Alterations in Phospholipid Composition in Ethanol Tolerance and Dependence John M. Littleton, B.Sc., M.B.B.S., Ph.D., Geryk Mammalian cells may retain a limited capaclty to alter membrane phosphollpld fatty acid composition. This may play a role in adaptatlon to the presence of ethanol (cellular tolerance) and In the response to the removal of ethanol and the withdrawal syndrome.
LCOHOLS and general anesthetics may A have a common mechanism of action at the cellular level. Both groups can be shown to increase the fluid nature of biologic and model membranes,' and this may be the basis of the central depressant properties of these drugs. Recently, Chin and Goldstein2showed that ethanol, in concentrations associated with intoxication rather than anesthesia, can induce increased fluidity in cell membranes obtained from mice. Using a spin-label technique, these investigators showed that addition of ethanol in vitro increased the physical fluidity of membranes from brain synaptosomes, mitochondria, and erythrocytes. If the mechanism of action of ethanol at the cellular level is by alteration of synaptic membrane fluidity, then this may have implications for tolerance and dependence on ethanol. Changes in temperature also can produce alterations in membrane fluidity; poikilotherms are known' to adapt to alterations in environmental temperature by changing the intrinsic fluidity of their cell membranes in the opposite direction to that induced by the temperature change. The mechanism of this change is thought to be primarily by alteration of membrane phospholipid fatty acid composition. As an example of this process Cossins' has recently shown that synaptic membranes of brain from warm-acclimated goldfish are less fluid than those of cold-acclimated fish when measured at an inter-
From the Department of Pharmacology, King's College, London, England. Supported by grants from the Medical Council on Alee holism and the Mental Health Research Foundation. Reprint request should be addressed to John M.Littleton. Ph.D.. Department of Pharmacology, King's College, London WCZR 213. England. 1979 by Grune & Stratton, Inc. 0145-6008/79/0301-0011$01.00/0
50
R. John, B.Sc., M.Sc., and Susan J. Grieve, B.Sc.
mediate temperature. This reduced intrinsic fluidity was correlated to a decreased proportion of polyunsaturated fatty acids in membrane phospholipids. The major change was in the choline phosphogylcerides; the cholesterol content of the synaptic membrane was unaffected. There is also evidence that unicellular organisms can adapt to pharmacologic agents by a similar mechanism. Thus Nandini-Kishore, Kitajuna, and Thompson' have demonstrated that Tetrahymena pyriformis responds to the membrane-fluidizing effects of the general anesthetic methoxyflurane by decreasing fatty acid desaturase activity in much the same fashion as is observed in response to increased temperature. If such organisms can adapt to the presence of fluidizing agents by altering membrane composition, it seems possible that a similar mechanism may occur in mammals and may underlie the development of cellular tolerance to ethanol. Hill and Banghams proposed such a mechanism for the cellular basis of tolerance to central depressants and extended the argument to include a hypothesis for physical dependence based on alterations in intrinsic membrane fluidity. Since then, work has appeared that partly supports their suggestion with respect to the development of tolerance to ethanol. In 1976, Ingram6 showed that bacteria, grown in media containing alcohols, showed an alteration in phospholipid fatty acid composition. The results were puzzling in that only relatively long-chain alcohols produced the expected decrease in unsaturated fatty acids. Short-chain alcohols, such as ethanol, produced an increase in membrane phospholipid unsaturated fatty acids. This paradox is still unresolved, although Ingram suggested that short-chain alcohols may reduce the fluidity of bacterial membranes, but this is at variance with the results obtained on mammalian membranes cited earlier.2 There may be some fundamental difference between the response to ethanol of bacterial membrane phospholipids, which contain a far lower proportion of polyunsaturated fatty acids, and the membranes of higher organisms. However, Ingram's work does demonstrate clearly that alteration of
Alcoholism: Clinical and Experimental Research. Vol. 3. No. 1 (January). 1879
PHOSPHOLIPID COMPOSITION
membrane fatty acid composition can occur in response to the continued presence of alcohols. Hill and Bangham's hypothesisSsuggests that tolerance and physical dependence on central depressant drugs should be associated with a reduction in the intrinsic fluidity of membranes from the dependent organism. Chin and Goldstein' investigated the fluidity of membranes from mice of the DBA strain that had been rendered tolerant and physically dependent on ethanol. Although they found that cell membranes (including synaptic membranes) from these animals were resistant to the in vitro fluidizing effects of ethanol, they were unable to detect any differences between the intrinsic fluidity of membranes from control mice and those tolerant and dependent on ethanol. Their work therefore supports the Hill and Bangham hypothesisS to the extent that some form of ethanol tolerance does appear to reside in the cell membrane, but does not support the idea that tolerance and dependence are accompanied by a reduction in the intrinsic fluidity of the cell membrane. As Chin and Goldstein point out, their failure to detect a difference in intrinsic fluidity may reflect methodological inadequacy to detect small differences between groups where variation is large. An alternative explanation is that the strain of mice used show rather unusual properties with regard to the development of tolerance and dependence on ethanol and may not be suitable for demonstration of alterations in membrane fluidity. This point will be returned to in the Discussion section. The effect of continuous ethanol administration to mice of the T O Swiss strain was investigated by Littleton and John.s In these preliminary experiments, we found a small but significant reduction in polyunsaturated fatty acids from phospholipids of the crude synaptosomal fraction of brains from ethanol-tolerant and dependent mice. It was suggested that, under the conditions of these experiments, an adaptive alteration in membrane phospholipid composition occurred in response to the continuous administration of ethanol. The experiments described here extend these observations to other tissues of ethanol-dependent mice and offer preliminary findings on the time course of the changes observed and their localization to different synaptosomal fractions.
51
MATERIALS AND METHODS The animals used in these experiments were young adult male mice of the TO Swiss strain, 20-22 g in weight, obtained from L.A.C. Dagenham, Essex. Administration of ethanol was by inhalation in the apparatus described previously.9 In all experiments mice had free access to food (41 B diet) and water. Mice described as being physically dependent on ethanol were exposed to blood concentrations of ethanol between 2 and 3 mg/ml-' for 10 days before sacrifice (whole body immersion in liquid nitrogen). In our experience, this produces signs of physical withdrawal in all animals of this strain when ethanol is removed. In some experiments, mice were exposed to very high concentrations of ethanol vapor for shorter periods. A concentration of 30 mg/liter-' ethanol in air produced a blood ethanol concentration of approximately 2 mg/ml-' in 2 hr; animals exposed in this way were used to investigate the short-term effects of ethanol. In a subsequent experiment. mice were first injected with 2.5 g/kg-' ethanol i.p. and then exposed intermittently to ethanol vapor, 25 mg/liter-' for 5 hr. This produced blood ethanol concentrations in the range of 3 - 6 mg/ml-'. The last regimen was chosen because it had been shown'O to produce rapid and marked cellular tolerance to ethanol. The fatty acid composition of phospholipids was analyzed in essentially the same way described by Littleton and John.' Briefly, the method consists of Folch extraction of membrane lipids, separation of phospholipids onto a slurry of activated silicic acid, hydrolysis and saponification of phospholipids with sodium hydroxide, and methylesterification of fatty acids with boron trifluoride/methanol reagent. The chloroform extract of methylesterified fatty acids is then analyzed by gas liquid chromatography on a polyethylene glycol adipate column. Quantification of peak areas was by electronic-integration. When subcellular fractions from sucrose density gradient centrifugation (see below) were to be extracted, at least two washes in chloroform/methanol were performed. For whole tissue, such as heart and liver, the tissue was initially weighed, sliced. and then homogenized (glass: Teflon Potter homogenizer) directly in chloroform/methanol. For separation of subcellular fractions of brain, the following methods were used: Either single brains (for crude synaptosomal fraction) or pooled brains of 4 animals (for sucrose density gradient fractionation) were homogenized in 10 volumes of ice-cold 0.32M sucrose using a motor-driven homogenizer. The homogenate was centrifuged at lOOOg ( 2 0 min) and the pellet discarded. The supernatant was centrifuged at 20,OOOg for 30 min to obtain the crude synaptosomal (P2)fraction. The pellet was resuspended in ice-cold 0.32M sucrose before layering onto the surface of the sucrose density gradient described below (see Fig. 1). A 4-step sucrose density gradient was prepared in transparent 25 ml centrifuge tubes by layering on sucrose of concentrations 1.4, 1.2. 1 .O, and 0.8M with a roller pump (Fig. 1). Gradients were spun a t 100,OOOg for 2 hr in a 3 X 25 swing-out head. Four fractions were formed at the interfaces of the sucrose layers and a mitochondria1 pellet also separated (Fig. 1). The fractions were pumped off using a roller pump, starting with fraction C, and stored on ice prior to Folch extraction. The mitochondria1 pellet was resuspended in 2 ml of 0.32M sucrose before extraction.
LITTLETON, JOHN, AND GRIEVE
52
le
H
-I.OM
SYNAPTOSOMES
-1.2M
I
U
c
ID
q I . 4 M
MITOCHONDRIA-
e e lOO.Oo0 G. 2HR. Fig. 1.
Sucrose density fractions.
In all experiments, control animals were kept in the same conditions as experimental animals but with ethanol absent from the inspired air. In experiments where ethanol was injected, isotonic saline was used as a control injcction. All biochemical procedures were carried out on a paired basis (test sample versus control sample) and results were analyzed using a paired Student's t test. RESULTS
Fatty Acid Composition of Phospholipids from Tissues of Ethanol-Dependent Mice The continuous administration of ethanol by inhalation for 10 days produced alterations in the fatty acid composition of brain (crude synaptosomal fraction), heart, and liver. These results are shown in Table 1 . Table 1 shows that there are similarities in the ethanol-induced change in phospholipid composition on brain and heart. In both there is a reduction in polyunsaturated fatty acids. In the brain there is a corresponding increase in saturated and mono-unsaturated fatty acids, whereas
in the heart the increase is in oleic (18:l) and linoleic (18:2) acids. The changes in the liver seem rather different in that there is a tendency to decrease in all fatty acids except oleic (18:l) and linoleic (18:2). There is in fact a marked and significant increase in the proportion of oleic acid from hepatic phospholipids. When recovery of phospholipids from these tissues was estimated by comparing the total integrated counts of fatty acids obtained, it was observed that there was an increase in the phospholipids obtained from liver of ethanol-dependent animals. The change, expressed as concentration per liver net weight, was estimated to be from 2.2 f 0.28 mg/g-' fatty acids from phospholipids (control, N=5)to 3.9 k0.60 mg/g-' fatty acids from phospholipids (ethanol-dependent, N = 5 ) . No significant change in phospholipid concentration was observed in brain or heart. Time Course of Changes in Fatty Acid Composition of Tissue Phospholipids during Continuous Administration of Ethanol These experiments were begun in order to establish whether the change in brain occurred only after the chronic administration of ethanol. It was found that administration of ethanol at high concentrations for only 2 hr could produce a change of only slightly less magnitude than the change produced by administration for 10 days (see Table 2). In the heart, no alteration in cardiac phospholipids was seen after 2 hr; and after ethanol administration in high concentrations for 10 hr, only a small alteration in polyunsaturated fatty acids was observed (Table 2).
Table 1. Fatty Acid Composition of Phospholipids from Tissues of Ethanol-Dependent Mice (TO Swiss Strain) Brain (P, Fraction)
Liver
Heart
-
Fatty Acid
Control N - 7
Dependent N 10
Control N - 5
Dependent N - 5
Control N - 5
Dependent N - 5
16:O 16:1 18:O 18:1 18:2 20:1 20:4 22:4 22:6
24.2 f 0.9
25.4f 1.1
22.6 f 0.3 18.2 f 0.5 1 .O f 0.3 2.1 f 0.1 9.9 f 0.2 1.9f 0.1 14.5f 0.6
24.4' f 0.6 19.3f 0.5 1.3 f 0.2 2.2f 0.3 8.0' f 0.3 1.7f 0.1 11.9f 1.4
17.9 f 1.3 0.9f 0.1 20.4f 0.2 13.4f 0.4 15.6 f 0.5
18.5 + 1.9 2.3 f 0.7 19.0f 1.1 18.0' + 0.6 19.3' f 0.8
30.5 f 2.4 2.2 f 0.3 16.1 f 0.4 19.Of 1.5 16.8f 1.1
22.2' f 1.1 2.3 f 0.7 14.9 f 1.1 26.9' f 1.6 18.6 f 1.3
4.5 f 0.5
-
10.7 f 2.0
8.5f 1.3
24.3 f 1.6
13.9' f 1.0
5.5f 0.7
5.3 f 0.9
5.8 f 0.5
-
-
-
The main fatty acids present in phospholipids of mouse tissue are shown in the lefthand column: figures represent chain 1ength:number of double bonds. Values given in other columns represent the mean percentage (+ standard error) of the total fatty acids present. *Indicates a value significantly ( p < 0.05in paired Student's t test) different from the appropriate control.
PHOSPHOLIPID COMPOSITION
53
Table 2. Time Course of Alteration of Fatty Acid Composition of Brain and Heart by Continuous Administration of Ethanol to Mice (TO Swiss Strain) ~ ~ _ _ _ _ ~
Brain (PI Fraction)
-
Ethanol 2 hr N - 5
Fatty Acid
Control N 7
16:O 16:l 18:O 18:1 18:2 2O:l 20:4 22:4 22:6
24.2 f 0.9
22.6 f 0.3 18.2 0.5 1 .O f- 0.3 2.1 f 0.1 9.9 f 0.2 1.9 0.1 14.5 f 0.6
Heart Control N - 5
Ethanol 10 hr
N - 5
26.3 f 1 . 1
17.9 f 1.3 17.4 f 0.4 0.9 f 0.1 0.8 f 0.1 20.1 f 0.4 24.1 f 0.7 20.4 f 0.2 18.6 f 0.5 13.4 f 0.4 12.1 f- 0.6 14.1 f 1.5 1.2 f 0.2 15.6 f 0.5 1.7 f 0.2 6.0 f 0.4 5.8 f 0.5 8.7 f 0.3 1.6 f 0.2 12.9' f 0.3 24.3 f 1.6 19.6' f 0.7
The main fatty acids present in phospholipids of mouse tissue are shown in the lefthand column: figures represent chain 1ength:number of double bonds. Values given in other columns represent the mean percentage (f standard error) of the total fatty acids present. 'Indicates a value significantly ( p < 0.05 in paired Student's t test) different from the appropriate control.
Materials and Methods section. The results of analysis of these fractions are shown in Tables 3 and 4. Table 3 shows the results obtained with fractions A and E (myelin and mitochondria, respectively) from the sucrose density gradient. In neither fraction are significant changes seen in the fatty acid composition of membrane phospholipids. The fatty acid composition of the three synaptosomal fractions B, C, and D are shown in Table 4. Only in fraction D, presumably containing the largest and heaviest synaptosomes, is there a significant change in phospholipid composition. As with the crude synaptosomal fraction, there is a reduction. in polyunsaturated fatty acids, specifically docosahexaenoic (22:6), and a tendency to increased saturated fatty acids. DISCUSSION
Fatty Acid Composition of Phospholipids of Subcellular Fractions of Brain during Development of Rapid Tolerance to Ethanol Experiments carried out in parallel with those described here had indicated that TO Swiss mice, when first injected with ethanol and then exposed intermittently to high concentrations of ethanol vapor, could be demonstrated to rapidly acquire cellular tolerance.1° Therefore, we replicated these conditions with 24 mice exposed to ethanol vapor for 5 hr. Brains of four mice were then pooled and subjected to sucrose density gradient centrifugation as described in the Table 3. Fatty Acid Composition of Phospholipids of Myelin and Mitochondria from Mouse (TO Swiss) Brain during Induction of Rapid Cellular Tolerance to Ethanol Fraction E IMitochondria)
Fraction A (Myelin) Fatty Acid
-
Control N - 3
Ethanol 5 hr N - 2
24.7 f 1.6 23.5 f 1.9 25.1 f 2.1 3.0 f 0.3 9.1 f 0.7 6.1 f 0.1 2.1 0.5 5.8 f 1.5
22.2 f 1.7 21.8 f 1 . 1 24.8 f 0.2 2.3 f 0.3 9.3 f 0.7 8.4 + 1.3 2.1 0.1 9.3 f 0.1
Control N - 6
Ethanol 5 hr N - 6
~
16:O 18:O 18:l 18:2 20: 1 2014 22:4 22:6
*
*
23.1 f 1 . 1 22.4 f 0.9 22.9 f 0.4 22.6 f 0.3 16.0fO.5 17.1 fO.5 0.9 f 0.2 0.9 f 0.1 0.8 f 0.1 0.9 0.1 1 1.5 f 0.3 1 1.9 f- 0.2 2.3 f 0.1 2.2 f 0.2 22.5 f 0.6 22.0 f 0.7
The main fatty acids present in phospholipids of mouse tissue are shown in the lefthand column: figures represent chain 1ength:number of double bonds. Values given in other columns represent the mean percentage ( + standard error) of the total fatty acids present.
The results demonstrate that a reduction in the proportion of polyunsaturated fatty acids entering into phospholipid composition occurs in some tissues during the continuous administration of ethanol to TO Swiss mice. In the brain small reductions in arachidonic (20:4) and docosahexaenoic (22:6) acids were found in phospholipids of the crude synaptosomal fraction. The time course of this change seemed to be surprisingly rapid, the fall in polyunsaturated fatty acids after 2 hr being nearly as great as that observed after 10 days. When the crude synaptosomal fraction was fractionated by sucrose density centrifugation, the main change in fatty acid composition was seen in the lowest (heaviest) synaptosomal fraction. No significant change was observed in myelin or mitochondria1 phospholipids of brain. The phospholipids of the heart showed a qualitatively similar change to that seen in brain, but the magnitude was greater and the time course slower. In liver, there was an apparent increase in the hepatic phospholipid content, which made interpretation of the relative proportions of fatty acids difficult. The results suggested increased hepatic formation of phospholipids containing oleic acid ( 1 8 : l ) . Contamination of the sample by increased amounts of triglyceride fatty acid is an alternative explanation. The results reported here of changes in peripheral lipid metabolism associated with
LITTLETON, JOHN, AND GRIEVE
54
Tabla 4. Fa*
Acid Composition of Phospholipidsof Syneptosornal Fractions of Mouse (TO Swiss) Brain during Induction of Rapid Cellular Tolerance to Ethanol Fraction B
FaW Acid
16:O 18:O 18:l 1a:2 20:1 20:4 22:4 22:6
Fraction D
Fraction C
Control
Tolerant
Control
Tolerant
Control
N - 6
N - 6
N - 6
N - 6
N - 6
26.3 f 1.1 24.2 f 0.6 19.9f 0.9 1.2f 0.5 2.0 f 0.3 8.0f 0.8 2.8f 0.2 15.7f 0.9
26.3 f 0.5 25.4f 0.8 20.4 f 0.5 1.5f 0.3 1.8f 0.3 7.8f 0.6 3.0 f 0.2 14.4f 0.9
29.5 f 1.1 24.7 f 0.9 17.0f 0.3 2.2 f 0.2 1.7 f 0.4 7.9 f 0.4 2.7 f 0.4 15.6 f 1.0
28.6f 1.9 23.1 f 1.1 17.4f 0.1 1.8t 0.3 1.9f 0.4 8.4& 0.3 4.0f 0.8 16.1 f 1.6
25.9f 1.2 23.9 f 1.0 16.0f 0.5 2.0 f 0.3 2.1 f 0.8 9.6f 0.8 2.8f 0.4 19.5f 0.7
Tolerant
N - 6
28.1 f 2.1 23.7 f 0.5 16.8f 0.7 2.5 f 0.9 2.2 f 0.8 10.1f 1.0 2.3 f 0.3 15.7' f 0.9
The main fatty acids present in phospholipids of mouse tissue are shown in the lefthand column; figures represent chain 1ength:number of double bonds. Values given in other columns represent the mean percentage (tstandard error) of the total fatty acids present. *Indicates a value significantly ( p < 0.05in paired Student's t test) different from the appropriate control.
-
chronic ethanol administration show similarities to results reported previously for heart" and liver mitochondria and erythrocytes.'* This seems to be the first report in which such changes have been demonstrated in animals made physically dependent on ethanol. The differences between the tissues studied in these experiments are of interest. The reduction in polyunsaturated fatty acids produced in the brain is very small, possibly reflecting the large amount of relatively inert phospholipid associated with myelin in brain. On the other hand, the synaptosomal fraction appears to show a very rapid alteration in phospholipid composition in response to ethanol, perhaps related to the rapid turnover of fatty acids in synaptic membrane." Similarly, the slower appearance of the ethanolinduced change in cardiac phospholipids may reflect a slower turnover of membrane phospholipid fatty acids in this tissue, whereas the greater magnitude of the change may indicate that all cardiac membranes take part. The increase in phospholipids found in liver of ethanol-dependent animals may be related to increased synthesis of endoplasmic reticulum in response to chronic ethanol administration. Alternatively, the increase may represent one aspect of the grossly abnormal hepatic lipid metabolism associated with ethanol dependence.14 Increased hepatic triglycerides may contribute to the change. The preliminary evidence presented, of differences in the extent to which different synapses show the ethanol-induced alteration of membrane phospholipids, may be important. The synaptosomal fraction that showed the greatest
change is that fraction associated with the enzymes involved in y-amino butyric acid (GABA) synthesis and breakd0~n.l~ This may indicate that synapses utilizing GABA play an important part in the rapid development of tolerance to ethanol. Some evidence linking GABA metabolism and the development of cellular tolerance already exists.16 We are currently investigating the effect of drugs that alter GABA metabolism on the development of rapid cellular tolerance to ethanol. It has been implied throughout this article that the ethanol-induced reduction in membrane phospholipid polyunsaturated fatty acids may play some part in cellular adaptation to ethanol, but this is clearly not necessarily so. The alteration in fatty acid composition may be a result of direct inhibition by ethanol of desaturase activity in brain or it may result from increased lipid peroxidation produced by ethanol. It is also conceivable that changes in peripheral fatty acid metabolism produced by ethanol may lead to the changes observed in brain. In this connection it is of interest that cold resistance of the brain of hibernating mammals seems to be associated with an increase in desaturase activity of the liver rather than the brain,!' the change probably being communicated to the brain via an increase in circulating unsaturated free fatty acids. With this possibility in mind, we have measured the concentration and composition of nonesterified fatty acids in plasma of ethanoldependent mice (unpublished). There is an increase in the concentration of nonesterified fatty acids but no significant difference in the relative proportions of fatty acids seen. This
PHOSPHOLIPID COMPOSITION
mechanism therefore seems unlikely. Nutritional disturbances, too, seem unlikely as a cause. While they may contribute to the longterm effects of ethanol, it would be surprising if nutritional influences could explain an alteration occurring in the brain within hours of ethanol administration. From this evidence we cannot be certain of the reasons for the ethanol-induced change in fatty acid composition of phospholipids. If it is a genuine adaptive mechanism, the change may be produced by a reduction in fatty acid elongation/desaturation processes in brain, perhaps linked to membrane-associated enzyme activity, which is determined by membrane fluidity. Regardless of the mechanism of the change, it could clearly affect the intrinsic fluidity of synaptosomal membranes in the brain and could therefore modify the cellular effects of ethanol. If the change reported does modify the effects of ethanol, it is important to consider how it may be involved in ethanol tolerance and dependence. The possibility of a relationship with cellular tolerance to ethanol seems obvious. One objection is that the time course of the alteration of phospholipid composition in synaptosomes is very rapid, occurring within hours of continuous administration of ethanol. Most work on the time course of development of ethanol tolerance suggests that it may take several days.Is However, we have recently shown that, when mice of the TO Swiss strain are exposed to high concentrations of ethanol vapor, as in these experiments, cellular tolerance develops very rapidly and may reach a maximum in as little as 4 hr.Io The time course of acquisition of cellular tolerance may therefore be similar to the time course of the change in phospholipid composition. It is still not clear whether the biochemical changes that we report here can explain the resistance to the fluidizing effects of ethanol on “tolerant” membranes reported by Chin and Goldstein.’ These workers were unable to detect any alteration in intrinsic fluidity of membranes from ethanol-tolerant and dependent mice of the DBA strain, whereas our results suggest that such an alteration, albeit small, should occur in mice of the TO Swiss strain. In preliminary experiments designed to investigate this anomaly, we find that mice of the DBA strain show no change in phospholipid composition in brain or heart after induction of tolerance and depen-
55
dence. Interestingly, mice of the DBA strain also do not show the rapid development of cellular tolerance on exposure to ethanol vapor. It seems that these animals may adapt to ethanol by a slower mechanism, not associated with alteration in .the lipid portion of the membrane. One possibility may be a change in the composition or configuration of structural proteins in the membrane conferring resistance to the fluidizing effects of ethanol. This leads to consideration of the relationship between the proposed mechanism of membrane lipid adaptation and the development of ethanol dependence. The hypothesis of Hill and Bangham,s in common with all mechanistic theories linking tolerance and dependence, suggests that the physical withdrawal syndrome is an expression of the altered functional state of neurons associated with cellular tolerance. If so, the withdrawal syndrome should last as long as it takes to reverse the state of cellular tolerance. If, as suggested here, cellular tolerance in TO Swiss mice is related to membrane lipid adaptation and can occur very rapidly, then it should also be capable of relatively rapid reversal, thereby limiting the duration of the withdrawal syndrome. It is of note that mice of the DBA strain, which appear to show neither rapid cellular tolerance nor ethanol-induced change in membrane phospholipids, have a particularly severe and prolonged physical withdrawal syndrome.19J0It seems possible that the rate of cellular adaptation may be genetically determined and control both the rate of acquisition of cellular tolerance and the susceptibility to physical dependence. If rapid membrane lipid adaptation is a determinant of the severity and duration of the ethanol withdrawal syndrome, it may be possible to affect this with drugs that alter lipid metabolism. One possibility that we have investigated,” is that administration of DL-carnitine may modify the withdrawal syndrome. DL-carnitine is an important carrier for fatty acid groups across cell membranes in tissues, including brain.*I Administration of DL-carnitine may promote utilization of fatty acids in ethanoldependent animals. We found that dietary supplementation with DL-carnitine during the induction of ethanol dependence in TO mice significantly attenuated the subsequent withdrawal syndrome.
LITTLETON, JOHN, AND GRIEVE
56
In conclusion, we suggest that mammalian cell membranes, including those of central synapses, retain a limited capacity for altering their phospholipid fatty acid composition in response to agents, including ethanol, that alter membrane fluidity. This mechanism may form the basis for rapid adaptation at the synaptic
level. As such, it may be both the basis for rapid cellular tolerance to ethanol and the mechanism for limiting the ethanol physical withdrawal syndrome. It is possible that this mechanism has a genetic background and that it is susceptible to pharmacologic manipulation.
REFERENCES I . Metcalfe JC, Seeman P, Burgen ASV: The proton relaxation of benzyl alcohol in erythrocyte membranes. Mol Pharmacol 4:87-95, 1968 2. Chin JH, Goldstein DB: Effects of low concentrations of ethanol on the fluidity of spin-labeled erythrocyte and brain membranes. Mol Pharmacol 13:435-441, 1977 3. Cossins AR: Adaptation of biological membranes to temperature. The effect of temperature acclimation of goldfish upon the viscosity of synaptosomal membranes. Biochim Biophys Acta 470395-41 I , 1977 4. Nandini-Kishore SG, Kitajuna Y, Thompson GAJ: Membrane fluidizing effects of the general anaesthetic methoxyflurane elicit an acclimation response in Tefruhymenu. Biochim Biophys Acta 471:157-161, 1977 5 . Hill MW, Bangham A D General depressant drug dependency: A biophysical hypothesis. Adv Exp Med Biol 59:1-9, 1975 6. lngram LO: Adaptation of membrane lipids to alcohols. J Bacteriol 12567-78, 1976 7. Chin JH. Goldstein DB: Drug tolerance in biomembranes: A spin label study of the effects of ethanol. Science 196:684485, 1977 8. Littleton JM, John GR: Synaptosomal membrane lipids of mice during continuous exposure to ethanol. J Pharm Pharmacol 29579-580. 1977 9. Griffiths PJ. Littleton JM. Ortiz A: Changes in monoamino concentrations in mouse brain associated with ethanol dependence and withdrawal. Br J Pharmacol 50:489498. 1974 10. Grieve SJ, Littleton JM: Rapid development of cellular tolerance during continuous administration of ethanol to mice by inhalation. Br J Pharmacol63:375P-376P. 1978 1 I . Reitz RC. Helsabeck E, Mason D P Effects of chronic alcohol ingestion on the fatty acid composition of the heart. Lipids 8:8&84. 1973
12. Ihrig TJ, French SW.Morin RJ: Lipid composition of cellular membranes after ethanol feeding. Fed Proc 28:626, 1969 13. Sun GY, Yau TM; Incorporation of l-"C oleic acid and I-"C arachidonic acid into lipids of the subcellular fractions of mouse brain. J Neurochem 27337-92, 1976 14. Abu Murad C. Begg SJ. Griffiths PJ, et al: Hepatic triglyceride accumulation and the ethanol physical withdrawal syndrome in mice. Br J Exp Pathol 58:606-615, 1977 15. De Robertis E Ultrastructure and cytochemistry of the synaptic region. Science 156:907-914, 1967 16. Leitch GJ, Backes DJ, Siegman FS, et al: Possible role of GABA in the development of tolerance to alcohol. Experientia 33:496-498, 1977 17. Goldman SS: Cold resistance of the brain during hibernation: The role of stearyl CoA desaturase in brain and liver as the source for monoenes. J Neurochem 30:397400, 1978 18. Majchrowicz E, Hunt WA: Temporal relationship of the induction of tolerance and physical dependence after continuous intoxication with maximum tolerable doses of ethanol in rats. Psychopharmacology 50:107-112. 1976 19. Goldstein DB, Kakihana R: Alcohol withdrawal reactions and reserpine effects in inbred strains of mice. Life Sci 15:415425, 1974 20. Griffiths PJ. Abu Murad C, Littleton JM: Ethanolinduced hepatic triglyceride accumulation in mice and genetic differences in the ethanol physical withdrawal syndrome. Br J Addict (in press) 21. AWel Latif AA, Roberts MB, Karp WB et al: Metabolism of phosphatidylcholine. phosphatidyl inositol and palmityl carnitine in synaptosomes from rat brain. J Neurochem 20: 189-202, 1973
LCOHOLS and general anesthetics may A have a common mechanism of action at the cellular level. Both groups can be shown to increase the fluid nature of biologic and model membranes,' and this may be the basis of the central depressant properties of these drugs. Recently, Chin and Goldstein2showed that ethanol, in concentrations associated with intoxication rather than anesthesia, can induce increased fluidity in cell membranes obtained from mice. Using a spin-label technique, these investigators showed that addition of ethanol in vitro increased the physical fluidity of membranes from brain synaptosomes, mitochondria, and erythrocytes. If the mechanism of action of ethanol at the cellular level is by alteration of synaptic membrane fluidity, then this may have implications for tolerance and dependence on ethanol. Changes in temperature also can produce alterations in membrane fluidity; poikilotherms are known' to adapt to alterations in environmental temperature by changing the intrinsic fluidity of their cell membranes in the opposite direction to that induced by the temperature change. The mechanism of this change is thought to be primarily by alteration of membrane phospholipid fatty acid composition. As an example of this process Cossins' has recently shown that synaptic membranes of brain from warm-acclimated goldfish are less fluid than those of cold-acclimated fish when measured at an inter-
From the Department of Pharmacology, King's College, London, England. Supported by grants from the Medical Council on Alee holism and the Mental Health Research Foundation. Reprint request should be addressed to John M.Littleton. Ph.D.. Department of Pharmacology, King's College, London WCZR 213. England. 1979 by Grune & Stratton, Inc. 0145-6008/79/0301-0011$01.00/0
50
R. John, B.Sc., M.Sc., and Susan J. Grieve, B.Sc.
mediate temperature. This reduced intrinsic fluidity was correlated to a decreased proportion of polyunsaturated fatty acids in membrane phospholipids. The major change was in the choline phosphogylcerides; the cholesterol content of the synaptic membrane was unaffected. There is also evidence that unicellular organisms can adapt to pharmacologic agents by a similar mechanism. Thus Nandini-Kishore, Kitajuna, and Thompson' have demonstrated that Tetrahymena pyriformis responds to the membrane-fluidizing effects of the general anesthetic methoxyflurane by decreasing fatty acid desaturase activity in much the same fashion as is observed in response to increased temperature. If such organisms can adapt to the presence of fluidizing agents by altering membrane composition, it seems possible that a similar mechanism may occur in mammals and may underlie the development of cellular tolerance to ethanol. Hill and Banghams proposed such a mechanism for the cellular basis of tolerance to central depressants and extended the argument to include a hypothesis for physical dependence based on alterations in intrinsic membrane fluidity. Since then, work has appeared that partly supports their suggestion with respect to the development of tolerance to ethanol. In 1976, Ingram6 showed that bacteria, grown in media containing alcohols, showed an alteration in phospholipid fatty acid composition. The results were puzzling in that only relatively long-chain alcohols produced the expected decrease in unsaturated fatty acids. Short-chain alcohols, such as ethanol, produced an increase in membrane phospholipid unsaturated fatty acids. This paradox is still unresolved, although Ingram suggested that short-chain alcohols may reduce the fluidity of bacterial membranes, but this is at variance with the results obtained on mammalian membranes cited earlier.2 There may be some fundamental difference between the response to ethanol of bacterial membrane phospholipids, which contain a far lower proportion of polyunsaturated fatty acids, and the membranes of higher organisms. However, Ingram's work does demonstrate clearly that alteration of
Alcoholism: Clinical and Experimental Research. Vol. 3. No. 1 (January). 1879
PHOSPHOLIPID COMPOSITION
membrane fatty acid composition can occur in response to the continued presence of alcohols. Hill and Bangham's hypothesisSsuggests that tolerance and physical dependence on central depressant drugs should be associated with a reduction in the intrinsic fluidity of membranes from the dependent organism. Chin and Goldstein' investigated the fluidity of membranes from mice of the DBA strain that had been rendered tolerant and physically dependent on ethanol. Although they found that cell membranes (including synaptic membranes) from these animals were resistant to the in vitro fluidizing effects of ethanol, they were unable to detect any differences between the intrinsic fluidity of membranes from control mice and those tolerant and dependent on ethanol. Their work therefore supports the Hill and Bangham hypothesisS to the extent that some form of ethanol tolerance does appear to reside in the cell membrane, but does not support the idea that tolerance and dependence are accompanied by a reduction in the intrinsic fluidity of the cell membrane. As Chin and Goldstein point out, their failure to detect a difference in intrinsic fluidity may reflect methodological inadequacy to detect small differences between groups where variation is large. An alternative explanation is that the strain of mice used show rather unusual properties with regard to the development of tolerance and dependence on ethanol and may not be suitable for demonstration of alterations in membrane fluidity. This point will be returned to in the Discussion section. The effect of continuous ethanol administration to mice of the T O Swiss strain was investigated by Littleton and John.s In these preliminary experiments, we found a small but significant reduction in polyunsaturated fatty acids from phospholipids of the crude synaptosomal fraction of brains from ethanol-tolerant and dependent mice. It was suggested that, under the conditions of these experiments, an adaptive alteration in membrane phospholipid composition occurred in response to the continuous administration of ethanol. The experiments described here extend these observations to other tissues of ethanol-dependent mice and offer preliminary findings on the time course of the changes observed and their localization to different synaptosomal fractions.
51
MATERIALS AND METHODS The animals used in these experiments were young adult male mice of the TO Swiss strain, 20-22 g in weight, obtained from L.A.C. Dagenham, Essex. Administration of ethanol was by inhalation in the apparatus described previously.9 In all experiments mice had free access to food (41 B diet) and water. Mice described as being physically dependent on ethanol were exposed to blood concentrations of ethanol between 2 and 3 mg/ml-' for 10 days before sacrifice (whole body immersion in liquid nitrogen). In our experience, this produces signs of physical withdrawal in all animals of this strain when ethanol is removed. In some experiments, mice were exposed to very high concentrations of ethanol vapor for shorter periods. A concentration of 30 mg/liter-' ethanol in air produced a blood ethanol concentration of approximately 2 mg/ml-' in 2 hr; animals exposed in this way were used to investigate the short-term effects of ethanol. In a subsequent experiment. mice were first injected with 2.5 g/kg-' ethanol i.p. and then exposed intermittently to ethanol vapor, 25 mg/liter-' for 5 hr. This produced blood ethanol concentrations in the range of 3 - 6 mg/ml-'. The last regimen was chosen because it had been shown'O to produce rapid and marked cellular tolerance to ethanol. The fatty acid composition of phospholipids was analyzed in essentially the same way described by Littleton and John.' Briefly, the method consists of Folch extraction of membrane lipids, separation of phospholipids onto a slurry of activated silicic acid, hydrolysis and saponification of phospholipids with sodium hydroxide, and methylesterification of fatty acids with boron trifluoride/methanol reagent. The chloroform extract of methylesterified fatty acids is then analyzed by gas liquid chromatography on a polyethylene glycol adipate column. Quantification of peak areas was by electronic-integration. When subcellular fractions from sucrose density gradient centrifugation (see below) were to be extracted, at least two washes in chloroform/methanol were performed. For whole tissue, such as heart and liver, the tissue was initially weighed, sliced. and then homogenized (glass: Teflon Potter homogenizer) directly in chloroform/methanol. For separation of subcellular fractions of brain, the following methods were used: Either single brains (for crude synaptosomal fraction) or pooled brains of 4 animals (for sucrose density gradient fractionation) were homogenized in 10 volumes of ice-cold 0.32M sucrose using a motor-driven homogenizer. The homogenate was centrifuged at lOOOg ( 2 0 min) and the pellet discarded. The supernatant was centrifuged at 20,OOOg for 30 min to obtain the crude synaptosomal (P2)fraction. The pellet was resuspended in ice-cold 0.32M sucrose before layering onto the surface of the sucrose density gradient described below (see Fig. 1). A 4-step sucrose density gradient was prepared in transparent 25 ml centrifuge tubes by layering on sucrose of concentrations 1.4, 1.2. 1 .O, and 0.8M with a roller pump (Fig. 1). Gradients were spun a t 100,OOOg for 2 hr in a 3 X 25 swing-out head. Four fractions were formed at the interfaces of the sucrose layers and a mitochondria1 pellet also separated (Fig. 1). The fractions were pumped off using a roller pump, starting with fraction C, and stored on ice prior to Folch extraction. The mitochondria1 pellet was resuspended in 2 ml of 0.32M sucrose before extraction.
LITTLETON, JOHN, AND GRIEVE
52
le
H
-I.OM
SYNAPTOSOMES
-1.2M
I
U
c
ID
q I . 4 M
MITOCHONDRIA-
e e lOO.Oo0 G. 2HR. Fig. 1.
Sucrose density fractions.
In all experiments, control animals were kept in the same conditions as experimental animals but with ethanol absent from the inspired air. In experiments where ethanol was injected, isotonic saline was used as a control injcction. All biochemical procedures were carried out on a paired basis (test sample versus control sample) and results were analyzed using a paired Student's t test. RESULTS
Fatty Acid Composition of Phospholipids from Tissues of Ethanol-Dependent Mice The continuous administration of ethanol by inhalation for 10 days produced alterations in the fatty acid composition of brain (crude synaptosomal fraction), heart, and liver. These results are shown in Table 1 . Table 1 shows that there are similarities in the ethanol-induced change in phospholipid composition on brain and heart. In both there is a reduction in polyunsaturated fatty acids. In the brain there is a corresponding increase in saturated and mono-unsaturated fatty acids, whereas
in the heart the increase is in oleic (18:l) and linoleic (18:2) acids. The changes in the liver seem rather different in that there is a tendency to decrease in all fatty acids except oleic (18:l) and linoleic (18:2). There is in fact a marked and significant increase in the proportion of oleic acid from hepatic phospholipids. When recovery of phospholipids from these tissues was estimated by comparing the total integrated counts of fatty acids obtained, it was observed that there was an increase in the phospholipids obtained from liver of ethanol-dependent animals. The change, expressed as concentration per liver net weight, was estimated to be from 2.2 f 0.28 mg/g-' fatty acids from phospholipids (control, N=5)to 3.9 k0.60 mg/g-' fatty acids from phospholipids (ethanol-dependent, N = 5 ) . No significant change in phospholipid concentration was observed in brain or heart. Time Course of Changes in Fatty Acid Composition of Tissue Phospholipids during Continuous Administration of Ethanol These experiments were begun in order to establish whether the change in brain occurred only after the chronic administration of ethanol. It was found that administration of ethanol at high concentrations for only 2 hr could produce a change of only slightly less magnitude than the change produced by administration for 10 days (see Table 2). In the heart, no alteration in cardiac phospholipids was seen after 2 hr; and after ethanol administration in high concentrations for 10 hr, only a small alteration in polyunsaturated fatty acids was observed (Table 2).
Table 1. Fatty Acid Composition of Phospholipids from Tissues of Ethanol-Dependent Mice (TO Swiss Strain) Brain (P, Fraction)
Liver
Heart
-
Fatty Acid
Control N - 7
Dependent N 10
Control N - 5
Dependent N - 5
Control N - 5
Dependent N - 5
16:O 16:1 18:O 18:1 18:2 20:1 20:4 22:4 22:6
24.2 f 0.9
25.4f 1.1
22.6 f 0.3 18.2 f 0.5 1 .O f 0.3 2.1 f 0.1 9.9 f 0.2 1.9f 0.1 14.5f 0.6
24.4' f 0.6 19.3f 0.5 1.3 f 0.2 2.2f 0.3 8.0' f 0.3 1.7f 0.1 11.9f 1.4
17.9 f 1.3 0.9f 0.1 20.4f 0.2 13.4f 0.4 15.6 f 0.5
18.5 + 1.9 2.3 f 0.7 19.0f 1.1 18.0' + 0.6 19.3' f 0.8
30.5 f 2.4 2.2 f 0.3 16.1 f 0.4 19.Of 1.5 16.8f 1.1
22.2' f 1.1 2.3 f 0.7 14.9 f 1.1 26.9' f 1.6 18.6 f 1.3
4.5 f 0.5
-
10.7 f 2.0
8.5f 1.3
24.3 f 1.6
13.9' f 1.0
5.5f 0.7
5.3 f 0.9
5.8 f 0.5
-
-
-
The main fatty acids present in phospholipids of mouse tissue are shown in the lefthand column: figures represent chain 1ength:number of double bonds. Values given in other columns represent the mean percentage (+ standard error) of the total fatty acids present. *Indicates a value significantly ( p < 0.05in paired Student's t test) different from the appropriate control.
PHOSPHOLIPID COMPOSITION
53
Table 2. Time Course of Alteration of Fatty Acid Composition of Brain and Heart by Continuous Administration of Ethanol to Mice (TO Swiss Strain) ~ ~ _ _ _ _ ~
Brain (PI Fraction)
-
Ethanol 2 hr N - 5
Fatty Acid
Control N 7
16:O 16:l 18:O 18:1 18:2 2O:l 20:4 22:4 22:6
24.2 f 0.9
22.6 f 0.3 18.2 0.5 1 .O f- 0.3 2.1 f 0.1 9.9 f 0.2 1.9 0.1 14.5 f 0.6
Heart Control N - 5
Ethanol 10 hr
N - 5
26.3 f 1 . 1
17.9 f 1.3 17.4 f 0.4 0.9 f 0.1 0.8 f 0.1 20.1 f 0.4 24.1 f 0.7 20.4 f 0.2 18.6 f 0.5 13.4 f 0.4 12.1 f- 0.6 14.1 f 1.5 1.2 f 0.2 15.6 f 0.5 1.7 f 0.2 6.0 f 0.4 5.8 f 0.5 8.7 f 0.3 1.6 f 0.2 12.9' f 0.3 24.3 f 1.6 19.6' f 0.7
The main fatty acids present in phospholipids of mouse tissue are shown in the lefthand column: figures represent chain 1ength:number of double bonds. Values given in other columns represent the mean percentage (f standard error) of the total fatty acids present. 'Indicates a value significantly ( p < 0.05 in paired Student's t test) different from the appropriate control.
Materials and Methods section. The results of analysis of these fractions are shown in Tables 3 and 4. Table 3 shows the results obtained with fractions A and E (myelin and mitochondria, respectively) from the sucrose density gradient. In neither fraction are significant changes seen in the fatty acid composition of membrane phospholipids. The fatty acid composition of the three synaptosomal fractions B, C, and D are shown in Table 4. Only in fraction D, presumably containing the largest and heaviest synaptosomes, is there a significant change in phospholipid composition. As with the crude synaptosomal fraction, there is a reduction. in polyunsaturated fatty acids, specifically docosahexaenoic (22:6), and a tendency to increased saturated fatty acids. DISCUSSION
Fatty Acid Composition of Phospholipids of Subcellular Fractions of Brain during Development of Rapid Tolerance to Ethanol Experiments carried out in parallel with those described here had indicated that TO Swiss mice, when first injected with ethanol and then exposed intermittently to high concentrations of ethanol vapor, could be demonstrated to rapidly acquire cellular tolerance.1° Therefore, we replicated these conditions with 24 mice exposed to ethanol vapor for 5 hr. Brains of four mice were then pooled and subjected to sucrose density gradient centrifugation as described in the Table 3. Fatty Acid Composition of Phospholipids of Myelin and Mitochondria from Mouse (TO Swiss) Brain during Induction of Rapid Cellular Tolerance to Ethanol Fraction E IMitochondria)
Fraction A (Myelin) Fatty Acid
-
Control N - 3
Ethanol 5 hr N - 2
24.7 f 1.6 23.5 f 1.9 25.1 f 2.1 3.0 f 0.3 9.1 f 0.7 6.1 f 0.1 2.1 0.5 5.8 f 1.5
22.2 f 1.7 21.8 f 1 . 1 24.8 f 0.2 2.3 f 0.3 9.3 f 0.7 8.4 + 1.3 2.1 0.1 9.3 f 0.1
Control N - 6
Ethanol 5 hr N - 6
~
16:O 18:O 18:l 18:2 20: 1 2014 22:4 22:6
*
*
23.1 f 1 . 1 22.4 f 0.9 22.9 f 0.4 22.6 f 0.3 16.0fO.5 17.1 fO.5 0.9 f 0.2 0.9 f 0.1 0.8 f 0.1 0.9 0.1 1 1.5 f 0.3 1 1.9 f- 0.2 2.3 f 0.1 2.2 f 0.2 22.5 f 0.6 22.0 f 0.7
The main fatty acids present in phospholipids of mouse tissue are shown in the lefthand column: figures represent chain 1ength:number of double bonds. Values given in other columns represent the mean percentage ( + standard error) of the total fatty acids present.
The results demonstrate that a reduction in the proportion of polyunsaturated fatty acids entering into phospholipid composition occurs in some tissues during the continuous administration of ethanol to TO Swiss mice. In the brain small reductions in arachidonic (20:4) and docosahexaenoic (22:6) acids were found in phospholipids of the crude synaptosomal fraction. The time course of this change seemed to be surprisingly rapid, the fall in polyunsaturated fatty acids after 2 hr being nearly as great as that observed after 10 days. When the crude synaptosomal fraction was fractionated by sucrose density centrifugation, the main change in fatty acid composition was seen in the lowest (heaviest) synaptosomal fraction. No significant change was observed in myelin or mitochondria1 phospholipids of brain. The phospholipids of the heart showed a qualitatively similar change to that seen in brain, but the magnitude was greater and the time course slower. In liver, there was an apparent increase in the hepatic phospholipid content, which made interpretation of the relative proportions of fatty acids difficult. The results suggested increased hepatic formation of phospholipids containing oleic acid ( 1 8 : l ) . Contamination of the sample by increased amounts of triglyceride fatty acid is an alternative explanation. The results reported here of changes in peripheral lipid metabolism associated with
LITTLETON, JOHN, AND GRIEVE
54
Tabla 4. Fa*
Acid Composition of Phospholipidsof Syneptosornal Fractions of Mouse (TO Swiss) Brain during Induction of Rapid Cellular Tolerance to Ethanol Fraction B
FaW Acid
16:O 18:O 18:l 1a:2 20:1 20:4 22:4 22:6
Fraction D
Fraction C
Control
Tolerant
Control
Tolerant
Control
N - 6
N - 6
N - 6
N - 6
N - 6
26.3 f 1.1 24.2 f 0.6 19.9f 0.9 1.2f 0.5 2.0 f 0.3 8.0f 0.8 2.8f 0.2 15.7f 0.9
26.3 f 0.5 25.4f 0.8 20.4 f 0.5 1.5f 0.3 1.8f 0.3 7.8f 0.6 3.0 f 0.2 14.4f 0.9
29.5 f 1.1 24.7 f 0.9 17.0f 0.3 2.2 f 0.2 1.7 f 0.4 7.9 f 0.4 2.7 f 0.4 15.6 f 1.0
28.6f 1.9 23.1 f 1.1 17.4f 0.1 1.8t 0.3 1.9f 0.4 8.4& 0.3 4.0f 0.8 16.1 f 1.6
25.9f 1.2 23.9 f 1.0 16.0f 0.5 2.0 f 0.3 2.1 f 0.8 9.6f 0.8 2.8f 0.4 19.5f 0.7
Tolerant
N - 6
28.1 f 2.1 23.7 f 0.5 16.8f 0.7 2.5 f 0.9 2.2 f 0.8 10.1f 1.0 2.3 f 0.3 15.7' f 0.9
The main fatty acids present in phospholipids of mouse tissue are shown in the lefthand column; figures represent chain 1ength:number of double bonds. Values given in other columns represent the mean percentage (tstandard error) of the total fatty acids present. *Indicates a value significantly ( p < 0.05in paired Student's t test) different from the appropriate control.
-
chronic ethanol administration show similarities to results reported previously for heart" and liver mitochondria and erythrocytes.'* This seems to be the first report in which such changes have been demonstrated in animals made physically dependent on ethanol. The differences between the tissues studied in these experiments are of interest. The reduction in polyunsaturated fatty acids produced in the brain is very small, possibly reflecting the large amount of relatively inert phospholipid associated with myelin in brain. On the other hand, the synaptosomal fraction appears to show a very rapid alteration in phospholipid composition in response to ethanol, perhaps related to the rapid turnover of fatty acids in synaptic membrane." Similarly, the slower appearance of the ethanolinduced change in cardiac phospholipids may reflect a slower turnover of membrane phospholipid fatty acids in this tissue, whereas the greater magnitude of the change may indicate that all cardiac membranes take part. The increase in phospholipids found in liver of ethanol-dependent animals may be related to increased synthesis of endoplasmic reticulum in response to chronic ethanol administration. Alternatively, the increase may represent one aspect of the grossly abnormal hepatic lipid metabolism associated with ethanol dependence.14 Increased hepatic triglycerides may contribute to the change. The preliminary evidence presented, of differences in the extent to which different synapses show the ethanol-induced alteration of membrane phospholipids, may be important. The synaptosomal fraction that showed the greatest
change is that fraction associated with the enzymes involved in y-amino butyric acid (GABA) synthesis and breakd0~n.l~ This may indicate that synapses utilizing GABA play an important part in the rapid development of tolerance to ethanol. Some evidence linking GABA metabolism and the development of cellular tolerance already exists.16 We are currently investigating the effect of drugs that alter GABA metabolism on the development of rapid cellular tolerance to ethanol. It has been implied throughout this article that the ethanol-induced reduction in membrane phospholipid polyunsaturated fatty acids may play some part in cellular adaptation to ethanol, but this is clearly not necessarily so. The alteration in fatty acid composition may be a result of direct inhibition by ethanol of desaturase activity in brain or it may result from increased lipid peroxidation produced by ethanol. It is also conceivable that changes in peripheral fatty acid metabolism produced by ethanol may lead to the changes observed in brain. In this connection it is of interest that cold resistance of the brain of hibernating mammals seems to be associated with an increase in desaturase activity of the liver rather than the brain,!' the change probably being communicated to the brain via an increase in circulating unsaturated free fatty acids. With this possibility in mind, we have measured the concentration and composition of nonesterified fatty acids in plasma of ethanoldependent mice (unpublished). There is an increase in the concentration of nonesterified fatty acids but no significant difference in the relative proportions of fatty acids seen. This
PHOSPHOLIPID COMPOSITION
mechanism therefore seems unlikely. Nutritional disturbances, too, seem unlikely as a cause. While they may contribute to the longterm effects of ethanol, it would be surprising if nutritional influences could explain an alteration occurring in the brain within hours of ethanol administration. From this evidence we cannot be certain of the reasons for the ethanol-induced change in fatty acid composition of phospholipids. If it is a genuine adaptive mechanism, the change may be produced by a reduction in fatty acid elongation/desaturation processes in brain, perhaps linked to membrane-associated enzyme activity, which is determined by membrane fluidity. Regardless of the mechanism of the change, it could clearly affect the intrinsic fluidity of synaptosomal membranes in the brain and could therefore modify the cellular effects of ethanol. If the change reported does modify the effects of ethanol, it is important to consider how it may be involved in ethanol tolerance and dependence. The possibility of a relationship with cellular tolerance to ethanol seems obvious. One objection is that the time course of the alteration of phospholipid composition in synaptosomes is very rapid, occurring within hours of continuous administration of ethanol. Most work on the time course of development of ethanol tolerance suggests that it may take several days.Is However, we have recently shown that, when mice of the TO Swiss strain are exposed to high concentrations of ethanol vapor, as in these experiments, cellular tolerance develops very rapidly and may reach a maximum in as little as 4 hr.Io The time course of acquisition of cellular tolerance may therefore be similar to the time course of the change in phospholipid composition. It is still not clear whether the biochemical changes that we report here can explain the resistance to the fluidizing effects of ethanol on “tolerant” membranes reported by Chin and Goldstein.’ These workers were unable to detect any alteration in intrinsic fluidity of membranes from ethanol-tolerant and dependent mice of the DBA strain, whereas our results suggest that such an alteration, albeit small, should occur in mice of the TO Swiss strain. In preliminary experiments designed to investigate this anomaly, we find that mice of the DBA strain show no change in phospholipid composition in brain or heart after induction of tolerance and depen-
55
dence. Interestingly, mice of the DBA strain also do not show the rapid development of cellular tolerance on exposure to ethanol vapor. It seems that these animals may adapt to ethanol by a slower mechanism, not associated with alteration in .the lipid portion of the membrane. One possibility may be a change in the composition or configuration of structural proteins in the membrane conferring resistance to the fluidizing effects of ethanol. This leads to consideration of the relationship between the proposed mechanism of membrane lipid adaptation and the development of ethanol dependence. The hypothesis of Hill and Bangham,s in common with all mechanistic theories linking tolerance and dependence, suggests that the physical withdrawal syndrome is an expression of the altered functional state of neurons associated with cellular tolerance. If so, the withdrawal syndrome should last as long as it takes to reverse the state of cellular tolerance. If, as suggested here, cellular tolerance in TO Swiss mice is related to membrane lipid adaptation and can occur very rapidly, then it should also be capable of relatively rapid reversal, thereby limiting the duration of the withdrawal syndrome. It is of note that mice of the DBA strain, which appear to show neither rapid cellular tolerance nor ethanol-induced change in membrane phospholipids, have a particularly severe and prolonged physical withdrawal syndrome.19J0It seems possible that the rate of cellular adaptation may be genetically determined and control both the rate of acquisition of cellular tolerance and the susceptibility to physical dependence. If rapid membrane lipid adaptation is a determinant of the severity and duration of the ethanol withdrawal syndrome, it may be possible to affect this with drugs that alter lipid metabolism. One possibility that we have investigated,” is that administration of DL-carnitine may modify the withdrawal syndrome. DL-carnitine is an important carrier for fatty acid groups across cell membranes in tissues, including brain.*I Administration of DL-carnitine may promote utilization of fatty acids in ethanoldependent animals. We found that dietary supplementation with DL-carnitine during the induction of ethanol dependence in TO mice significantly attenuated the subsequent withdrawal syndrome.
LITTLETON, JOHN, AND GRIEVE
56
In conclusion, we suggest that mammalian cell membranes, including those of central synapses, retain a limited capacity for altering their phospholipid fatty acid composition in response to agents, including ethanol, that alter membrane fluidity. This mechanism may form the basis for rapid adaptation at the synaptic
level. As such, it may be both the basis for rapid cellular tolerance to ethanol and the mechanism for limiting the ethanol physical withdrawal syndrome. It is possible that this mechanism has a genetic background and that it is susceptible to pharmacologic manipulation.
REFERENCES I . Metcalfe JC, Seeman P, Burgen ASV: The proton relaxation of benzyl alcohol in erythrocyte membranes. Mol Pharmacol 4:87-95, 1968 2. Chin JH, Goldstein DB: Effects of low concentrations of ethanol on the fluidity of spin-labeled erythrocyte and brain membranes. Mol Pharmacol 13:435-441, 1977 3. Cossins AR: Adaptation of biological membranes to temperature. The effect of temperature acclimation of goldfish upon the viscosity of synaptosomal membranes. Biochim Biophys Acta 470395-41 I , 1977 4. Nandini-Kishore SG, Kitajuna Y, Thompson GAJ: Membrane fluidizing effects of the general anaesthetic methoxyflurane elicit an acclimation response in Tefruhymenu. Biochim Biophys Acta 471:157-161, 1977 5 . Hill MW, Bangham A D General depressant drug dependency: A biophysical hypothesis. Adv Exp Med Biol 59:1-9, 1975 6. lngram LO: Adaptation of membrane lipids to alcohols. J Bacteriol 12567-78, 1976 7. Chin JH. Goldstein DB: Drug tolerance in biomembranes: A spin label study of the effects of ethanol. Science 196:684485, 1977 8. Littleton JM, John GR: Synaptosomal membrane lipids of mice during continuous exposure to ethanol. J Pharm Pharmacol 29579-580. 1977 9. Griffiths PJ. Littleton JM. Ortiz A: Changes in monoamino concentrations in mouse brain associated with ethanol dependence and withdrawal. Br J Pharmacol 50:489498. 1974 10. Grieve SJ, Littleton JM: Rapid development of cellular tolerance during continuous administration of ethanol to mice by inhalation. Br J Pharmacol63:375P-376P. 1978 1 I . Reitz RC. Helsabeck E, Mason D P Effects of chronic alcohol ingestion on the fatty acid composition of the heart. Lipids 8:8&84. 1973
12. Ihrig TJ, French SW.Morin RJ: Lipid composition of cellular membranes after ethanol feeding. Fed Proc 28:626, 1969 13. Sun GY, Yau TM; Incorporation of l-"C oleic acid and I-"C arachidonic acid into lipids of the subcellular fractions of mouse brain. J Neurochem 27337-92, 1976 14. Abu Murad C. Begg SJ. Griffiths PJ, et al: Hepatic triglyceride accumulation and the ethanol physical withdrawal syndrome in mice. Br J Exp Pathol 58:606-615, 1977 15. De Robertis E Ultrastructure and cytochemistry of the synaptic region. Science 156:907-914, 1967 16. Leitch GJ, Backes DJ, Siegman FS, et al: Possible role of GABA in the development of tolerance to alcohol. Experientia 33:496-498, 1977 17. Goldman SS: Cold resistance of the brain during hibernation: The role of stearyl CoA desaturase in brain and liver as the source for monoenes. J Neurochem 30:397400, 1978 18. Majchrowicz E, Hunt WA: Temporal relationship of the induction of tolerance and physical dependence after continuous intoxication with maximum tolerable doses of ethanol in rats. Psychopharmacology 50:107-112. 1976 19. Goldstein DB, Kakihana R: Alcohol withdrawal reactions and reserpine effects in inbred strains of mice. Life Sci 15:415425, 1974 20. Griffiths PJ. Abu Murad C, Littleton JM: Ethanolinduced hepatic triglyceride accumulation in mice and genetic differences in the ethanol physical withdrawal syndrome. Br J Addict (in press) 21. AWel Latif AA, Roberts MB, Karp WB et al: Metabolism of phosphatidylcholine. phosphatidyl inositol and palmityl carnitine in synaptosomes from rat brain. J Neurochem 20: 189-202, 1973
