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Effects of ethanol on the incorporation of free fatty acids into cerebral membrane phospholipids
~
Pergamon
Neurochem. Int. Vol. 28, No. 5/6, pp. 551-555,1996 Copyright © 1996ElsevierScienceLtd Printedin Great Britain.All rightsreserved 01974)186/96 $15.00+0.00
0197-0186(95)00131-X
EFFECTS OF ETHANOL ON THE INCORPORATION OF FREE FATTY ACIDS INTO CEREBRAL M E M B R A N E PHOSPHOLIPIDS Z H I H O N G ZHENG, AMIRAM I. BARKAI and BASALINGAPPA L. H U N G U N D * New York State Psychiatric Institute and Department of Psychiatry, College of Physicians and Surgeons, Columbia University, New York, NY 10032, U.S.A. (Received 11 August 1995; accepted 20 October 1995)
Abstract---Chronic ethanol exposure is known to affect deacylation-reacylation of membrane phospholipids (PL). In our earlier studies we have demonstrated that chronic exposure to ethanol (EtOH) leads to a progressive increase in membrane phospholipase A2 (PLA2) activity. In the current study, we investigated the effects of chronic EtOH exposure on the incorporation of different free fatty acids (FFAs) into membrane PL. The results suggest that the incorporation of fatty acids into four major PL varied from 9.6 fmol/min/mg protein for docosahexaenoic acid (DHA) into phosphatidylinositol (PI) to 795.8 fmol/min/mg protein for linoleic acid (LA) into phosphatidylcholine (PC). These results also suggest a preferential incorporation of DHA into PC; arachidonic acid (AA) into PI; oleic acid into phosphatidylethanolamine (PE) and PC ; LA into PC and stearic acid into PE. Chronic EtOH exposure affected the incorporation of unsaturated fatty acid into PI, phosphatidylserine (PS) and PC. However, EtOH did not affect significantly the incorporation of any of the fatty acids (FA) studied into PE. No significant differences were observed with the stearic acid. It is suggested that acyltransferases may play an important role in the membrane adaptation to the injurious effects of EtOH. Copyright © 1996 Elsevier Science Ltd
Accumulating evidence suggests that both acute and chronic administration of ethanol (EtOH) to experimental animals induce functional changes in neuronal membranes (Goldstein and Chin, 1981 ; Hunt, 1985). EtOH exposure has been shown to disorganize the neuronal membrane structure at the molecular level (Chin and Goldstein, 1977). The adaptation (tolerance) observed after chronic EtOH exposure was explained by the development of resistance to such disordering effects on the membrane (Johnson et al., 1979 ; Waring et al., 1982 ; Harris et al., 1984). Various studies have shown that EtOH exposure induced changes in various lipid constituents of the membrane, such as cholesterol (Chin et al., 1978 ; Johnson et al., 1979), cholesterol esters (Wing et al., 1982), fatty acid ethyl esters (Hungund et al., 1988) and particularly the fatty acyl composition of membrane phospholipids (PL) (Littleton and John, 1977; Ailing et al., 1982; Smith and Gerhart, 1982; Sun and Sun, 1979). It is conceivable that many of the actions of EtOH leading to the disordering of PL acyl chains in the membrane and the subsequent development of resistance to EtOH are associated with changes in
enzymes that govern PL metabolism in cellular membranes, such as phospholipase A2 (PLA2) and acyltransferases. These enzymes may participate in several key events that determine the turnover of PL in cell membranes, i.e. the deacylation and reacylation cycle (Van Den Bosch, 1980 ; Waite, 1987), the biosynthesis of eicosanoids (Van Den Bosch, 1980; Waite, 1987) and the signal transduction across the membrane (Winkler, 1976; Creutz, 1981; Frye and Holz, 1984, 1985 ; Karli et al., 1990). An increase of PLA2 activity in synaptosomal membranes after exposure to EtOH was reported by John et al. (1985) and by our group (Hungund et aL, 1994). Since EtOH increases PLA2 activity in synaptic membranes, the resulting lysophospholipids may impair cellular functions and need to be reacylated. Therefore, it is reasonable to expect an increase in the activity of acyltransferase enzymes in synaptosomal membranes from animals that have been exposed to EtOH. Thus Sun et al. (1985) have shown that chronic EtOH treatment resulted in an increased incorporation of arachidonic acid (AA) into phosphatidylinositol (PI) and phosphatidylcholine (PC) in plasma membranes of rat brain, but the effects of * Author to whom all correspondence should be addressed. EtOH on the incorporation of other fatty acids have 551
552
Zhihong Zheng et al.
not been investigated in detail. The question as to whether e n h a n c e m e n t of reacylation by E t O H may involve specific acyltransferases should be addressed because alterations in the composition of fatty acids in the Sn-2 position of individual PL could play an i m p o r t a n t role in the a d a p t a t i o n of the m e m b r a n e s to EtOH exposure. In the present study we directed our a t t e n t i o n to the question as to whether there are differential effects o f EtOH on the i n c o r p o r a t i o n of various acyl moieties into individual PL in s y n a p t o s o m a l m e m b r a n e . O u r results indicate that there are preferential effects of EtOH on the reacylation of some PL with selected unsaturated fatty acids.
EXPERIMENTAL PROCEDURES
Chemieals [4,5-3H]Docosahexaenoic acid (DHA) (60 Ci/mmol), [5,6,8,9,11,12,14,15-3H]arachidonic acid (AA) (100 Ci/ mmol) and [9,10-~H]oleic acid (10 Ci/mmol) were obtained from New England Nuclear Corp. (Boston, MA, U.S.A.). [9,10,12,13-3H]Linoleic acid (LA) (90 Ci/mmol) and [9,103H]stearic acid (60 Ci/mmol) were obtained from American Radiochemicals (St. Louis, MO, U.S.A.). ATP, coenzyme A (CoA) and PL standards were purchased from Sigma (St. Louis. MO, U.S.A.). Animals and ethanol administration Male Swiss Webster mice (25 30 g) were chronically exposed to EtOH by inhalation procedure for 3 days (Goldstein, 1972; Hungund et al., 1988, 1994). An i.p. injection of pyrazole (68 mg/kg) was given daily to maintain a relatively constant blood EtOH level. Control animals were housed under identical conditions except for the absence of EtOH from the inspired air. All the animals including the controls received daily pyrazole injections. Blood EtOH levels were determined with the enzymatic method (Lundquist, 1959). Animals were killed by decapitation, their brains were removed and immediately processed for the preparation of synaptosomes. Svnaptosomal preparation Crude synaptosomal fraction was prepared according to described procedure (Jones and Matus, 1974). Whole brain including cerebellum was homogenized in 9 vol. of 0.32 M sucrose and centrifuged at 800 g for 20 rain. The supernatant was removed and centrifuged at 15,000 g for 20 min to obtain pellet (P2). The P2 fraction was washed and resuspended with Krebs-Ringer buffer (KRB). Protein was estimated by the method of Lowry et al. (1951). Free.laity acid incorporation and lipid extraction Incorporation was assayed as described previously (Barkai and M urthy, 1988). Briefly, each assay tube contained freshly prepared membrane suspension (l mg protein) in 0.45 ml KRB (pH 7.4) and was preincubated at 3 7 C for 10 min. Incorporation of fatty acid was initiated by the addition of 50 #1 KRB containing isotope-labeled fatty acid (0.25/zCi), ATP (25 mM), Mg (10 raM) and CoA (1 mM) and the
mixture was incubated at 3 7 C in a shaking water bath for additional 10 min. The reaction was terminated by adding 1.5 ml chloroform : methanol (1 : 2), followed by the addition of I ml chloroform and 0.5 ml distilled water. The tubes were then vortexed and centrifuged at 180 g for 10 min for the separation of organic and aqueous phases. The lower organic phase was removed and evaporated to dryness under a stream of N, and the lipids were redissolved in 50/~1 chloroform. Antioxidant (BHT) was present in the extraction medium and during storage throughout the procedure. Phospholipid separation and radioaeticiO' determination PL were purified by column chromatography according to the procedure described by Vance and Sweeley (1967). Briefly, 1 g of silicic acid (Bio-Rad, Richmond, CA, U.S.A.) was activated for at least 12 h at 80'C and suspended immediately in diethyl ether. The slurry was applied into a small column, the adsorbent was washed with 10 ml of chloroform before the solution of crude total lipids was applied to the column. The column was then eluted with 15 ml of chloroform and 15 ml of acetone : methanol (9 : 1), respectively. Finally, the column was eluted with 15 ml of methanol to obtain the fraction of total PL. The eluate containing PL was dried under N2 and the PL residue was dissolved in chlorotk)rm:methanol (2: 1) and chromatographed on HPTLC plates (E. Merck, Germany) for the separation of individual PL. The solvent system used was chloroform:methyl acetate:n-propanol:methanol:0.25% KCI, 2 5 : 2 5 : 2 5 : 1 0 : 9 (Vitiello and Zanetta, 1978). This solvent system was found to separate all the major membrane PL. After visualization of PL with iodine vapor, PL spots were scraped into scintillation vials and mixed with 100/A tissue solubilizer. The vials were incubated a! 50'~C for 30 rain and cooled to 4 C . Radioactivity was estimated by scintillation counting after correction for efficiency and quenching. The specific activity values of the exogenously added substrates were used to convert the incorporated radioactivity to mass. RESULTS Incorporation 0 [ various" FA into P L o f synaptos'omal membrane U n d e r the incubation conditions employed the incorporation of various F A into the four m a j o r PL varied from 9.6 f m o l / m i n / m g protein for D H A into Pl to 795.8 f m o l / m i n / m g protein for L A into PC (Fig. 1). These results also show a preferential incorp o r a t i o n of D H A into PC, A A into PI, oleic acid into PC and p h o s p h a t i d y l e t h a n o l a m i n e (PE), L A into PC and stearic acid into PE (Fig. 2). Eli ect 0[' chronic E t O H exposure on FA incorporation into membrane P L C h r o n i c exposure to E t O H led to alteration in the i n c o r p o r a t i o n patterns of u n s a t u r a t e d fatty acids into PI, phosphatidylserine (PS) a n d PC, but not into PE. Chronic alcohol exposure increased the i n c o r p o r a t i o n of oleic acid into PI and PS c o m p a r e d to control membranes. In addition there was increased incor-
553
Ethanol affects the incorporation of fatty acids
800 Q) o_ o)
E .c_ E
[] • [] [] []
Docosahexaenoic Arachidonic Oleic Linoleic Stearic
600
400"
E 200"
0
PE
~
PI PS PC Fig. l. The rate of incorporation of various fatty acids into the four major phospholipids in crude synaptosomes from control mouse brain. Values represent mean + SEM, n = 4-6 preparations.
80J c o
• PE [] PI BPS ~PC
60"
i,0
I
20'
0
Docosahexaen0ic Arachid0nic
01eic
Unoleie
Stearlc
Fig. 2. Distribution of the incorporated fatty acids in different phospholipids of crude synaptosomal preparation from control mouse brain. Values represent mean _ SEM, n = 4-6 preparations.
poration of LA into PS and PC, DHA into PS, AA into PC and decreased incorporation of AA into PI compared to control group. No significant differences were observed for stearic acid (Fig. 3). DISCUSSION
Many studies have suggested that alterations in PL acyl chains of neuronal membrane may play an important role in the adaptive mechanism to chronic EtOH exposure. For example, Ailing et al. (1982) reported an increase in the proportion of oleic acid in PC and a decrease of AA in PE in the synaptosomal fraction of EtOH exposed rats. A decrease in unsaturated acyl groups of PS but not other PL in EtOH tolerant brain membrane has been reported by Harris et al. (1984). Moreover, Sun and Sun (1979) also reported that
acyl group changes were related to a decrease in the proportion of monoenes in both PC and PE and an increase in polyenes in PE due to chronic EtOH administration. It can be inferred from these studies that changes in enzymes governing the PL metabolism may partially explain the adaptation mechanism. While there are several studies directed towards analysis of alterations in PL acyl composition, such studies are mainly concerned with total PL and do not provide information about reacylation patterns of individual PL after chronic EtOH exposure (La Droitte et al., 1984; Wing et al., 1984). The results described here suggest that the exposure to alcohol affects the incorporation of unsaturated FA into PI, PS and PC but not into PE. No significant differences were observed with the stearic acid. The preferential incorporation of several FA might be explained by our earlier findings
554
Zhihong Zheng et al.
1601 150 t 1401
[] Docosahexaenoic •
Arachidonic
[] Unoleic I~10leic [] Stearic
O 1304 O "5 120E(D ,o Q_
110
100
i
90 80 PE
PI
PS
PC
Fig. 3. EffEct of EtOH on the incorporation of various l:atty acids into different phospholipids of crude synaptosomal preparation. Values represent mean ++SEM, paired t-test, n = 4~6 preparations, * P < 0.05, • * P < 0.01.
where we showed that chronic alcohol exposure results in activation of membrane PLA2, an enzyme responsible for deacylation of Sn-2 fatty acyl chain of PL. These studies support the possibility that the reacylation of lyso-PL with different fatty-acyl chains is dependent on the activation of a specific acyltransferase which facilitates the incorporation of a given F A to the Sn-2 position of a given PL. Thus alcohol can induce activation of PLA2 as an adaptation to the continued presence of EtOH. The induction of deacylation in the presence of EtOH may further lead to activation of selective acyl transferase enzymes that participate in the adaptation to EtOH. There have been several reports detailing the specificity of acyl transferases for the transfer of fatty acid to PI and PC (Deka et al., 1986; Sanjanwala et al., 1989; Sun and MacQuarrie, 1989). It is of interest to note that linoleic acid, which is not a major constituent of mouse brain membrane PL, appears to have higher affinity for incorporation into PL compared to other FA when presented exogenously. The significance of this finding with reference to repair mechanism needs further investigation. The isolation and purification of a PLA2 from cytosolic fraction of rabbit platelet which preferentially hydrolyses the arachiodonyl residue has been reported (Kim et al., 1990). In the same study PLA2 from another fraction of cytosol was shown to have preference for the linoleyl moiety compared to the arachidonyl acyl group (Kim et al., 1990). Similarly, preferential incorporation of certain FA, in particular, unsaturated F A into lysophospholipids is
shown to occur in guinea pig liver microsome (Badiani et al., 1993). Therefore it is not unreasonable to
assume that such selective activation of enzymes may occur and may play a role in the adaptation process. It is inferred from these results that acyl transferases specific for transfer of different F A play an important role in adaptation to E t O H exposure. However, additional studies are needed with purified synaptosomal membranes and with other polyunsaturated F A substrates to establish further the importance of the various acyl transferases in repair of cellular damage. Since E t O H is known to produce different effects depending on the brain region, it would be interesting to study whether acyl transferases are affected differently in defined regions. Acknowledgements The authors wish to thank Thomas B. Cooper, Chief, Department of Analytical Pscyhopharmacology at NYS Psychiatric Institute for his support of this work. This work was supported in part by NIH grant AA 07525.
REFERENCES
Ailing C., Liljequist S. and Engel J. (1982) The effect of chronic ethanol administration on lipids and fatty acids in subcellular fractions of rat brain. Med. Biol. 60, 149-154. Badiani K., Lu X. and Arthur G. (1993) Effect of A9-tetrahydrocannabinol and merthiolate on acyltransferase activities in guinea pig liver microsomes. Lipids 28, 299 303. Barkai A. ]. and Murthy L. R. (1988) Arachidonate incorporation into phospholipids in rat brain: a comparison
Ethanol affects the incorporation of fatty acids between slice and membrane preparation. Neurochem. Int. 12, 505-512. 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. Chin J. H., Parsons L. M. and Goldstein D. B. (1978) Increased cholesterol content of erythrocyte and brain membranes in ethanol-tolerant mice. Biochim. biophys. Acta 513, 358-363. Creutz C. E+ (1981) Cis-unsaturated fatty acid induce the fusion of chromaffin granules aggregated by synexin. J. Cell Biol. 91,247-256. Deka N., Sun G. Y. and MacQuarrie R. (1986) Purification of acyl-CoA : 1-acyl-sn-glycero-3-phosphocholine-O-acyltransferase from bovine brain microsomes. Arch. Biochem. Biophys. 246, 554-563. Frye R. A. and Holz R. W. (1984) The relationship between arachidonic acid release and catecholamine secretion from cultured bovine adrenal chromaffin cell. J. Neurochem. 43, 146-150. Frye R. A. and Holz R. W. (1985) Arachidonic acid release and catecholamine secretion from digitonin-treated chromarlin cells : 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. Goldstein D. B. and Chin J. H. (1981) Interaction of ethanol with biological membrane. Fedn Proc. 40, 2073 2076. Harris R. A., Baxter D. M., Mitchell M. A. and Hitzemann R. (1984) Physical properties and lipid composition of brain membranes from ethanol tolerant-dependent mice. Molec. Pharmac. 25, 401-409. Hungund B. L., Goldstein D. B., Villegas F. and Cooper T. B. (1988) Formation of fatty acid ethyl esters during chronic ethanol treatment in mice. Biochem. Pharmac. 37, 3001-3004. Hungund B. L., Zheng Z., Lin L. and Barkai A. I. (1994) Ganglioside GM1 reduces ethanol induced phospholipase A2 activity in synaptosomal preparations from mice. Neurochem. Int. 25, 321-325. Hunt W. A. (1985) Alcohol and Bioloyical Membrane. Guilford Press, New York. John G. R., Littleton J. M. and Nhamburo P. T. (1985) Increased activity of CaZ+-dependent enzymes of membrane lipid metabolism in synaptosomal preparations from ethanol-dependent rats. J. Neurochem. 44, 12351241. Jones D. H. and Matus A. J. (1974) Isolation of synaptic plasma membrane from brain by combined flotation-sedimentation density gradient centrifugation. Biochim. biophys. Acta 356, 276-287. Johnson D. A., Lee N. M., Cooke R. and Loh H. H. (1979) Ethanol-induced fluidization of brain lipid bilayers: required presence of cholesterol in membrane for the expression of tolerance. Molec. Pharmac. 15, 739-746. Karli O., Schafer T. and Burger M. M. (1990) Fusion of neurotransmitter vesicles with target membrane is calcium independent in cell-free system. Proc. natn. Acad. Sci. U.S.A. 87, 5912-5915. Kim D., Kudo I., Fujimori Y., Mizushima H., Masuda M.,
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Kikuchi R., Ikizawa K. and Inoue K. (1990) Detection and subcellular localization of rabbit platelet phospholipase A2 which preferentially hydrolyzes an arachidonyl residue. J. Biochem. 108, 903-906. La Droitte P. H., Lamboeuf Y. and De Saint Blanquet G. (1984) Membrane fatty acid changes and ethanol tolerance in rat and mouse. Life Sci. 35, 1221-1229. Littleton J. K. and John G. (1977) Synaptosomal membrane lipids of mice during continuous exposure to ethanol. J. Pharm. Pharmac. 29, 579-580. Lowry O. H., Rosebrough 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. Anal. 7, 217-251. Sanjanwala M., Sun G. Y. and MacQuarrie R. A. (1989) Purification and kinetic properties of lysophosphatidylinositol acyltransferase from bovine heart muscle microsomes and comparison with lysophosphatidylcholine acyltransferase. Arch. Biochim. Biophys. 271, 407-4 13. Smith T. L. and Gerhart M. J. (1982) Alterations in brain lipid composition of mice made physically dependent to ethanol. Life Sci. 31, 1419 1425. Sun G. Y. and MacQuarrie R. A. (1989) Deacylation-reacylation of arachidonoyl groups in cerebral phospholipids. Ann. N. Y. Acad. Sci. 559, 37-55. Sun G. Y. and Sun A. Y. (1979) Effects of chronic ethanol administration on phospholipid acyl groups of synaptic plasma membrane fraction isolated from guinea pig brain. Res. Commun. Chem. path. Pharmac. 24, 405-408. Sun G. Y., Kelleher J. A. and Sun A. Y. (1985) Effects of chronic ethanol administration and withdrawal on incorporation of arachidonate into membrane phospholipids. Neurochem. Int. 7, 491-495. Vance D. E. and Sweeley C. C. (1967) Quantitative determination of the neutral glycosyl ceramides in human blood. J. Lipid Res. 8, 621~30. Van Den Bosch H. (1980) Intracellular phospholipases A. Biochim. biophys. Acta 604, 291-246. Vitiello F. and Zanetta J. P. (1978) Thin-layer chromatography of phospholipids. J. Chromat. 166, 637-640. Waite M. (1987) In: Phospholipases (Hanahan D. J., ed.), pp. 111-133. Plenum Press, New York. Waring A. J., Rottenberg, H., Ohnishi T. and Rubin E. (1982) Membranes and phospholipids of liver mitochondria from chronic alcoholic rats are resistant to membrane disordering by alcohol. Proc. natn. Acad. Sci. U.S.A. 78, 2582 2586. Wing D. R., Harvey D. J., Hughes J., Dunbar P+ G., McPherson K. A. and Paton W. D. M. (1982) Effects of chronic ethanol administration on the composition of membrane lipids in the mouse. Biochem. Pharmac. 31, 3431-3439. Wing D. R., Harvey D. J., Belcher S. J. and Paton W. D. (1984) Changes in membrane lipid content after chronic ethanol administration with respect to fatty acid compositions and phospholipid type. Biochem. Pharmac. 33, 1625-1632. Winkler H. (1976) The composition of adrenal chromaffin granules: an assessment of controversial results. Neuroscience 1, 65-80.
Pergamon
Neurochem. Int. Vol. 28, No. 5/6, pp. 551-555,1996 Copyright © 1996ElsevierScienceLtd Printedin Great Britain.All rightsreserved 01974)186/96 $15.00+0.00
0197-0186(95)00131-X
EFFECTS OF ETHANOL ON THE INCORPORATION OF FREE FATTY ACIDS INTO CEREBRAL M E M B R A N E PHOSPHOLIPIDS Z H I H O N G ZHENG, AMIRAM I. BARKAI and BASALINGAPPA L. H U N G U N D * New York State Psychiatric Institute and Department of Psychiatry, College of Physicians and Surgeons, Columbia University, New York, NY 10032, U.S.A. (Received 11 August 1995; accepted 20 October 1995)
Abstract---Chronic ethanol exposure is known to affect deacylation-reacylation of membrane phospholipids (PL). In our earlier studies we have demonstrated that chronic exposure to ethanol (EtOH) leads to a progressive increase in membrane phospholipase A2 (PLA2) activity. In the current study, we investigated the effects of chronic EtOH exposure on the incorporation of different free fatty acids (FFAs) into membrane PL. The results suggest that the incorporation of fatty acids into four major PL varied from 9.6 fmol/min/mg protein for docosahexaenoic acid (DHA) into phosphatidylinositol (PI) to 795.8 fmol/min/mg protein for linoleic acid (LA) into phosphatidylcholine (PC). These results also suggest a preferential incorporation of DHA into PC; arachidonic acid (AA) into PI; oleic acid into phosphatidylethanolamine (PE) and PC ; LA into PC and stearic acid into PE. Chronic EtOH exposure affected the incorporation of unsaturated fatty acid into PI, phosphatidylserine (PS) and PC. However, EtOH did not affect significantly the incorporation of any of the fatty acids (FA) studied into PE. No significant differences were observed with the stearic acid. It is suggested that acyltransferases may play an important role in the membrane adaptation to the injurious effects of EtOH. Copyright © 1996 Elsevier Science Ltd
Accumulating evidence suggests that both acute and chronic administration of ethanol (EtOH) to experimental animals induce functional changes in neuronal membranes (Goldstein and Chin, 1981 ; Hunt, 1985). EtOH exposure has been shown to disorganize the neuronal membrane structure at the molecular level (Chin and Goldstein, 1977). The adaptation (tolerance) observed after chronic EtOH exposure was explained by the development of resistance to such disordering effects on the membrane (Johnson et al., 1979 ; Waring et al., 1982 ; Harris et al., 1984). Various studies have shown that EtOH exposure induced changes in various lipid constituents of the membrane, such as cholesterol (Chin et al., 1978 ; Johnson et al., 1979), cholesterol esters (Wing et al., 1982), fatty acid ethyl esters (Hungund et al., 1988) and particularly the fatty acyl composition of membrane phospholipids (PL) (Littleton and John, 1977; Ailing et al., 1982; Smith and Gerhart, 1982; Sun and Sun, 1979). It is conceivable that many of the actions of EtOH leading to the disordering of PL acyl chains in the membrane and the subsequent development of resistance to EtOH are associated with changes in
enzymes that govern PL metabolism in cellular membranes, such as phospholipase A2 (PLA2) and acyltransferases. These enzymes may participate in several key events that determine the turnover of PL in cell membranes, i.e. the deacylation and reacylation cycle (Van Den Bosch, 1980 ; Waite, 1987), the biosynthesis of eicosanoids (Van Den Bosch, 1980; Waite, 1987) and the signal transduction across the membrane (Winkler, 1976; Creutz, 1981; Frye and Holz, 1984, 1985 ; Karli et al., 1990). An increase of PLA2 activity in synaptosomal membranes after exposure to EtOH was reported by John et al. (1985) and by our group (Hungund et aL, 1994). Since EtOH increases PLA2 activity in synaptic membranes, the resulting lysophospholipids may impair cellular functions and need to be reacylated. Therefore, it is reasonable to expect an increase in the activity of acyltransferase enzymes in synaptosomal membranes from animals that have been exposed to EtOH. Thus Sun et al. (1985) have shown that chronic EtOH treatment resulted in an increased incorporation of arachidonic acid (AA) into phosphatidylinositol (PI) and phosphatidylcholine (PC) in plasma membranes of rat brain, but the effects of * Author to whom all correspondence should be addressed. EtOH on the incorporation of other fatty acids have 551
552
Zhihong Zheng et al.
not been investigated in detail. The question as to whether e n h a n c e m e n t of reacylation by E t O H may involve specific acyltransferases should be addressed because alterations in the composition of fatty acids in the Sn-2 position of individual PL could play an i m p o r t a n t role in the a d a p t a t i o n of the m e m b r a n e s to EtOH exposure. In the present study we directed our a t t e n t i o n to the question as to whether there are differential effects o f EtOH on the i n c o r p o r a t i o n of various acyl moieties into individual PL in s y n a p t o s o m a l m e m b r a n e . O u r results indicate that there are preferential effects of EtOH on the reacylation of some PL with selected unsaturated fatty acids.
EXPERIMENTAL PROCEDURES
Chemieals [4,5-3H]Docosahexaenoic acid (DHA) (60 Ci/mmol), [5,6,8,9,11,12,14,15-3H]arachidonic acid (AA) (100 Ci/ mmol) and [9,10-~H]oleic acid (10 Ci/mmol) were obtained from New England Nuclear Corp. (Boston, MA, U.S.A.). [9,10,12,13-3H]Linoleic acid (LA) (90 Ci/mmol) and [9,103H]stearic acid (60 Ci/mmol) were obtained from American Radiochemicals (St. Louis, MO, U.S.A.). ATP, coenzyme A (CoA) and PL standards were purchased from Sigma (St. Louis. MO, U.S.A.). Animals and ethanol administration Male Swiss Webster mice (25 30 g) were chronically exposed to EtOH by inhalation procedure for 3 days (Goldstein, 1972; Hungund et al., 1988, 1994). An i.p. injection of pyrazole (68 mg/kg) was given daily to maintain a relatively constant blood EtOH level. Control animals were housed under identical conditions except for the absence of EtOH from the inspired air. All the animals including the controls received daily pyrazole injections. Blood EtOH levels were determined with the enzymatic method (Lundquist, 1959). Animals were killed by decapitation, their brains were removed and immediately processed for the preparation of synaptosomes. Svnaptosomal preparation Crude synaptosomal fraction was prepared according to described procedure (Jones and Matus, 1974). Whole brain including cerebellum was homogenized in 9 vol. of 0.32 M sucrose and centrifuged at 800 g for 20 rain. The supernatant was removed and centrifuged at 15,000 g for 20 min to obtain pellet (P2). The P2 fraction was washed and resuspended with Krebs-Ringer buffer (KRB). Protein was estimated by the method of Lowry et al. (1951). Free.laity acid incorporation and lipid extraction Incorporation was assayed as described previously (Barkai and M urthy, 1988). Briefly, each assay tube contained freshly prepared membrane suspension (l mg protein) in 0.45 ml KRB (pH 7.4) and was preincubated at 3 7 C for 10 min. Incorporation of fatty acid was initiated by the addition of 50 #1 KRB containing isotope-labeled fatty acid (0.25/zCi), ATP (25 mM), Mg (10 raM) and CoA (1 mM) and the
mixture was incubated at 3 7 C in a shaking water bath for additional 10 min. The reaction was terminated by adding 1.5 ml chloroform : methanol (1 : 2), followed by the addition of I ml chloroform and 0.5 ml distilled water. The tubes were then vortexed and centrifuged at 180 g for 10 min for the separation of organic and aqueous phases. The lower organic phase was removed and evaporated to dryness under a stream of N, and the lipids were redissolved in 50/~1 chloroform. Antioxidant (BHT) was present in the extraction medium and during storage throughout the procedure. Phospholipid separation and radioaeticiO' determination PL were purified by column chromatography according to the procedure described by Vance and Sweeley (1967). Briefly, 1 g of silicic acid (Bio-Rad, Richmond, CA, U.S.A.) was activated for at least 12 h at 80'C and suspended immediately in diethyl ether. The slurry was applied into a small column, the adsorbent was washed with 10 ml of chloroform before the solution of crude total lipids was applied to the column. The column was then eluted with 15 ml of chloroform and 15 ml of acetone : methanol (9 : 1), respectively. Finally, the column was eluted with 15 ml of methanol to obtain the fraction of total PL. The eluate containing PL was dried under N2 and the PL residue was dissolved in chlorotk)rm:methanol (2: 1) and chromatographed on HPTLC plates (E. Merck, Germany) for the separation of individual PL. The solvent system used was chloroform:methyl acetate:n-propanol:methanol:0.25% KCI, 2 5 : 2 5 : 2 5 : 1 0 : 9 (Vitiello and Zanetta, 1978). This solvent system was found to separate all the major membrane PL. After visualization of PL with iodine vapor, PL spots were scraped into scintillation vials and mixed with 100/A tissue solubilizer. The vials were incubated a! 50'~C for 30 rain and cooled to 4 C . Radioactivity was estimated by scintillation counting after correction for efficiency and quenching. The specific activity values of the exogenously added substrates were used to convert the incorporated radioactivity to mass. RESULTS Incorporation 0 [ various" FA into P L o f synaptos'omal membrane U n d e r the incubation conditions employed the incorporation of various F A into the four m a j o r PL varied from 9.6 f m o l / m i n / m g protein for D H A into Pl to 795.8 f m o l / m i n / m g protein for L A into PC (Fig. 1). These results also show a preferential incorp o r a t i o n of D H A into PC, A A into PI, oleic acid into PC and p h o s p h a t i d y l e t h a n o l a m i n e (PE), L A into PC and stearic acid into PE (Fig. 2). Eli ect 0[' chronic E t O H exposure on FA incorporation into membrane P L C h r o n i c exposure to E t O H led to alteration in the i n c o r p o r a t i o n patterns of u n s a t u r a t e d fatty acids into PI, phosphatidylserine (PS) a n d PC, but not into PE. Chronic alcohol exposure increased the i n c o r p o r a t i o n of oleic acid into PI and PS c o m p a r e d to control membranes. In addition there was increased incor-
553
Ethanol affects the incorporation of fatty acids
800 Q) o_ o)
E .c_ E
[] • [] [] []
Docosahexaenoic Arachidonic Oleic Linoleic Stearic
600
400"
E 200"
0
PE
~
PI PS PC Fig. l. The rate of incorporation of various fatty acids into the four major phospholipids in crude synaptosomes from control mouse brain. Values represent mean + SEM, n = 4-6 preparations.
80J c o
• PE [] PI BPS ~PC
60"
i,0
I
20'
0
Docosahexaen0ic Arachid0nic
01eic
Unoleie
Stearlc
Fig. 2. Distribution of the incorporated fatty acids in different phospholipids of crude synaptosomal preparation from control mouse brain. Values represent mean _ SEM, n = 4-6 preparations.
poration of LA into PS and PC, DHA into PS, AA into PC and decreased incorporation of AA into PI compared to control group. No significant differences were observed for stearic acid (Fig. 3). DISCUSSION
Many studies have suggested that alterations in PL acyl chains of neuronal membrane may play an important role in the adaptive mechanism to chronic EtOH exposure. For example, Ailing et al. (1982) reported an increase in the proportion of oleic acid in PC and a decrease of AA in PE in the synaptosomal fraction of EtOH exposed rats. A decrease in unsaturated acyl groups of PS but not other PL in EtOH tolerant brain membrane has been reported by Harris et al. (1984). Moreover, Sun and Sun (1979) also reported that
acyl group changes were related to a decrease in the proportion of monoenes in both PC and PE and an increase in polyenes in PE due to chronic EtOH administration. It can be inferred from these studies that changes in enzymes governing the PL metabolism may partially explain the adaptation mechanism. While there are several studies directed towards analysis of alterations in PL acyl composition, such studies are mainly concerned with total PL and do not provide information about reacylation patterns of individual PL after chronic EtOH exposure (La Droitte et al., 1984; Wing et al., 1984). The results described here suggest that the exposure to alcohol affects the incorporation of unsaturated FA into PI, PS and PC but not into PE. No significant differences were observed with the stearic acid. The preferential incorporation of several FA might be explained by our earlier findings
554
Zhihong Zheng et al.
1601 150 t 1401
[] Docosahexaenoic •
Arachidonic
[] Unoleic I~10leic [] Stearic
O 1304 O "5 120E(D ,o Q_
110
100
i
90 80 PE
PI
PS
PC
Fig. 3. EffEct of EtOH on the incorporation of various l:atty acids into different phospholipids of crude synaptosomal preparation. Values represent mean ++SEM, paired t-test, n = 4~6 preparations, * P < 0.05, • * P < 0.01.
where we showed that chronic alcohol exposure results in activation of membrane PLA2, an enzyme responsible for deacylation of Sn-2 fatty acyl chain of PL. These studies support the possibility that the reacylation of lyso-PL with different fatty-acyl chains is dependent on the activation of a specific acyltransferase which facilitates the incorporation of a given F A to the Sn-2 position of a given PL. Thus alcohol can induce activation of PLA2 as an adaptation to the continued presence of EtOH. The induction of deacylation in the presence of EtOH may further lead to activation of selective acyl transferase enzymes that participate in the adaptation to EtOH. There have been several reports detailing the specificity of acyl transferases for the transfer of fatty acid to PI and PC (Deka et al., 1986; Sanjanwala et al., 1989; Sun and MacQuarrie, 1989). It is of interest to note that linoleic acid, which is not a major constituent of mouse brain membrane PL, appears to have higher affinity for incorporation into PL compared to other FA when presented exogenously. The significance of this finding with reference to repair mechanism needs further investigation. The isolation and purification of a PLA2 from cytosolic fraction of rabbit platelet which preferentially hydrolyses the arachiodonyl residue has been reported (Kim et al., 1990). In the same study PLA2 from another fraction of cytosol was shown to have preference for the linoleyl moiety compared to the arachidonyl acyl group (Kim et al., 1990). Similarly, preferential incorporation of certain FA, in particular, unsaturated F A into lysophospholipids is
shown to occur in guinea pig liver microsome (Badiani et al., 1993). Therefore it is not unreasonable to
assume that such selective activation of enzymes may occur and may play a role in the adaptation process. It is inferred from these results that acyl transferases specific for transfer of different F A play an important role in adaptation to E t O H exposure. However, additional studies are needed with purified synaptosomal membranes and with other polyunsaturated F A substrates to establish further the importance of the various acyl transferases in repair of cellular damage. Since E t O H is known to produce different effects depending on the brain region, it would be interesting to study whether acyl transferases are affected differently in defined regions. Acknowledgements The authors wish to thank Thomas B. Cooper, Chief, Department of Analytical Pscyhopharmacology at NYS Psychiatric Institute for his support of this work. This work was supported in part by NIH grant AA 07525.
REFERENCES
Ailing C., Liljequist S. and Engel J. (1982) The effect of chronic ethanol administration on lipids and fatty acids in subcellular fractions of rat brain. Med. Biol. 60, 149-154. Badiani K., Lu X. and Arthur G. (1993) Effect of A9-tetrahydrocannabinol and merthiolate on acyltransferase activities in guinea pig liver microsomes. Lipids 28, 299 303. Barkai A. ]. and Murthy L. R. (1988) Arachidonate incorporation into phospholipids in rat brain: a comparison
Ethanol affects the incorporation of fatty acids between slice and membrane preparation. Neurochem. Int. 12, 505-512. 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. Chin J. H., Parsons L. M. and Goldstein D. B. (1978) Increased cholesterol content of erythrocyte and brain membranes in ethanol-tolerant mice. Biochim. biophys. Acta 513, 358-363. Creutz C. E+ (1981) Cis-unsaturated fatty acid induce the fusion of chromaffin granules aggregated by synexin. J. Cell Biol. 91,247-256. Deka N., Sun G. Y. and MacQuarrie R. (1986) Purification of acyl-CoA : 1-acyl-sn-glycero-3-phosphocholine-O-acyltransferase from bovine brain microsomes. Arch. Biochem. Biophys. 246, 554-563. Frye R. A. and Holz R. W. (1984) The relationship between arachidonic acid release and catecholamine secretion from cultured bovine adrenal chromaffin cell. J. Neurochem. 43, 146-150. Frye R. A. and Holz R. W. (1985) Arachidonic acid release and catecholamine secretion from digitonin-treated chromarlin cells : 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. Goldstein D. B. and Chin J. H. (1981) Interaction of ethanol with biological membrane. Fedn Proc. 40, 2073 2076. Harris R. A., Baxter D. M., Mitchell M. A. and Hitzemann R. (1984) Physical properties and lipid composition of brain membranes from ethanol tolerant-dependent mice. Molec. Pharmac. 25, 401-409. Hungund B. L., Goldstein D. B., Villegas F. and Cooper T. B. (1988) Formation of fatty acid ethyl esters during chronic ethanol treatment in mice. Biochem. Pharmac. 37, 3001-3004. Hungund B. L., Zheng Z., Lin L. and Barkai A. I. (1994) Ganglioside GM1 reduces ethanol induced phospholipase A2 activity in synaptosomal preparations from mice. Neurochem. Int. 25, 321-325. Hunt W. A. (1985) Alcohol and Bioloyical Membrane. Guilford Press, New York. John G. R., Littleton J. M. and Nhamburo P. T. (1985) Increased activity of CaZ+-dependent enzymes of membrane lipid metabolism in synaptosomal preparations from ethanol-dependent rats. J. Neurochem. 44, 12351241. Jones D. H. and Matus A. J. (1974) Isolation of synaptic plasma membrane from brain by combined flotation-sedimentation density gradient centrifugation. Biochim. biophys. Acta 356, 276-287. Johnson D. A., Lee N. M., Cooke R. and Loh H. H. (1979) Ethanol-induced fluidization of brain lipid bilayers: required presence of cholesterol in membrane for the expression of tolerance. Molec. Pharmac. 15, 739-746. Karli O., Schafer T. and Burger M. M. (1990) Fusion of neurotransmitter vesicles with target membrane is calcium independent in cell-free system. Proc. natn. Acad. Sci. U.S.A. 87, 5912-5915. Kim D., Kudo I., Fujimori Y., Mizushima H., Masuda M.,
555
Kikuchi R., Ikizawa K. and Inoue K. (1990) Detection and subcellular localization of rabbit platelet phospholipase A2 which preferentially hydrolyzes an arachidonyl residue. J. Biochem. 108, 903-906. La Droitte P. H., Lamboeuf Y. and De Saint Blanquet G. (1984) Membrane fatty acid changes and ethanol tolerance in rat and mouse. Life Sci. 35, 1221-1229. Littleton J. K. and John G. (1977) Synaptosomal membrane lipids of mice during continuous exposure to ethanol. J. Pharm. Pharmac. 29, 579-580. Lowry O. H., Rosebrough 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. Anal. 7, 217-251. Sanjanwala M., Sun G. Y. and MacQuarrie R. A. (1989) Purification and kinetic properties of lysophosphatidylinositol acyltransferase from bovine heart muscle microsomes and comparison with lysophosphatidylcholine acyltransferase. Arch. Biochim. Biophys. 271, 407-4 13. Smith T. L. and Gerhart M. J. (1982) Alterations in brain lipid composition of mice made physically dependent to ethanol. Life Sci. 31, 1419 1425. Sun G. Y. and MacQuarrie R. A. (1989) Deacylation-reacylation of arachidonoyl groups in cerebral phospholipids. Ann. N. Y. Acad. Sci. 559, 37-55. Sun G. Y. and Sun A. Y. (1979) Effects of chronic ethanol administration on phospholipid acyl groups of synaptic plasma membrane fraction isolated from guinea pig brain. Res. Commun. Chem. path. Pharmac. 24, 405-408. Sun G. Y., Kelleher J. A. and Sun A. Y. (1985) Effects of chronic ethanol administration and withdrawal on incorporation of arachidonate into membrane phospholipids. Neurochem. Int. 7, 491-495. Vance D. E. and Sweeley C. C. (1967) Quantitative determination of the neutral glycosyl ceramides in human blood. J. Lipid Res. 8, 621~30. Van Den Bosch H. (1980) Intracellular phospholipases A. Biochim. biophys. Acta 604, 291-246. Vitiello F. and Zanetta J. P. (1978) Thin-layer chromatography of phospholipids. J. Chromat. 166, 637-640. Waite M. (1987) In: Phospholipases (Hanahan D. J., ed.), pp. 111-133. Plenum Press, New York. Waring A. J., Rottenberg, H., Ohnishi T. and Rubin E. (1982) Membranes and phospholipids of liver mitochondria from chronic alcoholic rats are resistant to membrane disordering by alcohol. Proc. natn. Acad. Sci. U.S.A. 78, 2582 2586. Wing D. R., Harvey D. J., Hughes J., Dunbar P+ G., McPherson K. A. and Paton W. D. M. (1982) Effects of chronic ethanol administration on the composition of membrane lipids in the mouse. Biochem. Pharmac. 31, 3431-3439. Wing D. R., Harvey D. J., Belcher S. J. and Paton W. D. (1984) Changes in membrane lipid content after chronic ethanol administration with respect to fatty acid compositions and phospholipid type. Biochem. Pharmac. 33, 1625-1632. Winkler H. (1976) The composition of adrenal chromaffin granules: an assessment of controversial results. Neuroscience 1, 65-80.