Ganglioside GM1 reduces ethanol induced phospholipase A2 activity in synaptosomal preparations from mice

Neurochem. Int. Vol.25, No. 4, pp. 321 325, 1994

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Pergamon

0197-0186(94)E0072-2

Copyright ~' 1994ElsevierScienceLtd Printedin Great Britain.All rightsreserved 0197 -0186/94$7.00+ 0.00

G A N G L I O S I D E GM1 R E D U C E S E T H A N O L I N D U C E D PHOSPHOLIPASE A2 ACTIVITY IN S Y N A P T O S O M A L P R E P A R A T I O N S F R O M MICE BASALINGAPPA L. HUNGUND .1'2, ZHIHONG ZHENG 1, LING LIN ~ a n d AMIRAM I. BARKAI1'2 ~Divisionof Analytical Psychopharmacology, New York State Psychiatric Institute, 722 West 168th Street, NY 10032 2Department of Psychiatry, College of Physicians and Surgeons of Columbia University, NY 10032, U.S.A. (Received 1 March 1994 ; accepted 8 April 1994)

Abstract---The adaptation (tolerance) to chronic EtOH exposure was explained by the development of resistance to the disordering of the membrane phospholipids (PL). This phenomenon may be associated with changes in enzymessuch as phospholipase A2 (PLA2) that govern PL metabolism. The data presented here, using the mouse inhalation model, supports and confirms previously reported findings that chronic exposure to EtOH substantially increased PLA2 activity in synaptosomal preparations from rat brain. We have previously reported that pretreatment with ganglioside GM1 reduced the intoxicating effect of EtOH in mice. The present study indicates that GMI pretreatment both & vivo and in vitro reduced the EtOHinduced activation of PLA2 in synaptosomal preparations. Thus GM 1 may exert its neuroprotective effects by influencingdeacylation/reacylation of membrane phospholipids.

One persistent explanation of the mechanism underlying the pharmacological action of ethanol (EtOH) has been the disordering of phospholipid (PL) acyl chains in the cell membrane (Hunt, 1985; Sun and Sun, 1979). The adaptation (tolerance) after chronic exposure to EtOH was explained by the development of resistance to such disordering of the membrane PL (Chin and Goldstein, 1977; Johnson et al., 1979; Hunt, 1985; Taraschi et al., 1987; Hungund and Mahadik, 1993). It is conceivable that the action of EtOH leading to the disordering of PL acyl chains in the membrane and the development of resistance after chronic EtOH exposure are associated with changes in the enzymes that govern PL metabolism in cellular membranes. Particular attention has to be given to the family of phospholipase A2 (PLA2) enzymes that hydrolyse the sn2 fatty acyl ester bond of phosphoglycerides, producing free fatty acids and lysophospholipids (Dennis, 1983; Waite, 1987). These *Author to whom all correspondence should be addressed at the NYS Psychiatric Institute. Abbreviations: PLA2, phospholipase A2 ; EIOH, ethanol; GM1, ganglioside GM1; SPM, synaptosomal plasma membranes ; PL, phospholipids. 321

enzymes are known to participate in several key events that determine the turnover of PL in biological membranes including the deacylation-reacylation cycle (Van den Bosch, 1980; Waite, 1987), the biosynthesis of eicosanoids (Van den Bosch, 1980; Waite, 1987) and signal transduction across the biomembrane (Winkler, 1976; Creutz, 1981 ; Frye and Holz, 1984, 1985; Karli et al., 1990). John et al. (1985) had investigated the effects of EtOH on PLA2 and demonstrated a progressive increase of the activity of this enzyme in synaptosomal preparations from animals exposed to EtOH. Such an increase in PLA2 activity might be expected to reduce the proportion of unsaturated acyl chains in certain PL and thus influence the development of resistance to the disordering effect by EtOH. In addition, Waring et al. 0981) have shown that liver microsomes from animals chronically exposed to EtOH were increasingly resistent to the hydrolytic action of exogenous PLA2. This resistance has been shown to persist in lipid vesicles that were reconstituted from the PL of the microsomal membranes of such animals (Stubbs et al., 1988). Assuming that PLA2 plays an important role in mediating the effects of EtOH on cell membranes, it is conceivable that some of the adverse effects of EtOH

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could be altered by m o d u l a t i n g the activity of this enzyme. Recent studies from o u r laboratory, a n d studies by others, indicated t h a t a d m i n i s t r a t i o n of the monosialoganglioside G M 1 to mice considerably reduced the intoxicating effects of acute E t O H treatment ( K l e m m a n d Foster, 1988 ; H u n g u n d et al., 1990, 1991 ). Since the gangliosides G M 1, G D 1a a n d G T 1b have been s h o w n /n vitro to inhibit the activity of PLA2 at relatively low m o l a r c o n c e n t r a t i o n s (Bianco et al., 1989, 1990) it is possible that the ganglioside action in reducing the intoxicating effects o f E t O H might be linked to its inhibition of PLA2 activity. In the present study we have used GM1 to determine whether or not the a d m i n i s t r a t i o n of this ganglioside in vivo would interact with the activity o f P L A 2 in s y n a p t o s o m a l p r e p a r a t i o n s from the brains of mice t h a t were chronically exposed to E t O H . We have also investigated the effects o f E t O H o n the activity of PLA2 in vitro in the presence a n d absence o f exogenously added G M 1. The results of these studies s u p p o r t the finding of J o h n et al. (1985) that exposure of mice to E t O H is associated with a n increase of P L A 2 activity in their synaptic m e m b r a n e s a n d indicate t h a t a d m i n i s t r a t i o n of G M 1 a t t e n u a t e d the E t O H - i n d u c e d activation of PLA2 in b r a i n synaptosomes.

crystalline bovine serum albumin as standard (Lowry et al., 1951). Estimation of P L A 2 activity

The PLA2 activity was assayed according to the procedures described by Katsumata et al., (1986) and Quik (1987). The substrate, 1-stearoyl-2[l 14C]-arachidonyl3phosphatidylcholine (SA 56 Ci/mol, Amersham) was dried under nitrogen and resuspended in the incubation buffer with the aid ofa sonicator. Incubation was carried out in 100 mM Tris-HCl buffer containing 0.1% Triton X-100 and 2 mM CaCI2 at pH 9.0. All emulsions of the [14C] substrate were used within 1 h of preparation. Freshly prepared P2 fraction (10/d; 200/~g protein) was preincubated at 3 7 C for 5 min. Then a 10 pl aliquot of buffer containing 1.78 nmol (100 nCi) of the radioactive substrate was added and the reaction mixture incubated in a shaking water bath at 3 7 C for additional 10 min. The reaction was terminated by the addition of 0.5 ml of 0.05N HCI containing 1% Triton X100 and the fatty acids released were extracted with 3 ml hexane. The activity of PLA2 was determined from the radioactivity in the hexane layer and the results are expressed as pmol of substrate hydrolysed per mg protein per h. Preliminary experiments were conducted with increasing amounts of tissue (ranging from 50 to 300 /~g protein) or increasing time of incubation (2-15 min). In both cases there was a linear relationship between amount of tissue, or incubation time, and the PLA2 activity. Ethanol treatment in vitro

EX PER1MENTAL P R O C E D U R E S

Ethanol treatment in vivo

Male Swiss Webster mice (25-30 g body weight, 6-8 weeks old) were chronically exposed to EtOH by inhalation procedure for periods of up to 4 days. (Goldstein, 1972; Hungund et al., 1988) An intraperitoneal (i.p.) injection of the alcohol dehydrogenase inhibitor, pyrazole (68 mg/kg) was given daily to the animal to maintain relatively constant blood EtOH levels. Using this procedure, it has been demonstrated that animals become tolerant to EtOH after 3 days of continuous exposure (Goldstein, 1972). Ganglioside GM 1 (10 mg/kg in saline, 3 mg/ml) was administered (i.m.) 24 h prior to the initial exposure to EtOH and then once daily 1 h before the injection of pyrazole. Blood EtOH levels were determined with the enzymatic method of Lundquist (1959) after collecting 10 /~1 tail blood prior to the GMI administration. Controls were housed under identical conditions except for the absence of EtOH from the inspired air. Animals were sacrificed by decapitation after varying periods of EtOH exposure (1-4 days), their brains removed and immediately processed for the preparation of synaptosomes. Synaptosomal preparation

Crude synaptosomal fraction was prepared according to described procedure (Jones and Matus, 1974). Brains were homogenized with a glass homogenizer in 9 vol of 0.32 M sucrose and centrifuged for 20 rain at 800 g. The supernatant was removed and centrifuged for 20 min at 9000 g to obtain the P2 pellet. This P2 fraction was washed with phosphate buffer (pH 7) and then resuspended in Tris-HC1 buffer (pH 9). Protein was estimated by the method of Lowry et al. with

The P2 fraction (200 /~g protein) in a volume (20 /~1) from an untreated mouse brain was incubated with varying concentrations of EtOH (0--500 raM) at 3 7 C for 15 min after a preincubation period of 15 min. Effects of pretreatment with GMI on EtOH-induced changes in PLA2 activity were assessed by incubation of the P2 fraction with 5 pg GM 1 for 15 rain before the addition of EtOH.

RESULTS

B l o o d E t O H durin 9 e x p o s u r e

Exposure of mice to E t O H vapors for a period o f up to 4 days had no significant effect on the body weight or b r a i n weight c o m p a r e d to either control or G M l - t r e a t e d animals. The b l o o d E t O H level after 1 day of E t O H exposure reached a m e a n value of 2.6_+ 0.05 m g / m l a n d then remained stable upto 4 days o f exposure. P r e t e a t m e n t with G M 1 h a d no significant effect on the blood E t O H level c o m p a r e d to the untreated animals. Eff~,cts on P L A 2 activity

The P L A 2 activity in s y n a p t o s o m a l p r e p a r a t i o n increased with increasing time of exposure to E t O H a n d reached a significantly higher level after the third day of exposure c o m p a r e d to the control mice (Fig.

GMI treatment reduces EtOH induced PLA2 activity l). Pretreatment with G M l attenuated the increase in PLA2 activity induced by E t O H but pretreatment with G M 1 without E t O H exposure had no significant effect on PLA2 activity compared to control untreated mice (Fig. 1). Addition of E t O H in vitro to synaptosomal preparations caused significant increases in PLA2 activities when the final concentration of E t O H was 200 m M or higher. Lower concentrations of 50 m M or 100 m M had no apparent effect. Incubation of the synaptosomal preparations with G M I prior to the addition of E t O H had a clear inhibitory effect on the EtOH-induced increase of PLA2 activity (Fig. 2).

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The increase in PLA2 activity seen in the present study alter chronic exposure to EtOH is consistent with the finding of John et al. (1985) and is possibly caused by EtOH-induced alterations of the synaptosomal membrane lipids. This increase in PLA2 activity may represent, in part, an adaptation mechanism to the continuous presence of EtOH. Our results also show that pretreatment with the ganglioside GM1 significantly reduces the activation of PLA2 in synaptosomal membranes from mice that

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DISCUSSION

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Days Fig. 1. Changes in synaptosomal PLA2 activity with time of exposure of mice to EtOH (open circles), treatment with GMI ganglioside (triangles) and pretreatment with GMI of EtOH-exposed mice (squares). Shaded area represents __+3 SEM of the mean control value (199+ 15 pmol/h/mg protein = 100%) calculated for untreated controls of the three groups. Each group represents 4 mice. A significant increase (P < 0.01, pairmatched t-test) of 33% and 61% was seen after 3 and 4 days, respectively, of EtOH exposure compared to pair-matched controls.

Fig. 2. Effect of varying EtOH concentrations on PEA2 activity in vitro. EtOH concentrations above 200mM caused a significant increase in PLA2 activity compared to pairmatched controls without EtOH. Such EtOH effect was not seen when GMI (5 /tg) was present in the incubation medium. *P < 0.05; n = 4. were chronically exposed to EtOH. Earlier studies in our laboratory and others have proven GM1 to be more effective compared to other ganglioside species (Hungund and Mahadik, 1993). There are several plausible mechanisms which can explain this inhibitory effect of GM1. One possible mechanism for the ganglioside effect is that G M I inhibits PLA2 activation by its direct insertion into the membrane and thereby altering membrane properties. Our studies on placental transfer using radiolabeled GM1, suggests that a significant portion of injected GM1 gets incorporated into the brain (Hungund et al., 1993). Such membrane alterations may lead to the rigidization of the membrane resulting in reduced penetration of E t O H into the membrane. A similar effect has been demonstrated in in vitro studies with synthetic lipid bilayers and a PLA2 preparation, from pig pancreas. In those studies, the addition of the gangliosides G M 1, G D 1a and G T 1b was found to strongly inhibit enzyme activity at relatively low molar concentrations (Bianco et al., 1989). Other plausible mechanisms might involve changes in the surface chemistry leading to alterations in protein receptors, ion channels and protein kinases which could result in changes of ionic concentrations and thus affect PLA2 activity. In addition, chronic exposure to E t O H has been shown to affect endogenous ganglioside composition (Hueso et al., 1979 ; Vrabaski et al., 1984 ; Klemm and Foster, 1986) which may be associated with the mechanism of adaptation to the continuous presence of EtOH.

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]]ASALINGAPPAL. HUNGUNDet al.

The adaptation may also involve reacylation of lysophospholipids produced from ethanol induced PLA2 activation. There is new evidence now which suggests that in vivo GM1 administration alters reacylation of membrane phospholipids (Hungund and Barkai, unpublished data). Thus it may alter physical properties of the membrane leading to inhibition of PLA2. The addition of EtOH to membrane preparation in vitro had no significant effect on membrane PLA2 when the final E t O H concentration was 100 m M or less. However, higher concentrations of E t O H (200 500 mM) significantly enhanced PLA2 activity. The blood levels of ethanol maintained in the mice (approx. 50 mM) are four times lower than the levels of ethanol that produce an effect on PLA-2 in vitro. The probable explanation would be that the tissue level of ethanol may be much smaller than when ethanol is added in vitro. John et al. (1985) showed a slight inhibitory effect on membrane PLA2 after addition of EtOH in vitro to reach a final concentration of 50 m M but those investigators have not studied effects of higher EtOH concentrations. The question as to what extent the action of E t O H was due to alterations in cytosolic PLA2 activities (Hirashima et al., 1992, Horrocks et al., 1994) has not been addressed in the present study. It is most likely, however, that under our PLA2-assay conditions (pH 9 : 2 m M Ca 2+) the majority of cytosolic PLA2 available in the synaptosomes would be translocated to membranes (Yoshihara and Watanabe, 1990). Thus another plausible mechanism by which G M 1 may act is by inhibiting the translocation of PLA-2 from cytosol. Since much of the PLA2 activity in the cytosol is selectively directed to the release of arachidonic acid, it is important to examine E t O H effects on PLA2 using various phospholipid substrates, John et al. (1985) used PC with 3H-oleate in the sn-2 position and obtained results which are in good agreement with ours (using 3H-arachidonic in sn-2), thus supporting the possibility that EtOH acted on membranebound PLA2. Chronic E t O H exposed, E t O H dependent and EtOH withdrawn animals are reported to have almost identical PLA2 activities and were all significantly different from the control group (John et al., 1985). Thus withdrawal from ethanol does not appear to affect the PLA2 activity. In conclusion, our studies indicate that chronic E t O H treatment could induce PLA2 activation and pretreatment with GM1 may prevent the E t O H induced activation of this enzyme. Such interaction of GM1 on EtOH-induced activation of PLA2 may be

closely related to the mechanisms involved in reducing the toxicity effects of E t O H by G M 1 as demonstrated by Klemm et al. (1988) and by Hungund et al. (1990, 1991). Additional studies are required, however, to establish unequivocally whether or not the modulation of PLA2 by G M l would attenuate the deletarious effects of E t O H and prevent E t O H withdrawal symptoms. The authors wish to thank Fidia Corp. Italy, for the generous supply of GMI and Mr Thomas B. Cooper, Chief, Department of Analytical Psychopharmacology at N.Y.S. Psychiatric Institute for his support of this work. This work was supported by PHS grant # R01AA07525 (BLH). Acknowledgements

REFERENCES

Bianco I. D., Fidelio G. D. and Maggio B. (1989) Modulation of phospholipase A2 activity by neutral and anionic glycosphingolipids in monolayer. Biochem. J. 258, 95-99. Bianco 1. D., Fidelio G. D. and Maggio B. (1990) Effect of sulfatide and gangliosides on phospholipase C and phospholipase A2 activity: a monolayer study. BBA, 1026, 179-185. Chin J. H. and Goldstein D. B. (1977) Drug tolerance in biomembrane : a spin label study of the effects of ethanol. Science 196, 684 685. Creutz C. E. (1981) Cis-unsaturated fatty acid induce the fusion of chromaffin granules aggregated by synexin. J. Cell. Biol. 91,247 256. Dennis E. A. (1983) Phospholipases. In: The Enzymes (Boyer P. D., ed.), Vol+ 16, pp. 307 353. Academic Press, New York. Frye R. A. and Holz R. W. (1984) The relationship between arachidonic acid release and cetacholamine secretion from cultured bovine adrenal chromaffin cell. J. Neurochem. 43, 146 150. Frye R. A. and Holz R. W. (1985) Arachidonic acid release and catecholamin secretion from digitonin-treated chromaffin ceils : effects of micromolar calcium, phorbol ester and protein alkylating agents. J. Neurochem. 44, 265 273. Goldstein D. B. (1972) Relationship of alcohol dose to intensity of withdrawal sign in mice. J. Pharmac. exp. Ther. 180, 203 215. Hirashima Y., Farooqui A. A., Mills J. S. and Horrocks L. A. (1992) Identification and purification of calciumindependant phospholipase A_~from bovine brain cytosol+ J. Neurochem. 59, 708 714. Horrocks L. A., Yang H. C., Hirashima Y. and Farooqui A. A. (1994) Biochemistry and characterization of phospholipase A2. Trans. Am. Soc. Neurochem. 25, 320. Hueso P., Rodrigo M. and Cabezas J. A. (1987) Ganglioside alterations in various brain areas, liver and kidney from chronin alcoholic rats. Neurochem. Int. 11,383-388. Hungund B. L+ and Mahadik S. P. (1993) Role of gangliosides in behavioral and biochemical actions of alcohol: cell membrane structure and function. Alcohol. Clin. exp. Res. 17, 329 339. Hungund B. L., Goldstein D. B., Villegas F. and Cooper T. B. (1988) Formation of fatty acid ethyl esters during

GMI treatment reduces EtOH induced PLA2 activity chronic ethanol treatment in mice. Biochem. Pharmac. 37, 3001-3004. Hungund B. L., Reddy M. V., Bharucha V. A. and Mahadik S. P. (1990) Monosialogangliosides (GM1 and AGF2) reduce acute ethanol intoxication: sleep time, mortality and cerebral cortical Na ÷, K+-ATPase. Drug Dev. Res. 19, 443 -451. Hungund B. L., Gokhale V. S., Cooper T. B. and Mahadik S. P. (1991) Prenatal ganglioside GMI treatment protects ethanol-induced sleep time in rats exposed to ethanol in utero during gestation days 7 and 8. Drug Dez~. Res. 24, 261 267. Hungund B.L., Morishima H. O., Gokhale V. S. and Cooper T. B. (1993) Placental transfer of (3H)-GM1 and its distribution to maternal and fetal tissues of the rat. L([~, Sci. 53, 113 119. Hunt W. A. (1985) Alcohol and Biological Membranes. The Guilford Press, New York. John G, R., Littleton J. M. and Nhamburo P. T. (1985) Increased activity of CA -,+ dependent enzymes of membrane lipid metabolism in synaptosomal preparations from ethanol-dependent rats. J. Neurochem. 44, 1235 1241. Johnson D. A., Lee N. M., Cooke R. and Lob H. H. (1979) Ethanol-induced fluidization of brain lipid bilayers: required presence of cholesterol in membranes for the expression of tolerance. Molec. Pharmac. 15, 739-746. Jones D. H. and Matus A. J. (1974) Isolation of synaptic plasma membrane from brain by combined flotation sedimentation density gradient centrifugation. BBA 356, 276 287. Karli O., Schafer T. and Burger M. M. (1990) Fusion of neurotransmitter vesicles with target membrane is calcium independent in a cell-free system. Proc. natn. Acad. Sci. U.S.A. 87, 5912-5915. Katsumata M., Gupta C. and Goldman A. S. (1986) A rapid assay for activity of phospholipase A2 using radioactive substrate. Analyt. Biochem. 134, 676-681. Klemm W. R. and Foster D. M. (1986) Effect of chronic alcohol consumption in weanling rats on brain gangliosides. Prog. Neuro. Psychopharmac. Biol. P,~:vch. 10, 697 702. Klemm W.R. and Foster D. M. (1988) Gangliosides or sialic

325

acid, antagonize ethanol intoxication. L([~" Sci. 63, 1837 1843. Lowry O. H., Rosenbrough N. J,, Farr A. L. and Randall R. J. (1951) Protein measurement with the folin phenol reagent. J. biol. Chem. 193, 265-275. Lundquist F. (1959) The determination of ethyl alcohol in blood and tissues. Meth. biochem. Analysis. 7, 217-251. Quik M. (1987) Characterization and localization of phospholipase A2 activity in synapthetic ganglia. J. Neurochem. 48, 217 224. Stubbs C. D., Williams B. W., Pryor C. L. and Rubin E. (1988) Ethanol-induced modifications to membrane lipid structure: effect on phospholipase A2-membrane interactions. Arch. Biochem. Biophys. 262, 560 573. Sun G. Y. and Sun A. Y. (1979) Effect of chronic ethanol administration on phospholipid acyl groups of synaptic plasma membrane fractions from guinea pig brain. Res. Commun. Chem. path. Pharmac. 24, 405-408. Taraschi T. F., Ellingson J. S. and Rubin E. (1987) Membrane structural alterations caused by chronic ethanol consumption: the molecular basis of membrane tolerance. Ann. N.Y. Acad. Sci. 492, 171 180. Van Den Bosch H. (1980) Intracellular phospholipase A. Biochem, biophys. Acta. 604, 191 246. Vrabaski S. R., Grujic-lnjac B. and Ristic M. (1984) Phospholipid and ganglioside composition in rat brain after chronic intake of ethanol. J. Neurochem. 42, 1235 1239. Vrabaski S. R. and Ristic M. (1985) Cerebellum lipids in rats after chronic ethanol treatment. J. Neurochem. 44, 1868 1872. Waite M. (1987) In: Phospholipases (Hanahan D. J., ed.), pp. I 11 133. Plenum Press. New York. Waring A. J., Rottenberg H., Ohnishi T. and Rubin E. ( 1981 ) Membranes and phospholipids of liver mitochondria from chronic alcoholic rats are resistant to membrane dissordering by alcohol. Proc. nam. Acad. Sci. U.S.A. 78, 2582-2586, Winkler H. (1976) The composition of adrenal chromaffin granules: an assessment of controversial results. Neuroscience 1, 65 80. Yoshihira Y. and Watanabe Y. (1990) Translocation of phospholipase A2 from cytosol to membranes in rat brain induced by calcium ions. Biochim. Biophys. res. Commun. ! 70, 484-490.