Role of CYP2E1 in the pathogenesis of alcoholic liver disease: modifications by cAMP and ubiquitin-proteasome pathway.

The ethanol inducible isoform of cytochrome P450, CYP2E1, may play a role in ethanol-induced liver injury. Therefore, the factors which govern CYP2E1 degradation and turnover were investigated. These factors include cAMP, ubiquitin, proteasomal enzymes and CYP2E1 mRNA. Rats fed ethanol or pair-fed isocaloric dextrose were pair-fed with rats fed ethanol or dextrose treated with cAMP for 2 months. The liver pathology, regenerative activity, fatty acid composition, NFkappaB activation, ubiquitin conjugates and proteasomal enzymes were measured as were the apoprotein levels of CYP2E1, CYP3A, CYP4A and mRNA levels for CYP2E1 and ubiquitin expression. The results showed, that the cAMP treatment ameliorated the increase liver fat storage and changes in the fatty acid composition in the livers of ethanol fed rats. Other histologic features of alcoholic liver disease were not changed. Western blot quantitation showed that the amount of ubiquitin and ubiquitin conjugates were markedly reduced by ethanol treatment. Similarly, ethanol decreased the level of ubiquitin mRNA. cAMP ameliorated the inhibition of the proteasomal enzyme proteolysis caused by ethanol feeding. The ethanol-induced increase in the CYP2E1 protein was partially inhibited by cAMP treatment. cAMP treatment decreased CYP2E1 mRNA levels in both ethanol-fed and pair fed control rats. Likewise NFkappaB activation was not increased by ethanol but cAMP reduced the level of NFkappaB activation. CAMP treatment also reduced CYP4A but not CYP3A. The results support the concept that cAMP treatment partially protects the liver from ethanol-induced fatty liver by reducing CYP2E1 induction through cAMP's effects on CYP2E1 synthesis.

Comparison of hexadecylphosphocholine with fish oil as an antitumor agent.

Hexadecylphosphocholine (HePC) reduced the growth of the human mammary tumor, MX-1, in the athymic nude mouse similar to the fish oil, MaxEPA. When used together, HePC and MaxEPA were additive towards reducing tumor growth. An unsaturated alkylphosphocholine mixture, ShisoPC, was not as effective as HePC in reducing tumor growth. MaxEPA reduced tumor PGE2 levels greater than 90%, while HePC and the ShisoPC only reduced tumor PGE2 40-60% with HePC being slightly better than ShisoPC. MaxEPA markedly increased the cellular omega 3 fatty acids and decreased 20:4 omega 6, the substrate for PGE2. HePC did not alter the tumor fatty acid composition, but it significantly lowered the total fatty acid concentration of the tumor by about 47%. In addition, phosphatidylcholine and sphingomyelin decreased in tumors from animals treated with HePC, and alterations in other phospholipids also were noted. These data suggest that different mechanisms exist for HePC and fish oil in reducing tumor growth.

Lipid peroxidation, CYP2E1 and arachidonic acid metabolism in alcoholic liver disease in rats.

The role of cytochrome P450 metabolism of fatty acids and lipid peroxidation in the alterations of the fatty acid composition of the liver and liver pathology was investigated. The CYP2E1 inhibitors partially prevented CYP2E1 induction by ethanol and completely blocked lipid peroxidation. However, the liver pathology induced by ethanol was only partially prevented as was the decrease in arachidonic acid in total liver lipid, triglycerides and cholesterol esters. This means that liver peroxidation induced by ethanol can not completely account for the liver pathology or the decrease in arachidonic acid caused by ethanol. Lauric acid omega-1 hydroxidation by the liver microsomes in vitro was increased by ethanol and partially blocked by CYP2E1 inhibitors. However, although ethanol feeding increased the total hydroxidation and epoxidation of arachidonic acid, these were not inhibited by CYP2E1 inhibitors. Thus the ethanol-induced arachidonic acid depletion is not likely due to CYP2E1 metabolism of arachidonic acid, since the severity of liver pathology correlated negatively with the decrease in arachidonic acid in the ethanol-fed rats. The increase in its metabolism by microsomes and decrease in synthesis may be an important mechanism of ethanol-induced liver injury.

Effects of omega 3 fatty acids on receptor tyrosine kinase and PLC activities in EMT6 cells.

The effects of omega 3 fatty acids and epidermal growth factor (EGF) on the activity of receptor tyrosine kinase (RTK) and phospholipase C (phosphatidylinositol (PI)-specific PLC) were examined in EMT6 cells. The non-omega 3 treated, non-EGF stimulated cells served as controls. Treatment of the EMT6 cells with omega 3 fatty acids resulted in a 62% increase in RTK activity and a 67% increase in PI-specific PLC activity. When EGF was added to incubations for RTK activity, it stimulated the RTK activity 40% in the control cells and 130% in the omega 3-treated cells. When EGF was added to incubations for PI-specific PLC activity, a 54% increase in PI-specific PLC activity was observed in control cells and a 94% increase in the omega 3-treated cells. Thus, treating EMT6 cells with omega 3 fatty acids seems to increase RTK activity and PI-specific PLC activity to a similar extent, but has differential effects on the ability of these enzyme activities to be stimulated by EGF.

Proposed mechanism for the cytochrome P450-catalyzed conversion of aldehydes to hydrocarbons in the house fly, Musca domestica.

Experiments were performed to elucidate the mechanism of hydrocarbon formation in microsomal preparations from the house fly, Musca domestica. Antibody to both house fly cytochrome P450 reductase and a purified cytochrome P450 (CYP6A1) from the house fly inhibited (Z)-9-tricosene (Z9-23:Hy) formation from [15,16-3H]-(Z)-15-tetracosenal (24:1 aldehyde). Chemical ionization-gas chromatography-mass spectrometry (CI-GC-MS) analyses of the n-tricosane formed by microsomal preparations from [2,2-2H2,2-13C]- and [3,3-2H2,3-13C]tetracosanoyl-CoA demonstrated that the deuteriums on the 2,2- and 3,3-positions were retained in the conversion to the hydrocarbon product. Likewise, CI-GC-MS analysis of the Z9-23:Hy formed from [1-2H]tetracosenal by microsomal preparations demonstrated that the aldehydic proton on the 1-carbon was transferred to the hydrocarbon product. Hydrogen peroxide, cumene hydroperoxide, and iodosobenzene were able to support hydrocarbon production from [3H]24:1 aldehyde in place of O2 and NADPH for short incubation times. From these data, a cytochrome P450 mechanism is proposed in which the perferryl iron-oxene, resulting from heterolytic cleavage of the O-O bond of the iron-peroxy intermediate, abstracts an electron from the C=O double bond of the carbonyl group of the aldehyde. The reduced perferryl attacks the 1-carbon of the aldehyde to form a thiyl-iron-hemiacetal diradical. The latter intermediate can fragment to form an alkyl radical and a thiyl-iron-formyl radical. The alkyl radical then abstracts the formyl hydrogen to produce the hydrocarbon and CO2.

CYP-2E1 inhibitors partially ameliorate the changes in hepatic fatty acid composition induced in rats by chronic administration of ethanol and a high fat diet.

The objective of this study was to determine if ethanol-induced cytochrome P450 2E1 (CYP2E1) was responsible for the changes in hepatic fatty acids observed in rats fed ethanol intragastrically. We hypothesized that if CYP2E1 was responsible for these changes then CYP2E1 inhibitors fed with ethanol should prevent the ethanol-induced changes in fatty acids. We compared the fatty acid composition of the liver in rats fed ethanol alone with that in rats fed ethanol with the CYP2E1 inhibitors, diallyl sulfide and phenethyl isothiocyanate. In each experiment, rats pair-fed isocaloric glucose were included to determine the effect of the inhibitors alone on the hepatic fatty acid composition. The lobular distribution of succinic dehydrogenase was determined histochemically because the lobular distribution of CYP2E1 shifts to the periportal area in livers of rats fed CYP2E1 inhibitors. The CYP2E1 inhibitors ameliorated both the ethanol-induced changes in fatty acids and the shift in succinic dehydrogenase. Rats fed ethanol but no inhibitors had significantly greater hepatic total fatty acids and triglyceride fractions than when inhibitors were fed ethanol. Ethanol altered the fatty acid composition compared with rats fed ethanol with CYP2E1 inhibitors. The ratio of 20:4/18:2 was significantly lower and that of 18:1/18:0 was greater in alcohol-fed rats compared with their pair-fed controls. The CYP2E1 inhibitors inhibited many of the above effects of alcohol. The data suggest that the changes in the fatty acid composition due to ethanol ingestion are the result of CYP2E1-dependent lipid peroxidation and fatty acid metabolism.

Regulation of enzymatic activity involved in sex pheromone production in the housefly, Musca domestica.

Ovarian produced ecdysteroids regulate sex pheromone production in the female housefly, inducing the synthesis of (Z)-9-tricosene (Z9-23:Hy), cis-9,10-epoxytricosane, (Z)-14-tricosen-10-one and methylalkanes. Experiments were performed to gain a detailed understanding of the processes affected by 20-hydroxyecdysone (20-HE) that result in sex pheromone production as the female becomes reproductively mature. A novel microsomal fatty acid synthetase (FAS) is present in the epidermal tissue and plays a role in producing the methyl-branched fatty acid precursors to the methylalkanes. This FAS is released from the microsomes in the presence of 3 M KCl. A major enzyme activity influenced by 20-HE is the fatty acyl-CoA elongation system. A shift in the chain length specificity of the products of the elongation system causes the change in the chain lengths of the alkenes produced to switch from C27 and longer in the previtellogenic female to C23 in the mature female. Data is presented indicating that it is the condensation activity of the elongation system that is affected. Z9-23:Hy arises from a 24 carbon acyl group which is reduced to an aldehyde, and then converted to the hydrocarbon. Data is presented demonstrating that it is the fatty acyl-CoA derivative and not the free fatty acid that is the substrate. There does not appear to be a chain length specificity which regulates the conversion of fatty acyl-CoAs to hydrocarbons as both 24 and 28 carbon fatty acyl-CoAs are converted to hydrocarbon by both males and females of all ages.

Structural basis of the phospholipid acyltransferase enzyme substrate specificity: a computer modeling study of the phospholipid acceptor molecule.

The activity of the 1-acyl-sn-glycero-3-phosphocholine acyltransferase enzyme (E.C. 2.3.1.??) was measured with three radically different acceptor substrates: 1-palmitoyl-sn-glycero-3-phosphocholine (P-sn-G3PC), 1-palmitoyl-sn-glycero-2-phosphocholine (P-sn-G2PC), and 1-hexadecyl-sn-glycero-3-phosphocholine (He-sn-G3PC). It was found that the enzyme had similar activity with P-sn-G3PC, the natural acceptor substrate, and with P-sn-G2PC. The enzyme showed no detectable activity toward He-sn-G3PC. These results are much different than would be expected from simple examination of the structures. Computer-assisted molecular modeling was done to study the geometrical configurations and to focus upon the similarities and differences of the three substrate acceptor molecules. Three bond distances were selected as important for enzyme recognition: the distance between the oxygen of the acceptor hydroxyl group and 1) the phosphorus; 2) the nitrogen; and 3) the oxygen bridge to the hydrocarbon chain. There were striking similarities for the bond distances of two of the three acceptor substrates, P-sn-G3PC and P-sn-G2PC. These were the two molecules that were shown to have activity with the enzyme. The bond distances found for the enzymically inactive acceptor substrate, He-sn-G3PC, differed significantly from P-sn-G3PC and P-sn-G2PC. Therefore, this latter molecule probably does not fit into the active site of the enzyme. The modeling data are also consistent with the experimental observation that He-sn-G3PC is not an inhibitor.

Unusual mechanism of hydrocarbon formation in the housefly: cytochrome P450 converts aldehyde to the sex pheromone component (Z)-9-tricosene and CO2.

An unusual mechanism for hydrocarbon biosynthesis is proposed from work examining the formation of (Z)-9-tricosene (Z9-23:Hy), the major sex pheromone component of the female housefly, Musca domestica. Incubation of (Z)-15-[1-14C]- and (Z)-15-[15,16-3H2]tetracosenoic acid (24:1 fatty acid) with microsomes from houseflies gave equal amounts of [3H]Z9-23:Hy and 14CO2. The formation of CO2 and not CO, as reported for hydrocarbon formation in plants, animals, and microorganisms [Dennis, M. & Kolattukudy, P. E. (1992) Proc. Natl. Acad. Sci. USA 89, 5306-5310], was verified by trapping agents and by radio-GLC analysis. Incubation of (Z)-15-[15,16-3H2]tetracosenoyl-CoA with microsomal preparations in the presence of NADPH and O2 gave almost equal amounts of (Z)-15-3H2]tetrasosenal (24:1 aldehyde) and Z9-23:Hy. Addition of increasing amounts of hydroxylamine (aldehyde trapping agent) caused a decrease in hydrocarbon formation with a concomitant increase in oxime (aldehyde derivative) formation. The 24:1 aldehyde was efficiently converted to (Z)-9-tricosene only in the presence of both NADPH and O2. Bubbling carbon monoxide (20:80 CO/O2) or including an antibody against housefly cytochrome P450 reductase inhibited the formation Z9-23:Hy from 24:1 aldehyde. These data demonstrate an unusual mechanism for hydrocarbon formation in insects in which the acyl-CoA is reduced to the corresponding aldehyde and then carbon-1 is removed as CO2. The requirement for NADPH and O2 and the inhibition by CO and the antibody to cytochrome P450 reductase strongly implicate the participation of a cytochrome P450 in this reaction.

Phospholipid methylation in brain membrane preparations: kinetic mechanism.

The methylation reactions which convert phosphatidylethanolamine (PE) to phosphatidylcholine (PC) have been studied kinetically using exogenously added intermediates and crude membrane preparations from brain. The addition of exogenous PE resulted in no change in the methylation rates compared to that of endogenous PE. The addition of the two intermediates, monomethylphosphatidylethanolamine (PMME) and dimethylphosphatidylethanolamine (PDME), resulted in significantly increased rates of methylation and allowed the kinetic analysis of these latter two methylation reactions. The mechanism for this enzyme appears to be similar to human RBC (Reitz et al. (1989) J. Biol. Chem. 264, 8097-8106) which was a rapid-equilibrium random Bi-Bi sequential mechanism. There were some slight differences between the brain enzyme and that from the RBC, but there is little reason to suggest a fundamentally different mechanism. It is more likely that the differences may relate to an additional dead-end complex for the enzyme from brain such that saturation with AdoMet cannot eliminate AdoHcy inhibition. The KM values for the two phospholipid substrates were 41-44 microM and 39 microM for the methylation of PMME and PDME, respectively. The KM for S-adenosylmethionine (AdoMet) was 7-9 microM with PMME and 4 microM with PDME as the other substrates. The Ki(lipid) varied from 54 microM with PMME to 225 microM with PDME, and the Ki(AdoMet) was 11 microM with PMME and 21 microM with PDME. The product from the use of AdoMet, S-adenosylhomocysteine (AdoHcy), was shown to be a noncompetitive inhibitor of both lipid substrates as well as AdoMet. The methylation of PMME was somewhat higher in cerebellum and brain stem compared to cortex and striatum, but the methylation of PDME was similar in cerebellum, brain stem and cortex.

Dietary fatty acids and alcohol: effects on cellular membranes.

The consumption of ethanol has been shown to exert profound effects on cellular membranes which result in damage and/or adaptation. Both membrane lipids and proteins are affected, but because of the physicochemical properties of ethanol, many of the membrane effects are directly related to the interaction of ethanol with the lipid component of the membrane. In addition to the direct lipid-ethanol interaction, ethanol has been shown to dramatically alter lipid metabolism. Triacylglycerol accumulates dramatically in the liver, and biosynthesis of the polyunsaturated fatty acids seems to be altered via effects upon the acyl-CoA desaturases. Because precursors of both families of unsaturated fatty acids, i.e. omega 3 and omega 6 families, cannot be synthesized de novo, they must be supplied from dietary sources. Thus, the unsaturated membrane fatty acid composition depends upon these dietary fats and their metabolism via the desaturases. Further, the level of dietary fat seems to play a very important role in ethanol-induced damage to various cellular membranes. Diets with high levels of fat greatly enhance liver steatosis as well as liver membrane damage and liver fibrosis. By altering the composition of dietary fat to include either more saturated fatty acids, higher levels of a specific omega 6 fatty acid, alpha-linolenic acid, or higher levels of the omega 3 fatty acids, biochemical, physiological and neurobehavioral effects of ethanol have been shown to be modulated. Therefore, it appears that dietary fatty acids may play an important role in altering some of the deleterious effects of ethanol.

Regulation of sex pheromone biosynthesis in the housefly, Musca domestica: relative contribution of the elongation and reductive steps.

The regulation of production of the sex pheromone (Z)-9-tricosene (Z9-23:Hy) in the housefly, Musca domestica, was studied by examining the chain length specificity of the fatty acyl-CoA elongation reactions and the reductive conversion of fatty acyl-CoAs to alkenes in 1- and 4-day-old male and female houseflies. Microsomal preparations from 4-day-old female insects produced as the predominant alkene Z9-23:Hy when incubated with malonyl-CoA, NADPH, and [9,10-3H2]oleoyl-CoA (18:1-CoA), whereas microsomal preparations from 4-day-old male insects produced predominantly (Z)-9-heptacosene (Z9-27:Hy). These are the major alkenes produced in vivo by Day 4 females and males, respectively. Microsomes prepared from both Day 1 males and Day 1 females produced Z9-27:Hy as the major alkene from labeled 18:1-CoA. This is the major alkene produced in vivo by both sexes at Day 1. An examination of the chain length specificity of the elongation reactions showed that microsomes prepared from Day 4 male insects readily elongated both 18:1-CoA and 15-[15,16-3H2]tetracosenoyl-CoA (24:1-CoA) to 28-carbon moieties, whereas microsomes from Day 4 female insects did not efficiently elongate either substrate beyond 24 carbons. With high substrate concentrations, microsomes prepared from male insects converted 24:1-CoA to Z9-23:Hy more efficiently than did those from females, whereas under lower and presumably more physiological substrate concentrations, microsomes from females had slightly higher activity than did those from males. Taken together, these data show that the regulation of the chain length of the alkenes, and thus sex pheromone production, in the housefly resides predominantly in the elongation reactions and not in the step which converts the fatty acyl-CoA to hydrocarbon.

Effects of high alpha-linolenate and linoleate diets on erythrocyte deformability and hematological indices in rats.

Rats were fed either a high alpha-linolenate diet or a high linoleate diet from weaning to 4 mon of age. Soybean oil was used as a control. Phospholipid compositions of erythrocytes from the three dietary groups were not significantly different. However, the difference in the alpha-linolenate (18:3n-3)/linoleate (18:2n-6) ratio of the diets was reflected in the n-3/n-6 ratios of the 20 and 22 carbon highly unsaturated fatty acids except for docosahexaenoic acid (22:6n-3) in the phospholipids. Despite the significant differences in the fatty acid compositions of phospholipids, no measurable differences were detectable in erythrocyte deformability, whole blood viscosity and hematological indices of the three dietary groups. These results indicate that the beneficial effects of the high alpha-linolenate diet, as compared with the high linoleate diet, are exerted without significant changes in these parameters.

Phosphatidylethanolamine N-methyltransferase in human red blood cell membrane preparations. Kinetic mechanism.

The successive methylations of phosphatidylethanolamine to form phosphatidylcholine were measured using exogenously added intermediates and membrane preparations from human red blood cells. The addition of phosphatidylethanolamine resulted in no increase in methylation rate over that with endogenous substrate; however, the addition of monomethylphosphatidylethanolamine (PME) and dimethylphosphatidylethanolamine (PDE) markedly increased the reaction rate and allowed studies into the kinetic mechanism for the second and third methylation reactions. The data are consistent with catalysis of the last two methylations being by a single enzyme with a random Bi-Bi sequential mechanism. Analysis of PDE:phosphatidylcholine product ratios indicates that the enzyme can conduct multiple methylations of enzyme-bound phospholipid. The nature of the acyl chain (16:0 versus 18:1) of the phospholipid had only a small effect on the value of the kinetic constants. The maximal velocities obtained with the 18:1 substrate were less than 5% lower than those obtained with the 16:0 substrate. The Km values for the two phospholipids were 20-45 and 10-14 microM for the methylation of PME and PDE, respectively. The Km for S-adenosylmethionine (AdoMet) was 5-9 microM with PME and 4 microM with PDE as substrates. Depending on the acyl chain and the phospholipid, the Ki(AdoMet) varied from 8 to 19 microM, the Ki(PME) from 41 to 82 microM, and the Ki(PDE) from 35 to 61 microM. The Ki for S-adenosylhomocysteine (AdoHcy) was between 1.0 and 1.4 microM depending upon the variable substrate. The endogenous concentrations of PME and PDE in red blood cell membranes were estimated to be 0.49 and 0.24 mumol/liter packed cells, respectively. The product from the utilization of AdoMet, S-adenosylhomocysteine (AdoHcy), was shown to be a competitive inhibitor of its precursor, AdoMet, and a noncompetitive inhibitor of the two phospholipid substrates.

Effects of dietary fish oil on human mammary carcinoma and on lipid-metabolizing enzymes.

The growth rate of a human mammary carcinoma, MX-1, was significantly reduced in athymic "nude" mice fed fish oil. Tumors from the fish oil-fed animals also showed a greater sensitivity to two anti-neoplastic agents, mitomycin C and doxorubicin. Mitochondria were isolated from control livers, host livers and tumors from fish oil- and corn oil-fed animals, and increased levels of 20:5n-3 and 22:6n-3 were found in mitochondrial lipids in all three tissues from the fish oil-fed animals. To investigate the effect of dietary n-3 fatty acids on lipid metabolism, the activity of the acyl-CoA:carnitine acyltransferase and three acyl-CoA desaturases were measured. Carnitine acyltransferase activity toward all four acyl-CoA substrates tested was markedly increased in mitochondria from liver by feeding fish oil. In mitochondria from tumors, feeding fish oil resulted in an increased activity toward only 18:3n-3. These data suggest that fish oil may induce an increase in the oxidation of fatty acids. The delta 9-desaturase activity was decreased in microsomes from liver and tumor from fish oil-fed animals. However, both the delta 6 and delta 5 desaturases were increased in tumor and in control liver as a result of feeding fish oil. The delta 5 desaturase was not altered in microsomes from the host animals. The effect of fish oil on the delta 5 and delta 6 desaturases may involve alterations to metabolism of specific polyunsaturated fatty acids especially in the tumor tissue.

Tissue and chain length specificity of the fatty acyl-CoA elongation system in the American cockroach.

The elongation of fatty acyl-CoAs, reactions involved in hydrocarbon biosynthesis, was examined in the cockroach, Periplaneta americana. Products were analyzed by radio-HPLC and radio-GLC. The majority of the elongation activity was observed in microsomes prepared from abdominal epidermal tissue. Linoleoyl-CoA (18:2-CoA) was elongated most efficiently followed by stearoyl-CoA (18:0-CoA), linolenoyl-CoA (18:3-CoA; n-3) and oleoyl-CoA (18:1-CoA). The products of 18:2-CoA elongation included all even numbered acyl groups up to 28 carbons, and the products of 18:0-CoA included all even numbered acyl groups to 26 carbons. The 18:3-CoA was elongated only to 20 and 22 carbons. Radioactivity from both 18:2-CoA (5.4%) and 18:0-CoA (1.2%) was recovered in the hydrocarbon fraction. Analysis of this hydrocarbon fraction showed that the radio-activity from 18:2-CoA was present in (Z,Z)-6,9-heptacosadiene and that the radioactivity from 18:0-CoA was present in n-pentacosane. These data demonstrate for the first time in an in vitro insect system that the fatty acid elongation reactions are coupled with the conversion of the elongated product to hydrocarbon. Thus, each of the expected intermediates in the conversion of 18:0 and 18:2 to 25 and 27 carbon hydrocarbons, respectively, was observed, and the results demonstrate high tissue, substrate, and product specificity.

Ethanol-induced alterations in rat synaptosomal plasma membrane phospholipids. Relationship to changes in the phospholipid methyltransferases.

The effects of ethanol ingestion on the lipids of the synaptic plasma membrane (SPM) have been measured and correlated with the time frame for the development of physical dependence. Alterations were observed in three of the phospholipid fractions: phosphatidylcholine (PC) increased, and the phosphatidylethanolamine (PE) and phosphatidylserine (PS) plus phosphatidylinositol (PI) fractions decreased. These alterations occurred after the animals showed signs of dependence. Because PC can be synthesized from PE by the methyltransferase pathway, synaptosomal methyl group incorporation was measured. Rats were fed ethanol for 6 days before an increase was observed in methyl incorporation, a shorter length of time than was necessary to demonstrate physical dependence or phospholipid alterations (10 to 14 days). After ethanol withdrawal, 7 days of control diet feeding were required for methyl group incorporation to return to control values. In vitro ethanol (10-250 mM) additions to the methyltransferase incubations resulted in a slight increase in methyl incorporation. These data suggest that synaptic membrane lipid alterations may be related to ethanol dependence and that changes in the PC/PE ratio may be the result of an increase in the incorporation of methyl groups into synaptosomal phospholipids.

Effects of chronic ethanol ingestion on male and female rat liver glycogen phosphorylase phosphatase.

The effects of chronic ethanol ingestion on the interconversion of the active to the inactive form of glycogen phosphorylase by phosphorylase phosphatase was studied. Male and female rats also were compared. Chronic ethanol feeding decreased phosphorylase a and total phosphorylase activity in male rats. In females, no change was observed in phosphorylase a, whereas total phosphorylase activity was increased 73%. This was found to correlate with the relative activities of phosphorylase phosphatase. The data show differences between the two sexes with regard to the AMP inhibition of phosphorylase phosphatase and the caffeine stimulation of the phosphatase. Ethanol markedly enhanced the AMP inhibition of the phosphatase in males but had no effect in females. Further studies in females showed that ethanol completely obliterated the well documented stimulation of the phosphatase by caffeine; however, it did not alter the caffeine effect in males. These data suggest possible alterations in the tertiary structure of phosphorylase a.