Chronic and acute ethanol treatment modifies fluidity and composition in plasma membranes of a human hepaic cell line (WRL-68)

The aim of this study was to compare the effects of chronic (0.1 mol/L ethanol exposure during 30 days) and acute (0.5 mol/L ethanol exposure during 24 h) ethanol treatment on the physical properties and the lipid composition of plasma membranes of the WRL-68 cells (fetal human hepatic cell line). Using fluorescence polarization we found that ethanol treatment reduced membrane anisotropy due to disorganization of acyl chains in plasma membranes and consequently increased fluidity, as measured with the diphenylhexatriene probe. Addition of ethanolin vitro reduced anisotropy in control plasma membranes, whereas chronically ethanol-treated plasma membranes were relatively tolerant to thein vitro addition of ethanol. Acutely ethanol-treated plasma membranes exhibited a smaller anisotropy parameter value than control plasma membranes. We found a decrease in total phospholipid content in acute ethanol WRL-68 plasma membranes. Cholesterol content was increased in both ethanol treatments, and we also found a significant decrease in phosphatidylinositol and phosphatidylcholine and an increase in phosphatidylethanolamine content in ethanol-treated plasma membranes. Our data showed that ethanol treatment decreased the anisotropy parameter consistently with increased fluidity, while increasing the cholesterol/phospholipid ratio of plasma membranes of WRL-68 cells, but only chronically ethanol-treated plasma membranes exhibited tolerance to thein vitro addition of ethanol. It is important to note that some changes that were interpreted as a result of chronic ethanol treatment were also present in short-period ethanol treatments.Abbreviations DPH diphenylhexatriene - PC phosphatidylcholine - PE phosphatidylethanolamine - PI phosphatidylinositol - PS phosphatidylserine - SPH sphingomyelin     

Increased cholesterol content of erythrocyte and brain membranes in ethanol-tolerant mice

Mice were treated with ethanol for eight or nine days, using a liquid diet regimen known to produce physical dependence. In previous experiments, synaptosomal plasma membranes and erythrocyte ghosts from such ethanoltreated animals were found to be resistant to the fluidizing effects of ethanol in vitro, as measured by electron paramagnetic resonance. In the present experiments, corresponding membranes were analysed for phospholipid and cholesterol. The ratio of cholesterol to phospholipid was found to be significantly increased in both types of membrane after chronic ethanol treatment. The changed ratio was produced by an increase in cholesterol. There was little or no change in phospholipid content of the membranes. Increased cholesterol may explain the previously observed alteration of physical properties of the membranes.     

SYNAPTOSOMAL PLASMA MEMBRANES. ACYL GROUP COMPOSITION OF PHOSPHOGLYCERIDES and (Na++ K+)-ATPase ACTIVITY DURING FATTY ACID DEFICIENCY

Abstract— Essential fatty acid deficiency was induced in mice after feeding a fatty acid deficient diet for 6 months. Activity of the (Na⁺+ K⁺)-ATPase in the total brain homogenates and in isolated synaptosomal plasma membranes was significantly higher ( P & lt; 0 05) in the deficient mice than the controls. Analysis of the acyl group composition of phosphoglycerides in brain as well as in the synaptosomal plasma membranes showed that mice fed the deficient diet had increased levels of 20:3(n-9) and 22:3(n-9) and decreased levels of 20:4(n-6) and 22:4(n-6). However, acyl group changes varied among individual phosphoglycerides and were most obvious in the two species of ethanolamine phosphoglycerides. A decrease in 22:6(n-3) level was also observed in some phosphoglycerides of the synaptosomal plasma membranes especially the diacyl- sn -glycerophosphorylserine. In this experiment, a new solvent system for chromatographic separation of the diacyl- sn -glycerophosphorylserine and diacyl- sn -glycerophosphorylinositol was reported. The separation technique was suitable for analysis of acyl group composition of individual phosphoglycerides by gas-liquid chromatography. The results were consislent with a positive correlation of the non-polar acyl groups of brain membranes with the active ion transport activity. The increase in enzymic activity during deficient state may be the result of a biological adaptation due to structural alteration of the brain membranes.     

Influence of dietary fat on the lipid composition of rat brain synaptosomal and microsomal membranes.

The modulation of rat brain microsomal and synaptosomal membrane lipid by diet fat was examined. Brain synaptosomal and microsomal membrane composition was compared for rats fed on diets containing either soya-bean oil (SBO), SBO plus choline, SBO lecithin, sunflower oil (SFO), chow or low-erucic acid rape-seed oil (LER) for 24 days. Cholesterol and phosphatidylcholine levels in both membranes were altered by diet. Diet fat also affected the microsomal content of sphingomyelin. Change in membrane phosphatidylcholine level was related to the relative balance of omega-6, omega-3 and monounsaturated fatty acids within the diets fed. The highest phosphatidylcholine levels appeared in membranes of animals fed on SBO lecithin and the lowest in those fed on LER. Microsomal membrane cholesterol and sphingomyelin content increased by feeding on SBO lecithin. In both synaptosomal and microsomal membranes a highly significant correlation was observed between membrane phosphatidylcholine and cholesterol content. The fatty acyl composition of phospholipids from both membranes also altered with diet and age. Alteration in fatty acid composition was observed in response to dietary levels of omega-6, omega-3 and monounsaturated fatty acids, but the unsaturation index of each phospholipid remained constant for all diet treatments. These changes in lipid composition suggest that dietary fat may be a significant modulator in vivo of the physicobiochemical properties of brain synaptosomal and microsomal membranes.     

Alterations induced by chronic ethanol treatment on lipid composition of microsomes, mitochondria and myelin from neonatal chick liver and brain

The effect of 18 days ethanol consumption on the lipid composition of microsomes, mitochondria and myelin have been studied in neonatal chick liver and brain. Neither cholesterol nor phospholipid content was modified in both liver microsomes and mitochondria. However, cholesterol content of brain microsomes, mitochondria and myelin was clearly increased, mainly due to an enhancement of free cholesterol. Likewise, ethanol consumption induced a clear increase of phospholipid content in brain mitochondria and myelin. As a consecuence of these changes, the cholesterol/phospholipid molar ratio strongly increased only in the myelin fraction. The myelin phospholipid composition markedly varied by ethanol treatment. Our results indicate that the maximal modifications were induced by ethanol in membranes with a high cholesterol content, suggesting that differences in the chemical composition of membranes could be responsible for differences in the response to the ethanol consumption.     

Fluorescence Polarization Analysis, Lipid Composition, and Na+, K+-ATPase Kinetics of Synaptosomal Membranes in Feline GM1 and GM2 Gangliosidosis

Neurochemical studies were performed on synaptosomal membranes from cats with GM1 or GM2 gangliosidosis to examine possible mechanisms of neuronal dysfunction in these disorders. The basic hypothesis tested was that deficient ganglioside catabolism causes increased ganglioside content of synaptosomal plasma membrane which in turn disrupts normal function. Fluidity characteristics of synaptosomal membranes were examined using fluorescence polarization. Results showed markedly reduced membrane fluidity in both GM1 and GM2 gangliosidosis. These results were supported by a second study which revealed that isolated synaptosomal membranes of GM1 gangliosidosis cats had a 24-fold increase in total ganglioside content caused predominantly by excess GM1, a 2.3-fold increased cholesterol content, and a 1.4-fold increased phospholipid content. Finally, kinetic analysis of synaptosomal plasma membrane Na+,K+-ATPase from cats with GM1 gangliosidosis showed negligible differences in kinetic parameters compared with controls. Thus, the enzyme appeared protected from the global membrane changes in fluidity and composition. These observations provide evidence for a pathogenetic mechanism of neuronal dysfunction in the gangliosidoses while demonstrating protection of certain vital functional components, such as Na+,K+-ATPase.     

Modification of the fatty acid composition of Ehrlich ascites tumor cell plasma membranes

The fatty acyl group composition of Ehrlich ascites tumor cell plasma membranes was modified by feeding the tumor-bearing mice diets rich in either coconut or sunflower oil. When coconut oil was fed, the oleate content of the membrane phospholipids was elevated and the linoleate content reduced. The opposite occurred when sunflower oil was fed. Qualitatively similar changes were observed in the plasma membrane phosphatidylethanolamine, phosphatidylcholine and mixed phosphatidylserine plus phosphatidylinositol fractions. These diets also produced differences in the sphingomyelin fraction, particularly in the palmitic and nervonic acid contents. Unexpectedly, the saturated fatty acid content of the plasma membrane phospholipids was somewhat greater when the highly polyunsaturated sunflower oil was fed. The small quantities of neutral lipids contained in the plasma membrane exhibited changes in acyl group composition similar to those observed in the phospholipids. These fatty acyl group changes were not accompanied by any alteration in the cholesterol or phospholipid contents of the plasma membranes. Therefore, the lipid alterations produced in this experimental model system are confined to the membrane acyl groups.     

Modification of the fatty acid composition of Ehrlich ascites tumor cell plasma membranes.

The fatty acyl group composition of Ehrlich ascites tumor cell plasma membranes was modified by feeding the tumor-bearing mice diets rich in either coconut or sunflower oil. When coconut oil was fed, the oleate content of the membrane phospholipids was elevated and the linoleate content reduced. The opposite occurred when sunflower oil was fed. Qualitatively similar changes were observed in the plasma membrane phosphatidylethanolamine, phosphatidylcholine and mixed phosphatidylserine plus phosphatidylinositol fractions. These diets also produced differences in the sphingomyelin fraction, particularly in the palmitic and nervonic acid contents. Unexpectedly, the saturated fatty acid content of the plasma membrane phospholipids was somewhat greater when the highly polyunsaturated sunflower oil was fed. The small quantities of neutral lipids contained in the plasma membrane exhibited changes in acyl group composition similar to those observed in the phospholipids. These fatty acyl group changes were not accompanied by any alteration in the cholesterol or phospholipid contents of the plasma membranes. Therefore, the lipid alterations produced in this experimental model system are confined to the membrane acyl groups.     

Fluidity and lipid composition of oat and rye shoot plasma membrane: effect of sterol perturbation by xenobiotics

Oat and rye plants were treated with either tetcyclacis (an experimental plant growth regulator), nuarimol (a fungicide) or gamma-ketotriazole (an experimental herbicide). These treatments reduced shoot growth and changed the lipid composition of the shoot plasma membranes. In oat, both tetcyclacis and nuarimol treatments increased plasma membrane cholesterol and increased the phosphatidylethanolamine/phosphatidylcholine (PE/PC) ratio, whereas gamma-ketotriazole treatment reduced cholesterol and the PE/PC ratio. In rye, all treatments reduced the PE/PC ratio. Generally, the sterol/phospholipid ratio was less in oat than in rye but the cholesterol/phospholipid ratio was greater. With all treatments in oat and rye, increases were observed in unsaturation of the phospholipid acyl chains. The fluidity of membranes was measured by steady-state fluorescence polarisation of the probe diphenylhexatriene; oat membranes were more fluid than rye. Membrane fluidity was greater in plasma membranes from plants treated with the xenobiotics than the controls. The results are discussed in the context of the effect of plasma membrane lipid composition on membrane fluidity, and it is concluded that there appears to be no overall simple relationship between membrane lipid composition and fluidity that holds for all treatments in both species.     

Differential response of chick liver and brain membranes to short ethanol treatment

The effect of 60 hr ethanol ingestion on lipid composition of liver and brain membranes from 2-day-old chicks was investigated. Analysis of hepatic membrane cholesterol shows that ethanol induced a slight increase in microsomes exclusively due to free cholesterol while mitochondria was not affected. In brain, both fractions showed a clear increase in their cholesterol content, while a high decrease was observed in myelin. Free cholesterol was also the main responsible for the changes found in brain. The ethanol-treated animals showed an alteration in their phospholipid composition exclusively in brain microsomes and myelin. Despite all these changes, the values of cholesterol/phospholipid molar ratio in both liver and brain membranes remained unaltered after short ethanol treatment. Our results indicate that neonatal chick brain membranes appears to be especially sensitive to the presence of ethanol.     

Changes in lipid composition of cortical synaptosomes from different age groups of mice

Lipid composition of cortical synaptosomes differed with age in C57BL/6NNIA mice. Significant age differences were observed for cholesterol and the ratio of cholesterol to total phospholipid phosphorus content. The phospholipid to protein ratio of individual phospholipids also increased with age with diacyl-$\text{sn}$-glycero-3-phosphocholine (PC) increasing the most. Acyl group composition of individual phospholipids, however, showed little age difference. The double bond index for PC decreased significantly with age. Changes in membrane composition may help explain differences in the effects of ethanol on the physical and biochemical properties of membranes from different age groups that have been reported previously.     

A new role for apolipoprotein E: modulating transport of polyunsaturated phospholipid molecular species in synaptic plasma membranes

Phospholipids and their acyl group composition are important in providing the proper membrane environment for membrane protein structure and function. In particular, the highly unsaturated phospholipids in synaptic plasma membranes in the CNS are known to play an important role in modulating receptor function and neurotransmitter release processes. Apolipoprotein E (apoE) is a major apolipoprotein in the CNS, mediating the transport of cholesterol, phospholipids and their fatty acids, particularly in reparative mechanisms during neuronal injury. This study was performed to determine whether deficiency in the apoE gene contributes to an alteration of the phospholipids in synaptic plasma membranes. Phospholipid molecular species were identified and quantitated by HPLC/electrospray ionization-mass spectrometry. Analysis of the different phospholipid classes in membranes of apoE-deficient and C57BL/6 J mice indicated no obvious differences in the distribution of different phospholipid classes but substantial differences in composition of phospholipid molecular species. Of special interest was the prevalence of phospholipids (phosphatidylcholine, diacyl-phosphatidylethanolamine, and phosphatidylserine) with 22:6n-3 in both the sn-1 and sn-2 positions of SPM and these phospholipid species were significantly higher in apoE-deficient mice as compared to control mice. Since polyunsaturated fatty acids in neurons are mainly supplied by astrocytes, these results revealed a new role for apoE in regulating polyunsaturated phospholipid molecular species in neuronal membranes.     

Variations in dietary triacylglycerol saturation alter the lipid composition and fluidity of rat intestinal plasma membranes

Rats were maintained on nutritionally complete diets enriched in unsaturated (corn oil) or saturated (butter fat) triacylglycerols. After 6 weeks, significant differences in the lipid composition and fluidity of a number of intestinal membranes were observed. The corn oil diet (enriched mainly in linoleic acid) increased the overall unsaturation of the acyl chains and enhanced the lipid fluidity, as assessed by the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene, of enterocyte microvillus and basolateral membranes and of colonocyte basolateral membranes. Concomitantly, the cholesterol content and the cholesterol/phospholipid molar ratio were increased in the microvillus but not in the basolateral membranes. The increased cholesterol in ileal microvillus membranes can result from enhanced cellular biosynthesis, since ileal slices from rats fed the unsaturated diet incorporated [14C]octanoate more rapidly into digitonin-precipitable sterol. Increased fluidity of the enterocyte microvillus and basolateral membranes, respectively, enhanced the enzyme specific activities of p-nitrophenylphosphatase and (Na+ + K+)-dependent adenosine triphosphatase. The results indicate that the lipid composition, fluidity and enzyme activities of intestinal plasma membranes can be altered by dietary means. Moreover, rat enterocytes possess regulatory mechanisms which modulate the cholesterol content of the microvillus membranes so as to mitigate changes in lipid fluidity.     

Chronic ethanol administration induced an increase in phosphatidylserine in guinea pig synaptic plasma membranes

Chronic ethanol administration to guinea pigs via intragastric intubation elicited a specific increase (50%) in phosphatidylserine in the synaptic plasma membrane. The ethanol-treated group also showed a 53% increase in synaptosomal (Na,K)-ATPase activity. Analysis of the acyl group composition of individual phospholipids in the same membrane fraction revealed only small changes which varied depending on the type of phospholipids. Since the (Na,K)-ATPase is known to be specifically activated by phosphatidylserine, the adaptive increase in enzyme activity during chronic ethanol treatment may be related to the increase in this type of negatively charged phospholipid.     

Chronic ethanol increases liver plasma membrane fluidity

Purified plasma membrane fractions of cultured well-differentiated Reuber H35 hepatoma cells were studied after growth in the presence or absence of ethanol. Growth of cells in the presence of ethanol significantly increased plasma membrane 5'-nucleotidase activity but did not influence sodium-potassium adenosinetriphosphatase activity. Fluorescence polarization of lipophilic probes was used to study membrane lipid structure. Steady-state polarization of diphenylhexatriene (DPH), a probe of the hydrophobic core, was significantly lower in plasma membranes from cells grown in 80 mM ethanol for 3 weeks, compared to controls. Decreased polarization of DPH in plasma membranes was observed after 3-weeks growth of cells in as little as 1 mM ethanol. A 1-h exposure to 80 mM ethanol had no effect. Altered DPH polarization was due to a decrease in the order parameter of the probe. The rotational correlation time of the probe was virtually unchanged. Chronic ethanol treatment of cells did not alter the polarization of the membrane surface probe trimethylammoniodiphenylhexatriene. Plasma membranes from cells grown in 80 mM ethanol had decreased contents of both phospholipid and unesterified cholesterol, but the cholesterol to phospholipid ratio was unchanged. The percentages of sphingomyelin and phosphatidylserine in plasma membrane phospholipids were significantly decreased after ethanol treatment, while the phosphatidylcholine/sphingomyelin ratio was increased by 42%. Vesicles prepared from total plasma membrane lipids of ethanol-treated cells, as well as vesicles prepared from polar lipids alone, showed the same alterations in DPH polarization as did plasma membranes. The importance of ethanol metabolism in the observed plasma membrane changes was demonstrated in two ways.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phospholipid composition of myelin and synaptosomal proteolipid from vertebrate brain

1. The phospholipid composition of the main proteolipid complexes of the nervous system was studied in myelin and synaptosomal membranes from brains of representatives of various vertebrate classes. 2. The relative content of acid phospholipids was much higher in proteolipid complexes from myelin and synaptosomal membranes of all vertebrates studied as compared to their content in the initial lipid extract (28-80% and 11-20% of total phospholipid content, respectively). 3. The relative content of acid phospholipids in proteolipid complexes of myelin membranes was much lower in brain of fishes and amphibia as compared to higher vertebrates. 4. The main acid phospholipids of proteolipid complexes was phosphatidylserine, phosphatidic acid being characteristic for myelin proteolipids and diphosphatidyl glycerol for synaptosomal proteolipids of all vertebrates studied.     

Phospholipid, cholesterol, polypeptide and glycoprotein composition of hepatic endosome subfractions.

The composition of hepatic endosome subfractions was compared directly with that of plasma membranes and Golgi-apparatus fractions. The neutral lipid and phospholipid composition of two endosome subfractions separated on Nycodenz gradients from a parent endosome fraction was similar to that of plasma membranes. The phospholipid/cholesterol ratios and the sphingomyelin contents were high, as in plasma membranes. However, the phosphatidylserine content was low. Endosomal subfractions contained a simpler polypeptide profile than did plasma membranes. However, a large number of glycoproteins were common to both fractions. Two endosome-specific glycoproteins, of Mr 59 000 and 38 000, were identified. Sialic acid was present at concentrations higher than in plasma membranes. The results indicate that endosomal membranes have a similar composition to plasma membranes that probably reflects their functional interaction during endocytosis and receptor recycling.     

Change of synaptic membrane lipid composition and fluidity by chronic administration of lithium

The effect of chronic administration of lithium salts on the lipid composition and physical properties of the synaptosomal plasma membrane was examined in rat brain. The effect of lithium treatment has been studied on the fluorescence polarization of synaptosomal plasma membrane and artificial lipid vesicles and on the lipid composition of the membranes. Fluorescence polarization of lipophilic probes was used to study membrane lipid structure. Steady-state polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH), a probe of the hydrophobic core, was significantly lower in plasma membranes from lithium-treated animals. Altered DPH polarization was due to a decrease in the order parameter of the probe. The lithium-treatment also changed the fluorescence of 1-anilino-8-naphthalene sulfonate (ANS), a probe that binds to the polar head group of the phospholipids and to proteins on the membrane surface. Synaptic plasma membranes from treated rats presented no significant changes on the cholesterol-to-phospholipid ratio, although the phospholipid class distribution was altered and the membrane phospholipid unsaturation increased. In summary, the neural plasma membranes became disorder after chronic lithium administration at therapeutic levels. This structural change may be due to changes in plasma membrane phospholipid distribution and to the degree of unsaturation of phospholipid fatty acids.     

Alterations in lipid composition and fluidity of liver plasma membranes in copper-deficient rats

In view of the importance of membrane fluidity on cell functions, the influence of phospholipid acyl groups on membrane fluidity, and the changes in lipid metabolism induced by copper (Cu) deficiency, this study was designed to examine the influence of dietary Cu on the lipid composition and fluidity of liver plasma membranes. Male Sprague-Dawley rats were divided into two dietary treatments, namely Cu deficient and Cu adequate. After 8 weeks of treatment, liver plasma membranes were isolated by sucrose density gradient centrifugation. The lipid fluidity of plasma membranes, as assessed by the intramolecular eximer fluorescence of 1,3-di(1-pyrenyl) propane, was significantly depressed by Cu deficiency. In addition, Cu deficiency significantly reduced the content of arachidonic and palmitoleic acids but increased the docosatetraenoic and docosahexaenoic acids of membrane phospholipids. This alteration in unsaturated phospholipid fatty acid composition, especially the large reduction in arachidonic acid, may have contributed to the depressed membrane fluidity. Furthermore, Cu deficiency also markedly altered the fatty acid composition of the triacylglycerols associated with the plasma membranes. Thus, the lipid composition and fluidity of liver plasma membranes are responsive to the animal's Cu status.     

Mature and immature synaptosomal membranes have a different lipid composition

Subfractionation of the optic tectum in chick embryos results in the isolation of two fractions enriched in synaptosomes (fraction A and fraction B). In chicks after hatching, this fractionation results in the isolation of a single synaptosomal fraction (fraction B) and of a fraction enriched in myelin membranes devoid of synaptosomes (fraction A). The lipid composition of synaptosomal fractions (A and B) and corresponding synaptosomal plasma membranes has been analyzed and compared to the lipid composition of similar fractions isolated from 2–3 day-old chicks. The phospholipid composition of fraction A in embryos was mainly represented by phosphatidylcholine (PC) and phosphatidylethanolamine (PE). The PE content was significantly lower than that of PC, which accounted for by approximately 50%. Sphingomyelin (SP) and phosphatidylinositol (PI) accounted for by only 6% of the total membrane phopsholipids. Fraction A isolated from the young chicks showed many significant changes. PC accounted for by approximately 40% and PE made up 35%. The amount of phosphatidylserine (PS) and SP increased. These data parallel our previous morphological observations, which showed that fraction A contains immature synaptosomes in embryos but myelin membranes and no synaptosomes in the young chicks. Fraction B has been shown to contain synaptosomes at all stages considered. It possessed in embryos a lipid composition similar to fraction A, except that PC content was higher in young embryos. The analyses on membrane fractions confirmed these results. On the contrary, this fraction showed many significant changes after hatching. The content of PC was significantly reduced, PE/PC ratio was significantly increased as well as ethanolamine plasmalogen (PLE) content. The percentage of PS, PI and SP were increased. The composition of fatty acids of the total fraction of phospholipids was also examined. The results parallel the observations on phospholipid classes.