BRAIN RESEARCH

E L S E V I E R Brain Research 713 (1996) 269-277

Research report

Contribution of plasma membrane and endoplasmic reticulum Ca 2 +-ATPases to the synaptosomal [Ca2+] i increase during oxidative stress

Clfiudia Pereira, Cristina Ferreira, Caetana Carvalho, Catarina Oliveira *

Center for Neurosciences of Coimbra, Department of Zoology and Faculty of Medicine, University of Coimbra, 3000 Coimbra, Portugal

Accepted 5 December 1995

Abstract

In the present study we analyzed the effect of ascorbate (0.8 m M ) / F e 2+ (2.5 /zM)-induced membrane lipid peroxidation on the levels of intracellular free calcium, [Ca 2+ ]i, and on the possible mechanisms involved in the perturbation of intracellular calcium homeostasis during oxidative stress. For this purpose, the influence of the ascorbate/iron oxidant system on the plasma membrane and endoplasmic reticulum Ca2+-dependent ATPases of brain cortical synaptosomes was studied. In addition, the influence of the peroxidalive process on the uptake of calcium (45Ca2+) and on the N a + / C a 2+ exchange activity at the plasma membrane was evaluated. After ascorbate/Fe 2 +- induced membrane lipid peroxidation of the order of 18.05 + 4.20 nmol T B A R S / m g protein, an increase in [Ca 2+ ]i occured, under basal or depolarizing conditions (30 mM KC1), which was dependent on the extracellular calcium concentration. Thus, for 1 and 3 mM extracellular calcium concentration, an increase of the resting [Ca2+] i values of 19.8% and 33.7% was observed, while after the K+-depolarization the enhancement of the [Ca2+] i was 18.4% and 29.5%, respectively. The N a + / C a 2+ exchange activity and the time-dependent influx of 45Ca2+, observed in basal conditions and after the 30 mM K+-depolarization, were not affected under the peroxidative conditions. The Ca2+-dependent ATPase activity of the synaptosomal plasma membrane was significantly depressed following peroxidation of membrane lipids, decreasing the Vma x by 48.1%, without significant changes in the affinity of the enzyme for calcium (K m for Ca 2+ was 0.54 -t- 0.04 /xM in control conditions and 0.56 + 0.034 /xM in peroxidized conditions). The Ca2+-ATPase activity of the endoplasmic reticulum was also affected during ascorbate/iron-induced oxidative stress; thus, an inhibition of 45.2% was observed 5 min after adding ATP. These data suggest that the increase in synaptosomal [Ca 2+ ]i due to oxidative stress may result from the inhibition of the plasma membrane and the endoplasmic reticulum membrane Ca2+-ATPase activities, probably as a result of the alteration of the lipid environment required for the maximal activity of these membrane enzymes. The consequent increase in [Ca2+]i may be responsible for the injury of the nervous tissue observed during several pathological conditions in which free radical generation seems to be involved.

Keywords: Cerebrocortical synaptosome; Lipid peroxidation; Intracellular calcium; Calcium uptake; Calcium-dependent ATPase

1. Introduction

During the last decade it was demonstrated that the oxidative damage of the cell, which occurs during the aging process and in some related disorders, is associated

Abbreviations: MDA, malonyldialdehyde; TBA, thiobarbituric acid; TBARS, thiobarbituric acid reactive substance; Pi, inorganic phosphate; [ Ca2÷ ]i, intracellular calcium concentration; [Ca 2÷ ]o, extracellular cal- cium concentration; ATP, adenosine 5' triphosphate; ATPase, adenosine triphosphatase; BSA, bovine serum albumin; Ci, Curie; Hepes, N-[2-hy- droxyethyl]piperazine N'-[2-ethanosulphonic] acid; EGTA, N-etilenog- lycerol-bis-(aminoethylic)N,N'-tetracetic acid; SDS, sodium dodecylsul- phate; TCA, trichloroacetic acid; CNS, central nervous system; SPM, synaptic plasma membrane

* Corresponding author. Fax: (351) (39) 26-798.

0006-8993/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0006-8993(95)01554-X

with the perturbation of intracellular calcium homeostasis [30]. Calcium seems to play an important role in neuronal lesions that occur in hypoxia-ischemia [9], and the alter- ation of intracellular calcium homeostasis is a common step to the cellular death caused by many toxic agents [42]. This process may be a consequence of the alteration of the calcium translocation systems of the plasma membrane, stimulation of calcium channels or inhibition of calcium sequestration by endoplasmic reticulum or mitochondria, resulting in the inability of the cell to maintain the cytoso- lic free calcium concentration, [Ca 2÷ ]i, at the physiological concentration range [5]. In neurons, the mechanisms used for regulating [Ca2+]i include: calcium extrusion at the plasma membrane (Na+/Ca 2+ exchanger and Ca 2÷- ATPase) and calcium sequestration in intracellular or-

270 C. Pereira et al./ Brain Research 713 (1996) 269-277

ganelles (mitochondria and endoplasmic reticulum), all these mechanisms being present in presynaptic terminals [51.

There is evidence that one of the major physiological targets of free radicals is polyunsaturated fatty acids of cell membranes, and that their degradation results in alteration of membrane structure and function [50]. The central ner- vous system with its high rate of oxygen consumption and increased content in polyunsaturated fatty acids is a tissue specially vulnerable to oxidative stress [22]. Membrane lipid peroxidation results in changes of membrane lipids fluidity [50], permeability to ions [32] and activities of membrane-bound enzymes [44,57]. Ca2+-dependent AT- Pases activity from different types of muscles [12,26] and red blood cells [24] has been shown to be inhibited by membrane lipid peroxidation. In neuronal cells, the rela- tion between [Ca 2÷ ]i and peroxidative damage of plasma membrane is unclear, and only a few reports are concerned with the effect of oxidative stress on the Ca2÷-pump ATPases activity [37,51].

In order to gain a better insight into how membrane lipid peroxidation influences the neuronal calcium home- ostasis, we studied the effect of membrane lipid peroxida- tion induced by ascorbate/Fe 2+ on the [Ca2+] i of synaptosomes isolated from rat brain cortex. Since an increase on [Ca 2+ ]i was observed under these experimental conditions, the mechanisms involved in the alteration of intrasynaptosomal calcium levels were analyzed, namely the 45Ca2+ uptake and the Ca 2+ extrusion mediated by the Na+/Ca 2 + exchanger and by the Ca2+-dependent ATPase present at the plasma membrane. The endoplasmic reticu- lum Ca ~- +-ATPase activity, responsible for Ca 2 + sequestra- tion by this intracellular organelle was also evaluated. The results show that oxidative stress induces an increase in intrasynaptosomal free calcium concentration. An impair- ment in plasma membrane ATP-dependent Ca 2+ extrusion mechanisms and endoplasmic reticulum Ca 2+ sequestra- tion was shown to be associated with the rise in [Ca2+]~ observed in ascorbate/iron peroxidative conditions, during which the generation of reactive oxygen species is thought to occur.

2. Materials and methods

2.1. Materials

Acetoxymethyl ester of Indo-1 (Indo-1/AM) was pur- chased from Molecular Probes, Inc., USA and 4SCaC12 (11.2 Ci/mmol) was from Amersham International (UK); vanadium-free Mg-ATP and oligomycin were obtained from Sigma Chemical Co. (USA), ionomycin from Cal- biochem-Boehringer Corp. San Diego (USA) and thapsi- gargin from LC Laboratories (USA). All other reagents used were of analytical grade.

2.2. Preparation of synaptosomal fractions and synaptic plasma membrane t,esicles

Synaptosomes were prepared from rat brain cortex ho- mogenates according to the method of Haj6s [21], slightly modified [6]. The synaptosomal pellets were washed and resuspended in 0.32 M sucrose buffered with 10 mM HEPES-Tris, pH 7.4. The protein content of the synaptoso- mal suspension was estimated by the biuret method [28] using bovine serum albumin as standard.

Synaptic plasma membrane vesicles were obtained after osmotic lysis of rat brain cortex synaptosomes according to the method described by Michaelis and Michaelis [34], with some modifications [10]. The pellets used for the Ca2+-ATPase activity determination were resuspended in 0.32 M buffered sucrose, whereas the pellets to be used for 45Ca2+ uptake (Na+/Ca 2+ exchange or ATP-dependent Ca 2+ uptake) were washed and resuspended in ionic medium, usually containing 150 mM NaCI or KC1, 1 mM MgC12, 10 mM glucose and 10 mM HEPES-Tris, pH 7.4. The protein concentration was determined by the Sedmak method [54] using bovine serum albumin as standard.

2.3. Induction and quantification of lipid peroxidation

Ascorbic acid and Fe 2+ were used to induce lipid peroxidation [58], and the extent of the peroxidative pro- cess was determined by the thiobarbituric acid (TBA) test [15]. Ascorbic acid (0.8 raM) and ferrous sulphate (2.5 /xM) were added to 5 ml of the synaptosomal fraction resuspended in a Na + medium containing (in mM): 132 NaCI, 1 KC1, 1 MgC12, 10 glucose and 10 HEPES-Na, pH 7.4, or to synaptic plasma membrane vesicles ressuspended in ionic medium (1 mg/ml of protein, final concentration). Samples were then incubated at 30°C for 15 min in a water bath with continuous stirring. Controls were incubated at 30°C during the same period of time, in the absence of ascorbic acid and ferrous sulphate. The reaction was stopped by lowering the temperature to 0-4°C by placing the tubes in ice. To measure the extent of lipid peroxida- tion, 0.5 ml of cold 40% trichloroacetic acid (TCA) and 2 ml of 0.67% TBA were added to 0.5 ml of sample material, and the tubes were treated for 10 rain in a boiling water bath. The tubes were allowed to cool at room temperature, and centrifuged at 1500 × g for 10 min. The supernatant was collected and the absorbance measured at 530 nm in a Bausch and Lomb-Spectronic 70 spectro- photometer. The amount of thiobarbituric acid reactive substances (TBARS) formed was calculated using a molar extinction coefficient of 1.56 × 105 mol-1 . cm 1 and ex- pressed as nmol TBARS/mg of protein [15]. The TBARS levels presented correspond to the difference between TBARS levels quantified after incubation of synaptosomes in the presence or absence of the oxidant system.

C. Pereira et al. / Brain Research 713 (1996) 269-277 271

2.4. [Ca 2 + ]i measurements

Free intrasynaptosomal Ca 2 + concentration ([Ca 2 + ]i) was measured in non-peroxidized and peroxidized synapto- somes by using the Indo-1 fluorescent dye. Synaptosomes (1 m g / m l protein, final concentration) were incubated with 3 /zM of the precursor acetoxymethylester of INDO-1 (Indo-1/AM) in a medium containing (in mM): 132 NaC1, 1 KC1, 1 MgCI2, 0.1 CaCI2, 10 glucose, 1.2 H2PO4, 10 HEPES-Na, pH 7.4, and 1 m g / m l BSA free of fatty acids, at 25°C, for 20 min. In order to obtain complete hydrolysis of Indo-1/AM, synaptosomes were further incubated for 10 min at 30°C. Then they were pelleted (for 10 sec at 12,000 × g) and resuspended in Na + medium. The [Ca 2+ ]i was determined at 30°C by diluting samples of Indo-1- loaded synaptosomes (0.5 m g / m l protein final concentra- tion) into cuvettes containing Na + medium, and the fluo- rescence was measured in a Perkin-Elmer LS-50 lumines- cence spectrometer, with excitation at 335 nm and emis- sion at 410 nm, using 5 and 10 nm slits, respectively. The [Ca2+] i was calculated as described previously [2] in the presence of different extracellular Ca 2+ concentrations: 0 (100 /~M EGTA added previously), 0.02, 0.1, 1 and 3 raM. The Fma x was calculated upon addition of 2 /zM ionomycin, in the presence of saturating extracellular cal- cium concentrations, and the autofluorescence was mea- sured in the presence of 2 mM MnC12.

2.5. Calcium uptake assays

2.5.1. ATP-independent Ca 2 + uptake 45Ca2+ uptake studies were performed at 30°C as de-

scribed previously [11]. The uptake of 45Ca2+ was started by transferring samples (0.5 mg of protein) of peroxidized and non-peroxidized synaptosomes to Na + medium or to Na + medium containing 30 mM K +. Both media had previously been supplemented with CaCI 2 plus 45CAC12 (final concentration 1 mM, 1 /.~Ci/ml). After various periods of time the reaction was stopped by rapid filtration of samples (0.5 ml containing 0.25 mg of protein) through Whatman G F / B filters, followed by washing [11]. The radioactivity retained in the filters was measured by liquid scintillation spectrometry in a Packard 2000 scintillation counter programmed for automatic quenching correction. Results are expressed as nmol CaZ+/mg protein.

2.5.2. Na + / C a e + exchange The synaptic plasma membrane vesicles were prepared

for N a + / C a 2+ exchange assays by incubation overnight, at 0-4°C, in medium containing 150 mM NaC1, 1 mM MgCI2, 10 mM glucose and 10 mM HEPES-Tris, pH 7.4, in order to load the membrane vesicles with Na +. Na+ /Ca 2+ exchange was assayed in media containing 150 mM KCI or NaC1 and 20 ~M CaCI 2 supplemented with 45CAC12 (2.5 ~Ci/ /~mol) . The reaction was initiated by the dilution (20 fold) of the synaptic plasma membrane

vesicles (0.5 mg protein/ml) into the reaction medium at 30°C. At the desired times, the reaction was stopped by filtration as described above.

2.5.3. ATP-dependent Ca: + uptake ATP-dependent Ca 2+ uptake was assayed in a medium

containing 150 mM KC1 and 20 /xM CaC12 supplemented with 45CAC12 (2.5 /zCi//xmol). Synaptic plasma mem- brane vesicles suspended in the same medium, were di- luted (20 fold) into the reaction medium (final protein concentration, 0.5 mg/ml) , preequilibrated at 30°C. The reaction was initiated by adding 1 mM Mg-ATP and was terminated by filtration as described above.

2.6. Assay for Ca 2 +-dependent ATPases actiuity

The enzymatic activity of the Ca2+-dependent ATPases of the endoplasmic reticulum and of the plasma membrane of synaptosomes was monitored with an Ingold pH elec- trode, following the production of H + resulting from the ATP hydrolysis [31]. Frozen and thawed (3 times) synapto- somes were used in order to obtain the complete exposure of the catalytic site of the enzyme. Reactions were carried out at 30°C in 2 ml of Na + medium supplemented with 0.1 mM EGTA, 0.5 mM ouabain, 1 /xg /mg protein oligomycin and 100 /xg of control or peroxidized synaptosomes, in the presence or in the absence of 0.1 mM CaC12. Intrinsic Na+,K+-ATPase and contaminating mitochondrial Mg 2+- ATPase were eliminated by inclusion in the assay of ouabain and oligomycin, respectively, as refered. The reac- tions were started by the addition of 1 mM Mg2+-ATP and the ATP hydrolysis was followed for 5 min. At the end of each experiment, calibration was performed by addition of 100 nmol H +. The experiments were carried out in the presence or in the absence of 2 /zM thapsigargin, an inhibitor of the endoplasmic reticulum CaZ+-ATPase. The plasma membrane Ca2+-ATPase activity results from the difference between the activity determined in the presence of thapsigargin and in the presence or absence of Ca 2+. The difference between the activity determined in the presence of Ca 2+, and in the presence or absence of thapsigargin, corresponds to the endoplasmic reticulum (Ca2++ Mg2+)-ATPase activity. The difference between the activity determined in the absence of calcium and in the presence or absence of thapsigargin corresponds to the MgZ+-ATPase activity. The CaZ+-ATPase activity of the endoplasmic reticulum was obtained from the difference between these two values. Results were expressed as nmol H + / m g protein.

Determinations of the kinetic parameters (Km and Vma ×) for the plasma membrane Ca2+-ATPase were performed by Lineweaver-Burk analysis of the enzymatic activity of synaptic plasma membrane vesicles, obtained by the differ- ence between activities determined in the presence and absence of calcium, vs. increasing concentrations of free

272 C. Pereira et al. / Brain Research 713 (1996) 269-277

[Ca 2+ ] (in the 55 -340 nM range), determined as described previously [7,23].

2.7. Analysis of data and statistics

Results are presented as means _+ S.E.M. of the number of experiments indicated in the figures. Statistical signifi- cance between two groups (control vs. peroxidized sam- ples) was determined by means of an unpaired two-tailed Student's t-test. Statistical significant differences between groups were determined by means of an analysis of vari- ance (ANOVA) followed by Dunett 's test for multiple comparisons.

3. Results

3.1. Influence of lipid peroxidation on the intrasynaptoso- mal Ca 2 + concentration and synaptosoma145Ca2 + uptake

The influence of lipid peroxidation induced by ascor- ba te /Fe 2+ on intrasynaptosomal Ca 2+ levels, under basal and K+-depolarization conditions, was studied for different extracellular Ca 2+ concentrations (0 -3 mM). The extent of lipid peroxidation attained corresponded to 18.05 _+ 4.20 nmol T B A R S / m g protein and represents the difference between TBARS quantified after incubation with the oxi- dants and basal TBARS levels measured in synaptosomes incubated in the absence of ascorbate and iron Fig. 1 represents the [Ca2+] i levels under both basal (Fig. 1A) and K+-depolarization conditions (Fig. 1B) as a function of extracellular calcium concentration, for synaptosomes incubated in the presence or absence of ascorbate/Fe 2+. After lipid peroxidation, an increase in basal and K+-de - polarization-induced intrasynaptosomal free Ca 2+ levels was observed for extracellular Ca 2+ concentrations in the 0 .02-3 mM range (Fig. 1A and B), this difference being statistically significant ( P < 0.01) only for 1 and 3 mM extracellular Ca 2+ concentrations and the percentage of increase is 19.8% and 33.7%, under basal conditions, and 18.4% and 29.5%, after K+-depolarization, for 1 and 3 mM extracellular calcium concentrations, respectively. Ex- cept for a total absence of Ca 2+ in the extracellular medium, K + depolarization induces a significant increase ( P < 0.01) in [Ca2+]i (Fig. 1B), both in non-peroxidized (the percentage of rise in [Ca 2+ ]i observed in the presence of 0.02, 0.1, 1 and 3 mM Ca 2+ in the extracellular medium is 23.9%, 25.8%, 43.5% and 40.6%, respectively) and peroxidized synaptosomes (the percentage of rise in [Ca 2+ ]i observed in the presence of 0.02, 0.1, 1 and 3 mM Ca 2+ in the extracellular medium is 32.3%, 29.6%, 42.5% and 36.8%, respectively). The values of [Ca2+]i are signifi- cantly increased for extracellular [Ca 2+] of 0.1 mM or higher, either in the control or in the peroxidized synapto- somes (Fig. 1A and B), as determined by A N O V A fol-

A 1600 ]

1400 J

8004

600

400

2OO

0 0 0.02 0.1 1 3

[] CONTROL

• PEROXIDIZED

B 1600 1 1400 *

1200

E lOOO

e,++ 800

600

400

200

0 0 0.02 0.1 1 3

[Ca 2 +] o(mM)

Fig. 1. Effect of lipid peroxidation on basal (A) and K+-depolarization stimulated (B) intracellular calcium levels in non-peroxidized and peroxi- dized synaptosomes. Intrasynaptosomal Ca 2+ concentration was deter- mined with the fluorescent dye lndo-l, in control and peroxidized synaptosomes (1 mg/ml protein) resuspended in Na + medium contain- ing 150 mM NaCI, 1 mM MgC12, 10 mM glucose and 10 mM HEPES- Tris, pH 7.4, at 30°C, in the absence (A) or in the presence of 30 mM KC1 (B). Extracellular Ca 2+ concentrations varied from 0 to 3 raM, 100 ~M EGTA being added to Ca 2+ free samples. The results correspond to means+S.E.M, for 3 to 7 different experiments. * Values statistically different from control (P < 0.01) determined by means of an unpaired Student's t-test.

lowed by Dunett test ( P < 0 . 0 1 ) . However, the most proeminent effect is that related to the differences between control and peroxidized synaptosomes for [Ca 2+ ]o of 1 and 3 raM.

The results shown in Fig. 2, which represents the time course of basal and K+-depolarization-induced 45Ca2+ up- take, show that the peroxidation of synaptosomal mem- brane lipids does not significantly affects ( P > 0.05) the basal and K+-evoked influx of 45Ca2+ by the synapto- somes, although the extent of lipid peroxidation, measured by thiobarbituric acid reactive substances formation (18.05 _+ 4.20 nmol T B A R S / m g protein) was relatively high.

c. Pereira et al. /Brain Research 713 (1996) 269-277 273

3.2. Influence o f lipid peroxidation on the Na + /Ca 2+ exchange

In o rder to m i n i m i z e the c o n t r i b u t i o n of in t race l lu la r

o rgane l l e s on the r egu la t ion of [Ca2+]i , synap t i c p l a s m a

m e m b r a n e ves ic l e s we re used to ana lyze the in f luence of

l ipid pe rox ida t i on on the N a + / C a 2÷ exchange . T he inf lu-

ence o f l ip id pe rox i da t i on on N a ÷ / C a 2÷ e x c h a n g e was

d e t e r m i n e d in synap t i c p l a s m a m e m b r a n e ves ic l e s ob t a ined

af ter o s m o t i c lys is of the s y n a p t o s o m a l f rac t ion in the

p r e sence or ab sence of a Na ÷ grad ien t , the d i f f e rence

b e t w e e n these two va lues b e i n g r ep re sen t ed in Fig. 3. W e

o b s e r v e d that , in the p r e s e n c e of a Na ÷ grad ien t , Ca 2+

was t aken up in a t i m e - d e p e n d e n t m a n n e r r e a c h i n g a

m a x i m a l va lue w i t h i n 3 - 5 min , b o t h in con t ro l and peroxi -

d ized m e m b r a n e ves ic les . For a l ipid pe r ox i da t i on ex ten t

o f 18.05 ___ 4 .20 n m o l T B A R S / m g prote in , the up take of

45Ca2+, 5 m in af ter the add i t ion o f p ro te in to the r eac t ion

m e d i u m , dec rea sed f rom 5 .96 _+ 0 .352 n m o l / m g p ro te in in

con t ro l ves i c l e s to 5 .527_+ 0 .617 n m o l / m g p ro te in in

pe rox id i zed ves i c l e s a l t h o u g h these va lues are not s tat is t i -

cal ly d i f fe ren t ( P > 0.05). W e also o b s e r v e d that , in the

a b s e n c e of a Na + grad ien t , there is on ly a sma l l Ca 2+

re t en t ion o f the o rder of 1 .738 _+ 0 .158 n m o l / m g p ro te in

(no t shown) , not s ign i f i can t ly a f fec ted by a s c o r b a t e / i r o n

i ncuba t ion (1.91 _+ 0.141 n m o l / m g pro te in , P > 0.05),

w h i c h c o r r e s p o n d s to Ca 2+ b i n d i n g to the surface o f the

m e m b r a n e ves ic les .

y ~

z ~

o C O N T R O L

P E R O X I D I Z E D

i i i i i i i

0 1 2 3 4 5 6

Time (rain)

Fig. 3. Influence of lipid peroxidation on Na+/Ca 2÷ exchange in synaptic plasma membrane vesicles. The SPM were loaded overnight at 0-4°C in medium containing 150 mM NaCI, 1 mM MgC12, 10 mM glucose and 10 mM HEPES-Tris, pH 7.4, to load the vesicles with Na + and were then submitted to lipid peroxidation as described in Materials and Methods. The Na+/Ca 2+ exchange was assayed by transferring samples of peroxidized or non-peroxidized membrane vesicles (0.5 mg protein/ml) into medium containing 150 mM NaCI or 150 mM KCI and 20 ~M CaCI~ supplemented with 45CAC12 (2.5 p~Ci/p~mol). The reac- tion was conducted for 5 min, at 30°C, and was terminated by filtration of samples, containing 0.25 mg of protein, as described in Materials and Methods. Results represent means+S.E.M, for 4 different experiments and correspond to the difference between Ca 2+ uptake in K + and Na + medium. Statistical significance was determined by means of an unpaired Student's t-test (P > 0.05).

20

o C O N T R O L

o P E R O X I D I Z E D

15

10

N a ÷ m e d i u m + 30 m M K C I

Na + medium

i i i i i i i

0 1 2 3 4 5 6

Time (min)

Fig. 2. Effect of lipid peroxidation on basal and K÷-depolarization induced 45Ca2+ uptake by synaptosomes. 45Ca2+ uptake experiments were carried out at 30°C by transferring peroxidized or non-peroxidized synaptosomes (0.5 mg protein/ml) to Na ÷ medium containing 150 mM NaCI, 1 mM MgC12, 10 mM glucose and 10 mM HEPES-Tris, pH 7.4, or to Na ÷ medium containing 30 mM KC1, supplemented with 1 mM CaCI 2 plus 45CAC12 (1 p~Ci/ml). After various periods of time the reaction was stopped by rapid filtration of samples containing 0.25 mg of protein, as described in Materials and Methods. Results represent means + S.E.M. for 3 to 5 different experiments. Statistical significance between control and peroxidized synaptosomes was determined, for either Na + medium or Na + medium plus 30 mM KC1, by means of Student's t-test (P > 0.05).

3.3. Influence of lipid peroxidation on the Ca 2 +-ATPase activity o f the plasma membrane and on the Ca 2 +-ATPase activity o f the endoplasmic reticulum

The t ime course m e a s u r e m e n t of H + p r o d u c t i o n result-

ing f rom A T P hydro lys i s , in the p r e sence o f thaps iga rg in ,

a spec i f ic inh ib i to r of e n d o p l a s m i c r e t i cu lum Ca 2 + -ATPase

[56], s h o w s that the pe rox ida t ive a l te ra t ion o f the synap to -

soma l m e m b r a n e i nduces a s ign i f i can t inh ib i t ion ( P < 0 .05)

Table 1 Effects of ascorbate/iron-induced lipid peroxidation on the kinetical parameters of the plasma membrane Ca2+-ATPase

Vm~ x (nmol/min/mg protein) K m ( # M )

Control 425.5 +_ 38.7 0.5384 +_ 0.04 Ascorbate/Fe 2 + 220.9 _+ 16.0 * 0.5624 +_ 0.034

The ATPase activity of synaptic plasma membrane vesicles, pre-in- cubated in the presence or absence of ascorbate/iron for 15 min at 30°C, was determined as a function of free Ca 2+ concentration (55-340 nM) with a pH electrode, by measuring the H + production as described in Materials and Methods. Results were then ploted by Lineweaver-Burk analysis and the Vma x and K m were calculated. The results represent the means_+ S.E.M. for 3 to 7 different experiments. * Values statistically different from control (P < 0.01) determined by means of an unpaired Student's t-test.

274 c. Pereira et al. / Brain Research 713 (1996) 269-277

of the Ca2+-ATPase of the plasma membrane of about

36.7%, 5 min after the addit ion of A T P (Fig. 4A). A reduct ion in the ATP-dependen t 45Ca2+ uptake (Fig. 5),

de termined in membrane ves ic les obtained after osmot ic

lysis o f synaptosomes, was observed: a 58.3% inhibit ion

occurs, 5 min after the addit ion of ATP, in peroxidized as

compared with control ves ic les ( P < 0.01) for the same

extent of the peroxidat ive process (18.05 _+ 4.20 nmol

T B A R S / m g protein).

A 120

100

"~ .== 80

~ ~ 611

+' N % ~ 20

B 120

lOO

"7- ._= 80

~ 60

i

i i i i i i i

0 1 2 3 4 5 6

o CONTROL P E R O X I D I Z E D

j j f J

T ~ J

J

i i i i i ~ i

0 1 2 3 4 5 6

T i m e (m in )

Fig. 4. Effect of lipid peroxidation on plasma membrane Ca2+-ATPase (A) and endoplasmic reticulum Ca2+-ATPase (B) activities. The determi- nation of the enzymatic activities of peroxidized and non-peroxidized synaptosomes was carried out with a pH electrode by measuring the H + production as described in Materials and Methods. The plasma membrane Ca2+-ATPase activity results from the difference between the activity determined in the presence of thapsigargin and in the presence or absence of Ca 2+. The difference between the total activity determined in the presence of Ca 2+, and in the presence or absence of thapsigargin, corresponds to the endoplasmic reticulum (Ca 2+ + Mg 2+ )-ATPase activ- ity. The difference between the activity determined in the absence of calcium and in the presence or absence of thapsigargin corresponds to the MgZ+-ATPase activity. The Ca2+-ATPase activity of the endoplasmic reticulum was obtained from the difference these two values. Results were expressed as nmol H+/mg protein. The results represent the means+ S.E.M. for 3 to 11 different experiments. ~ Values statistically different from control (P < 0.05) determined by means of an unpaired Student's t-test.

l0

S

~+ .~ 6

~ E

[. o .<

o CONTROL tJ PEROXIDIZED

+

0 1 2 3 4 5 6

T i m e (min)

Fig. 5. Effect of lipid peroxidation on ATP-dependent 45Ca2+ uptake by synaptic plasma membrane vesicles. 45Ca2+ uptake experiments were carried out at 30°C by transferring peroxidized or non-peroxidized mem- brane vesicles (0.5 mg protein/ml) to K + medium containing 150 mM KCI, 1 mM MgCI2, 10 mM glucose and 10 mM HEPES-Tris, pH 7.4 supplemented with 20 /xM CaCI 2 plus 45CaC12 (2.5 /xCi//zmol). At various periods of time after adding ATP-Mg, the reaction was stopped by rapid filtration of samples, containing 0.25 mg of protein as described in Materials and Methods. Results represent means+ S.E.M. for 3 to 4 different experiments. * Values statistically different from control (P < 0.01) determined by means of an unpaired Student's t-test.

T a b l e 1 s u m m a r i z e s the c h a n g e s caused by

a s c o r b a t e / i r o n - i n d u c e d membrane lipid peroxidat ion on

the kinetical parameters (Vma x and K m) of plasma mem- brane Ca2+-ATPase determined by L i n e w e a v e r - B u r k

analysis of the enzymat ic act ivi ty in the free [Ca 2 + ] range

of 5 5 - 3 4 0 nM. A s c o r b a t e / i r o n leds to a 48.1% decrease

of Vm, x values ( P < 0.01) without s ignif icant changes in

the affinity for Ca 2+ ( P > 0.05). An inhibit ion of the endoplasmic ret iculum Ca 2+-

ATPase of synaptosomes (Fig. 4B) was also observed after

lipid peroxidat ion, the percentage of inhibit ion being 45.2%

at 5 min after the addit ion of A T P ( P < 0.05). Similar

values for the Ca2+-ATPase activit ies were obtained when

the enzyme act ivi ty was measured by the product ion of

inorganic phosphate [27] (data not shown).

4 . D i s c u s s i o n

It has been recent ly suggested that the impairment of

Ca 2+ homeostas is in the C N S may be the t r iggering event

which leads to the deve lopment of neurodegenera t ion dur-

ing oxidat ive stress condi t ions [43]. The aim of the present

study was to evaluate the inf luence of oxidat ive stress on in t rasynaptosomal ca lc ium concentra t ion and on the mech-

anisms regulat ing the cytosol ic ca lc ium levels, part icularly

the ca lc ium influx and extrusion at the plasma membrane and the endoplasmic ret iculum Ca 2+ sequestration.

In this study, the a s c o r b a t e / F e 2+ pair was used to

induce oxidat ive injury o f synaptosomes, since this c o r n -

c. Pereira et al. / Brain Research 713 (1996) 269-277 275

plex is active in the formation of free radicals [16] and has long been shown to be effective as a suitable model of lipid peroxidation in biological systems [17]. The extent of lipid peroxidation was followed by measuring the TBARS production using the thiobarbituric acid test, a simple and sensitive procedure [48].

Our results showed that, after lipid peroxidation, an increase in intrasynaptosomal Ca 2+ concentration occurs when Ca 2+ is present in the extracellular medium in the physiological range of concentrations. Control and peroxi- dized synaptosomes were able to respond to K+-depolari - zation with an increase in [Ca2+]i, suggesting that voltage-gated calcium channels, present in the plasma membrane, are still activate under our experimental oxida- tive conditions. The difference observed in the uptake of 45Ca2+ by control and peroxidized synaptosomes was not statistically significant, reinforcing the concept that the mechanisms of Ca 2+ influx into synaptosomes are not altered. Furthermore, under these oxidative conditions, only a slight decrease of membrane potential was observed (data not shown) [25] suggesting that the integrity and permeability of synaptosomal plasma membrane are pre- served.

Although an increase in intracellular Ca 2+ concentra- tion has been commonly associated with oxidative injury of neuronal cells [14], the molecular mechanisms by which this increase occurs are far from being elucidated. Evi- dence for a positive interaction between calcium and ox- idative damage comes from the demonstration of a rise in [Ca2+] i and an increase in peroxidation during brain hy- poxia [19]. However, hypoxia has also been reported to decrease the [Ca2+]i and the uptake of 45Ca2+ in synapto- somes [47]. Seemingly conflicting results have been ob- tained for the uptake of 45Ca2+ by synaptosomes submit- ted to peroxidative conditions. While a reduction in 45Ca2+ uptake stimulated by K+-depolarization has been reported [4,46], an increase in calcium uptake was also shown to occur. The results we obtained for calcium uptake by synaptosomes, under ascorbate/Fe 2+ induced oxidative conditions, are in agreement with those reported by other authors showing that no significant change occurs both in basal [4] and K+-induced 4SCa2+ uptake [33] by synapto- somes. Also during ischemia conditions [39] where lipid peroxidation is involved, the influx of 45Ca2+ induced by K+-depolarization was shown to be maintained within the control levels.

Intrasynaptosomal calcium concentration represents the balance between different mechanisms. Under resting con- ditions, the plasma membrane CaZ+-ATPase, which pumps Ca 2+ out of the cell [52,53], and the sequestration of Ca 2+ by the Ca2+-ATPase in the synaptosomal endoplasmic reticulum [3] are the main mechanisms responsible for keeping the cytosolic free calcium concentrations low. The influx of Ca 2+ stimulated by membrane depolarization occurs through the activation of voltage-sensitive Ca 2+ channels [40] and may additionally activate two mecha-

nisms for Ca 2+ removal from the cytosol of nerve termi- nals: the Na+ /Ca 2+ exchanger, which utilizes the energy of Na + gradient [53], and the sequestration of Ca 2+ in mitochondria [1]. We demonstrated that either basal or K+-induced cytosolic free calcium concentration, both in control and peroxidized synaptosomes, depends upon ex- ternal calcium. We also observed that an increase in [ Ca2+ ]i occurs in synaptosomes following ascorbate/Fe 2+ induced peroxidation, this increase being statistically sig- nificant for physiological external calcium concentrations. In the present study, in non-peroxidized (control) synapto- somes, resting and K+-depolarization induced [Ca>]i in- creased as the external calcium was elevated from 0 to 3 mM, a maximum being obtained at 1 mM with only small differences between 1 and 3 mM external calcium. In peroxidized synaptosomes, a linear increase in [Ca 2+ ]i was observed as the external calcium concentration was ele- vated from 0 to 3 mM, suggesting that oxidative stress reduces the ability of the synaptosomes to buffer the entering of calcium. A potential site of reduced buffering capacity is mitochondria, whose capacity to take up cal- cium operates in the presence of physiological concentra- tions of external calcium [55] and is impaired during oxidative stress conditions [8]. Since in the absence of external calcium no difference in [Ca2+]i was observed between control and peroxidized synaptosomes, the signifi- cant rise in intrasynaptosomal calcium levels, observed for higher [Ca 2+ ]o (1 and 3 mM), does not seem to result from the release of Ca 2+ from intracellular stores. It may be due to an alteration of Ca 2+ transport mechanisms through the plasma membrane, namely the Ca 2+ extrusion mecha- nisms, or to a failure of either the mitochondria or the endoplasmic reticulum to buffer the incoming of calcium, as suggested previously [19].

According to this hypothesis, we tested the effect of ascorbate/Fe 2+ induced oxidative stress on the plasma membrane and endoplasmic reticulum CaZ+-ATPase activ- ity. An 36.8% inhibition of the Ca2+-ATPase activity of the plasma membrane was observed following ascorbate/iron induced oxidation of synaptosomal mem- brane lipids. An inhibition of 45.2% of the CaZ+-ATPase responsible for Ca 2+ sequestration by the endoplasmic reticulum was also observed. A direct effect of iron on enzyme activity was excluded according to a previous work of Leclerc et al. [29], where is demonstrated that iron per se does not inhibit the activity of an ATPase responsi- ble for intracellular Ca 2+ regulation. Furthermore, under our experimental conditions iron per se is not an inductor of lipid peroxidation, since TBARS levels in the presence of Fe 2+ are similar to those in controls (data not shown).

We have previously shown [20] that during ascorbate/iron membrane lipid peroxidation a loss of polyunsaturated fatty acids occurs, specially arachidonic acid (20:4) and docosahexaenoic acid (20:6) and that this fact contributes to an increase in membrane order [45]. The results obtained by Moore et al. [36] and by Michelangeli

276 c. Pereira et al. / Brain Research 713 (1996) 269-277

et al. [35] indicate that for optimal function, the Ca 2+- ATPase requires a ' f l u id ' membrane conta in ing unsatu- rated phospholipid acyl chains, and a good correlation be tween changes in protein conformat ion and lipid fluidity of the membranes associated with lipid peroxidat ion have also been reported [18,41]. Therefore, it seems that the alteration in the CaZ+-ATPases activity, both in synaptoso- mal plasma membrane and endoplasmic ret iculum, induced by lipid peroxidation, is related to changes in l ipid-protein interactions due to alteration of the lipid env i ronment around the enzyme. The kinetic determinat ions for the plasma membrane Ca2+-ATPase activity, showing that lipid peroxidat ion does not s ignif icant ly alter the affinity of this enzyme for Ca 2+ supports the idea that the ascor- b a t e / i r o n induced oxidat ion of polyunsaturated acyl chains and consequent alteration of membrane phosphol ipids composi t ion and fluidity modula tes the Ca2+-ATPase ac- tivity [49].

Effiux of calcium from neurons is dependent not only on the plasma membrane Ca 2+ pump, but also on the N a + / C a 2+ exchanger [13]. Thus, the exchanger is another potential site for altered ionic regulat ion dur ing lipid per- oxidation. It was shown that be low the exchanger reversal potential ( - 2 5 mV in squid axons), it extrudes Ca 2 + from the cell, whereas depolarizat ion of the membrane , above the reversal potential, causes Ca 2+ influx into the cell [38]. In the present study, the membrane potential ( - 4 8 mV), measured fol lowing a sco rba t e / i ron lipid peroxidation, is still compat ible with the funct ion of the N a + / C a 2+ ex- changer as an extrusion mechanism. The observat ion that N a + / C a 2+ exchange was not statistically decreased after a sco rba t e / i ron treatment (Fig. 3), when measured in iso- lated synaptic plasma membrane vesicles, indicates that the N a + / C a 2+ exchanger efficiently extrudes Ca 2+ and there- fore does not contr ibute to the observed increase in [Ca 2+ ]~. However, the possibil i ty that in intact nerve terminals the activity of the N a + / C a 2+ exchanger may be affected by peroxidation, can not be ruled out.

Accord ing to the results presented we can conclude that the defects in calcium extrusion and calcium sequestrat ion mechanisms, namely the reduced Ca 2 +-dependent ATPases activities, may contr ibute to the alterations in calcium homeostasis and neuronal cell damage occurr ing under condi t ions where membrane lipid peroxidat ion is thought to be involved, such as in hypoxia- ischemia of the central nervous system.

Acknowledgements

This work has been supported by JNICT (Portuguese Research Counci l ) and by the European Capital Mobil i ty Program (EU). Proposal n ° ERB 4050 PL 932039.

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