Increasing Age Alters Transbilayer Fluidity and Cholesterol Asymmetry in Synaptic Plasma Membranes of Mice

Abstract: Previous studies examining age differences in membrane fluidity and cholesterol content have reported on the average or total change in membrane structure, respectively. However, a membrane consists of an exofacial leaflet and a cytofacial leaflet that differ in fluidity and cholesterol distribution. The purpose of the present experiments was to determine fluidity and cholesterol distribution of the exofacial and cytofacial leaflets of brain synaptic plasma membranes (SPMs) from 3–4-, 14–15-, and 24–25-month-old C57BL/6NNIA mice by using trinitrobenzenesulfonic acid (TNBS)-quenching techniques and fluorescent probes. The exofacial leaflet of SPMs from young mice was significantly more fluid compared with the cytofacial leaflet. The large difference in fluidity between the two leaflets was abolished in SPMs of the oldest age group. Total SPM cholesterol and the cholesterol-to-phospholipid molar ratio did not differ among the three different age groups of mice. However, considerable differences were observed in the distribution of cholesterol in the two SPM leaflets. The exofacial leaflet contained substantially less cholesterol than did the cytofacial leaflet (13 vs. 87%, respectively) in SPMs of young mice. This asymmetric distribution of cholesterol was significantly modified with increasing age. There was an approximately twofold increase in exofacial leaflet cholesterol in the oldest group compared with the youngest age group. Transbilayer fluidity and cholesterol asymmetry were altered in SPMs of older mice. This approach is a new and different way of viewing how aging modifies membrane structure. Age differences in SPM leaflet structure may be an important factor regulating activity of certain membrane proteins.     

Cholesterol asymmetry in synaptic plasma membranes

Lipids are essential for the structural and functional integrity of membranes. Membrane lipids are not randomly distributed but are localized in different domains. A common characteristic of these membrane domains is their association with cholesterol. Lipid rafts and caveolae are examples of cholesterol enriched domains, which have attracted keen interest. However, two other important cholesterol domains are the exofacial and cytofacial leaflets of the plasma membrane. The two leaflets that make up the bilayer differ in their fluidity, electrical charge, lipid distribution, and active sites of certain proteins. The synaptic plasma membrane (SPM) cytofacial leaflet contains over 85% of the total SPM cholesterol as compared with the exofacial leaflet. This asymmetric distribution of cholesterol is not fixed or immobile but can be modified by different conditions in vivo: (i) chronic ethanol consumption; (ii) statins; (iii) aging; and (iv) apoE isoform. Several potential candidates have been proposed as mechanisms involved in regulation of SPM cholesterol asymmetry: apoE, low-density lipoprotein receptor, sterol carrier protein-2, fatty acid binding proteins, polyunsaturated fatty acids, P-glycoprotein and caveolin-1. This review examines cholesterol asymmetry in SPM, potential mechanisms of regulation and impact on membrane structure and function.     

Transbilayer effects of ethanol on fluidity of brain membrane leaflets

Previous work on membrane effects of ethanol focused on fluidization of the bulk membrane lipid bilayer. That work was extended in the present study to an examination of ethanol's effect on lipid domains. Two independent methods were developed to examine the effects of ethanol on the inner and outer leaflets of synaptic plasma membranes (SPM). First, differential polarized phase and modulation fluorometry and selective quenching of diphenyl-1,3,5-hexatriene (DPH) were used to examine individual leaflets. Both limiting anisotropy and rotational relaxation time of DPH in SPM indicated that the outer leaflet was more fluid than the inner leaflet. Second, plasma membrane sidedness selective fluorescent DPH derivatives, cationic 1-[4-(trimethylammonio)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH) and anionic 3-[p-6-phenyl)-1,3,5-hexatrienyl]phenylpropionic acid (PRO-DPH), confirmed this transmembrane fluidity difference. TMA-DPH and PRO-DPH preferentially localized in the inner and outer leaflets of SPM, respectively. Ethanol in vitro had a greater fluidizing effect in the outer leaflet as compared to the inner leaflet. Thus, ethanol exhibits a specific rather than nonspecific fluidizing action within transbilayer SPM domains. This preferential fluidization of the SPM outer leaflet may have a role in ethanol affecting transmembrane signaling in the nervous system.     

Asymmetric distribution of a fluorescent sterol in synaptic plasma membranes: effects of chronic ethanol consumption

Ethanol-induced structural changes in membranes have in some studies been attributed to an increase in total membrane cholesterol. Consistent changes in cholesterol content, however, have not been observed in membranes of ethanol consuming animals and alcoholic patients. This study examined the hypotheses that cholesterol was asymmetrically distributed in synaptic plasma membranes (SPM) and that chronic ethanol consumption alters the transbilayer distribution of cholesterol. Dehydroergosterol, a fluorescent cholesterol analogue was used to examine sterol distribution and exchange in chronic ethanol-treated and pair-fed control groups. The cytofacial leaflet was found to have significantly more dehydroergosterol as compared to the exofacial leaflet. This asymmetric distribution was significantly reduced by chronic ethanol consumption as was sterol transport. Total cholesterol content did not differ between the two groups. Chronic ethanol consumption appeared to alter transbilayer sterol distribution as determined by the incorporation and distribution of dehydroergosterol in SPM. The changes in transbilayer sterol distribution are consistent with recent reports on the asymmetric effects of ethanol in vitro ((1988) Biochim. Biophys. Acta 946, 85-94) and in vivo ((1989) J. Neurochem. 52, 1925-1930) on membrane leaflet structure. The results of this study also underscore the importance of examining membrane lipid domains in addition to the total content of different lipids.     

Role of polyunsaturated fatty acids and lipid peroxidation in LM fibroblast plasma membrane transbilayer structure

The effects of polyunsaturated fatty acids and lipid peroxidation on LM fibroblast plasma membrane individual leaflet sterol distribution and structural order were examined. The cytofacial (inner) leaflet was more rigid and contained more sterol than the exofacial (outer) leaflet. The static (limiting anisotropy) and dynamic (rotational relaxation time) structural components of diphenylhexatriene (DPH) motion in each leaflet were determined by phase and modulation fluorometry measurements combined with leaflet-specific quenching by trinitrophenyl groups. Polyunsaturated fatty acids, incorporated into the membrane phospholipids by culture medium supplementation, decreased the limiting anisotrophy of DPH in the cytofacial but not the exofacial leaflet thereby abolishing the transbilayer difference in fluidity. Peroxidation by Fe(II) + H2O2 resulted in a rigidification (increase in limiting anisotropy and rotational relaxation time) of the plasma membrane exofacial leaflet, regardless of whether the membranes contained saturated and monounsaturated fatty acids or were enriched in either linoleate or linolenate. The structure of the cytofacial leaflet reported by DPH was unaffected. Plasma membrane transbilayer sterol distribution, measured by leaflet-specific quenching of dehydroergosterol fluorescence, indicated that 20-28% of the sterol was localized in the exofacial leaflet. Polyunsaturated fatty acid supplementation of LM fibroblasts resulted in a complete reversal of plasma membrane transbilayer sterol distribution (72-76% exofacial leaflet). Sterol transbilayer distribution between the membrane leaflets was completely resistant to alteration by exposure to crosslinking agents and peroxidation in control plasma membranes and by peroxidation in linoleate- or linolenate-supplemented membranes.     

Charged anesthetics selectively alter plasma membrane order

Although indirect evidence supporting differential lipid fluidity in the two monolayers of plasma membranes has accumulated, unambiguous demonstration of this difference has been difficult to obtain. In the present study, the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH), selective quenching of fluorescence by trinitrophenyl groups, and differential polarized phase fluorescence techniques were used to directly examine the static (order) and dynamic (rotational rate) components of lipid motion in the exofacial and cytofacial leaflets of LM fibroblast plasma membranes. The limiting anisotropy (0.137), the order parameter (0.590), and the rotational relaxation time (1.20 ns) of DPH in the plasma membranes (inner plus outer leaflet) indicated rapid but restricted probe motion in the lipid environment. However, the statics and dynamics of DPH motion in the individual monolayers were significantly (p less than 0.025) different. The limiting anisotropy, order parameter, and rotational relaxation time of DPH in the cytofacial monolayer were 0.036, 0.08, and 0.16 ns, respectively, greater than calculated for the exofacial monolayer of the LM plasma membrane. At appropriate concentrations, phenobarbital and, to a lesser degree, pentobarbital preferentially reduced the limiting anisotropy of DPH calculated for the exofacial leaflet while prilocaine reduced the limiting anisotropy of DPH in the cytofacial leaflet of LM fibroblast plasma membranes. In contrast, the putative cytofacial anesthetic procaine failed to show any preference for either leaflet. Arrhenius plots of DPH fluorescence in LM plasma membranes showed a prominent characteristic break point near 30-32 degrees C. Phenobarbital, pentobarbital, and procaine did not affect this break point while prilocaine selectively abolished it. The break point was therefore assigned to the inner monolayer of the LM plasma membrane.     

Differential modulation of human small intestinal brush-border membrane hemileaflet fluidity affects leucine aminopeptidase activity and transport of D-glucose and L-glutamate.

The fluidity of the exofacial (outer) and cytofacial (inner) leaflets of human proximal small intestinal brush-border membrane vesicles was studied by selective quenching by trinitrophenyl groups, steady-state fluorescence polarization, and differential polarized phase fluorometry techniques, utilizing the lipid soluble fluorophore 1,6-diphenyl-1,3,5-hexatriene. Differences in the hemileaflet's phospholipid composition were also analyzed by trinitrophenylation of aminophospholipids and phospholipase A2 treatment of these preparations. The results of these studies demonstrated that the inner leaflet of these membranes was less fluid than its outer counterpart. Phosphatidylserine was located mainly in the inner hemileaflet, whereas phosphatidylethanolamine and phosphatidylcholine were more symmetrically distributed between the hemileaflets of this membrane. Moreover, in vitro addition of 2-[(2-methoxyethoxy)ethyl]-cis-8-(2-octylcyclopropyl)octanoate (final concentration, 7.5 microM) preferentially fluidized the cytofacial leaflet and concomitantly increased Na(+)-gradient-dependent D-glucose uptake, but decreased Na+, K+-dependent L-glutamic acid uptake in these membrane vesicles. In vitro addition of benzyl alcohol (final concentration, 25 mM) preferentially fluidized the exofacial leaflet and decreased leucine aminopeptidase activity in these preparations. These results, therefore, demonstrate that the hemileaflets of human small intestinal brush-border membranes have different phospholipid compositions and fluidities. Alterations of either the exofacial or cytofacial leaflet fluidity, moreover, modulate protein-mediated activities in a distinct manner.     

Charged anaesthetics alter LM-fibroblast plasma-membrane enzymes by selective fluidization of inner or outer membrane leaflets.

The functional consequences of the differences in lipid composition and structure between the two leaflets of the plasma membrane were investigated. Fluorescence of 1,6-diphenylhexa-1,3,5-triene(DPH), quenching, and differential polarized phase fluorimetry demonstrated selective fluidization by local anaesthetics of individual leaflets in isolated LM-cell plasma membranes. As measured by decreased limiting anisotropy of DPH fluorescence, cationic (prilocaine) and anionic (phenobarbital and pentobarbital) amphipaths preferentially fluidized the cytofacial and exofacial leaflets respectively. Unlike prilocaine, procaine, also a cation, fluidized both leaflets of these membranes equally. Pentobarbital stimulated 5'-nucleotidase between 0.1 and 5 mM and inhibited at higher concentrations, whereas phenobarbital only inhibited, at higher concentrations. Cationic drugs were ineffective. Two maxima of (Na+ + K+)-ATPase activation were obtained with both anionic drugs. Only one activation maximum was obtained with both cationic drugs. The maximum in activity below 1 mM for all four drugs clustered about a single limiting anisotropy value in the cytofacial leaflet, whereas there was no correlation between activity and limiting anisotropy in the exofacial leaflets. Therefore, although phenobarbital and pentobarbital below 1 mM fluidized the exofacial leaflet more than the cytofacial leaflet, the smaller fluidization in the cytofacial leaflet was functionally significant for (Na+ + K+)-ATPase. Mg2+-ATPase was stimulated at 1 mM-phenobarbital, unaffected by pentobarbital and slightly stimulated by both cationic drugs at concentrations fluidizing both leaflets. Thus the activity of (Na+ + K+)-ATPase was highly sensitive to selective fluidization of the leaflet containing its active site, whereas the other enzymes examined were little affected by fluidization of either leaflet.     

Transbilayer Distribution of Cholesterol Is Modified in Brain Synaptic Plasma Membranes of Knockout Mice Deficient in the Low-Density Lipoprotein Receptor, Apolipoprotein E, or Both Proteins

Abstract: Both apolipoprotein E (apoE) and the low-density lipoprotein (LDL) receptor are present in brain; however, little is known regarding the function of these proteins in brain, in particular with respect to brain cholesterol. The role of apoE and the LDL receptor in modulating the transbilayer or asymmetric distribution of cholesterol in the exofacial and cytofacial leaflets of synaptic plasma membranes (SPMs) was examined in mutant mice deficient in apoE, the LDL receptor, or both proteins by using the fluorescent sterol dehydroergosterol and fluorescent quenching procedures. Fluidity of the exofacial and cytofacial leaflets was also measured. Cholesterol asymmetry of SPMs was altered in the mutant mice, with the largest effect observed in the LDL receptor-deficient mice. There was an approximately twofold increase in the percent distribution of cholesterol in the exofacial leaflet of the LDL receptor-deficient mice (32%) compared with C57BL/6J mice (15%). Mice deficient in apoE or both proteins also showed a significantly higher percent distribution of cholesterol (23 and 26%, respectively) in the exofacial leaflet compared with the C57BL/6J mice. Although the percent distribution of cholesterol was highest in the exofacial leaflet of the LDL receptor-deficient mice, fluidity of the exofacial leaflet of that group was significantly lower. However, the cholesterol-to-phospholipid ratio of SPMs of the LDL receptor-deficient mice was significantly lower, and this difference was largely the result of a significant increase in the total amount of SPM phospholipid. This study demonstrates for the first time that SPM lipid structure is altered in mice deficient in apoE or the LDL receptor. Although the mechanism that maintains the asymmetric distribution of cholesterol in plasma membranes is not well understood, data of the present experiments indicate that both apoE and the LDL receptor are involved in maintaining the transbilayer distribution of cholesterol.     

Embedding Aβ42 in Heterogeneous Membranes Depends on Cholesterol Asymmetries

Using a coarse-grained lipid and peptide model, we show that the free energy stabilization of amyloid-β in heterogeneous lipid membranes is predicted to have a dependence on asymmetric distributions of cholesterol compositions across the membrane leaflets. We find that a highly asymmetric cholesterol distribution that is depleted on the exofacial leaflet but enhanced on the cytofacial leaflet of the model lipid membrane thermodynamically favors membrane retention of a fully embedded Aβ peptide. However, in the case of cholesterol redistribution that increases concentration of cholesterol on the exofacial layer, typical of aging or Alzheimer’s disease, the free energy favors peptide extrusion of the highly reactive N-terminus into the extracellular space that may be vulnerable to aggregation, oligomerization, or deleterious oxidative reactivity.     

Embedding Aβ42 in Heterogeneous Membranes Depends on Cholesterol Asymmetries

Using a coarse-grained lipid and peptide model, we show that the free energy stabilization of amyloid-β in heterogeneous lipid membranes is predicted to have a dependence on asymmetric distributions of cholesterol compositions across the membrane leaflets. We find that a highly asymmetric cholesterol distribution that is depleted on the exofacial leaflet but enhanced on the cytofacial leaflet of the model lipid membrane thermodynamically favors membrane retention of a fully embedded Aβ peptide. However, in the case of cholesterol redistribution that increases concentration of cholesterol on the exofacial layer, typical of aging or Alzheimer’s disease, the free energy favors peptide extrusion of the highly reactive N-terminus into the extracellular space that may be vulnerable to aggregation, oligomerization, or deleterious oxidative reactivity.     

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     

Polyunsaturated fatty acids alter sterol transbilayer domains in LM fibroblast plasma membrane

Sterols are asymmetrically distributed between the leaflets of animal cell plasma membranes. Although transbilayer migration of sterols is extremely rapid, s to min, previous experimental manipulations have not altered their transmembrane steady-state distribution. However, the effect of polyunsaturated fatty acids has not been reported. When cultured in a lipid-free, chemically defined culture medium, LM fibroblasts do not synthesize polyunsaturated fatty acids but will incorporate polyunsaturated fatty acids into their plasma membranes if supplied in the medium. Sterol transbilayer distribution in LM plasma membranes was determined from quenching of fluorescence of dehydroergosterol by trinitrophenyl groups selectively attached to the exofacial leaflet. When cells are cultured in lipid-free media, 28.1% of the plasma membrane sterol is located in the exofacial (outside) leaflet. In contrast, when cells are cultured with linoleate- or linolenate-supplemented medium, 71.8% and 75.5% of the plasma membrane sterol is exofacial, respectively.     

Exofacial membrane composition and lipid metabolism regulates plasma membrane P4-ATPase substrate specificity

The plasma membrane of a cell is characterized by an asymmetric distribution of lipid species across the exofacial and cytofacial aspects of the bilayer. Regulation of membrane asymmetry is a fundamental characteristic of membrane biology and is crucial for signal transduction, vesicle transport, and cell division. The type IV family of P-ATPases, or P4-ATPases, establishes membrane asymmetry by selection and transfer of a subset of membrane lipids from the lumenal or exofacial leaflet to the cytofacial aspect of the bilayer. It is unclear how P4-ATPases sort through the spectrum of membrane lipids to identify their desired substrate(s) and how the membrane environment modulates this activity. Therefore, we tested how the yeast plasma membrane P4-ATPase, Dnf2, responds to changes in membrane composition induced by perturbation of endogenous lipid biosynthetic pathways or exogenous application of lipid. The primary substrates of Dnf2 are glucosylceramide (GlcCer) and phosphatidylcholine (PC, or their lyso-lipid derivatives), and we find that these substrates compete with each other for transport. Acutely inhibiting sphingolipid synthesis using myriocin attenuates transport of exogenously applied GlcCer without perturbing PC transport. Deletion of genes controlling later steps of glycosphingolipid production also perturb GlcCer transport to a greater extent than PC transport. In contrast, perturbation of ergosterol biosynthesis reduces PC and GlcCer transport equivalently. Surprisingly, application of lipids that are poor transport substrates differentially affects PC and GlcCer transport by Dnf2, thus altering substrate preference. Our data indicate that Dnf2 exhibits exquisite sensitivity to the membrane composition, thus providing feedback onto the function of the P4-ATPases.     

Modulation of prolactin binding sites in vitro by membrane fluidizers. Effects on male prostatic and female hepatic membranes in alcohol-fed rats

The objectives of this study were (i) to determine if in vivo administration of ethanol to rats produced changes in apparent lipid fluidity and prolactin binding capacity of male prostatic and female hepatic membranes and (ii) to compare the effects of membrane fluidizers (aliphatic alcohols) in vitro on prolactin binding of prostatic and hepatic membranes in control and alcohol-fed animals. In vitro ethanol has been shown by us previously to increase prolactin receptor levels presumably by unmasking cryptic prolactin receptors. The degree of fluidization was monitored by a fluorescence polarization method using 1,6-diphenylhexatriene. Adult male and female rats were given either water or 4% ethanol as the sole source of drinking fluid for a period of 6 weeks. No significant changes in plasma prolactin were observed between control and ethanol-treated groups of either sex. However, the microviscosity parameter, inversely related to lipid fluidity, was increased approx. 34% and 40%, respectively, in male prostatic and female rat hepatic membranes after ethanol feeding. Furthermore, 125I-prolactin binding capacity was decreased approx. 30% and 26%, respectively, in prostatic and hepatic membranes of alcohol fed animals. In vitro treatment with aliphatic alcohols had no effect on either microviscosity or prolactin binding in hepatic or prostatic membranes from ethanol-fed rats, but both fluidized and increased prolactin binding in the same membrane preparations from control rats. Our observations are consistent with the direct relationship between membrane fluidity and prolactin receptor levels. The changes in prostatic and hepatic membranes after alcohol feeding, namely decreased prolactin receptor levels, decreased fluidity and increased resistance to the fluidizing effects of in vitro aliphatic alcohols may reflect a fundamental membrane defect.     

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)     

Variations in Membrane Sensitivity of Brain Region Synaptosomes to the Effects of Ethanol In Vitro and Chronic In Vivo Treatment

The effects of chronic ethanol treatment on the membrane order of synaptosomes from the cerebral cortex, striatum, cerebellum, brainstem, and hippocampus of rats were determined by measuring the fluorescence polarization of diphenylhexatriene (DPH) that had been incorporated into the synaptosomal membranes. Fischer-344 rats either were fed a nutritionally complete ethanol-containing liquid diet for 5 months or pair-fed with a diet that contained sucrose substituted isocalorically for ethanol. Polarization values for synaptosomes from all the brain regions studied were similar except for those from cerebral cortical synaptosomal membranes, which were significantly less ordered. Ethanol in vitro (30-500 mM) decreased the polarization values in synaptosomes from sucrose-control rats for all brain regions, although the sensitivity of cerebellar synaptosomes to the membrane disordering effects of ethanol in vitro was significantly greater that of synaptosomes from other brain regions. Chronic ethanol treatment did not alter baseline polarization for any brain region. Cerebellar and brainstem synaptosomes from the ethanol-fed rats were significantly less susceptible to the membrane disordering effects of ethanol in vitro compared to their sucrose controls, suggesting that chronic ethanol administration results in tolerance to ethanol's membrane effects. Striatal synaptosomes exhibited intermediate tolerance, whereas the sensitivities of cortical and hippocampal synaptosomes to membrane disordering by ethanol in vitro were not significantly affected by the chronic ethanol treatment. These results suggest that synaptosomal membranes have different membrane order requirements depending on the brain region from which they are prepared. Variations in brain regional neuronal membrane sensitivity to ethanol and differential tolerance development may contribute to some of the acute and chronic behavioral effects of ethanol.     

Conserved mechanism of phospholipid substrate recognition by the P4-ATPase Neo1 from Saccharomyces cerevisiae

The type IV P-type ATPases (P4-ATPases) thus far characterized are lipid flippases that transport specific substrates, such as phosphatidylserine (PS) and phosphatidylethanolamine (PE), from the exofacial leaflet to the cytofacial leaflet of membranes. This transport activity generates compositional asymmetry between the two leaflets important for signal transduction, cytokinesis, vesicular transport, and host-pathogen interactions. Most P4-ATPases function as a heterodimer with a β-subunit from the Cdc50 protein family, but Neo1 from Saccharomyces cerevisiae and its metazoan orthologs lack a β-subunit requirement and it is unclear how these proteins transport substrate. Here we tested if residues linked to lipid substrate recognition in other P4-ATPases also contribute to Neo1 function in budding yeast. Point mutations altering entry gate residues in the first (Q209A) and fourth (S457Q) transmembrane segments of Neo1, where phospholipid substrate would initially be selected, disrupt PS and PE membrane asymmetry, but do not perturb growth of cells. Mutation of both entry gate residues inactivates Neo1, and cells expressing this variant are inviable. We also identified a gain-of-function mutation in the second transmembrane segment of Neo1 (Neo1[Y222S]), predicted to help form the entry gate, that substantially enhances Neo1's ability to replace the function of a well characterized phospholipid flippase, Drs2, in establishing PS and PE asymmetry. These results suggest a common mechanism for substrate recognition in widely divergent P4-ATPases.     

Optimisation of plant sterols incorporation in human keratinocyte plasma membrane and modulation of membrane fluidity.

The in vitro effects of plant sterols were investigated with regard to their uptake and membrane lipid fluidity in human keratinocytes. Among the different media tested to transport sterols (liposomes, micelles and organic solvents), the best results in terms of incorporation and viability were obtained by the use of the organic solvents dimethylsulfoxide and ethanol. After 48 h incubation exogenous sterol can account for about 30% of the total cell sterol content. The total sterol amount in plasma membranes increased 2-fold after incubation with cholesterol, whereas it was not altered when phytosterols were incorporated. The incorporation of cholesterol, sitosterol and stigmasterol led to an increase in the percent of unsaturated fatty acid C18:1 in the plasma membrane. The effect of this uptake on membrane fluidity was studied by means of fluorescence polarisation using DPH and TMA-DPH as fluorescent probes. Whereas cholesterol and sitosterol had no significant effect on the DPH fluorescence anisotropy (rs), the presence of stigmasterol induced a 12% decrease of rs reflecting an increase in membrane fluidity. We can conclude from this study that in the presence of sitosterol, the mean fluidity of the membrane is regulated whereas stigmasterol triggers a looseness of molecular packing of phospholipids acyl chains, in accordance with previous results obtained on purely lipid model membranes.     

Interactive effects of dietary (n-3) polyunsaturated fatty acids and chronic ethanol intoxication on synaptic membrane lipid composition and fluidity in rats.

The influence of dietary polyunsaturated fatty acids on fatty acid composition, cholesterol and phospholipid content as well as 'fluidity' (assessed by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) probes) of brain synaptic plasma membranes (SPM) and their interactions with chronic ethanol effects were studied in rats fed for two generations with diets either devoid of (n-3) fatty acids (sunflower oil diet), rich in alpha-linolenic acid (soya oil diet) or in long chain (n-3) fatty acids (sunflower + cod liver oil diet). Results were compared with rats fed standard lab chow. Sunflower oil led to an increase in the (n-6)/(n-3) ratio in the membranes with an increase of the 'fluidity' at membrane apolar level; sunflower + cod liver oil decreased the (n-6)/(n-3) ratio without affecting membrane 'fluidity' while no difference was seen between the SPM of rats fed soya oil and standard diet. After 3 weeks alcohol intoxication in rat fed the standard diet: oleic alpha-linoleic acids and cholesterol levels were increased, arachidonic acid and the double bond index/saturated fatty acids were decreased and there was a decrease of 'fluidity' in the lipid core of the SPM. Soya oil almost totally abolished these usually observed changes in the SPM fatty acids composition but increased oleic acid and cholesterol without any change in fluidity. Sunflower oil led to the same general alterations of fatty acid as seen with standard diet but to a greater extent, with decrease of the 'fluidity" at the apolar level and in the region probed by TMA-DPH. When sunflower oil was supplemented with cod liver oil, oleic and alpha-linoleic acids were increased while the 'fluidity' of the apolar core of SPM was decreased. So, the small changes in fatty acid pattern seem able to modulate neural properties i.e. the responses to a neurotoxic like ethanol. A structurally specific role of PUFA is demonstrated by the pernicious effects of the alpha-linolenic acid deficient diet which are not totally prevented by the supply of long chain (n-3) PUFA.