Goldfish Calmodulin Molecular Cloning, Tissue Distribution and Regulation of Gene Expression in...

8/8/2019 Goldfish Calmodulin Molecular Cloning, Tissue Distribution and Regulation of Gene Expression in Goldfish Pituitary

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Goldfish Calmodulin: Molecular Cloning, TissueDistribution, and Regulation of Transcript Expression in

Goldfish Pituitary Cells

LONGFEI HUO, ERIC K. Y. LEE, P. C. LEUNG, AND ANDERSON O. L. WONG

Department of Zoology, University of Hong Kong, Hong Kong, Peoples Republic of China

Calmodulin (CaM) is a Ca2-binding protein essential for bi-ological functions mediated through Ca2-dependent mecha-nisms. In thegoldfish, CaM is involved in the signaling eventsmediating pituitary hormone secretion induced by hypotha-lamic factors. However, the structural identity of goldfishCaM has not been established, and the neuroendocrine mech-anisms regulating CaM gene expression at the pituitary levelarestillunknown. Here we clonedthe goldfish CaMand testedthe hypothesis that pituitary expression of CaM transcriptscan be the target of modulation by hypothalamic factors.Three goldfish CaM cDNAs, namely CaM-a, CaM-bS, and CaM-bL, were isolated by library screening. These cDNAs carry a450-bp open reading frame encoding the same 149-amino acidCaM protein, the amino acid sequence of which is identicalwith that of mammals, birds, and amphibians and is highlyhomologous (>90%) to that in invertebrates. In goldfish pitu-

itary cells, activation of cAMP- or PKC-dependent pathwaysincreased CaM mRNA levels, whereas the opposite was truefor induction of Ca2 entry. Basal levels of CaM mRNA wasaccentuated by GnRH and pituitary adenylate cyclase-acti-vating polypeptide but suppressed by dopaminergic stimula-tion. Pharmacological studies using D1 and D2 analogs re-vealed that dopaminergic inhibition of CaM mRNAexpressionwas mediated through pituitary D2 receptors. At the pituitarylevel, D2 activation was also effective in blocking GnRH- andpituitary adenylate cyclase-activating polypeptide-stimu-lated CaMmRNAexpression.As a whole,the present study hasconfirmed that the molecular structure of CaM is highly con-served, and its mRNA expression at the pituitary level can beregulated by interactions among hypothalamic factors. (En-docrinology 145: 50565067, 2004)

CALMODULIN (CAM), a heat-stable acidic protein ex-pressed in eukaryotes, serves as a major intracelluarCa2 sensor in living cells. The functional roles of CaM inregulating cell division and differentiation, gene expression,

programmed cell death, DNA replication and repairing, andexocytosis of hormone/neurotransmitter are well docu-mented (13). The molecular structure of CaM is character-ized by the presence of four Ca2-binding motifs known asthe helix-loop-helix EF-hands. Three-dimensional structuralanalysis has revealed that CaM is dumbbell shaped with twosimilar domains located at either end, each with two EF-hands for Ca2-binding. In the absence of Ca2, these EF-hands are in a closed conformation. This Ca2-free form ofCaM (or ApoCaM) is not functional but can still bind to asubset of target proteins, e.g. Nuf1p and Spc110p (4, 5). UponCa2 binding, CaM can shift to an open conformation. As aresult, two hydrophobic surfaces are exposed and allowed

for CaM interactions with Ca2

-sensitive target proteins (6,7). CaM is known to be encoded by members of a multigenefamily. At present, three CaM genes (CaM I, II, and III) inmammals (813) and two CaM genes (CaM I and II) in birds

have been reported (1416). Although these CaM genes havevariations in their nucleotide sequences, all of them encodethe same CaM protein with identical a.a. sequence. Whencompared with CaM molecules reported in lower verte-

brates, like the fish [e.g. electric eel (17)], only one amino acid(a.a.) substitution in the primary sequence can be noted.These findings indicate that CaM is highly conserved at theprotein level during vertebrate evolution.

In the goldfish, gonadotropin (GTH), and GH secretion areregulated by a multitude of neuroendocrine factors (18, 19).Among these regulators, most of them are hypothalamicfactors that can exert regulatory actions on GTH and GHrelease simultaneously. For examples, GnRH and pituitaryadenylate cyclase-activating polypeptide (PACAP) areknown to stimulate GTH-II (or fish LH) and GH releasedirectly from goldfish pituitary cells (2023). Dopamine, onthe contrary, can exert opposite effects on the release of these

two hormones, being stimulatory to GH secretion via pitu-itary D1 receptors (24, 25) and inhibitory to GTH-II releasethrough D2 receptors (26). Apparently, the availability ofextracellular Ca2 and its entry through voltage-sensitiveCa2 channels are essential for both basal and stimulatedGTH-II and GH release in goldfish pituitary cells (27). Usinga pharmacological approach, it has been shown that CaMantagonists and CaM kinase II inhibitors can block the reg-ulatory effects on GTH-II and GH release by GnRH, PACAP,and dopamine (28, 29), suggesting that CaM may be a keycomponent of the postreceptor signaling mechanisms forthese regulators. Although neuroendocrine regulation ofCaMexpression at the pituitary level has not been previously

Abbreviations: a.a., Amino acid; [Ca2]i, intracellular Ca2; CaM,calmodulin; DIG, digoxigenin; DMSO, dimethylsulfoxide; GTH, gonad-otropin; LSD, least significance difference; ORF, open reading frame;PACAP, pituitary adenylate cyclase-activating polypeptide; PKC, pro-tein kinase C; SDS, sodium dodecyl sulfate; SSC, saline sodium ci-trate; TPA, 12-O-tetra-decanoyl-phorbol-13-acetate; UTR, untranslatedregion.

Endocrinology is published monthly by TheEndocrine Society (http://www.endo-society.org), the foremost professional society serving theendocrine community.

0013-7227/04/$15.00/0 Endocrinology 145(11):5056 5067 Printed in U.S.A. Copyright 2004 by The Endocrine Society

doi: 10.1210/en.2004-0584

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examined, the findings in the goldfish have prompted us tospeculatethat CaMgene expression at thepituitary levelmay

be the target of modulation by hypophysiotropic factors,which may have direct consequences on pituitary hormonesynthesis and secretion.

In the present study, the structural identity of goldfishCaM was established by molecular cloning using the stan-dard techniques of cDNA library screening. The transcriptexpression of CaM in various tissues and brain areas wasexamined by Northern blot. Based on the nucleotide se-quences of CaM cDNAs obtained, a slot blot system was setup to quantify total CaM mRNA expression in primary cul-tures of goldfish pituitary cells. Using this assay system, theeffects of hypophysiotropic factors in fish models, includingGnRH, PACAP, and dopamine, on CaM gene expression ingoldfish pituitary cells were investigated using a static in-cubation approach. In parallel experiments, the effects ofactivating cAMP-, protein kinase C (PKC)-, and Ca2-depen-dent pathways on CaM mRNA expression at the pituitarylevel were also examined.

Materials and Methods

Animals

Goldfish (Carassius auratus) with body weight ranging from 35 to 50 gwere purchasedfrom local petstores andmaintained in 200-literaquariaat 20 2 C for at least 7 d before pituitary cell preparation. During theholding period, the fish were fed to satiation daily with commercial fishfeed. After the acclimation, pituitaries were collected for cell dispersionfrom goldfish anesthetized in MS222 followed by spinosectomy accord-ing to the regulations of animal use at the University of Hong Kong.

Reagents and test substances

TRIZOL, medium M199, and horse serum for cell culture were pur-chased from Gibco Life Technologies (Grand Island, NY). PCR-digoxi-genin (DIG) labeling kit, CDP-Star, and Anti-DIG-AP (Fab fragments)were obtained from Roche Molecular Biochemicals (Mannheim, Ger-many). Salmon GnRH and PACAP-38 were obtained from PeninsulaLaboratories Inc. (Belmont, CA). LY171555, () SKF38393, apomor-phine, domperidone, SCH23390, forskolin, and 12-O-tetra-decanoyl-phorbol-13-acetate (TPA) were purchased from RBI Sigma (St. Louis,MO). A23187 was obtained from Calbiochem (La Jolla, CA). GnRH andPACAP were dissolved in double-distilled deionized water to prepare1-mm stock solutions, aliquoted in small volume, and stored frozen at80 C. Stock solutions of forskolin, TPA, and A23187 were prepared ina similar manner except that they were dissolved in dimethylsulfoxide(DMSO)to give a stock concentration at 10 mm. Ly171555,() SKF38393,apomorphine, domperidone, and SCH23390 were freshly prepared onthe day of experiments. These pharmaceutical agents were first dis-solved in DMSO to give a 10-mm stock solution, which was then diluted

with culture medium to appropriated concentrations 15 min before drugtreatment. Thefinalconcentration of DMSO in themedium wasless than0.1% and had no effect on CaM mRNA expression.

Screening of goldfish pituitary cDNA library

A 32P-labeled probe prepared from bovine CaM cDNA was used forthe screening of a goldfish pituitary Zap Express cDNA library accord-ing to the instructions by the manufacturer (Stratagene, La Jolla, CA).Briefly, the 32P-labeled probe was allowed to hybridize with nylonmembranes lifted from agar plates with phage colonies of the goldfishpituitary cDNA library. The areas corresponding to positive signals onthe membranes were identified on the original plates. The plaques inthese areas were cored out, extracted, and spread on agar plates forsecondary and tertiary screening until single colonies were isolated. Asa result of library screening, three positive clones of goldfish CaM,

namely CaM-a, CaM-bS, and CaM-bL, were isolated. The cDNA insertsin these clones were sequenced using a BigDye terminator cycle se-quencing kit (Applied Biosystems, Foster City, CA) in a 310 Geneticanalyzer (PerkinElmer, Boston, MA). Based on the sequences obtained,a pair of primers (PU1, 5-CAGATATGGCTGACCAACTCAC-3 andPD1, 5-ACAGAAGAGCTTCACTTTGCCG-3) were designed to covera common sequence of CaM-a, CaM-bS, and CaM-bL. After that, aDIG-labeled cDNA probe that can recognize the mRNA transcripts

corresponding to these CaM clones was prepared for subsequent studiesusinga PCR-DIG labeling kit (Roche Diagnostics, Mannheim,Germany).

Northern blot of CaM mRNA

Total RNA was extracted with TRIzol from selected tissues and brainareas of the goldfish. After that, mRNA was purified from total RNAusing a PolyATract mRNA isolation system III (Promega, Madison, WI).These mRNA samples were denatured, size fractionated in 1% agarosegel with 0.22 m formaldehyde, and blotted onto a positively chargednylon membrane (Roche) using a VacuGene vacuum blotting system(Pharmacia Biotech, Piscataway, NJ). The membrane was UV cross-linked using a Stratalinker 2400 (Stratagene), prehybridized for 3 h in50% formamide-containing hybridization buffer, and incubated with theDIG-labeled CaM cDNA probe overnight at 42 C. On the following day,the membrane was washed two times at 68 C in 0.5 saline sodium

citrate (SSC) with 0.1% sodium dodecyl sulfate (SDS) and hybridizationsignals were visualized using a DIG luminescent detection kit (Roche)with CPD-Star as the substrate. Unless stated otherwise, Northern blotwas conducted using the DIG-labeled probe common for CaM-a, CaM-

bS, and CaM-bL. In this study, Northern blot of -actin was used as aninternal control.

Southern blot of genomic DNA

Genomic DNA was isolated from whole blood freshly collected fromthe goldfish. Briefly, blood cells were collected from 0.5 ml whole blood

by centrifugation and washed three times with ice-cold PBS. After that,blood cells was resuspended in 10 ml freshly prepared digestion buffer[100 mmNaCl, 10 mm Tris-HCl, 25 mm EDTA, 0.5% SDS, and 0.1 mg/mlproteinase K (pH8.0)] andincubated at 50 C with gentleshaking for18 h.The final solution was extracted two times with equal volume of phenol

(pH 8.0) and one time with chloroform. After that, the genomic DNA inthe aqueous phase was precipitated with equal volume of isopropanoland 1:3 volume of 3 m sodium acetate (pH 5.2). The DNA pellet washarvested, soakedin 70%ethanol for5 h, anddriedin a fume hood. Afterthat, the genomic DNA obtained was dissolved in 1 ml TE buffer anddigested with restriction enzymes including PvuII, HindIII, PstI, and

HincII, respectively. The digested products were size fractionized in a0.7% agarose gel and transferred onto a positively charged nylon mem-

brane (Roche) by vacuum blotting. After UV cross-linking, the mem-brane was incubated in 6 SSC with 1blocking reagent (Roche) for3 hfollowed by hybridization overnight with the DIG-labeled CaM cDNAprobe at 42 C. On the following day, the membrane was washed, andsignal development was carried out as described for Northern blot.

Static incubation of goldfish pituitary cells

Primary cultures of pituitary cells were prepared from the goldfishas described previously (30). Briefly, pituitaries were excised from gold-fish and washed three times in washing medium [Medium M199 withHanks salts, 25 mm HEPES, 26 mm NaHCO3, 100 U/ml penicillin, 100g/ml streptomycin, 250 g/ml Fungizone, and 0.3% BSA (pH 7.2)] toremove blood clots. After that, pituitaries were diced into 0.5-mm frag-ments using a McILwain tissue chopper (Mickle Laboratory Engineer-ing, Gomshall, UK) and exposed to trypsin (40 mg/10 ml, Sigma) for 30min at 28 C. After the incubation, trypsin digestion was terminated byadding soybean trypsin inhibitor (25 mg/10 ml, Sigma) and pituitaryfragments were rinsed with DNase II (0.1 mg/10 ml, Sigma) followed

by a two-step washing with EDTA (2 mm and 1 mm, respectively) inCa2-free medium [Medium M199 with Hanks salts without CaCl2,supplemented with 25 mm HEPES, 26 mm NaHCO3, 100 U/ml peni-cillin, 100 g/ml streptomycin, and 0.3% BSA (pH 7.2)]. Pituitary frag-ments were then dispersed in Ca2-free medium with DNase II (0.1mg/50 ml) by gentle trituration using a DPTP transfer pipette (Bio-Rad

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Laboratories, Richmond, CA).Pituitarycells wereharvestedby filtrationthrough a nylon mesh (30 m pore size) followed by centrifugation at200 g for 10 min. The cell pellet obtained was resuspended in 5 ml

Ca2-free medium, and the cell yield and percentage viability wereestimated by cell counting in the presence of trypan blue. The averagecell yield was 0.5 0.7 million cells per pituitary and cell viability wasalways 94% or greater. After cell counting, pituitary cells were pelleted

by centrifugation and resuspended in plating medium [Medium M199with Earles salts, 25 mm HEPES, 26 mm NaHCO3, 100 U/ml penicillin,and100 g/ml streptomycin (pH 7.2)] to give a concentration of ap-proximately 3 million cells/ml. The cell suspension was then dispensedinto 24-well culture plates (0.8 ml/well) precoated with poly-d-lysine

(Sigma). After 3 hr incubation at 28 C, 200l of5% horse serum inplatingmedium was added to individual wells, and pituitary cells were thenincubated overnight at 28 C under 5% CO2 and saturated humidity. On

the following day, test substances including GnRH, PACAP, and do-pamine D1/D2 analogs were prepared in testing medium [MediumM199 with 25 mm HEPES, 26 mm NaHCO

3, 100 U/ml penicillin, 100

g/ml streptomycin, and 0.1% BSA (pH 7.2)] at appropriate concen-trations and gently added onto pituitary cells after removing the platingmedium. Based on the results of our initial validation, the optimalduration of drug treatment was routinely fixed at 48 h. After drugtreatment, total RNA was extracted from individual wells using TRIzolaccording to the instructions of the manufacturer.

FIG. 2. Alignment of goldfish CaM with the corresponding a.a. sequences reported in other species. Sequence alignment was conducted usingClustal W with MacVector 6.5.3 program and the conserved a.a. residues in these protein sequences were shaded for identification. The fourEF-hands (i.e. EF-I, EF-II, EF-III, and EF-IV) were marked by arrows, and the Ca2-binding loop in the helix-loop-helix structure of individualEF-hands was boxed. The a.a. sequences of CaM in representative species were downloaded from the GenBank.

Fig. 1. Nucleotideand deduceda.a. sequencesof goldfish CaM. Thenucleotide sequencesof three goldfish CaMcDNAs,namely CaM-a,CaM-bS,and CaM-bL, were aligned using Clustal W with MacVector 6.5.3. Conserved nucleotides in these three clones were shaded for identification.The a.a. sequences of goldfish CaM deduced from the ORFs of these CaM cDNAs were found to be identical. Sequence analysis has revealedthat CaM-bS is a truncated form of CaM-bL, probably caused by differential use of polyadenylation signals in 3UTR. The nucleotidesubstitutions in the ORF of CaM-a are all silence mutations and do not alter the a.a. sequence of goldfish CaM. The putative polyadenylationsignals (AATAAA or AACAAA) are underlined and an asterisk (*) represents the stop codon at the end of the coding sequence.




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Measurement of total CaM mRNA by slot blot

Total RNA samples were extracted from goldfish pituitary cells, heatdenaturedat 70 C for15 min, and vacuum blotted onto nylon membraneusinga Bio-Dot SF microfiltration unit (Bio-Rad Laboratories).The mem-

brane was UV cross-linked, incubated for 3 h at 42 C in 50% formamide-containing 5 SSC with 1 blocking reagent (Roche), and hybridizedovernight (18 h or longer) under the same conditions with the DIG-

labeled CaM cDNA probe. After hybridization, the membrane was sub- jected to two washes in 2 SSC with 0.1% SDS at room temperaturefollowed by two washes in 0.5 SSC with 0.1% SDS at 68 C. After that,the membrane was washed two times in maleic acid buffer[0.1mmaleicacid,0.1mNaCl, and0.03%Tween20 (pH7.5)]and incubatedfor at least5 min in Tris detection buffer [100 mm Tris-HCl, 100 mmNaCl (pH 9.5)].Diluted solution (1:100) of CPD-Star was then added and hybridizationsignals werequantified usinga 440 image station (Kodak Digital Science,Rochester, NY). In these experiments, slot blots of -actin mRNA or 18SrRNA were used as the internal control.

Data transformation and statistical analysis

For quantitation of CaM gene expression in goldfish pituitary cells,CaM mRNA levels were measured in terms of arbitrary density units

and normalized against -actin mRNA or 18S rRNA data of the samesample. These ratio data were then transformed into a percentage of themean value in the control group without drug treatment for statisticalanalysis (as percent control). This data transformation was performedtoallow for pooling of data from separate experiments together withoutincreasing the overall variability of the data in individual groups. In thisstudy,data were analyzed using Students t test or ANOVA followed byFishers least significance difference (LSD) test. Differences were con-sidered significant at P 0.05.

Results and Discussion

After three rounds of library screening, three positiveclones, namely CaM-a, CaM-bS, and CaM-bL, were isolatedfrom the goldfish -phage pituitary cDNA library. The in-serts in these positive clones were sequenced and found to

be 722, 690, and 1530 bp in size for CaM-a, CaM-bS, andCaM-bL, respectively (Fig. 1). The open reading frames(ORFs) of these inserts encode a 149 a.a. CaM protein withidentical a.a. sequence. Alignment of nucleotide sequences ofthese three positive clones has revealed that CaM-bS is a

truncated form of CaM-bL, probably caused by differentialuse of polyadenylation signals in 3 untranslated region(UTR). There are 32 mismatches in the nucleotide sequenceof CaM-a, compared with that of CaM-bS andCaM-bL. Thesemismatches are not clustered together in a particular region

but randomly distributed throughout the nucleotide se-quence of CaM-a, indicating that the transcripts of CaM-aand CaM-b (S and L forms) may be originated from twoseparate genes. When compared with the coding sequence ofCaM-b, the 14-bp substitutions found in the ORF of CaM-aare all silent mutations and do not alter the primary structureof goldfish CaM.

Sequence alignment at the a.a. level (Fig. 2) has revealed

that the primary structure of goldfish CaM is identical withthat of mammals (e.g. human, bovine, mouse, and rat), birds(e.g. chicken and duck), amphibians (e.g. xenopus), and mod-ern bony fish (e.g. perch and medaka) and highly homolo-gous to that reported in early evolved bony fish (e.g. eel),cyclostome (e.g. hagfish), mollusca (e.g. aplysia), cephalo-chordate (e.g. brachiostoma), urochordate (e.g. ciona), nem-atode (e.g. Caenorhabditis elegans), porifera (e.g. sponge), andprotozoa (e.g. trypanosome). Except for a single a.a. substi-tution in the hagfish and eel CaMs, the primary structure ofCaM is conserved in vertebrates. The a.a. sequences of CaMsin invertebrates, however,are more diverse. Whencomparedwith the mammalian counterpart, multiple substitutions(315 a.a.) could be noted in the CaMs of brachiostoma,

FIG. 3. Phylogenetic analysis of CaM cDNAs from the goldfish andother animals. The cDNA sequences of representative species weredownloaded from the GenBank and unrooted analysis was conductedusing the neighbor-joining method after 100 bootstraps. The guidetree wasconstructed withPHYLIP and TreeView V.32 programs. Thestippled oval indicates the branch of CAM I gene subfamily, whereasthe scale bar represents the genetic distance for evolution.

FIG. 4. Interdomain comparison of EF-hands in goldfish CaM. A,Homology analysis based on Clustal W alignment of a.a. sequences ofEF-I, EF-II, EF-III, and EF-IV domains. Identical a.a. residues weremarked by dark gray color and conserved substitutions were shadedby lightgray. B, Comparisonof the nucleotidesequences of EF-hands.

Percentage homology was calculated using the Haggin & Sharp al-gorithm with MacDNAsis 3.1 program.




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aplysia, ciona, sponge, and trypanosoma, respectively. Theseresults, as a whole, indicate that the molecular structure ofCaM is highly conserved during the course of evolution. Thisstructural conservation may also reflect a high level of evo-lutionary constraints/selection pressure imposed by the es-sential functions of CaM in eukaryotic cells.

Although the protein structure of CaM is highly con-served, the genes coding for CaM tend to vary quite a bitamong different species or even within the same species.Multiple copies of CaM genes encoding the same CaM pro-tein have been reported in the human (8, 9, 31), rat (13, 32),chicken (15, 16), and frog (33), respectively. Based on se-quence comparison, these nonallelic CaM genes can be di-vided into CaM I, CaM II, and CaM III subfamilies (8 13),which form the basis of the multiple genes, single proteinmodel for CaM evolution (9, 34). This genetic redundancy iscommonly accepted to be the result of gene duplication oc-curred 1400 million years ago (35, 36). Using phylogeneticanalysis based on nucleotide sequence comparison (Fig. 3),the goldfish CaM-a and CaM-b cDNAs were grouped with

perchCaM andfoundto be related to thebranch of vertebrateCaM I gene but less related to CaM II and CaM III genes.Furthermore, sequence analysis of the helix-loop-helix EF-

hands in goldfish CaM has confirmed that these Ca2-bind-ings domains are highly conserved at the protein level (Fig.4A). Parallel comparison of nucleotide sequences of theseEF-hands (Fig. 4B) also revealed that the sequence homology

between EF-I and EF-III (55.17%) and EF-II and EF-IV(44.82%) are in general higher than that between other EFrepeats (31.03% to 34.48%). These findings are consistentwith the idea that CaM was evolved from an ancestral genewith a single EF-hand followed by two rounds of tandemduplication and divergence into the common ancestor ofCaM gene family with four EF hands (34, 37, 38). Because ahigh degree of sequence homology ( 95%) was noted be-tween CaM-a and CaM-b cDNAs, it raises the possibility thatthese two genes may be the products of a more recent geneduplication event, e.g. tetraploidization in the Cyprinid lin-eage (39).

A DIG-labeled probe covering the common sequence ofCaM-a and CaM-b was used to examine the tissue distribu-tion of CaM gene expression in the goldfish (Fig. 5A). Exceptfor the testes, a 1.6-kb transcript was predominantly ex-

pressed in all the tissues examined, including the pituitary,brain, liver, gill, intestine, kidney, spleen, muscle, and ovary.The highest levels of CaM mRNA expression were found in

FIG. 5. Tissue distribution of CaM transcripts inthe goldfish. A, Northern blot of CaM mRNA inselected tissues of the goldfish, including thebrain, pituitary, liver, gill, intestine, kidney,spleen, muscle, ovary, and testes. B, Northern blotof CaM mRNA in the ovary and testes of the gold-fish using a DIG-labeled probe flanking the distalregion of 3UTR in CaM-bL, which is absent inCaM-a and CaM-bS. C, Northern blot of CaMmRNA in selected brain areas of the goldfish, in-cluding the telencephalon, olfactory bulbs, hypo-thalamus, cerebellum, optic tectum, medulla ob-longata, and spinal cord. Unless stated otherwise,Northern blot was conducted using a DIG-labeled

probe covering a common region in the ORF ofCaM-a, CaM-bS, and CaM-bL. In these experi-ments, parallel blotting of-actin mRNA was usedas an internal control.




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the brain and liver; to a lesser extent in the pituitary, gills,kidney, muscle, and ovary; and to a lower level in the in-testine, spleen, and testes. An additional transcript of 0.8 kbin size was also detected in the gonads. In the case of thetestes, the dominant transcript for CaM was the one with 0.8kb but not 1.6 kb in size, which was quite the opposite as inthe case of the ovary. Although the physiological relevanceof this phenomenon is unclear, these results suggest that theexpression levels of different CaM genes (e.g. CaM-a andCaM-b) and/or posttranscriptional processing of CaM tran-scripts (e.g. by differential use of polyadenylation signals)may be tissue-specific in fish models. Using a DIG-labeledprobe covering the distal region of 3UTR of CaM-bL (whichis absent in CaM-a and CaM-bS), only the 1.6-kb transcriptcould be detected in the gonads (Fig. 5B), confirming that thistranscript was originated from CaM-bL. In parallel studies,the probe was also effective in detecting the 1.6-kb transcriptin other tissues previously examined (data not shown), in-dicating that, except in the testes, CaM-bL mRNA is pre-dominantly expressed in the goldfish.

In mammals, CaM is highly expressed in the central ner-vous system (40, 41) and accounts for up to 0.5% of brainproteins (42, 43). In the goldfish, the brain is among thetissues with the highest expression of CaM mRNA. To ex-amine the brain distribution of CaM mRNA, Northern blotwas performed in different brain areas of the goldfish, in-cluding the olfactory bulbs, optic tectum, telencephalon, hy-pothalamus, cerebellum, medulla oblongata, and spinal cord(Fig. 5C). In this case, the 1.6-kb transcript was found to beubiquitously expressed in all thebrain areas, with thehighestlevels in the hypothalamus, optic tectum, and medulla ob-longata and to a lesser extent in the telencephalon, cerebel-lum, olfactory bulbs, and spinal cord. These findings are in

agreement with the previous reports that CaM is involved inneuronal excitability (44, 45) and synthesis and/or secretion

of neurotransmitters (46 49). Besides, CaM is also known toincrease nitric oxide production in various neuronal path-ways by activating neuronal NO synthase coupled to Ca2

influx through N-methyl-d-aspartate receptors (50). Thisfunctional coupling can be modulated by CaM kinase II-

FIG. 6. Southern blot of goldfish genomic DNA for CaM genes.Genomic DNA prepared from the goldfish was digested with restric-tion enzymes including PvuII, HindIII, PstI, and HincII. After sizefractionation in 0.7% agarose gel and transblotting onto a nylon mem-brane, positive signals for goldfish CaM genes were detected by hy-bridization with a DIG-labeled CaM cDNA probe.

FIG. 7. Stimulation of CaM mRNA expression in goldfish pituitarycells by the adenylate cyclase activator forskolin and PKC activatorTPA. Pituitary cells were exposed for 48 h with increasing doses offorskolin (0.130 M, A) and TPA (10 300 nM, B). In these experi-ments, parallel blotting of 18S rRNA was used as an internal control.Data presented are expressed as mean SEM (n 9) and are thepooled results of three separate experiments. Individual groups de-noted by the same letter represent a similarmagnitude of CaM mRNAexpression (P 0.05, ANOVA followed by Fishers LSD test).




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induced phosphorylation of neuronal NO synthase associ-ated with N-methyl-D-aspartate receptors via the postsyn-aptic density protein PSD-95 (51). To shed light on the copynumber of CaM gene(s) in the goldfish, Southern blot wasconducted using genomic DNA digested with restrictionenzymes including PvuII, HindIII, PstI, and HincII, respec-tively (Fig. 6). In the samples examined, multiple bands (fiveto seven bands) were consistently detected, implying thatthere are multiple copies of CaM genes in the goldfish. Inmammals, CaMgenes, including CaM I, CaMII, and CaM III,are consisted of six exons interrupted by introns of varyingsizes (52), and multiple bands are commonly observed in theresults of Southern blot (13, 53). As mentioned in the pre-ceding section, the scattering pattern of nucleotide substitu-tions observed in the cDNAs of CaM-a and CaM-b argues forthe presence of at least two CaM genes in the goldfish. Giventhat the CaM-a and CaM-b genes have not been cloned in thisstudy, a detailed analysis of exon-intron structures related tothe interpretation of the results in Southern blot was notperformed.

In this study, using goldfish pituitary cells as a cell model,signal transduction mechanisms regulating CaM gene ex-pression at the pituitary level were examined using a phar-macological approach. In this case, pituitary cells were ex-posed to increasing doses of the adenylate cyclase activatorforskolin (0.130 m, Fig. 7A), PKC-activator TPA (10 300nm, Fig. 7B) and Ca2 ionophore A23187 (1 nm, 10 nm, and10 m, Fig. 8). Treatment with forskolin and TPA dose-dependently increased CaM mRNA levels in goldfish pitu-itary cells, suggesting that cAMP- and PKC-dependent path-ways can activate pituitary expression of CaM gene in fishspecies. In mammalian cell models, e.g. PC12 cells, cAMPanalogs can up-regulate CaM I and CaM II mRNA levels

without affecting CaM III transcripts (54). In NRK-44F cells,CaM mRNA levels can be elevated by TPA-induced PKCactivation (55). Our findings in the goldfish may indicate thatthe involvement of cAMP- and PKC-dependent mechanismsin CaM gene expression is highly conserved in vertebrates.

Unlike forskolin and TPA, A23187 was effective in reducingCaM mRNA expression in the nanomolar dose range (Fig.8A). When a high dose of A23187 (10 m) was used, basallevels of CaM mRNA were almost undetectable (Fig. 8B).These results, however, are at variance with the stimulatoryeffects of Ca2 ionophores on CaM expression reported in

mammalian cells, e.g. in NRK-44F cells (55). Although a pro-longed elevation of intracellular Ca2 ([Ca2]i) caused byCa2 ionophores is known to be toxic in cell cultures (56), thedrop in CaM mRNA levels as a result of cytotoxicity is ratherunlikely as the inhibitory effect of A23187 could be noted atnanomolar doses (i.e. the nontoxic dose range of Ca2 iono-phores). In previous studies by other research groups, mi-cromolar doses of A23187 and ionomycin have been reportedto induce GTH (57) and GH release (58) in goldfish pituitarycells without causing any noticeable levels of cytotoxic ef-fects. Because CaM is known to bind with L-type Ca2 chan-nel to inhibit its channel activity on Ca2 activation (59), wespeculate that a feedback control on CaM expression by

Ca2

-dependent mechanisms may be present in the fishmodel. This feedback mechanism may help to nullify thecytotoxic effects of high [Ca2]i because the cells will be-come less sensitive to Ca2 stimulation by reducing CaMexpression.

To examine whether CaM gene expression at the pituitarylevel could be the target of modulation by hypophysiotropicfactors, goldfish pituitary cells were exposed to increasingconcentrations of GnRH (0.00110 m, Fig. 9A) and PACAP(0.00110 m, Fig. 9B) under static incubation conditions. Inthis case, basal levels of CaM mRNA were elevated in adose-dependent manner by treatment with GnRH andPACAP. In parallel experiments, pituitary cells were also

exposed to increasing levels of the nonselective dopamineagonist apomorphine (0.00110 m, Fig. 10A). In contrast toGnRH and PACAP, apomorphine suppressed CaM mRNAlevels in a dose-related fashion. In the presence of apomor-phine (1 m), the stimulatory effects of GnRH (1 m) and

FIG. 8. Inhibition of CaM mRNA expression in goldfish

pituitary cells by the Ca

2

ionophore A23187. Pituitarycells were incubated for 48 h with nanomolar doses (110nM, A) or micromolar dose of A23187 (10 M, B). In theseexperiments, parallel blotting of 18S rRNA was used asan internal control. Data presented are expressed asmean SEM (n 9) and are the pooled results of threeseparate experiments. Individual groups denoted by thesame letter represent a similar magnitude of CaM mRNAexpression (P 0.05, ANOVA followed by Fishers LSDtest).




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PACAP (1 m) on CaM mRNA expression were totally abol-ished (Fig. 10B).

To elucidate the receptor specificity mediating apomor-phines action on CaM mRNA expression, goldfish pituitarycells were treated with increasing doses (0.0110 m) of thedopamine D1 agonist SKF38393 or D2 agonist Ly171555 (Fig.11A). The dose-dependence of apomorphine inhibition on

CaM mRNA expression was mimicked by the D2 agonistLy171555 but not by the D1 agonist SKF38393. Besides, theinhibitory action of apomorphine (1 m) on CaM mRNAexpression was blocked by the dopamine D2 antagonistdomperidone (1 m, Fig. 11B) and the D1 antagonistSCH23390 was not effective in this regard (data not shown).

FIG. 9. Stimulation of CaM mRNA expression in goldfish pituitarycells by GnRH and PACAP. Pituitary cells were exposed to increasingconcentrations of GnRH (0.00110 M, A) and PACAP (0.00110 M,B) for 48 h under static incubation. After drug treatment, total RNAsamples were extracted and assayed for CaM mRNA levels as de-scribed in Materials and Methods. In these experiments, parallelblotting of-actin mRNA was used as an internal control. Data pre-sented are expressed as mean SEM (n 9) and are the pooled resultsof three separate experiments. Individual groups denoted by thesameletter represent a similar magnitude of CaM mRNA expression (P 0.05, ANOVA followed by Fishers LSD test). Representative resultsof slot blots are also included in these figures for reference.

FIG. 10. Dopaminergic inhibition of CaM mRNA expression in gold-fish pituitary cells. A, Dopaminergic inhibition of basal CaM mRNAexpression. Pituitary cells were incubated for 48 h with increasingdoses (0.00110 M) of the nonselective dopamine agonist apomor-phine (Apo). B, Dopaminergic inhibition of GnRH- and PACAP-stim-

ulated CaM mRNA expression. Pituitary cells were incubated for 48 hwith GnRH (1 M) or PACAP (1 M) with or without simultaneoustreatment of apomorphine (1M). In these experiments, parallel blot-ting of 18S rRNA was used as an internal control. Data presented areexpressed as mean SEM (n 9) and are the pooled results of threeseparate experiments. Individual groups denoted by the same letterrepresent a similar magnitude of CaM mRNA expression (P 0.05,

ANOVA followed by Fishers LSD test).




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Similar to apomorphine, the D2 agonist Ly171555 (1m) alsoblocked the stimulatory effects of GnRH (1 m) and PACAP(1 m) on CaM mRNA expression (Fig. 12).

These results, taken together, confirm that dopaminesaction on CaM mRNA expression is mediated through pi-

tuitary D2 receptors. The present study has also demon-strated for the first time that CaM gene expression in thepituitary is under the control of hypothalamic factors. Ap-

parently, the stimulatory effects of GnRH and PACAP onCaM gene expression are negatively regulated by dopami-nergic input through activation of pituitary D2 receptors. Inthe goldfish, GnRH and PACAP are known to stimulateGTH-II and GH secretion at the pituitary level mainlythrough the PKC- (27) and cAMP-dependent mechanisms(21), respectively. In the same animal model, dopamine canexert differential effects on these two hormones, being stim-ulatory to GH release through D1 receptors coupled to theadenylyl cyclase/cAMP/protein kinase A pathway (25) andinhibitory to GTH-II release via D2 receptors coupled tosuppression of Ca2 channel activity and [Ca2]i mobiliza-tion (26, 60, 61). Because CaM and CaM kinase II are known

to be involved in GTH-II (29) and GH release in goldfishpituitary cells (28), it is tempting to speculate that modula-tion of CaM expression may represent a novel mechanism forGTH-II and GH regulation by hypothalamic factors. Thispossibility clearly warrants future investigations, especiallyto clarify whether GnRH, PACAP, and dopamine can reg-ulate CaM gene expression in goldfish gonadotrophs andsomatotrophs.

In summary, we have isolated two forms of goldfish CaMcDNA, CaM-a and CaM-b, by library screening. These twocDNAs encode the same CaM protein with identical a.a.sequence, compared with that reported in other vertebratesincluding mammals, birds, and amphibians. The nucleotidesequences of these cDNAs also reveal that goldfish CaM-a

FIG. 11. Receptor specificity of dopaminergic inhibition of CaMmRNA expression in goldfish pituitary cells. A, Effects of the dopa-mine D1 agonist SKF38393 and D2 agonist Ly171555 on CaM mRNAexpression. Pituitary cells were incubated for 48 h with increasingconcentrations of SKF38393 (0.0110 M) or Ly171555 (0.0110 M).

B, Blockade of dopaminergic inhibition of CaM mRNA expression bythe D2 antagonist domperidone (Domp). Pituitary cells were incu-bated for 48 h with the nonselective dopamine agonist apomorphine(Apo, 1M) in the presence or absence of domperidone (1M). In theseexperiments, parallel blotting of 18S rRNA was used as an internalcontrol. Data presented are expressed as mean SEM (n 9) and arethe pooled results of three separate experiments. Individual groupsdenoted by the same letter represent a similar magnitude of CaMmRNA expression (P 0.05, ANOVA followed by Fishers LSD test).

FIG. 12. Blockade of GnRH- and PACAP-stimulated CaM mRNA ex-pression in goldfish pituitary cells by the dopamine D2 agonistLy171555. Pituitary cells were incubated for 48 h with GnRH (1 M)or PACAP (1 M) in the presence or absence of Ly171555 (1 M). Inthese experiments, parallel blotting of 18S rRNA was used as aninternal control. Data presented are expressed as mean SEM (n 9) and are the pooled results of three separate experiments. Individ-ual groups denoted by the same letter represent a similar magnitudeof CaMmRNA expression (P 0.05, ANOVA followed by Fishers LSDtest).




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and CaM-b are putative members of the CaM I gene sub-family. Furthermore, multiple copies of CaM genes have

been implicated in the goldfish genome and CaM transcriptsare ubiquitously expressed in various tissues and brain areasof the goldfish. Using goldfish pituitary cells as a cell model,we have shown that CaM mRNA expression at the pituitarylevel can be induced by the cAMP- and PKC-dependentpathways but inhibited by Ca2-dependent mechanisms. Be-sides, pituitary expression of CaM mRNA is also under thecontrol of hypothalamic factors. GnRH and PACAP can up-regulate CaM mRNA expression in goldfish pituitary cells,and these stimulatory effects can be blocked by dopaminer-gic activation through D2 receptors. These results, as awhole, provide evidence that CaM gene expression at thepituitary levelcan be a target of modulation by hypothalamicfactors, which may have direct consequence on pituitaryfunctions in fish models.

Acknowledgments

Received May 10, 2004. Accepted July 28, 2004.Address all correspondence and requests for reprints to: Dr. Ander-

son O. L. Wong,Associate Professor, Departmentof Zoology,Universityof Hong Kong, Pokfulam Road, Hong Kong SAR, Peoples Republic ofChina. E-mail: [email protected].

This work was supported by Research Grant Council (Hong Kong)and Committee on Research and Conference Grants from University ofHong Kong (to A.O.L.W.). Financial support from the Department ofZoology, University of Hong Kong (to L.H.) in the form of a Ph.D.studentship is also acknowledged.

References

1. Van Eldik LJ, Roberts DM 1988 Calcium modulated proteins in pathophys-iology. In: Thompson MP, ed. Calcium-binding proteins. Vol II. Biologicalfunctions. Washington, DC: CRC Press; 401 403

2. Van Eldik LJ, Watterson, DM 1998 Calmodulin and signal transduction. In:Van Eldik LJ, Watterson DM, eds. Calmodulin and signal transduction. NewYork: Academic Press; 119

3. Bachs O,AgellN 1995 Calmodulin and calmoudlin-binding proteins in the cellnucleus. In:BachsO, Agell N, eds. Calcium andcalmodulin functionin thecellnucleus. New York: Springer-Verlag; 217240

4. Cyert MS 2001 Genetic analysis of calmodulin and its targets in Saccharomycescerevisiae. Annu Rev Genet 35:647 672

5. Jurado LA, Chockalingam PS, Jarrett HW 1999 Apocalmodulin. Physiol Rev79:661 682

6. Chin D, Means AR 2000 Calmodulin: a prototypical calcium sensor. TrendsCell Biol 10:322328

7. Nelson MR, Chazin WJ 1998 Calmodulin as a calcium sensor. In: Van EldikLJ, Watterson DM, eds. Calmodulin and signal transduction. New York: Ac-ademic Press; 17 65

8. SenGupta B, Friedberg F, Detera-Wadleigh SD 1987 Molecular analysis ofhuman and rat calmodulin complementary DNA clones. Evidence for addi-tional active genes in these species. J Biol Chem 262:1666316670

9. Fischer R, Koller M, Flura M, Mathews S, Strehler-Page MA, Krebs J,Penniston JT, Carafoli E, Strehler EE 1988 Multiple divergent mRNAs codefor a single human calmodulin. J Biol Chem 263:1705517062

10. Koller M, Schnyder B, Strehler EE 1990 Structural organization of the humanCaM III calmodulin gene. Biochim Biophys Acta 1087:180 189

11. BerchtoldMW,EgliR, RhynerJA,HameisterH, StrehlerEE 1993 Localizationof the human bona fide calmodulin genes CALM1, CALM2, and CALM3 tochromosomes 14q24 q31, 2p21.1-p21.3, and 19q13.2-q13.3. Genomics 16:461465

12. Toutenhoofd SL, Foletti D, Wicki R, Rhyner JA, Garcia F, Tolon R, StrehlerEE 1998 Characterization of the human CALM2 calmodulin gene and com-parison of the transcriptional activity of CALM1, CALM2 and CALM3. CellCalcium 23:323338

13. NojimaH 1989 Structuralorganization of multiple rat calmodulingenes. J MolBiol 208:269 282

14. Putkey JA, Tsui KF, Tanaka T, Lagace L, Stein JP, Lai EC, Means AR 1983Chicken calmodulin genes. A species comparison of cDNA sequences andisolation of a genomic clone. J Biol Chem 258:11864 11870

15. SimmenRC, TanakaT, TsuiKF, PutkeyJA, ScottMJ, LaiEC, MeansAR 1985

The structural organization of the chicken calmodulin gene. J Biol Chem260:907912

16. Ye Q, Berchtold MW 1997 Structure and expressionof the chicken calmodulinI gene. Gene 194:63 68

17. Lagace L, Chandra T, Woo SL, Means AR 1983 Identification of multiplespecies of calmodulin messenger RNA using a full length complementaryDNA. J Biol Chem 258:1684 1688

18. Trudeau VL 1997 Neuroendocrine regulation of gonadotrophin II release andgonadal growth in the goldfish, Carassius auratus. Rev Reprod 2:55 68

19. Peng C, Peter RE 1997 Neuropeptide regulation of growth hormone secretionand growth in fish. Zool Stud 36:79 89

20. Marchant TA, Chang JP, Nahorniak CS, Peter RE 1989 Evidence that gona-dotropin-releasing hormone also functions as a growth hormone-releasingfactor in the goldfish. Endocrinology 124:2509 2518

21. Wong AOL, Li WS, Lee EK, Leung MY, Tse LY, Chow BK, Lin HR, ChangJP 2000 Pituitary adenylate cyclase activating polypeptide as a novel hy-pophysiotropic factor in fish. Biochem Cell Biol 78:329 343

22. Chang JP,WirachowskyNR, Kwong P, Johnson JD 2001 PACAPstimulationof gonadotropin-II secretion in goldfish pituitary cells: mechanisms of actionand interaction with gonadotropin releasing hormone signalling. J Neuroen-docrinol 13:540 550

23. Wong AOL, Leung MY, Shea WL, Tse LY, Chang JP, Chow BK 1998 Hy-pophysiotropic action of pituitary adenylate cyclase-activating polypeptide(PACAP) in the goldfish: immunohistochemical demonstration of PACAP inthe pituitary, PACAP stimulation of growth hormone release from pituitarycells, and molecular cloning of pituitary type I PACAP receptor. Endocrinol-ogy 139:34653479

24. Wong AOL, Van Goor F, Chang JP 1994 Entry of extracellular calcium me-diates dopamine D1-stimulated growth hormone release from goldfish pitu-itary cells. Gen Comp Endocrinol 94:316 328

25. Wong AOL, van der Kraak G, Chang JP 1994 Cyclic 3,5-adenosine mono-phosphate mediates dopamine D1-stimulated growth hormone release fromgoldfish pituitary cells. Neuroendocrinology 60:410 417

26. Chang JP, Van Goor F, Lo A, Johnson JC, Jobin RM, Goldberg JI 1997 Signaltransduction in gonadotropin-II secretion in goldfish pituitary cells. In:Kikuyama Ska S, ed. Advances in comparative endocrinology. Bologna: Mon-duzzi Editore; 29 33

27. Chang JP, Johnson JD, Van Goor F, Wong CJ, Yunker WK, Uretsky AD,Taylor D, Jobin RM, Wong AOL, Goldberg JI 2000 Signal transductionmechanisms mediating secretion in goldfish gonadotropes and somatotropes.Biochem Cell Biol 78:139 153

28. Chang JP, Abele JT, Van Goor F, Wong AO, Neumann CM 1996 Role ofarachidonic acid and calmodulin in mediating dopamine D1- and GnRH-stimulated growth hormone release in goldfish pituitary cells. Gen CompEndocrinol 102:88 101

29. Jobin RM, Neumann CM, Chang JP 1996 Roles of calcium and calmodulin inthe mediation of acute and sustained GnRH-stimulated gonadotropin secre-tion from dispersed goldfish pituitary cells. Gen Comp Endocrinol 101:91106

30. Lee EK, Chan VC, Chang JP, Yunker WK, Wong AOL 2000 Norepinephrineregulation of growth hormone release from goldfish pituitary cells. I. Involve-ment of 2 adrenoreceptor and interactions with dopamine and salmon go-nadotropin-releasing hormone. J Neuroendocrinol 12:311322

31. Senterre-Lesenfants S, Alag AS, Sobel ME 1995 Multiple mRNA species aregenerated by alternate polyadenylation from the human calmodulin-I gene.

J Cell Biochem 58:445 45432. Nojima H, Sokabe H 1987 Structure of a gene for rat calmodulin. J Mol Biol

193:439 44533. Chien YH, Dawid IB 1984 Isolation and characterization of calmodulin genes

from Xenopus laevis. Mol Cell Biol 4:50751334. Nojima H 1987 Molecular evolution of the calmodulin gene. FEBS Lett 217:

18719035. Baba ML, Goodman M, Berger-Cohn J, Demaille JG, Matsuda G 1984 The

early adaptive evolution of calmodulin. Mol Biol Evol 1:442 45536. Hardy DO, Bender PK, Kretsinger RH 1988 Two calmodulin genes are ex-

pressed in Arbacia punctulata. An ancient gene duplication is indicated. J MolBiol 199:223227

37. Erickson BW, Watterson DM, Marshak DR 1980 Sequence alignment ofcalmodulin domains by metric analysis. Ann NY Acad Sci 356:378 379

38. Watterson DM, Sharief F, Vanaman TC 1980 The complete amino acid se-quence of the Ca2-dependent modulator protein (calmodulin) of bovine

brain. J Biol Chem 255:96297539. LarhammarD, Risinger C 1994 Moleculargenetic aspects of tetraploidy in the

common carp Cyprinus carpio. Mol Phylogenet Evol 3:59 6840. Brown IR, Gurd JW, Ni B 1992 Molecular cloning of neuronal calmodulin

mRNA species. Biochem Soc Trans 20:38238541. Palfi A, Kortvely E, FeketeE, KovacsB, Varszegi S, Gulya K 2002 Differential

calmodulin gene expression in the rodent brain. Life Sci 70:2829 285542. Toutenhoofd SL, Strehler EE 2000 The calmodulin multigene family as a

unique case of genetic redundancy: multiple levels of regulation to providespatial and temporal control of calmodulin pools? Cell Calcium 28:8396

43. Saimi Y, Kung C 2002 Calmodulin as an ion channel subunit. Annu RevPhysiol 64:289 311




12/12

44. Sola C,Barron S, Tusell JM, Serratosa J 1999 The Ca2/calmodulin signalingsystemin the neural response to excitability. Involvement of neuronal and glialcells. Prog Neurobiol 58:207232

45. Sola C, Barron S, Tusell JM, Serratosa J 2001 The Ca2/calmodulin systemin neuronal hyperexcitability. Int J Biochem Cell Biol 33:439 455

46. DeLorenzo RJ 1980 Role of calmodulin in neurotransmitter release and syn-aptic function. Ann NY Acad Sci 356:92109

47. DeLorenzo RJ 1982 Calmodulin in neurotransmitter release and synapticfunction. Fed Proc 41:22652272

48. Brailoiu E, Miyamoto MD, Dun NJ 2002 Calmodulin increases transmitterrelease by mobilizing quanta at the frog motor nerve terminal. Br J Pharmacol137:719 727

49. Fujisawa H, Yamauchi T, Nakata H, Okuno S 1984 Role of calmodulin inneurotransmitter synthesis. Fed Proc 43:30113014

50. Dawson VL, Dawson TM 1996 Nitric oxide neurotoxicity. J Chem Neuroanat10:179 190

51. Watanabe Y, Song T, Sugimoto K, Horii M, Araki N, Tokumitsu H, TezukaT, Yamamoto T, Tokuda M 2003 Post-synaptic density-95 promotes calcium/calmodulin-dependent protein kinase II-mediated Ser847 phosphorylation ofneuronal nitric oxide synthase. Biochem J 372:465 471

52. FriedbergF, RhoadsAR 2001 Evolutionary aspects of calmodulin. IUBMBLife51:215221

53. Rhyner JA, Ottiger M, Wicki R, Greenwood TM, Strehler EE 1994 Structure

of the human CALM1 calmodulin gene and identification of two CALM1-related pseudogenes CALM1P1 and CALM1P2. Eur J Biochem 225:71 82

54. Bai G, Nichols RA, Weiss B 1992 Cyclic AMP selectively up-regulates cal-modulin genes I and II in PC12 cells. Biochim Biophys Acta 1130:189196

55. Bosch M, Lopez-Girona A, Bachs O, Agell N 1994 Protein kinase C regulatescalmodulin expression in NRK cells activated to proliferate from quiescence.Cell Calcium 16:446 454

56. Clapham DE 1995 Calcium signaling. Cell 80:259 26857. Chang JP, de Leeuw R 1990 In vitro goldfish growth hormone responses to

gonadotropin-releasing hormone: possible roles of extracellular calcium andarachidonic acid metabolism? Gen Comp Endocrinol 80:155164

58. Chang JP, Freedman GL, de Leeuw R 1990 Use of a pituitary cell dispersionmethod and primary culture system for the studies of gonadotropin-releasinghormone action in the goldfish, Carassius auratus. II. Extracellular calciumdependence and dopaminergic inhibition of gonadotropin responses. GenComp Endocrinol 77:274 282

59. Mori MX, Erickson MG, Yue DT 2004 Functional stoichiometry and localenrichment of calmodulininteracting withCa2 channels.Science 304:432 435

60. Chang JP, Van Goor F, Jobin RM, Lo A 1996 GnRH signaling in goldfishpituitary cells. Biol Signals 5:70 80

61. Van Goor F, Goldberg JI, Chang JP 1998 Dopamine-D2 actions on voltage-dependent calcium current and gonadotropin-II secretion in cultured goldfishgonadotrophs. J Neuroendocrinol 10:175186

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