synthesisTNF-120572may be a good candidate Indeed TNF-R1 the TNF-120572 receptor that interacts with soluble TNF is present in thepineal gland reinforcing the importance of this signalingpathway [55] Acting as an inhibitor of TACE to prevent TNF-120572 release BB1101 was thus adequate in demonstrating the roleplayed byTNF-120572 In addition the hypothesis that the p50p50homodimer may induce TNF-120572 gene expression and releaseneeds to be clarified

In the CNS astrocytes are known to release gliotransmit-ters such as glutamate D-serine and ATP [21] Moreoverastrocytes can also synthesize and release proinflammatorycytokines in response to NF-120581B activation such as TNF-120572 and interleukin-1 and induce gene expression of iNOS

12 BioMed Research International

NE 1120583MGlu 100120583MGlu 600120583MPDTC 300120583M

Mela

toni

n (n

ggl

and)

20

15

10

5

0

lowastlowast

lowastlowast

+ +

+ + +

++

+

+

+

+

+ +

minus minus minusminus

minus minus minusminus

minus minus minus

Figure 8 Effect of PDTC an inhibitor of NF-120581B on melatoninsynthesis Pineal glands in culture were incubated with PDTC(300120583M) and stimulated by norepinephrine (NE 1120583M) in thepresence of glutamate (100 and 600120583M) andmelatonin content wasevaluated One-way ANOVA Bonferroni multiple comparison test119875 lt 001 versus NE 1 120583M lowastlowast119875 lt 001 versus NE + Glu 100 120583Mand NE + Glu 600 120583M plotted as the mean plusmn SEM 119899 = 12 for eachgroup 3 experiments

PinealocyteAstrocyte

NF-120581Bactivation

AMPANMDAmGluR15

Release of solublefactor

TNF-120572

Glu

TNF-120572 alone (recruiting AMPA)or in association with Glu

mGluR3TNF-R1 AMPA[iCa+2]

Melatoninsynthesis

Figure 9 Diagram illustrating the putative paracrine interactionbetween pinealocytes and astrocytes in the modulation of pinealmelatonin synthesis by glutamate Glutamate (Glu) induces intra-cellular Ca2+ increase in astrocytes with the subsequent activationof NF120581B and release of a soluble factor possibly TNF-120572 This factoralone or in association with Glu promotes melatonin synthesisreduction in pinealocytes

resulting in nitric oxide formation [56] In this work wesuggested that TNF-120572 is the factor that is released byastrocytes under glutamate stimulation because the TACEinhibitor antagonized glutamatersquos effects Reinforcing thisidea recent preliminary data obtained in our laboratorywith the L929 tumor cell lineage which is sensitive to TNF-120572 showed that astrocytes release TNF-120572 when stimulatedby glutamate (data not shown) The literature states that

TNF-120572 decreases the serotonin content and AANAT mRNAexpression resulting in the reduction of N-acetylserotonin[57 58] the immediate precursor of melatonin synthesis Itis not clear whether TNF-120572 acts by itself or in combinationwith glutamate because there are glutamate receptors in thepinealocyte membrane [12 15 16 18] Recent studies havereported the control of hippocampal synapses by TNF-120572 dueto the increased insertion of AMPA receptors in the surfaceof the neuronal soma [59]The same effect could take place inthe pineal gland in which TNF-120572 released by the astrocytesin response to NF-120581B activation could increase the numberof AMPA receptors in the pinealocyte membrane resultingin the inhibition of melatonin synthesis

A model of the putative paracrine interactions betweenastrocytes and pinealocytes in the modulation of pinealmelatonin synthesis by glutamate is shown in Figure 9

5 Conclusions

In summary according to our results we propose thatglutamate acts on glutamate receptors (AMPA NMDAandor mGluR15) in astrocytes increasing the intracellularcalcium concentration promoting NF-120581B activation anddriving the release of a soluble factor possibly TNF-120572 whichacts independently or with glutamate in the pinealocytesresulting in inhibition of melatonin synthesis

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

This work was supported by a grant from FAPESP (0404328-1) Darine Villela was supported by a master fellowshipfrom FAPESP (0556943-4) Anderson Augusto Moutinhoand Victoria Fairbanks Atherino were supported by CNPq(PIBIC)

References

[1] J Calvo J Boya A Borregon and J E Garcia-MaurinoldquoPresence of glial cells in the rat pineal gland a light andelectronmicroscopic immunohistochemical studyrdquoAnatomicalRecord vol 220 no 4 pp 424ndash428 1988

[2] L Vollrath The Pineal Organ Springer New York NY USA1981

[3] R Y Moore J C Speh and J P Card ldquoThe retinohypothalamictract originates from a distinct subset of retinal ganglion cellsrdquoJournal of Comparative Neurology vol 352 no 3 pp 351ndash3661995

[4] D Sugden ldquoMelatonin biosynthesis in the mammalian pinealglandrdquo Experientia vol 45 no 10 pp 922ndash932 1989

[5] A F Afeche D CMVillelaM V Abrahao R Peres J Cipolla-Neto et al ldquoMelatonin and pineal glandrdquo in New Researchon Neurosecretory Systems E Romano Ed pp 151ndash177 NovaScience Publishers Hauppauge NY USA 2008

[6] J Arendt Melatonin and the Mammalian Pineal Gland Chap-man amp Hall London UK 1995

BioMed Research International 13

[7] M Ehret P Pevet and M Maitre ldquoTryptophan hydroxylasesynthesis is induced by 3101584051015840-cyclic adenosine monophosphateduring circadian rhythm in the rat pineal glandrdquo Journal ofNeurochemistry vol 57 no 5 pp 1516ndash1521 1991

[8] D C Klein G R Berg and J Weller ldquoMelatonin synthesisadenosine 3101584051015840-monophosphate and norepinephrine stimulateN-acetyltransferaserdquo Science vol 168 no 3934 pp 979ndash9801970

[9] D C Klein N L Schaad M A A Namboordiri L Yu and JL Weller ldquoControl of N-acetyltransferaserdquo Biochemical SocietyTransactions vol 20 no 2 pp 299ndash304 1992

[10] V Simonneaux and C Ribelayga ldquoGeneration of the melatoninendocrine message in mammals a review of the complexregulation of melatonin synthesis by norepinephrine peptidesand other pineal transmittersrdquoPharmacological Reviews vol 55no 2 pp 325ndash395 2003

[11] S Ishio H Yamada C M Craft and Y MoriyamaldquoHydroxyindole-O-methyltransferase is another target forL-glutamate-evoked inhibition of melatonin synthesis in ratpinealocytesrdquo Brain Research vol 850 no 1-2 pp 73ndash78 1999

[12] H Yamada S Yatsushiro S Ishio et al ldquoMetabotropic gluta-mate receptors negatively regulate melatonin synthesis in ratpinealocytesrdquo Journal of Neuroscience vol 18 no 6 pp 2056ndash2062 1998

[13] H Yamada A Yamamoto M Takahashi H Michibata HKumon and Y Moriyama ldquoThe L-type Ca2+ channel isinvolved in microvesicle-mediated glutamate exocytosis fromrat pinealocytesrdquo Journal of Pineal Research vol 21 no 3 pp165ndash174 1996

[14] H Yamada A Yamamoto S Yodozawa et al ldquoMicrovesicle-mediated exocytosis of glutamate is a novel paracrine-likechemical transduction mechanism and inhibits melatoninsecretion in rat pinealocytesrdquo Journal of Pineal Research vol 21no 3 pp 175ndash191 1996

[15] H Yamada A Ogura S Koizumi A Yamaguchi and YMoriyama ldquoAcetylcholine triggers L-glutamate exocytosis vianicotinic receptors and inhibits melatonin synthesis in ratpinealocytesrdquo Journal of Neuroscience vol 18 no 13 pp 4946ndash4952 1998

[16] S Yatsushiro H Yamada M Hayashi A Yamamoto andY Moriyama ldquoIonotropic glutamate receptors trigger micro-vesicle-mediated exocytosis of L-glutamate in rat pinealocytesrdquoJournal of Neurochemistry vol 75 no 1 pp 288ndash297 2000

[17] M-HKim SUehara AMuroyama BHille YMoriyama andD-S Koh ldquoGlutamate transporter-mediated glutamate secre-tion in the mammalian pineal glandrdquo Journal of Neurosciencevol 28 no 43 pp 10852ndash10863 2008

[18] S Yatsushiro H Yamada M Hayashi S Tsuboi and YMoriyama ldquoFunctional expression of metabotropic glutamatereceptor type 5 in rat pinealocytesrdquo NeuroReport vol 10 no 7pp 1599ndash1603 1999

[19] C Kaur V Sivakumar and E A Ling ldquoExpression ofN-methyl-D-aspartate (NMDA) and 120572-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) GluR23 receptors inthe developing rat pineal glandrdquo Journal of Pineal Research vol39 no 3 pp 294ndash301 2005

[20] H Pabst and P Redecker ldquoInterstitial glial cells of the gerbilpineal gland display immunoreactivity for the metabotropicglutamate receptors mGluR23 and mGluR5rdquo Brain Researchvol 838 no 1-2 pp 60ndash68 1999

[21] M M Halassa T Fellin and P G Haydon ldquoThe tripartitesynapse roles for gliotransmission in health and diseaserdquoTrends in Molecular Medicine vol 13 no 2 pp 54ndash63 2007

[22] S M Finkbeiner ldquoGlial calciumrdquo Glia vol 9 no 2 pp 83ndash1041993

[23] E A Newman ldquoNew roles for astrocytes regulation of synaptictransmissionrdquo Trends in Neurosciences vol 26 no 10 pp 536ndash542 2003

[24] J T Porter and K D McCarthy ldquoAstrocytic neurotransmitterreceptors in situ and in vivordquo Progress in Neurobiology vol 51no 4 pp 439ndash455 1997

[25] V Parpura T A Basarsky F Liu K Jeftinija S Jeftinija and PG Haydon ldquoGlutamate-mediated astrocyte-neuron signallingrdquoNature vol 369 no 6483 pp 744ndash747 1994

[26] T D Hassinger P B Atkinson G J Strecker et al ldquoEvidencefor glutamate-mediated activation of hippocampal neurons byglial calcium wavesrdquo Journal of Neurobiology vol 28 no 2 pp159ndash170 1995

[27] R H Lipsky K Xu D Zhu et al ldquoNuclear factor 120581B is a criticaldeterminant in N-methyl-D-aspartate receptor-mediated neu-roprotectionrdquo Journal of Neurochemistry vol 78 no 2 pp 254ndash264 2001

[28] M P Mattson C Culmsee Z Yu and S Camandola ldquoRoles ofnuclear factor 120581B in neuronal survival and plasticityrdquo Journal ofNeurochemistry vol 74 no 2 pp 443ndash456 2000

[29] C DMunhoz B Garcıa-Bueno J L MMadrigal L B LepschC Scavone and J C Leza ldquoStress-induced neuroinflammationmechanisms and new pharmacological targetsrdquo Brazilian Jour-nal of Medical and Biological Research vol 41 no 12 pp 1037ndash1046 2008

[30] S Camandola andM PMattson ldquoNF-ΚB as a therapeutic targetin neurodegenerative diseasesrdquo Expert Opinion on TherapeuticTargets vol 11 no 2 pp 123ndash132 2007

[31] B Kaltschmidt D Widera and C Kaltschmidt ldquoSignaling viaNF-120581B in the nervous systemrdquo Biochimica et Biophysica Actavol 1745 no 3 pp 287ndash299 2005

[32] P T Massa H Aleyasin D S Park X Mao and S W BargerldquoNF120581B in neurons the uncertainty principle in neurobiologyrdquoJournal of Neurochemistry vol 97 no 3 pp 607ndash618 2006

[33] H Hacker and M Karin ldquoRegulation and function of IKK andIKK-related kinasesrdquo Sciencersquos STKE vol 2006 no 357 p re132006

[34] S Beinke and S C Ley ldquoFunctions of NF-120581B1 and NF-120581B2 inimmune cell biologyrdquo Biochemical Journal vol 382 no 2 pp393ndash409 2004

[35] N D Perkins ldquoIntegrating cell-signalling pathways with NF-120581Band IKK functionrdquo Nature Reviews Molecular Cell Biology vol8 pp 49ndash62 2007

[36] E Cecon P A Fernandes L Pinato Z S Ferreira and R PMarkus ldquoDaily variation of constitutively activated nuclear fac-tor 120581B (NFKB) in rat pineal glandrdquoChronobiology Internationalvol 27 no 1 pp 52ndash67 2010

[37] Z S Ferreira P A C M Fernandes D Duma J Assreuy MC W Avellar and R P Markus ldquoCorticosterone modulatesnoradrenaline-induced melatonin synthesis through inhibitionof nuclear factor 120581Brdquo Journal of Pineal Research vol 38 no 3pp 182ndash188 2005

[38] S C Afeche R Barbosa J H Scialfa I M Terra A CCassola and J Cipolla-Neto ldquoEffects of the blockade of highvoltage-activated calcium channels on in vitro pineal melatoninsynthesisrdquo Cell Biochemistry and Function vol 24 no 6 pp499ndash505 2006

14 BioMed Research International

[39] B Barlaam T G Bird C Lambert-Van Der Brempt DCampbell S J Foster and R Maciewicz ldquoNew 120572-substitutedsuccinate-based hydroxamic acids as TNF120572 convertaseinhibitorsrdquo Journal of Medicinal Chemistry vol 42 no 23 pp4890ndash4908 1999

[40] I Glezer C D Munhoz E M Kawamoto T MarcourakisM C Werneck Avellar and C Scavone ldquoMK-801 and 7-Ni attenuate the activation of brain NF-120581B induced by LPSrdquoNeuropharmacology vol 45 no 8 pp 1120ndash1129 2003

[41] Y Rong and M Baudry ldquoSeizure activity results in a rapidinduction of nuclear factor-120581B in adult but not juvenile ratlimbic structuresrdquo Journal of Neurochemistry vol 67 no 2 pp662ndash668 1996

[42] I Nicoletti G Migliorati M C Pagliacci F Grignani and CRiccardi ldquoA rapid and simple method for measuring thymocyteapoptosis by propidium iodide staining and flow cytometryrdquoJournal of Immunological Methods vol 139 no 2 pp 271ndash2791991

[43] Y F Jiang-Shieh C H Wu M L Chang J Y Shiehand C Y Wen ldquoRegional heterogeneity in immunoreactivemacrophagesmicroglia in the rat pineal glandrdquo Journal ofPineal Research vol 35 no 1 pp 45ndash53 2003

[44] K-O Soderstrom ldquoLectin binding to the human retinardquoAnatomical Record vol 220 no 2 pp 219ndash223 1988

[45] F Uehara M Sameshima T Muramatsu and N OhbaldquoLocalization of fluorescence-labeled lectin binding sites onphotoreceptor cells of the monkey retinardquo Experimental EyeResearch vol 36 no 1 pp 113ndash123 1983

[46] H-W Korf ldquoThe pineal organ as a component of the biologicalclock phylogenetic and ontogenetic considerationsrdquo Annals ofthe New York Academy of Sciences vol 719 pp 13ndash42 1994

[47] E Maronde and J H Stehle ldquoThe mammalian pineal glandknown facts unknown facetsrdquo Trends in Endocrinology andMetabolism vol 18 no 4 pp 142ndash149 2007

[48] X-M Zhang and J Zhu ldquoKainic acid-induced neurotoxicitytargeting glial responses and glia-derived cytokinesrdquo CurrentNeuropharmacology vol 9 no 2 pp 388ndash398 2011

[49] D Stellwagen and R CMalenka ldquoSynaptic scalingmediated byglial TNF-120572rdquo Nature vol 440 no 7087 pp 1054ndash1059 2006

[50] E Maronde M Pfeffer J Olcese et al ldquoTranscription factorsin neuroendocrine regulation rhythmic changes in pCREB andICER levels framemelatonin synthesisrdquo Journal of Neurosciencevol 19 no 9 pp 3326ndash3336 1999

[51] D M Bustos M J Bailey D Sugden et al ldquoGlobal dailydynamics of the pineal transcriptomerdquoCell and Tissue Researchvol 344 no 1 pp 1ndash11 2011

[52] A K Moorthy O V Savinova J Q Ho V Y-F Wang D VuandGGhosh ldquoThe 20S proteasome processesNF-120581B1 p105 intop50 in a translation-independent mannerrdquo EMBO Journal vol25 no 9 pp 1945ndash1956 2006

[53] L Lin G N DeMartino and W C Greene ldquoCotranslationalbiogenesis of NF-120581B p50 by the 26S proteasomerdquo Cell vol 92no 6 pp 819ndash828 1998

[54] H W L Ziegler-Heitbrock T Sternsdorf J Liese et alldquoPyrrolidine dithiocarbamate inhibits NF-120581B mobilization andTNF production in humanmonocytesrdquo Journal of Immunologyvol 151 no 12 pp 6986ndash6993 1993

[55] C E Carvalho-Sousa S S Cruz-Machado E K Tamura P AC M Fernandes L Pinato et al ldquoMolecular basis for definingthe pineal gland and pinealocytes as targets for tumor necrosisfactorrdquo Frontiers in Endocrinology vol 2 pp 1ndash11 2011

[56] M PMattson ldquoNF-120581B in the survival and plasticity of neuronsrdquoNeurochemical Research vol 30 no 6-7 pp 883ndash893 2005

[57] P A CM Fernandes E Cecon R PMarkus and Z S FerreiraldquoEffect of TNF-120572 on the melatonin synthetic pathway in the ratpineal gland basis for a rsquofeedbackrsquo of the immune response oncircadian timingrdquo Journal of Pineal Research vol 41 no 4 pp344ndash350 2006

[58] S-Y Tsai T E OrsquoBrien and J A McNulty ldquoMicroglia play arole in mediating the effects of cytokines on the structure andfunction of the rat pineal glandrdquo Cell and Tissue Research vol303 no 3 pp 423ndash431 2001

[59] J S Bains and S H R Oliet ldquoGlia they make your memoriesstickrdquo Trends in Neurosciences vol 30 no 8 pp 417ndash424 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

12 BioMed Research International

NE 1120583MGlu 100120583MGlu 600120583MPDTC 300120583M

Mela

toni

n (n

ggl

and)

20

15

10

5

0

lowastlowast

lowastlowast

+ +

+ + +

++

+

+

+

+

+ +

minus minus minusminus

minus minus minusminus

minus minus minus

Figure 8 Effect of PDTC an inhibitor of NF-120581B on melatoninsynthesis Pineal glands in culture were incubated with PDTC(300120583M) and stimulated by norepinephrine (NE 1120583M) in thepresence of glutamate (100 and 600120583M) andmelatonin content wasevaluated One-way ANOVA Bonferroni multiple comparison test119875 lt 001 versus NE 1 120583M lowastlowast119875 lt 001 versus NE + Glu 100 120583Mand NE + Glu 600 120583M plotted as the mean plusmn SEM 119899 = 12 for eachgroup 3 experiments

PinealocyteAstrocyte

NF-120581Bactivation

AMPANMDAmGluR15

Release of solublefactor

TNF-120572

Glu

TNF-120572 alone (recruiting AMPA)or in association with Glu

mGluR3TNF-R1 AMPA[iCa+2]

Melatoninsynthesis

Figure 9 Diagram illustrating the putative paracrine interactionbetween pinealocytes and astrocytes in the modulation of pinealmelatonin synthesis by glutamate Glutamate (Glu) induces intra-cellular Ca2+ increase in astrocytes with the subsequent activationof NF120581B and release of a soluble factor possibly TNF-120572 This factoralone or in association with Glu promotes melatonin synthesisreduction in pinealocytes

resulting in nitric oxide formation [56] In this work wesuggested that TNF-120572 is the factor that is released byastrocytes under glutamate stimulation because the TACEinhibitor antagonized glutamatersquos effects Reinforcing thisidea recent preliminary data obtained in our laboratorywith the L929 tumor cell lineage which is sensitive to TNF-120572 showed that astrocytes release TNF-120572 when stimulatedby glutamate (data not shown) The literature states that

TNF-120572 decreases the serotonin content and AANAT mRNAexpression resulting in the reduction of N-acetylserotonin[57 58] the immediate precursor of melatonin synthesis Itis not clear whether TNF-120572 acts by itself or in combinationwith glutamate because there are glutamate receptors in thepinealocyte membrane [12 15 16 18] Recent studies havereported the control of hippocampal synapses by TNF-120572 dueto the increased insertion of AMPA receptors in the surfaceof the neuronal soma [59]The same effect could take place inthe pineal gland in which TNF-120572 released by the astrocytesin response to NF-120581B activation could increase the numberof AMPA receptors in the pinealocyte membrane resultingin the inhibition of melatonin synthesis

A model of the putative paracrine interactions betweenastrocytes and pinealocytes in the modulation of pinealmelatonin synthesis by glutamate is shown in Figure 9

5 Conclusions

In summary according to our results we propose thatglutamate acts on glutamate receptors (AMPA NMDAandor mGluR15) in astrocytes increasing the intracellularcalcium concentration promoting NF-120581B activation anddriving the release of a soluble factor possibly TNF-120572 whichacts independently or with glutamate in the pinealocytesresulting in inhibition of melatonin synthesis

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

This work was supported by a grant from FAPESP (0404328-1) Darine Villela was supported by a master fellowshipfrom FAPESP (0556943-4) Anderson Augusto Moutinhoand Victoria Fairbanks Atherino were supported by CNPq(PIBIC)

References

[1] J Calvo J Boya A Borregon and J E Garcia-MaurinoldquoPresence of glial cells in the rat pineal gland a light andelectronmicroscopic immunohistochemical studyrdquoAnatomicalRecord vol 220 no 4 pp 424ndash428 1988

[2] L Vollrath The Pineal Organ Springer New York NY USA1981

[3] R Y Moore J C Speh and J P Card ldquoThe retinohypothalamictract originates from a distinct subset of retinal ganglion cellsrdquoJournal of Comparative Neurology vol 352 no 3 pp 351ndash3661995

[4] D Sugden ldquoMelatonin biosynthesis in the mammalian pinealglandrdquo Experientia vol 45 no 10 pp 922ndash932 1989

[5] A F Afeche D CMVillelaM V Abrahao R Peres J Cipolla-Neto et al ldquoMelatonin and pineal glandrdquo in New Researchon Neurosecretory Systems E Romano Ed pp 151ndash177 NovaScience Publishers Hauppauge NY USA 2008

[6] J Arendt Melatonin and the Mammalian Pineal Gland Chap-man amp Hall London UK 1995

BioMed Research International 13

[7] M Ehret P Pevet and M Maitre ldquoTryptophan hydroxylasesynthesis is induced by 3101584051015840-cyclic adenosine monophosphateduring circadian rhythm in the rat pineal glandrdquo Journal ofNeurochemistry vol 57 no 5 pp 1516ndash1521 1991

[8] D C Klein G R Berg and J Weller ldquoMelatonin synthesisadenosine 3101584051015840-monophosphate and norepinephrine stimulateN-acetyltransferaserdquo Science vol 168 no 3934 pp 979ndash9801970

[9] D C Klein N L Schaad M A A Namboordiri L Yu and JL Weller ldquoControl of N-acetyltransferaserdquo Biochemical SocietyTransactions vol 20 no 2 pp 299ndash304 1992

[10] V Simonneaux and C Ribelayga ldquoGeneration of the melatoninendocrine message in mammals a review of the complexregulation of melatonin synthesis by norepinephrine peptidesand other pineal transmittersrdquoPharmacological Reviews vol 55no 2 pp 325ndash395 2003

[11] S Ishio H Yamada C M Craft and Y MoriyamaldquoHydroxyindole-O-methyltransferase is another target forL-glutamate-evoked inhibition of melatonin synthesis in ratpinealocytesrdquo Brain Research vol 850 no 1-2 pp 73ndash78 1999

[12] H Yamada S Yatsushiro S Ishio et al ldquoMetabotropic gluta-mate receptors negatively regulate melatonin synthesis in ratpinealocytesrdquo Journal of Neuroscience vol 18 no 6 pp 2056ndash2062 1998

[13] H Yamada A Yamamoto M Takahashi H Michibata HKumon and Y Moriyama ldquoThe L-type Ca2+ channel isinvolved in microvesicle-mediated glutamate exocytosis fromrat pinealocytesrdquo Journal of Pineal Research vol 21 no 3 pp165ndash174 1996

[14] H Yamada A Yamamoto S Yodozawa et al ldquoMicrovesicle-mediated exocytosis of glutamate is a novel paracrine-likechemical transduction mechanism and inhibits melatoninsecretion in rat pinealocytesrdquo Journal of Pineal Research vol 21no 3 pp 175ndash191 1996

[15] H Yamada A Ogura S Koizumi A Yamaguchi and YMoriyama ldquoAcetylcholine triggers L-glutamate exocytosis vianicotinic receptors and inhibits melatonin synthesis in ratpinealocytesrdquo Journal of Neuroscience vol 18 no 13 pp 4946ndash4952 1998

[16] S Yatsushiro H Yamada M Hayashi A Yamamoto andY Moriyama ldquoIonotropic glutamate receptors trigger micro-vesicle-mediated exocytosis of L-glutamate in rat pinealocytesrdquoJournal of Neurochemistry vol 75 no 1 pp 288ndash297 2000

[17] M-HKim SUehara AMuroyama BHille YMoriyama andD-S Koh ldquoGlutamate transporter-mediated glutamate secre-tion in the mammalian pineal glandrdquo Journal of Neurosciencevol 28 no 43 pp 10852ndash10863 2008

[18] S Yatsushiro H Yamada M Hayashi S Tsuboi and YMoriyama ldquoFunctional expression of metabotropic glutamatereceptor type 5 in rat pinealocytesrdquo NeuroReport vol 10 no 7pp 1599ndash1603 1999

[19] C Kaur V Sivakumar and E A Ling ldquoExpression ofN-methyl-D-aspartate (NMDA) and 120572-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) GluR23 receptors inthe developing rat pineal glandrdquo Journal of Pineal Research vol39 no 3 pp 294ndash301 2005

[20] H Pabst and P Redecker ldquoInterstitial glial cells of the gerbilpineal gland display immunoreactivity for the metabotropicglutamate receptors mGluR23 and mGluR5rdquo Brain Researchvol 838 no 1-2 pp 60ndash68 1999

[21] M M Halassa T Fellin and P G Haydon ldquoThe tripartitesynapse roles for gliotransmission in health and diseaserdquoTrends in Molecular Medicine vol 13 no 2 pp 54ndash63 2007

[22] S M Finkbeiner ldquoGlial calciumrdquo Glia vol 9 no 2 pp 83ndash1041993

[23] E A Newman ldquoNew roles for astrocytes regulation of synaptictransmissionrdquo Trends in Neurosciences vol 26 no 10 pp 536ndash542 2003

[24] J T Porter and K D McCarthy ldquoAstrocytic neurotransmitterreceptors in situ and in vivordquo Progress in Neurobiology vol 51no 4 pp 439ndash455 1997

[25] V Parpura T A Basarsky F Liu K Jeftinija S Jeftinija and PG Haydon ldquoGlutamate-mediated astrocyte-neuron signallingrdquoNature vol 369 no 6483 pp 744ndash747 1994

[26] T D Hassinger P B Atkinson G J Strecker et al ldquoEvidencefor glutamate-mediated activation of hippocampal neurons byglial calcium wavesrdquo Journal of Neurobiology vol 28 no 2 pp159ndash170 1995

[27] R H Lipsky K Xu D Zhu et al ldquoNuclear factor 120581B is a criticaldeterminant in N-methyl-D-aspartate receptor-mediated neu-roprotectionrdquo Journal of Neurochemistry vol 78 no 2 pp 254ndash264 2001

[28] M P Mattson C Culmsee Z Yu and S Camandola ldquoRoles ofnuclear factor 120581B in neuronal survival and plasticityrdquo Journal ofNeurochemistry vol 74 no 2 pp 443ndash456 2000

[29] C DMunhoz B Garcıa-Bueno J L MMadrigal L B LepschC Scavone and J C Leza ldquoStress-induced neuroinflammationmechanisms and new pharmacological targetsrdquo Brazilian Jour-nal of Medical and Biological Research vol 41 no 12 pp 1037ndash1046 2008

[30] S Camandola andM PMattson ldquoNF-ΚB as a therapeutic targetin neurodegenerative diseasesrdquo Expert Opinion on TherapeuticTargets vol 11 no 2 pp 123ndash132 2007

[31] B Kaltschmidt D Widera and C Kaltschmidt ldquoSignaling viaNF-120581B in the nervous systemrdquo Biochimica et Biophysica Actavol 1745 no 3 pp 287ndash299 2005

[32] P T Massa H Aleyasin D S Park X Mao and S W BargerldquoNF120581B in neurons the uncertainty principle in neurobiologyrdquoJournal of Neurochemistry vol 97 no 3 pp 607ndash618 2006

[33] H Hacker and M Karin ldquoRegulation and function of IKK andIKK-related kinasesrdquo Sciencersquos STKE vol 2006 no 357 p re132006

[34] S Beinke and S C Ley ldquoFunctions of NF-120581B1 and NF-120581B2 inimmune cell biologyrdquo Biochemical Journal vol 382 no 2 pp393ndash409 2004

[35] N D Perkins ldquoIntegrating cell-signalling pathways with NF-120581Band IKK functionrdquo Nature Reviews Molecular Cell Biology vol8 pp 49ndash62 2007

[36] E Cecon P A Fernandes L Pinato Z S Ferreira and R PMarkus ldquoDaily variation of constitutively activated nuclear fac-tor 120581B (NFKB) in rat pineal glandrdquoChronobiology Internationalvol 27 no 1 pp 52ndash67 2010

[37] Z S Ferreira P A C M Fernandes D Duma J Assreuy MC W Avellar and R P Markus ldquoCorticosterone modulatesnoradrenaline-induced melatonin synthesis through inhibitionof nuclear factor 120581Brdquo Journal of Pineal Research vol 38 no 3pp 182ndash188 2005

[38] S C Afeche R Barbosa J H Scialfa I M Terra A CCassola and J Cipolla-Neto ldquoEffects of the blockade of highvoltage-activated calcium channels on in vitro pineal melatoninsynthesisrdquo Cell Biochemistry and Function vol 24 no 6 pp499ndash505 2006

14 BioMed Research International

[39] B Barlaam T G Bird C Lambert-Van Der Brempt DCampbell S J Foster and R Maciewicz ldquoNew 120572-substitutedsuccinate-based hydroxamic acids as TNF120572 convertaseinhibitorsrdquo Journal of Medicinal Chemistry vol 42 no 23 pp4890ndash4908 1999

[40] I Glezer C D Munhoz E M Kawamoto T MarcourakisM C Werneck Avellar and C Scavone ldquoMK-801 and 7-Ni attenuate the activation of brain NF-120581B induced by LPSrdquoNeuropharmacology vol 45 no 8 pp 1120ndash1129 2003

[41] Y Rong and M Baudry ldquoSeizure activity results in a rapidinduction of nuclear factor-120581B in adult but not juvenile ratlimbic structuresrdquo Journal of Neurochemistry vol 67 no 2 pp662ndash668 1996

[42] I Nicoletti G Migliorati M C Pagliacci F Grignani and CRiccardi ldquoA rapid and simple method for measuring thymocyteapoptosis by propidium iodide staining and flow cytometryrdquoJournal of Immunological Methods vol 139 no 2 pp 271ndash2791991

[43] Y F Jiang-Shieh C H Wu M L Chang J Y Shiehand C Y Wen ldquoRegional heterogeneity in immunoreactivemacrophagesmicroglia in the rat pineal glandrdquo Journal ofPineal Research vol 35 no 1 pp 45ndash53 2003

[44] K-O Soderstrom ldquoLectin binding to the human retinardquoAnatomical Record vol 220 no 2 pp 219ndash223 1988

[45] F Uehara M Sameshima T Muramatsu and N OhbaldquoLocalization of fluorescence-labeled lectin binding sites onphotoreceptor cells of the monkey retinardquo Experimental EyeResearch vol 36 no 1 pp 113ndash123 1983

[46] H-W Korf ldquoThe pineal organ as a component of the biologicalclock phylogenetic and ontogenetic considerationsrdquo Annals ofthe New York Academy of Sciences vol 719 pp 13ndash42 1994

[47] E Maronde and J H Stehle ldquoThe mammalian pineal glandknown facts unknown facetsrdquo Trends in Endocrinology andMetabolism vol 18 no 4 pp 142ndash149 2007

[48] X-M Zhang and J Zhu ldquoKainic acid-induced neurotoxicitytargeting glial responses and glia-derived cytokinesrdquo CurrentNeuropharmacology vol 9 no 2 pp 388ndash398 2011

[49] D Stellwagen and R CMalenka ldquoSynaptic scalingmediated byglial TNF-120572rdquo Nature vol 440 no 7087 pp 1054ndash1059 2006

[50] E Maronde M Pfeffer J Olcese et al ldquoTranscription factorsin neuroendocrine regulation rhythmic changes in pCREB andICER levels framemelatonin synthesisrdquo Journal of Neurosciencevol 19 no 9 pp 3326ndash3336 1999

[51] D M Bustos M J Bailey D Sugden et al ldquoGlobal dailydynamics of the pineal transcriptomerdquoCell and Tissue Researchvol 344 no 1 pp 1ndash11 2011

[52] A K Moorthy O V Savinova J Q Ho V Y-F Wang D VuandGGhosh ldquoThe 20S proteasome processesNF-120581B1 p105 intop50 in a translation-independent mannerrdquo EMBO Journal vol25 no 9 pp 1945ndash1956 2006

[53] L Lin G N DeMartino and W C Greene ldquoCotranslationalbiogenesis of NF-120581B p50 by the 26S proteasomerdquo Cell vol 92no 6 pp 819ndash828 1998

[54] H W L Ziegler-Heitbrock T Sternsdorf J Liese et alldquoPyrrolidine dithiocarbamate inhibits NF-120581B mobilization andTNF production in humanmonocytesrdquo Journal of Immunologyvol 151 no 12 pp 6986ndash6993 1993

[55] C E Carvalho-Sousa S S Cruz-Machado E K Tamura P AC M Fernandes L Pinato et al ldquoMolecular basis for definingthe pineal gland and pinealocytes as targets for tumor necrosisfactorrdquo Frontiers in Endocrinology vol 2 pp 1ndash11 2011

[56] M PMattson ldquoNF-120581B in the survival and plasticity of neuronsrdquoNeurochemical Research vol 30 no 6-7 pp 883ndash893 2005

[57] P A CM Fernandes E Cecon R PMarkus and Z S FerreiraldquoEffect of TNF-120572 on the melatonin synthetic pathway in the ratpineal gland basis for a rsquofeedbackrsquo of the immune response oncircadian timingrdquo Journal of Pineal Research vol 41 no 4 pp344ndash350 2006

[58] S-Y Tsai T E OrsquoBrien and J A McNulty ldquoMicroglia play arole in mediating the effects of cytokines on the structure andfunction of the rat pineal glandrdquo Cell and Tissue Research vol303 no 3 pp 423ndash431 2001

[59] J S Bains and S H R Oliet ldquoGlia they make your memoriesstickrdquo Trends in Neurosciences vol 30 no 8 pp 417ndash424 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

BioMed Research International 13

[7] M Ehret P Pevet and M Maitre ldquoTryptophan hydroxylasesynthesis is induced by 3101584051015840-cyclic adenosine monophosphateduring circadian rhythm in the rat pineal glandrdquo Journal ofNeurochemistry vol 57 no 5 pp 1516ndash1521 1991

[8] D C Klein G R Berg and J Weller ldquoMelatonin synthesisadenosine 3101584051015840-monophosphate and norepinephrine stimulateN-acetyltransferaserdquo Science vol 168 no 3934 pp 979ndash9801970

[9] D C Klein N L Schaad M A A Namboordiri L Yu and JL Weller ldquoControl of N-acetyltransferaserdquo Biochemical SocietyTransactions vol 20 no 2 pp 299ndash304 1992

[10] V Simonneaux and C Ribelayga ldquoGeneration of the melatoninendocrine message in mammals a review of the complexregulation of melatonin synthesis by norepinephrine peptidesand other pineal transmittersrdquoPharmacological Reviews vol 55no 2 pp 325ndash395 2003

[11] S Ishio H Yamada C M Craft and Y MoriyamaldquoHydroxyindole-O-methyltransferase is another target forL-glutamate-evoked inhibition of melatonin synthesis in ratpinealocytesrdquo Brain Research vol 850 no 1-2 pp 73ndash78 1999

[12] H Yamada S Yatsushiro S Ishio et al ldquoMetabotropic gluta-mate receptors negatively regulate melatonin synthesis in ratpinealocytesrdquo Journal of Neuroscience vol 18 no 6 pp 2056ndash2062 1998

[13] H Yamada A Yamamoto M Takahashi H Michibata HKumon and Y Moriyama ldquoThe L-type Ca2+ channel isinvolved in microvesicle-mediated glutamate exocytosis fromrat pinealocytesrdquo Journal of Pineal Research vol 21 no 3 pp165ndash174 1996

[14] H Yamada A Yamamoto S Yodozawa et al ldquoMicrovesicle-mediated exocytosis of glutamate is a novel paracrine-likechemical transduction mechanism and inhibits melatoninsecretion in rat pinealocytesrdquo Journal of Pineal Research vol 21no 3 pp 175ndash191 1996

[15] H Yamada A Ogura S Koizumi A Yamaguchi and YMoriyama ldquoAcetylcholine triggers L-glutamate exocytosis vianicotinic receptors and inhibits melatonin synthesis in ratpinealocytesrdquo Journal of Neuroscience vol 18 no 13 pp 4946ndash4952 1998

[16] S Yatsushiro H Yamada M Hayashi A Yamamoto andY Moriyama ldquoIonotropic glutamate receptors trigger micro-vesicle-mediated exocytosis of L-glutamate in rat pinealocytesrdquoJournal of Neurochemistry vol 75 no 1 pp 288ndash297 2000

[17] M-HKim SUehara AMuroyama BHille YMoriyama andD-S Koh ldquoGlutamate transporter-mediated glutamate secre-tion in the mammalian pineal glandrdquo Journal of Neurosciencevol 28 no 43 pp 10852ndash10863 2008

[18] S Yatsushiro H Yamada M Hayashi S Tsuboi and YMoriyama ldquoFunctional expression of metabotropic glutamatereceptor type 5 in rat pinealocytesrdquo NeuroReport vol 10 no 7pp 1599ndash1603 1999

[19] C Kaur V Sivakumar and E A Ling ldquoExpression ofN-methyl-D-aspartate (NMDA) and 120572-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) GluR23 receptors inthe developing rat pineal glandrdquo Journal of Pineal Research vol39 no 3 pp 294ndash301 2005

[20] H Pabst and P Redecker ldquoInterstitial glial cells of the gerbilpineal gland display immunoreactivity for the metabotropicglutamate receptors mGluR23 and mGluR5rdquo Brain Researchvol 838 no 1-2 pp 60ndash68 1999

[21] M M Halassa T Fellin and P G Haydon ldquoThe tripartitesynapse roles for gliotransmission in health and diseaserdquoTrends in Molecular Medicine vol 13 no 2 pp 54ndash63 2007

[22] S M Finkbeiner ldquoGlial calciumrdquo Glia vol 9 no 2 pp 83ndash1041993

[23] E A Newman ldquoNew roles for astrocytes regulation of synaptictransmissionrdquo Trends in Neurosciences vol 26 no 10 pp 536ndash542 2003

[24] J T Porter and K D McCarthy ldquoAstrocytic neurotransmitterreceptors in situ and in vivordquo Progress in Neurobiology vol 51no 4 pp 439ndash455 1997

[25] V Parpura T A Basarsky F Liu K Jeftinija S Jeftinija and PG Haydon ldquoGlutamate-mediated astrocyte-neuron signallingrdquoNature vol 369 no 6483 pp 744ndash747 1994

[26] T D Hassinger P B Atkinson G J Strecker et al ldquoEvidencefor glutamate-mediated activation of hippocampal neurons byglial calcium wavesrdquo Journal of Neurobiology vol 28 no 2 pp159ndash170 1995

[27] R H Lipsky K Xu D Zhu et al ldquoNuclear factor 120581B is a criticaldeterminant in N-methyl-D-aspartate receptor-mediated neu-roprotectionrdquo Journal of Neurochemistry vol 78 no 2 pp 254ndash264 2001

[28] M P Mattson C Culmsee Z Yu and S Camandola ldquoRoles ofnuclear factor 120581B in neuronal survival and plasticityrdquo Journal ofNeurochemistry vol 74 no 2 pp 443ndash456 2000

[29] C DMunhoz B Garcıa-Bueno J L MMadrigal L B LepschC Scavone and J C Leza ldquoStress-induced neuroinflammationmechanisms and new pharmacological targetsrdquo Brazilian Jour-nal of Medical and Biological Research vol 41 no 12 pp 1037ndash1046 2008

[30] S Camandola andM PMattson ldquoNF-ΚB as a therapeutic targetin neurodegenerative diseasesrdquo Expert Opinion on TherapeuticTargets vol 11 no 2 pp 123ndash132 2007

[31] B Kaltschmidt D Widera and C Kaltschmidt ldquoSignaling viaNF-120581B in the nervous systemrdquo Biochimica et Biophysica Actavol 1745 no 3 pp 287ndash299 2005

[32] P T Massa H Aleyasin D S Park X Mao and S W BargerldquoNF120581B in neurons the uncertainty principle in neurobiologyrdquoJournal of Neurochemistry vol 97 no 3 pp 607ndash618 2006

[33] H Hacker and M Karin ldquoRegulation and function of IKK andIKK-related kinasesrdquo Sciencersquos STKE vol 2006 no 357 p re132006

[34] S Beinke and S C Ley ldquoFunctions of NF-120581B1 and NF-120581B2 inimmune cell biologyrdquo Biochemical Journal vol 382 no 2 pp393ndash409 2004

[35] N D Perkins ldquoIntegrating cell-signalling pathways with NF-120581Band IKK functionrdquo Nature Reviews Molecular Cell Biology vol8 pp 49ndash62 2007

[36] E Cecon P A Fernandes L Pinato Z S Ferreira and R PMarkus ldquoDaily variation of constitutively activated nuclear fac-tor 120581B (NFKB) in rat pineal glandrdquoChronobiology Internationalvol 27 no 1 pp 52ndash67 2010

[37] Z S Ferreira P A C M Fernandes D Duma J Assreuy MC W Avellar and R P Markus ldquoCorticosterone modulatesnoradrenaline-induced melatonin synthesis through inhibitionof nuclear factor 120581Brdquo Journal of Pineal Research vol 38 no 3pp 182ndash188 2005

[38] S C Afeche R Barbosa J H Scialfa I M Terra A CCassola and J Cipolla-Neto ldquoEffects of the blockade of highvoltage-activated calcium channels on in vitro pineal melatoninsynthesisrdquo Cell Biochemistry and Function vol 24 no 6 pp499ndash505 2006

14 BioMed Research International

[39] B Barlaam T G Bird C Lambert-Van Der Brempt DCampbell S J Foster and R Maciewicz ldquoNew 120572-substitutedsuccinate-based hydroxamic acids as TNF120572 convertaseinhibitorsrdquo Journal of Medicinal Chemistry vol 42 no 23 pp4890ndash4908 1999

[40] I Glezer C D Munhoz E M Kawamoto T MarcourakisM C Werneck Avellar and C Scavone ldquoMK-801 and 7-Ni attenuate the activation of brain NF-120581B induced by LPSrdquoNeuropharmacology vol 45 no 8 pp 1120ndash1129 2003

[41] Y Rong and M Baudry ldquoSeizure activity results in a rapidinduction of nuclear factor-120581B in adult but not juvenile ratlimbic structuresrdquo Journal of Neurochemistry vol 67 no 2 pp662ndash668 1996

[42] I Nicoletti G Migliorati M C Pagliacci F Grignani and CRiccardi ldquoA rapid and simple method for measuring thymocyteapoptosis by propidium iodide staining and flow cytometryrdquoJournal of Immunological Methods vol 139 no 2 pp 271ndash2791991

[43] Y F Jiang-Shieh C H Wu M L Chang J Y Shiehand C Y Wen ldquoRegional heterogeneity in immunoreactivemacrophagesmicroglia in the rat pineal glandrdquo Journal ofPineal Research vol 35 no 1 pp 45ndash53 2003

[44] K-O Soderstrom ldquoLectin binding to the human retinardquoAnatomical Record vol 220 no 2 pp 219ndash223 1988

[45] F Uehara M Sameshima T Muramatsu and N OhbaldquoLocalization of fluorescence-labeled lectin binding sites onphotoreceptor cells of the monkey retinardquo Experimental EyeResearch vol 36 no 1 pp 113ndash123 1983

[46] H-W Korf ldquoThe pineal organ as a component of the biologicalclock phylogenetic and ontogenetic considerationsrdquo Annals ofthe New York Academy of Sciences vol 719 pp 13ndash42 1994

[47] E Maronde and J H Stehle ldquoThe mammalian pineal glandknown facts unknown facetsrdquo Trends in Endocrinology andMetabolism vol 18 no 4 pp 142ndash149 2007

[48] X-M Zhang and J Zhu ldquoKainic acid-induced neurotoxicitytargeting glial responses and glia-derived cytokinesrdquo CurrentNeuropharmacology vol 9 no 2 pp 388ndash398 2011

[49] D Stellwagen and R CMalenka ldquoSynaptic scalingmediated byglial TNF-120572rdquo Nature vol 440 no 7087 pp 1054ndash1059 2006

[50] E Maronde M Pfeffer J Olcese et al ldquoTranscription factorsin neuroendocrine regulation rhythmic changes in pCREB andICER levels framemelatonin synthesisrdquo Journal of Neurosciencevol 19 no 9 pp 3326ndash3336 1999

[51] D M Bustos M J Bailey D Sugden et al ldquoGlobal dailydynamics of the pineal transcriptomerdquoCell and Tissue Researchvol 344 no 1 pp 1ndash11 2011

[52] A K Moorthy O V Savinova J Q Ho V Y-F Wang D VuandGGhosh ldquoThe 20S proteasome processesNF-120581B1 p105 intop50 in a translation-independent mannerrdquo EMBO Journal vol25 no 9 pp 1945ndash1956 2006

[53] L Lin G N DeMartino and W C Greene ldquoCotranslationalbiogenesis of NF-120581B p50 by the 26S proteasomerdquo Cell vol 92no 6 pp 819ndash828 1998

[54] H W L Ziegler-Heitbrock T Sternsdorf J Liese et alldquoPyrrolidine dithiocarbamate inhibits NF-120581B mobilization andTNF production in humanmonocytesrdquo Journal of Immunologyvol 151 no 12 pp 6986ndash6993 1993

[55] C E Carvalho-Sousa S S Cruz-Machado E K Tamura P AC M Fernandes L Pinato et al ldquoMolecular basis for definingthe pineal gland and pinealocytes as targets for tumor necrosisfactorrdquo Frontiers in Endocrinology vol 2 pp 1ndash11 2011

[56] M PMattson ldquoNF-120581B in the survival and plasticity of neuronsrdquoNeurochemical Research vol 30 no 6-7 pp 883ndash893 2005

[57] P A CM Fernandes E Cecon R PMarkus and Z S FerreiraldquoEffect of TNF-120572 on the melatonin synthetic pathway in the ratpineal gland basis for a rsquofeedbackrsquo of the immune response oncircadian timingrdquo Journal of Pineal Research vol 41 no 4 pp344ndash350 2006

[58] S-Y Tsai T E OrsquoBrien and J A McNulty ldquoMicroglia play arole in mediating the effects of cytokines on the structure andfunction of the rat pineal glandrdquo Cell and Tissue Research vol303 no 3 pp 423ndash431 2001

[59] J S Bains and S H R Oliet ldquoGlia they make your memoriesstickrdquo Trends in Neurosciences vol 30 no 8 pp 417ndash424 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

14 BioMed Research International

[39] B Barlaam T G Bird C Lambert-Van Der Brempt DCampbell S J Foster and R Maciewicz ldquoNew 120572-substitutedsuccinate-based hydroxamic acids as TNF120572 convertaseinhibitorsrdquo Journal of Medicinal Chemistry vol 42 no 23 pp4890ndash4908 1999

[40] I Glezer C D Munhoz E M Kawamoto T MarcourakisM C Werneck Avellar and C Scavone ldquoMK-801 and 7-Ni attenuate the activation of brain NF-120581B induced by LPSrdquoNeuropharmacology vol 45 no 8 pp 1120ndash1129 2003

[41] Y Rong and M Baudry ldquoSeizure activity results in a rapidinduction of nuclear factor-120581B in adult but not juvenile ratlimbic structuresrdquo Journal of Neurochemistry vol 67 no 2 pp662ndash668 1996

[42] I Nicoletti G Migliorati M C Pagliacci F Grignani and CRiccardi ldquoA rapid and simple method for measuring thymocyteapoptosis by propidium iodide staining and flow cytometryrdquoJournal of Immunological Methods vol 139 no 2 pp 271ndash2791991

[43] Y F Jiang-Shieh C H Wu M L Chang J Y Shiehand C Y Wen ldquoRegional heterogeneity in immunoreactivemacrophagesmicroglia in the rat pineal glandrdquo Journal ofPineal Research vol 35 no 1 pp 45ndash53 2003

[44] K-O Soderstrom ldquoLectin binding to the human retinardquoAnatomical Record vol 220 no 2 pp 219ndash223 1988

[45] F Uehara M Sameshima T Muramatsu and N OhbaldquoLocalization of fluorescence-labeled lectin binding sites onphotoreceptor cells of the monkey retinardquo Experimental EyeResearch vol 36 no 1 pp 113ndash123 1983

[46] H-W Korf ldquoThe pineal organ as a component of the biologicalclock phylogenetic and ontogenetic considerationsrdquo Annals ofthe New York Academy of Sciences vol 719 pp 13ndash42 1994

[47] E Maronde and J H Stehle ldquoThe mammalian pineal glandknown facts unknown facetsrdquo Trends in Endocrinology andMetabolism vol 18 no 4 pp 142ndash149 2007

[48] X-M Zhang and J Zhu ldquoKainic acid-induced neurotoxicitytargeting glial responses and glia-derived cytokinesrdquo CurrentNeuropharmacology vol 9 no 2 pp 388ndash398 2011

[49] D Stellwagen and R CMalenka ldquoSynaptic scalingmediated byglial TNF-120572rdquo Nature vol 440 no 7087 pp 1054ndash1059 2006

[50] E Maronde M Pfeffer J Olcese et al ldquoTranscription factorsin neuroendocrine regulation rhythmic changes in pCREB andICER levels framemelatonin synthesisrdquo Journal of Neurosciencevol 19 no 9 pp 3326ndash3336 1999

[51] D M Bustos M J Bailey D Sugden et al ldquoGlobal dailydynamics of the pineal transcriptomerdquoCell and Tissue Researchvol 344 no 1 pp 1ndash11 2011

[52] A K Moorthy O V Savinova J Q Ho V Y-F Wang D VuandGGhosh ldquoThe 20S proteasome processesNF-120581B1 p105 intop50 in a translation-independent mannerrdquo EMBO Journal vol25 no 9 pp 1945ndash1956 2006

[53] L Lin G N DeMartino and W C Greene ldquoCotranslationalbiogenesis of NF-120581B p50 by the 26S proteasomerdquo Cell vol 92no 6 pp 819ndash828 1998

[54] H W L Ziegler-Heitbrock T Sternsdorf J Liese et alldquoPyrrolidine dithiocarbamate inhibits NF-120581B mobilization andTNF production in humanmonocytesrdquo Journal of Immunologyvol 151 no 12 pp 6986ndash6993 1993

[55] C E Carvalho-Sousa S S Cruz-Machado E K Tamura P AC M Fernandes L Pinato et al ldquoMolecular basis for definingthe pineal gland and pinealocytes as targets for tumor necrosisfactorrdquo Frontiers in Endocrinology vol 2 pp 1ndash11 2011

[56] M PMattson ldquoNF-120581B in the survival and plasticity of neuronsrdquoNeurochemical Research vol 30 no 6-7 pp 883ndash893 2005

[57] P A CM Fernandes E Cecon R PMarkus and Z S FerreiraldquoEffect of TNF-120572 on the melatonin synthetic pathway in the ratpineal gland basis for a rsquofeedbackrsquo of the immune response oncircadian timingrdquo Journal of Pineal Research vol 41 no 4 pp344ndash350 2006

[58] S-Y Tsai T E OrsquoBrien and J A McNulty ldquoMicroglia play arole in mediating the effects of cytokines on the structure andfunction of the rat pineal glandrdquo Cell and Tissue Research vol303 no 3 pp 423ndash431 2001

[59] J S Bains and S H R Oliet ldquoGlia they make your memoriesstickrdquo Trends in Neurosciences vol 30 no 8 pp 417ndash424 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom