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Chronic ethanol consumption alters transbilayer distribution of phosphatidylcholine in erythrocytes of Sinclair (S-1) miniature swine
Alcohol. Vol. 8, pp. 395-399. Pergamon Press plc, 1991. Printed in the U.S.A.
0741-8329/91 $3.00 + .00
Chronic Ethanol ConsumptiOn Alters Transbilayer Distribution of Phosphatidylcholine in Erythrocytes of Sinclair (S-I) Miniature Swine W . G I B S O N W O O D , *l C H R I S T I N E G O R K A , t J U L I E A. JOHNSON,'I" G R A C E Y, SUN,:~ A L B E R T Y. SUN~: A N D F R I E D H E L M S C H R O E D E R §
*Geriatric Research, Education and Clinical Center, Veterans Administration Medical Center and Department of Pharmacology, University of Minnesota, School of Medicine, Minneapolis, MN 55417 :'Veterans Administration Medical Center, St. Louis, MO 63125 .::Sinclair Comparative Medicine Research Farm and Department of Biochemistry University of Missouri, Columbia, MO 65203 §Department of Pharmacology and Medicinal Chemistry, College of Pharmacy, and Department of Pharmacology and Cell Biophysics, College.of Medicine, University of Cincinnati Medical Center, Cincinnati, OH 45267-0004 R e c e i v e d 6 D e c e m b e r 1990; A c c e p t e d 1 M a r c h 1991 WOOD, W. G., C. GORKA, J. A. JOHNSON, G. Y. SUN, A. Y. SUN AND F. SCHROEDER. Chronicethanol consumption alters transbilayerdistribution of phosphatidylcholine in er3,throcytesof Sinclair (S-1) miniatureswine. ALCOHOL 8(5) 395-399, 1991.--Effects of chronic ethanol consumption on transbilayer distribution of phospholipids in the exofacial and cytofacial leaflets of erythrocytes from chronic ethanol-consuming Sinclair (S-l) miniature swine were examined. Phosphatidylcholine (PCI was predominantly located in the exofacial leaflet and phosphatidylethanolamine (PE) and phosphatidylserine (PS) located primarily in the cytofacial leaflet. Chronic ethanol consumption significantly increased PC content in the exofacial leaflet without changing bulk membrane PC composition. Ethanol-induced changes in PC distribution were specific for PC and not detected in PE or PS. There was also a significant decrease in sphingomyelin in the ethanol group. Sphingomyelin is primarily an exofacial phospholipid. The specific ethanol-induced changes in the exofacial leaflet are consistent with recent studies showing that the exofacial membrane leaflet is more susceptible to effects of ethanol as compared to the cytofacial leaflet. Such specificity of action provides a new way of viewing how ethanol alters membrane structure and function. Chronic ethanol consumption Ethanol tolerance Lipid domains Phosphatidylcholine Membrane asymmetry Ethanol
Phospholipids
Erythrocytes
ing observed in bulk membrane fluidity and total cholesterol content. It has not been determined, however, whether chronic ethanol consumption may also modify the distribution of phospholipids in the exofacial and cytofacial leaflets. Phosphatidylcholine (PC) and sphingomyelin (SM) are considered exofacial phospholipids and phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI) are cytofacial phospholipids (14). Chronic ethanol consumption may alter the distribution of one or more phospholipids in the exofacial and cytofacial leaflets without changing the total amount of an individual phospholipid. Changes in transbilayer phospholipid distribution in erythrocytes have been reported as a result of treatment with specific drugs and other agents. The oxidant drug phenylhydrazine resulted in the transbilayer movement of PE and PS from the cytofacial to the exofacial leaflet in erythrocytes (9). Certain cationic amphiphilic drugs were found to alter PE and PC membrane distribution (7,23). Acyl group distribution of PC in. the exofacial leaflets of erythrocytes was found to be modified by
ALCOHOLS, including ethanol, have a specific effect on lipid domains of biological membranes (18, 24--26, 28). Two domains that have been examined are the membrane exofacial and cytofacial leaflets (18, 26, 28). Ethanol in vitro decreased the limiting anisotropy of diphenylhexatriene in the exofacial leaflet of synaptic plasma membranes (SPM) more than in the cytofacial leaflet (18). The in vitro effects of ethanol were reduced in the SPM exofacial leaflet of chronic ethanol-treated animals (26). It also was observed that baseline fluidity (i.e., in the absence of ethanol in vitro) of the two leaflets was altered in chronic ethanol-consuming animals. The exofacial leaflet became less fluid whereas the cytofacial leaflet became more fluid in SPM of chronic ethanol-consuming animals. In a subsequent study it was shown that there was a redistribution of cholesterol between the two SPM leaflets in chronic ethanol-treated animals (28). Those studies demonstrated that the exofacial leaflet was more susceptible t o the effects of ethanol and that specific changes could occur in membrane structure without changes be-
~Requests for reprints should be addressed to W. Gibson Wood, Ph.D., VA Medical Center, GRECC, 1 Veterans Drive, Minneapolis, MN 55417.
395
396
WOOD ET AL.
TABLE 1
diets that differed in fat content (11). It was the purpose of this study to determine if chronic ethanol consumption would modify the transbilayer distribution of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine in erythrocyte membranes of Sinclair (S-I) miniature swine.
PHOSPHOLIPID DISTRIBUTION IN THE EXOFACIAL LEAFLETS OF ERYTHROCYTES FROM CONTROL AND CHRONIC ETHANOL-CONSUMING SWINE
% Phospholipid in Exofacial Leaflet
Group
PC
PE
PS
PI
Control Ethanol
56.7 --- 2.5 66.9 _ 1.5"
7.2 __. 1.9 7.5 +- 2.9
n.d. n.d.
n.d. n.d.
METHOD
Chemicals All chemicals and reagents were purchased from Sigma Chemical Company, St, Louis, MO. Phospholipase A 2 from Naja mocambique mocambique (pI = 9.6) was used.
Ethanol Administration Adult Sinclair (S-I) miniature boars (12 months of age) with a mean body weight of 90 kg were used (Sinclair Comparative Medicine Research Farm, University of Missouri, Columbia, MO), Animals were given daily rations of a formulated diet containing corn, soybean meal, meat meal, alfalfa meal, cornstarch, salts and vitamin supplement as described previously (6,20). The ethanol group (n = 6) received 6 g/kg/day of a beer solution fortified to 10% ethanol for three months (6,20). The ethanol solution was administered in graduated bottles containing a drinking tube and the total amount of the ethanol solution administered was consumed each day. Animals also had free access to water. Control animals ( n = 6 ) were given the same dietary ration and also received glucose in water to approximate the calories supplied by ethanol. Ten ml of blood was drawn from the jugular vein of each animal and collected in heparinized tubes between 10 a.m. and 11 a.m. Animals had not been withdrawn from ethanol prior to the time when blood samples were taken.
Erythrocytes were incubated with phospholipase Az under nonpenetrating conditions, lipids extracted and chromatographed using two-dimensional thin-layer chromatography and phosphorus measured as described in the Method section. Distribution was calculated from the plateau reached during hydrolysis of the phospholipids in the presence of exogenous phospholipase A2. The percent phospholipid was determined by dividing the individual lysoPC or lysoPE phosphorus by the sum of lysoPC plus PC or lysoPE plus PE. Individual values represent the mean ___ SEM (n=6 per group). *Represents p<0.01 as compared to the control group by Student's t-test, n.d., not detectable.
tubes and phosphorus assayed (1). Standards of all phospholipids and lysophospholipids were chromatographed as described above and compared to the samples of the control group and ethanol groups. The percent hydrolysis of PC, PE, and PS was calculated by dividing the individual lysophospholipid phosphorus by the sum of the lysophospholipid and phospholipid phosphorus. In some experiments, erythrocyte ghosts were prepared (10,27) and incubated with phospholipase A 2 as described above. The experiments with erythrocyte ghosts were used as a control to show that when phospholipase A 2 has access to both leaflets, a high amount of hydrolysis occurs for PC but also for PE and PS (14).
Phospholipase Treatment Erythrocytes were isolated as previously described (10,27). Packed erythrocytes (125 p,1) were suspended in glycylglycine buffer (pH 7.4) containing 100 mM KC1, 50 mM NaCI, 0.25 mM MgC12, 44 mM sucrose, 10 mM glycyiglycine, and 0.25 mM CaCI 2 (9). Phospholipase A 2 (50 U) was added to the erythrocytes and incubated for different periods of time in a shaking water bath at 37°C under conditions where the phospholipase did not penetrate into the cytofacial leaflet (9). The react.ion was stopped with 90 mM EDTA and the samples placed on ice for 5 min. An aliquot of the supernatant was removed to measure hemoglobin using a cyanmethemoglobin assay (Sigma Chemical, St. Louis, MO). Hemoglobin was measured to determine the extent of cell lysis during phospholipase A 2 incubation (9). Erythrocytes were then washed twice with isotonic TRIS (172 mM pH 7.6). The pellet was resuspended in distilled water to lyse the cells. A lipid extraction was performed with isopropanol and chloroform (16). Erythrocytes of the control and ethanol groups were incubated under identical conditions in the absence of phospholipase A 2 and served as controls for each group. The lipid extracts were dried in a micro-rotary evaporator and then brought up in 2 ml of chloroform. Phospholipids and lysophospholipids were separated using two-dimensional thinlayer chromatography (TLC) on silica gel H plates (Analtech, Newark, DE) as described previously (9). The solvent system in the first direction consisted of chloroform/methanol/glacial acetic acid/water (50:25:8:4, by vol.) and the solvent system in the second direction was chloroform/methanol/water (5:10:1 v/v/v). The lipid spots were visualized with iodine, scraped into test
RESULTS
There was significantly more PC in the exofacial leaflet as compared to PE (Table 1). This conclusion was based on the significantly higher (p<0.001) plateau reached during hydrolysis of PC as compared to the plateau of PE (Fig. 1). The amount of PC hydrolyzed plateaued after 60 min and did not differ significantly between 120 and 150 min incubation. The majority of PE appears to be located in the cytofacial leaflet. There was very little PE hydrolyzed by phospholipase A 2 in intact erythrocytes where the phospholipase was hydrolyzing phospholipids in the exofacial leaflet (Fig. 1). When the phospholipase had access to both leaflets in erythrocyte ghosts, there was a large amount of PE hydrolyzed (Table 2). PS and PI were not detected in the exofacial leaflet. The absence of PS and PI in the exofacial leaflet was consistent with an earlier study on erythrocytes from swine of a different genetic strain (14). Marked hydrolysis of PS occurred in erythrocyte ghosts when phospholipase A 2 had access to the cytofacial leaflet (Table 2). Chronic ethanol consumption altered the distribution of PC in the exofacial leaflet. The percentage of PC in the exofacial leaflet of the ethanol group was significantly higher (p<0.01) as compared to the control group (Table 1). Figure 1 shows that PC hydrolysis plateaued at a significantly higher level in the ethanol as compared to the control group. Hydrolysis of PC plateaued after 60 min for both groups. Comparisons between the amounts of PC hydrolyzed at 120 and 150 rain within each group were not statistically significant. The transbilayer distribution of PE, PS, and PI were not altered by chronic ethanol con-
ETHANOL CONSUMPTION AND TRANSBILAYER DISTRIBUTION OF PC
TABLE 2 PHOSPHOLIPIDHYDROLYSISBY PHOSPHOL[PASEA2 IN ERYTHROCYTEGHOSTSOF MINIATURESWINE
_o O_J (:b -r ~L
Incubation Time (min) Phospholipid
1
5
21.6 - 0.4 25.0 - 2.2 0.0
C)--O
Control Elhonol
•
-6"o
66.6 - 3.4 62.8 --- 3.9 23.8 --- 1.2
"
79.0 ± 8.5 72.9 ± 6.3 50.6 ± 13.1
10
Ii Hi i
PC
PE
SM
PF
PS
FIG. 2. Phospholipid composition of erythrocytes (exofacial + cytofacial) of miniature swine administered ethanol for three months as compared to control swine. Erythrocyte phospholipids were determined using two-dimensional thin layer chromatography as described in the Method section. Values are the mean __.SEM (n =6 per group) of the percent composition of each phospholipid. *p<0.01 as compared to controls. PC (phosphatidylcholine); PE (phosphatidylethanolamine); SM (sphingomyelin); PI (phosphatidylinositol); PS (phosphatidylserine).
shown in the present study that PE is primarily located in the cytofacial leaflet of erythrocytes [reviewed in (I4)]. If the phospholipase had penetrated into the cytofacial leaflet a greater amount of PE would have been hydrolyzed. Less than 10 percent of PE was hydrolyzed during the 150-rain incubation with the phospholipase using intact erythrocytes (Table 1). However, when the phospholipase had access to both leaflets in erythrocyte ghosts the amount of PC and PE hydrolyzed was approximately equal after 15 min of incubation (Table 2). Additional support for the conclusion that the phospholipase was only acting on the exofacial leaflet of intact erythrocytes was that significant hydrolysis of PS only occurred in erythrocyte ghosts as compared to intact erythrocytes (Table 2). PS distribution in the erythrocyte exofacial leaflet has been described as ranging from 0 percent to 6 percent (14). Finally, the release of hemoglobin was used as an indicator of cell lysis occurring during phospholipase incubation of intact erythrocytes (9). Lysis was less than 2 percent over the 150-min incubation period and significant differences were not observed between the ethanol and control groups. The small amount of hemoglobin released was consistent with an earlier report using similar incubation procedures (9). The data presented support the conclusion that the phospholipase did not penetrate into the cytofacial leaflet when intact erythrocytes were used and that the erythrocytes were structurally stable. Generally, chronic ethanol consumption did not significantly change bulk (exofacial + cytofacial) phospholipid composition (Fig. 2). An exception was that the percent of sphingomyelin, a predominantly exofacial phospholipid, was significantly lower (p<0.01) in the ethanol group as compared to the control group.
P C Pc
-I- 0
~_~ 2 := 01"4
I
ol 40
30-
20-
sumption. The amount of PE hydrolyzed in the exofacial leaflet did not differ significantly between the ethanol and control groups (Fig. 1). The amount of PE hydrolyzed in the exofacial leaflet did not differ significantly between the ethanol and control groups (Fig. 1). Lysophospholipids of PS and PI were not detected following incubation with phospholipase A 2 in the intact erythrocytes of the ethanol and control groups. Data on transbilayer distribution of phospholipids in the intact erythrocyte are based on the requirement that the exogenous phospholipase A 2 would act only on the exofacial leaflet and not penetrate into the cytofacial leaflet during incubation. Phospholipase A 2 did not penetrate into the cytofacial leaflet and this conclusion was supported by the following results. One method used was to examine the amount of hydrolysis of PE in the exofacial leaflet. It has been well established in other studies and
" 0 - - 0
I - ~ control ethonol
i5
Erythrocyte ghosts were incubated with phospholipase A2 under conditions where both the exofacial and cytofacial leaflets were exposed to the enzyme. Values represent the mean --- SEM of the percent of each phospholipid hydrolyzed (n = 3 membrane preparations).
4
40-
:E
% Hydrolysis PC PE PS
397
80
120
160
Incubolion Time (Min)
FIG. l. Hydrolysis of phosphatidylcholine and phosphatidylethanolamine erythrocyte exofacial leaflets of miniature swine administered ethanol for three months as compared to control swine. Erythrocytes were incubated with phospholipase A2, lipids extracted and chromatographed using two-dimensional thin layer chromatography as described in the Method section. The amount of lysophosphatidylcholine and lysophosphatidylethanolamine phosphorus was quantitated by phosphorous assay. Values represent the mean ± SEM (n = 6 per group) of phospholipids hydrolyzed in the exofacial leaflet at each time point. *p<0.05; **p<0.02 as compared to the control group.
DISCUSSION Ethanol has recently been shown to have a selective effect on the structure of the exofacial and cytofacial leaflets of membranes (18, 26, 28). The present study extended those results b7 examining effects of chronic ethanol consumption on transbilayer distribution of phospholipids in the exofacial leaflet of erythrocytes from Sinclair (S-l) miniature swine. The transbilayer distribution of PC, PE and PS in the erythrocyte exofacial leaflet of miniature swine was similar to that
398
WOOD ET AL.
reported in erythrocytes of humans, rodents and a different swine strain (14). There was more PC located in the exofacial leaflet as compared to PE. PS was not detected in the exofacial leaflet. Chronic ethanol consumption altered the transbilayer distribution of PC in the erythrocyte exofacial leaflet. There was a significant increase in the amount of PC in the exofacial leaflet of the ethanol group as compared to the control group. The transbilayer distribution of PS and PE was not changed by chronic ethanol consumption. Previous studies that have examined the effects of chronic ethanol consumption on membrane phospholipids have focused on bulk changes in membrane phospholipids (21). However, ethanol-induced changes in bulk membrane phospholipids have not been consistently observed. The present study showed that chronic ethanol consumption can alter the distribution of a specific phospholipid within the two leaflets. The changes in PC distribution were observed in the absence of significant changes in total PC composition (exofacial + cytofacial) between the ethanol and control groups. It has been previously reported that other treatments.(e.g., dietary manipulation, certain drugs) can modify membrane lipid distribution without changing the total amount of a particular lipid (7, 11, 23). The transbilayer movement of PC, PE and PS between leaflets is thought to be protein mediated [reviewed in (4)]. Evidence indicates that different proteins are involved for PC as compared to the two aminophospholipids, PS and PE (4). PE and PS have been studied most extensively and it has been reported that transport requires the presence of ATP (4). PC transport does not appear to require ATP although such a conclusion is tentative based on the small number of studies (4). Chronic ethanol consumption may specifically act on the putative PC transporter without having an effect on the PS and PE transporter. The tt[2s (rain) for PC hydrolysis did not differ significantly between the ethanol (16.83 ± 2.97) and the control (18.83--- 1.35)
groups. It has been reported that the rates of PC and PE hydrolysis were significantly less in liver microsomes of ethanoltreated rats as compared to pair-fed controls (19). The present study and the study using liver microsomes differed on several obvious factors (e.g., cell type, species, methods used to assess effects of the phospholipase). An important difference between the two studies is that the lipid composition of liver microsomes is different as compared to erythrocytes (17,22). The percent of phosphatidylinositol is higher in liver microsomes as compared to erythrocytes. The ratio of cholesterol/phospholipid is lower in liver microsomes than the ratio in erythrocytes. The differences in lipid composition between liver microsomes and erythrocytes may have an effect on phospholipid hydrolysis. An effect of increased exofacial PC induced by ethanol may be to alter cell volume. Erythrocytes of alcoholic patients generally have an increased cell volume as compared to controls (2, 3, 8, 13). Changes in transbilayer phospholipid distribution altered the shape of human erythrocytes (5, 12, 15). Increasing PE content has been found to decrease cell volume (5), Ethanolinduced changes in exofacial PC may alter erythrocyte cell volume. The exofacial and cytofacial leaflets of plasma membranes are asymmetric in fluidity, lipid distribution, electrical charge and membrane function. Results of recent studies including the present study indicate that ethanol has an asymmetric effect on membrane leaflet lipid structure. This approach is a relatively new way of viewing how ethanol acts on membranes and has the potential for linking changes in membrane lipid domains with specific membrane functions. ACKNOWLEDGEMENTS This work was supported in part by NIAAA grant AA07292 (W.G.W.) and by the Medical Research Service and the Geriatric Research, Education, and Clinical Center of the Department of Veterans Affairs.
REFERENCES 1. Bartlett, G. R. Phosphorous assay in column chromatography. J. Biol. Chem. 234:466--468; 1959. 2. Clemens, M. R.; Kessler, W.; Schied, H. W.; Schupmann, A.; Waller, H. D. Plasma and red cell lipids in alcoholics with macrocytosis. Clin. Chim. Acta 156:321-328; 1986. 3. Cooper, R. A.; Diloy-Puray, M.; Lando, P.; Greenberg, M. S. An analysis of lipoproteins, bile acids, and red cell membranes associated with target cells and spur cells in patients with liver disease. J. Clin. Invest. 51:3182-3192; 1972. 4. Devaux, P. F. Phospholipid flippases. FEBS Lett. 234(1):8-12; 1988. 5. Disc, C. A.; Goodman, D. B. P.; Rasmussen, H. Selective stimulation of erythrocyte membrane phospholipid fatty acid turnover associated with decreased cell volume. J. Biol. Chem. 255:5201-5207; 1980. 6. Foudin, L.; Tumbleson, M. E.; Sun, A. Y.; Geisler, R. W.; Sun, G. Y. Ethanol consumption and serum lipid profiles in Sinclair (S-I) miniature swine. Life Sci. 34:819-826; 1984. 7. Gazitt, Y.; Loyter, A.; Ohad, I. Induction of ATP depletion, intrarnembrane particle aggregation and exposure of membrane phospholipids in chicken erythrocytes by local anesthetics and tranquilizers. Biochim. Biophys. Acta 471:361-371; 1977. 8. Halsall, S.; Peters, T. J. Erythrocyte and leukocyte lipids in alcoholic maerocytosis. Clin. Chem. Acta 149:165-173; 1985. 9. Jain, S. K. In vivo externalization of phosphatidylserine and phosphatidylethanolamine in the membrane bilayer and hypercoagulability by the lipid peroxidation of erythrocytes in rats. J. Clin. Invest. 76:281-286; 1985. 10. Kelly-Murphy, S.; Waring, A. J.; Rottenberg, H.; Rubin, E. Effects of chronic ethanol consumption on the partition of lipophilic compounds into erythrocyte membranes. Lab. Invest. 50:174-180; 1983.
11. Kuypers, F. A.; Abraham, S.; Lubin, B.; Chiu, D. Diet-induced asymmetry of the phosphatidylcholine fatty acid composition in rat erythrocytes. L Lab. Clin. Med. 111:529-536; 1988. 12. Kuypers, F. A.; Roelofsen, B.; Berendsen, W.; Op den Kamp, J. A. F.; van Deenen, L. L. M. Shape changes in human erythrocytes induced by replacement of the native phosphatidylcholine with species containing various fatty acids. J. Cell Biol. 99:2260-2267; 1984. 13. La Droitle, P.; Lamboeuf, Y.; de Saint Blanquat, G.; Bezaury. J. Sensitivity of individual erythrocyte membrane phospholipids to changes in fatty acid composition in chronic alcohol patients. Alcohol.: Clin. Exp. Res. 9:135-137; 1984. 14. Roelofsen, B. Phospholipases as tools to study the localization of phospholipids in biological membranes. A critical review. J. Toxicol. 1(1):87-197; 1982. 15. Roelofsen, B.; Kuypers, F. A.; Op den Kamp, J. A. F.; van Deenen, L. L. M. Influence of phosphatidylcholine molecular species composition on stability of the erythrocyte membrane. Biochem. Soc. Trans. 17:284-286; 1989. 16. Rose, H. G.; Oklander, M. Improved procedure for the extraction of lipids from human erythrocytes, i. Lipid Res. 6:428---431; 1965. 17. Rouser, G.; Kritchevsky, G. Lipids in the nervous system of different species as a function of age: brain, spinal cord, peripheral nerve, purified whole cell preparations and subcellular particulates:regulatory mechanisms and membrane structure. Adv. Lipid Res. 10:261361; 1972. 18. Schroeder, F.; Morrison, W. J.; Gorka, C.; Wood, W. G. Transbilayer effects of ethanol on fluidity of brain membrane leaflets. Biochim. Biophys. Acta 946:85-94; 1988. 19. Stubbs, C. D.; Williams, B. W.; Pryor, C. L.; Rubin, E. Ethanolinduced modifications to membrane lipid structure: effect of phospholipid A2-membrane interactions. Arch. Biochem. Biophys. 262(20):
E T H A N O L C O N S U M P T I O N A N D T R A N S B I L A Y E R DISTRIBUTION OF PC
561)-573; 1988. 20. Sun, A. Y.; Tumbleson, M.; Dexter, J.; Sun, G. Y. Miniature swine as an animal model for alcohol research. In: Sun, G. Y.; Rudeen, P. K.; Wood, W. G.; Wei, Y. H.; Sun, A. Y., eds. Molecular mechanisms of alcohol. Clifton, NJ: Humana Press; 1989: 371-388. 21. Sun, G. Y.; Sun, A. Y. Ethanol and membrane lipids. Alcohol.: Clin. Exp. Res. 9(2):164-180; 1985. 22. Sutter, M. A.; Wood, W. G.; Williamson, L. S.; Strong, R.; Pickham, K.; Richardson, A. Comparison of the hepatic mixed function oxidase system of young, adult, and old non-human primates (Macaca nemestrina). Biochem. Pharmacol. 34:2983-2987; 1985. 23. Tamura, A.; Moriwaki, N.; Fujii, T. Disturbing effect of cationic a.mphiphilic drugs on phospholipid asymmetry of the membrane lipid bilayer of human erythrocytes. Chem. Pharm. Bull. (Tokyo) 31:1692-1697; 1983. 24. Treistman, S.; Moynihan, M. M.; Wolf, D. Influence of alcohols,
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temperature, and region on the mobility of lipids in neuronal membrane. Biochim. Biophys. Acla 898:109-120; 1987. Treistman, S.; Wilson, A. Alkanol effects on early potassium currents in Aplysia neurons depend on chain length. Proc. Nad. Acad. Sci. USA 84:9299-9303; 1987. Wood, W. G.; Gorka, C.; Schroeder, F. Acute and chronic effects of ethanol on transbilayer membrane domains. J. Neurochem. 52: 1925-1930; 1989. Wood, W. G.; Lahiri, S.; Gorka, C.; Armbrecht, H. J.; Strong, R. In vitro effects of ethanol on erythrocyte membrane fluidity of alcoholic patients: An electron spin resonance study. Alcohol.: Clin. Exp. Res. 11:332-335; 1987. Wood, W. G.; Schroeder, F.; Hogy, L.; Ran, A. M.; Nemecz, G. Asymmetric distribution of a fluorescent sterol in synaptic plasma membranes: Effects of chronic ethanol consumption. Biochim. Biophys. Acta 1025:243-246; 1990.
0741-8329/91 $3.00 + .00
Chronic Ethanol ConsumptiOn Alters Transbilayer Distribution of Phosphatidylcholine in Erythrocytes of Sinclair (S-I) Miniature Swine W . G I B S O N W O O D , *l C H R I S T I N E G O R K A , t J U L I E A. JOHNSON,'I" G R A C E Y, SUN,:~ A L B E R T Y. SUN~: A N D F R I E D H E L M S C H R O E D E R §
*Geriatric Research, Education and Clinical Center, Veterans Administration Medical Center and Department of Pharmacology, University of Minnesota, School of Medicine, Minneapolis, MN 55417 :'Veterans Administration Medical Center, St. Louis, MO 63125 .::Sinclair Comparative Medicine Research Farm and Department of Biochemistry University of Missouri, Columbia, MO 65203 §Department of Pharmacology and Medicinal Chemistry, College of Pharmacy, and Department of Pharmacology and Cell Biophysics, College.of Medicine, University of Cincinnati Medical Center, Cincinnati, OH 45267-0004 R e c e i v e d 6 D e c e m b e r 1990; A c c e p t e d 1 M a r c h 1991 WOOD, W. G., C. GORKA, J. A. JOHNSON, G. Y. SUN, A. Y. SUN AND F. SCHROEDER. Chronicethanol consumption alters transbilayerdistribution of phosphatidylcholine in er3,throcytesof Sinclair (S-1) miniatureswine. ALCOHOL 8(5) 395-399, 1991.--Effects of chronic ethanol consumption on transbilayer distribution of phospholipids in the exofacial and cytofacial leaflets of erythrocytes from chronic ethanol-consuming Sinclair (S-l) miniature swine were examined. Phosphatidylcholine (PCI was predominantly located in the exofacial leaflet and phosphatidylethanolamine (PE) and phosphatidylserine (PS) located primarily in the cytofacial leaflet. Chronic ethanol consumption significantly increased PC content in the exofacial leaflet without changing bulk membrane PC composition. Ethanol-induced changes in PC distribution were specific for PC and not detected in PE or PS. There was also a significant decrease in sphingomyelin in the ethanol group. Sphingomyelin is primarily an exofacial phospholipid. The specific ethanol-induced changes in the exofacial leaflet are consistent with recent studies showing that the exofacial membrane leaflet is more susceptible to effects of ethanol as compared to the cytofacial leaflet. Such specificity of action provides a new way of viewing how ethanol alters membrane structure and function. Chronic ethanol consumption Ethanol tolerance Lipid domains Phosphatidylcholine Membrane asymmetry Ethanol
Phospholipids
Erythrocytes
ing observed in bulk membrane fluidity and total cholesterol content. It has not been determined, however, whether chronic ethanol consumption may also modify the distribution of phospholipids in the exofacial and cytofacial leaflets. Phosphatidylcholine (PC) and sphingomyelin (SM) are considered exofacial phospholipids and phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI) are cytofacial phospholipids (14). Chronic ethanol consumption may alter the distribution of one or more phospholipids in the exofacial and cytofacial leaflets without changing the total amount of an individual phospholipid. Changes in transbilayer phospholipid distribution in erythrocytes have been reported as a result of treatment with specific drugs and other agents. The oxidant drug phenylhydrazine resulted in the transbilayer movement of PE and PS from the cytofacial to the exofacial leaflet in erythrocytes (9). Certain cationic amphiphilic drugs were found to alter PE and PC membrane distribution (7,23). Acyl group distribution of PC in. the exofacial leaflets of erythrocytes was found to be modified by
ALCOHOLS, including ethanol, have a specific effect on lipid domains of biological membranes (18, 24--26, 28). Two domains that have been examined are the membrane exofacial and cytofacial leaflets (18, 26, 28). Ethanol in vitro decreased the limiting anisotropy of diphenylhexatriene in the exofacial leaflet of synaptic plasma membranes (SPM) more than in the cytofacial leaflet (18). The in vitro effects of ethanol were reduced in the SPM exofacial leaflet of chronic ethanol-treated animals (26). It also was observed that baseline fluidity (i.e., in the absence of ethanol in vitro) of the two leaflets was altered in chronic ethanol-consuming animals. The exofacial leaflet became less fluid whereas the cytofacial leaflet became more fluid in SPM of chronic ethanol-consuming animals. In a subsequent study it was shown that there was a redistribution of cholesterol between the two SPM leaflets in chronic ethanol-treated animals (28). Those studies demonstrated that the exofacial leaflet was more susceptible t o the effects of ethanol and that specific changes could occur in membrane structure without changes be-
~Requests for reprints should be addressed to W. Gibson Wood, Ph.D., VA Medical Center, GRECC, 1 Veterans Drive, Minneapolis, MN 55417.
395
396
WOOD ET AL.
TABLE 1
diets that differed in fat content (11). It was the purpose of this study to determine if chronic ethanol consumption would modify the transbilayer distribution of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine in erythrocyte membranes of Sinclair (S-I) miniature swine.
PHOSPHOLIPID DISTRIBUTION IN THE EXOFACIAL LEAFLETS OF ERYTHROCYTES FROM CONTROL AND CHRONIC ETHANOL-CONSUMING SWINE
% Phospholipid in Exofacial Leaflet
Group
PC
PE
PS
PI
Control Ethanol
56.7 --- 2.5 66.9 _ 1.5"
7.2 __. 1.9 7.5 +- 2.9
n.d. n.d.
n.d. n.d.
METHOD
Chemicals All chemicals and reagents were purchased from Sigma Chemical Company, St, Louis, MO. Phospholipase A 2 from Naja mocambique mocambique (pI = 9.6) was used.
Ethanol Administration Adult Sinclair (S-I) miniature boars (12 months of age) with a mean body weight of 90 kg were used (Sinclair Comparative Medicine Research Farm, University of Missouri, Columbia, MO), Animals were given daily rations of a formulated diet containing corn, soybean meal, meat meal, alfalfa meal, cornstarch, salts and vitamin supplement as described previously (6,20). The ethanol group (n = 6) received 6 g/kg/day of a beer solution fortified to 10% ethanol for three months (6,20). The ethanol solution was administered in graduated bottles containing a drinking tube and the total amount of the ethanol solution administered was consumed each day. Animals also had free access to water. Control animals ( n = 6 ) were given the same dietary ration and also received glucose in water to approximate the calories supplied by ethanol. Ten ml of blood was drawn from the jugular vein of each animal and collected in heparinized tubes between 10 a.m. and 11 a.m. Animals had not been withdrawn from ethanol prior to the time when blood samples were taken.
Erythrocytes were incubated with phospholipase Az under nonpenetrating conditions, lipids extracted and chromatographed using two-dimensional thin-layer chromatography and phosphorus measured as described in the Method section. Distribution was calculated from the plateau reached during hydrolysis of the phospholipids in the presence of exogenous phospholipase A2. The percent phospholipid was determined by dividing the individual lysoPC or lysoPE phosphorus by the sum of lysoPC plus PC or lysoPE plus PE. Individual values represent the mean ___ SEM (n=6 per group). *Represents p<0.01 as compared to the control group by Student's t-test, n.d., not detectable.
tubes and phosphorus assayed (1). Standards of all phospholipids and lysophospholipids were chromatographed as described above and compared to the samples of the control group and ethanol groups. The percent hydrolysis of PC, PE, and PS was calculated by dividing the individual lysophospholipid phosphorus by the sum of the lysophospholipid and phospholipid phosphorus. In some experiments, erythrocyte ghosts were prepared (10,27) and incubated with phospholipase A 2 as described above. The experiments with erythrocyte ghosts were used as a control to show that when phospholipase A 2 has access to both leaflets, a high amount of hydrolysis occurs for PC but also for PE and PS (14).
Phospholipase Treatment Erythrocytes were isolated as previously described (10,27). Packed erythrocytes (125 p,1) were suspended in glycylglycine buffer (pH 7.4) containing 100 mM KC1, 50 mM NaCI, 0.25 mM MgC12, 44 mM sucrose, 10 mM glycyiglycine, and 0.25 mM CaCI 2 (9). Phospholipase A 2 (50 U) was added to the erythrocytes and incubated for different periods of time in a shaking water bath at 37°C under conditions where the phospholipase did not penetrate into the cytofacial leaflet (9). The react.ion was stopped with 90 mM EDTA and the samples placed on ice for 5 min. An aliquot of the supernatant was removed to measure hemoglobin using a cyanmethemoglobin assay (Sigma Chemical, St. Louis, MO). Hemoglobin was measured to determine the extent of cell lysis during phospholipase A 2 incubation (9). Erythrocytes were then washed twice with isotonic TRIS (172 mM pH 7.6). The pellet was resuspended in distilled water to lyse the cells. A lipid extraction was performed with isopropanol and chloroform (16). Erythrocytes of the control and ethanol groups were incubated under identical conditions in the absence of phospholipase A 2 and served as controls for each group. The lipid extracts were dried in a micro-rotary evaporator and then brought up in 2 ml of chloroform. Phospholipids and lysophospholipids were separated using two-dimensional thinlayer chromatography (TLC) on silica gel H plates (Analtech, Newark, DE) as described previously (9). The solvent system in the first direction consisted of chloroform/methanol/glacial acetic acid/water (50:25:8:4, by vol.) and the solvent system in the second direction was chloroform/methanol/water (5:10:1 v/v/v). The lipid spots were visualized with iodine, scraped into test
RESULTS
There was significantly more PC in the exofacial leaflet as compared to PE (Table 1). This conclusion was based on the significantly higher (p<0.001) plateau reached during hydrolysis of PC as compared to the plateau of PE (Fig. 1). The amount of PC hydrolyzed plateaued after 60 min and did not differ significantly between 120 and 150 min incubation. The majority of PE appears to be located in the cytofacial leaflet. There was very little PE hydrolyzed by phospholipase A 2 in intact erythrocytes where the phospholipase was hydrolyzing phospholipids in the exofacial leaflet (Fig. 1). When the phospholipase had access to both leaflets in erythrocyte ghosts, there was a large amount of PE hydrolyzed (Table 2). PS and PI were not detected in the exofacial leaflet. The absence of PS and PI in the exofacial leaflet was consistent with an earlier study on erythrocytes from swine of a different genetic strain (14). Marked hydrolysis of PS occurred in erythrocyte ghosts when phospholipase A 2 had access to the cytofacial leaflet (Table 2). Chronic ethanol consumption altered the distribution of PC in the exofacial leaflet. The percentage of PC in the exofacial leaflet of the ethanol group was significantly higher (p<0.01) as compared to the control group (Table 1). Figure 1 shows that PC hydrolysis plateaued at a significantly higher level in the ethanol as compared to the control group. Hydrolysis of PC plateaued after 60 min for both groups. Comparisons between the amounts of PC hydrolyzed at 120 and 150 rain within each group were not statistically significant. The transbilayer distribution of PE, PS, and PI were not altered by chronic ethanol con-
ETHANOL CONSUMPTION AND TRANSBILAYER DISTRIBUTION OF PC
TABLE 2 PHOSPHOLIPIDHYDROLYSISBY PHOSPHOL[PASEA2 IN ERYTHROCYTEGHOSTSOF MINIATURESWINE
_o O_J (:b -r ~L
Incubation Time (min) Phospholipid
1
5
21.6 - 0.4 25.0 - 2.2 0.0
C)--O
Control Elhonol
•
-6"o
66.6 - 3.4 62.8 --- 3.9 23.8 --- 1.2
"
79.0 ± 8.5 72.9 ± 6.3 50.6 ± 13.1
10
Ii Hi i
PC
PE
SM
PF
PS
FIG. 2. Phospholipid composition of erythrocytes (exofacial + cytofacial) of miniature swine administered ethanol for three months as compared to control swine. Erythrocyte phospholipids were determined using two-dimensional thin layer chromatography as described in the Method section. Values are the mean __.SEM (n =6 per group) of the percent composition of each phospholipid. *p<0.01 as compared to controls. PC (phosphatidylcholine); PE (phosphatidylethanolamine); SM (sphingomyelin); PI (phosphatidylinositol); PS (phosphatidylserine).
shown in the present study that PE is primarily located in the cytofacial leaflet of erythrocytes [reviewed in (I4)]. If the phospholipase had penetrated into the cytofacial leaflet a greater amount of PE would have been hydrolyzed. Less than 10 percent of PE was hydrolyzed during the 150-rain incubation with the phospholipase using intact erythrocytes (Table 1). However, when the phospholipase had access to both leaflets in erythrocyte ghosts the amount of PC and PE hydrolyzed was approximately equal after 15 min of incubation (Table 2). Additional support for the conclusion that the phospholipase was only acting on the exofacial leaflet of intact erythrocytes was that significant hydrolysis of PS only occurred in erythrocyte ghosts as compared to intact erythrocytes (Table 2). PS distribution in the erythrocyte exofacial leaflet has been described as ranging from 0 percent to 6 percent (14). Finally, the release of hemoglobin was used as an indicator of cell lysis occurring during phospholipase incubation of intact erythrocytes (9). Lysis was less than 2 percent over the 150-min incubation period and significant differences were not observed between the ethanol and control groups. The small amount of hemoglobin released was consistent with an earlier report using similar incubation procedures (9). The data presented support the conclusion that the phospholipase did not penetrate into the cytofacial leaflet when intact erythrocytes were used and that the erythrocytes were structurally stable. Generally, chronic ethanol consumption did not significantly change bulk (exofacial + cytofacial) phospholipid composition (Fig. 2). An exception was that the percent of sphingomyelin, a predominantly exofacial phospholipid, was significantly lower (p<0.01) in the ethanol group as compared to the control group.
P C Pc
-I- 0
~_~ 2 := 01"4
I
ol 40
30-
20-
sumption. The amount of PE hydrolyzed in the exofacial leaflet did not differ significantly between the ethanol and control groups (Fig. 1). The amount of PE hydrolyzed in the exofacial leaflet did not differ significantly between the ethanol and control groups (Fig. 1). Lysophospholipids of PS and PI were not detected following incubation with phospholipase A 2 in the intact erythrocytes of the ethanol and control groups. Data on transbilayer distribution of phospholipids in the intact erythrocyte are based on the requirement that the exogenous phospholipase A 2 would act only on the exofacial leaflet and not penetrate into the cytofacial leaflet during incubation. Phospholipase A 2 did not penetrate into the cytofacial leaflet and this conclusion was supported by the following results. One method used was to examine the amount of hydrolysis of PE in the exofacial leaflet. It has been well established in other studies and
" 0 - - 0
I - ~ control ethonol
i5
Erythrocyte ghosts were incubated with phospholipase A2 under conditions where both the exofacial and cytofacial leaflets were exposed to the enzyme. Values represent the mean --- SEM of the percent of each phospholipid hydrolyzed (n = 3 membrane preparations).
4
40-
:E
% Hydrolysis PC PE PS
397
80
120
160
Incubolion Time (Min)
FIG. l. Hydrolysis of phosphatidylcholine and phosphatidylethanolamine erythrocyte exofacial leaflets of miniature swine administered ethanol for three months as compared to control swine. Erythrocytes were incubated with phospholipase A2, lipids extracted and chromatographed using two-dimensional thin layer chromatography as described in the Method section. The amount of lysophosphatidylcholine and lysophosphatidylethanolamine phosphorus was quantitated by phosphorous assay. Values represent the mean ± SEM (n = 6 per group) of phospholipids hydrolyzed in the exofacial leaflet at each time point. *p<0.05; **p<0.02 as compared to the control group.
DISCUSSION Ethanol has recently been shown to have a selective effect on the structure of the exofacial and cytofacial leaflets of membranes (18, 26, 28). The present study extended those results b7 examining effects of chronic ethanol consumption on transbilayer distribution of phospholipids in the exofacial leaflet of erythrocytes from Sinclair (S-l) miniature swine. The transbilayer distribution of PC, PE and PS in the erythrocyte exofacial leaflet of miniature swine was similar to that
398
WOOD ET AL.
reported in erythrocytes of humans, rodents and a different swine strain (14). There was more PC located in the exofacial leaflet as compared to PE. PS was not detected in the exofacial leaflet. Chronic ethanol consumption altered the transbilayer distribution of PC in the erythrocyte exofacial leaflet. There was a significant increase in the amount of PC in the exofacial leaflet of the ethanol group as compared to the control group. The transbilayer distribution of PS and PE was not changed by chronic ethanol consumption. Previous studies that have examined the effects of chronic ethanol consumption on membrane phospholipids have focused on bulk changes in membrane phospholipids (21). However, ethanol-induced changes in bulk membrane phospholipids have not been consistently observed. The present study showed that chronic ethanol consumption can alter the distribution of a specific phospholipid within the two leaflets. The changes in PC distribution were observed in the absence of significant changes in total PC composition (exofacial + cytofacial) between the ethanol and control groups. It has been previously reported that other treatments.(e.g., dietary manipulation, certain drugs) can modify membrane lipid distribution without changing the total amount of a particular lipid (7, 11, 23). The transbilayer movement of PC, PE and PS between leaflets is thought to be protein mediated [reviewed in (4)]. Evidence indicates that different proteins are involved for PC as compared to the two aminophospholipids, PS and PE (4). PE and PS have been studied most extensively and it has been reported that transport requires the presence of ATP (4). PC transport does not appear to require ATP although such a conclusion is tentative based on the small number of studies (4). Chronic ethanol consumption may specifically act on the putative PC transporter without having an effect on the PS and PE transporter. The tt[2s (rain) for PC hydrolysis did not differ significantly between the ethanol (16.83 ± 2.97) and the control (18.83--- 1.35)
groups. It has been reported that the rates of PC and PE hydrolysis were significantly less in liver microsomes of ethanoltreated rats as compared to pair-fed controls (19). The present study and the study using liver microsomes differed on several obvious factors (e.g., cell type, species, methods used to assess effects of the phospholipase). An important difference between the two studies is that the lipid composition of liver microsomes is different as compared to erythrocytes (17,22). The percent of phosphatidylinositol is higher in liver microsomes as compared to erythrocytes. The ratio of cholesterol/phospholipid is lower in liver microsomes than the ratio in erythrocytes. The differences in lipid composition between liver microsomes and erythrocytes may have an effect on phospholipid hydrolysis. An effect of increased exofacial PC induced by ethanol may be to alter cell volume. Erythrocytes of alcoholic patients generally have an increased cell volume as compared to controls (2, 3, 8, 13). Changes in transbilayer phospholipid distribution altered the shape of human erythrocytes (5, 12, 15). Increasing PE content has been found to decrease cell volume (5), Ethanolinduced changes in exofacial PC may alter erythrocyte cell volume. The exofacial and cytofacial leaflets of plasma membranes are asymmetric in fluidity, lipid distribution, electrical charge and membrane function. Results of recent studies including the present study indicate that ethanol has an asymmetric effect on membrane leaflet lipid structure. This approach is a relatively new way of viewing how ethanol acts on membranes and has the potential for linking changes in membrane lipid domains with specific membrane functions. ACKNOWLEDGEMENTS This work was supported in part by NIAAA grant AA07292 (W.G.W.) and by the Medical Research Service and the Geriatric Research, Education, and Clinical Center of the Department of Veterans Affairs.
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