Chronic ethanol ingestion modifies liver microsomal phosphatidylserine inducing resistance to hydrolysis by exogenous phospholipase A2.

Chronic ethanol ingestion leads to the acquisition of a tolerance to membrane lipid disordering, a lowered partition coefficient to hydrophobic compounds and a resistance to the hydrolysis of the phospholipids by exogenous phospholipase A2. Anionic phospholipids have been implicated as being responsible for the resistance to lipid disordering and a number of modifications to these phospholipids are known to occur as a result of chronic ethanol-ingestion. In this study the basis of the resistance to phospholipase A2 in hepatic microsomes was investigated. It was found that chronic ethanol-induced modifications to each of the major phospholipid classes was responsible to some extent for the resistance to phospholipase A2, however, PS was particularly potent considering it is a compositionally minor constituent. The effect was interpreted as a reduced ability to activate the phospholipase A2 since PS acts as an essential activator of phospholipase A2 (along with PI). Fatty acid analysis revealed that the chronic ethanol-treatment resulted in a elevated level of docosahexaenoate with a parallel reduction in arachidonate in phosphatidylserine. Lipid packing and organization is important in the regulating the level of exogenous phospholipase A2 activity but the activity was not found to correlate with lipid order of different phosphatidylserine species. It is concluded that subtle differences in the molecular species arrangement or disposition around the enzyme may be responsible for the altered phospholipase A2 interaction with the membrane induced by chronic ethanol-treatment. One implication of this study is that other anionic phospholipid dependent membrane proteins, of which there are many known examples, may also be modified as a result of chronic ethanol-ingestion.

The effects of phospholipid unsaturation and alcohol perturbation at the protein/lipid interface probed using fluorophore lifetime heterogeneity.

The influence of phospholipid unsaturation and perturbation by alcohols, on the membrane protein/lipid interface, was probed using the fluorescence decay properties of 1,6-diphenyl-1,3,5-hexatriene (DPH) and DPH attached to the sn-2 chain of phosphatidylcholine (DPH-PC), in lipid bilayers and microsomal membranes. With microsomal membranes it was found that it was appropriate to describe the fluorescence decay of DPH-PC as a range of decay rates, accomplished by fitting the data to a bimodal fluorescence lifetime distribution. The major lifetime center had a broad distributional width, indicative of excited state fluorophore heterogeneity. The effect was attributable to protein, and by inference, the protein/lipid interface, since in vesicles made from total microsomal lipids (i.e., without protein) the fluorescence decay was homogeneous. Upon addition of ethanol or hexanol the width of the lifetime distribution of the major lifetime center increased, indicating increased environmental heterogeneity. It was confirmed that the effect was manifest at the protein/lipid interface, and not due to lipid-reorganizational factors, since it could also be obtained using a simple lipid bilayer vesicle system with apocytochrome c as a model membrane protein, and DPH instead of DPH-PC. Environmental heterogeneity was also found to increase with increased phosphatidylcholine (sn-2) unsaturation. The environmental heterogeneity at the protein/lipid interface could arise from a combination of varying polarities of amino acid side chains and of water that may intercalate in packing defects on the hydrophobic surface of the protein. Therefore the results could be explained on the basis of an increased degree of hydration at the protein/lipid interface. Such an effect offers a route whereby acyl chain perturbation and increased unsaturation might influence protein conformation and hence function.

Analysis of cell membrane micro-heterogeneity using the fluorescence lifetime of DPH-type fluorophores.

Heterogeneity in the lipid organization in lipid bilayers and cell membranes was probed by using the fluorescence decay of 1,6-diphenyl-1,3,5-hexatriene (DPH) and DPH attached to the sn-2 position of phosphatidylcholine (DPH-PC). In the presence of protein, it is proposed that the bulk lipids and boundary lipids can potentially provide distinct enough fluorophore environments for two different lifetime centers to be recovered from the analysis of the fluorescence decay. To test this model experiments were performed with cytochrome b5 in 1-palmitoyl-2-oleoylphosphatidylcholine bilayers. The number of boundary lipids of cytochrome b5 is known from the literature or can be calculated from known dimensions, so that for a known protein:lipid ratio the fraction of lipids in the bulk and boundary lipid regions is known. These values were found to closely correspond to the fractions associated with the lifetime centers recovered from an analysis of the fluorescence decay assuming two major fluorophore populations. This indicated that the DPH distributed in a similar manner to the lipids and that its boundary lipid residency time was greater than the excited state lifetime, showing the validity of the approach. An important requirement was that the protein should influence the fluorophore decay sufficiently enough to enable separate lifetime centers for the bulk and boundary lipid fluorophores to be recovered by the analysis. Attempts were made to analyze the fluorescence decay of DPH in liver plasma membranes and microsomes as arising from two distinct fluorophore populations, however, the basic condition was not satisfied. By contrast, using DPH-PC it was possible to extract two separate lifetime centers. The limitations and potential of this approach are critically assessed and it is concluded that in certain circumstances information pertaining to the protein-lipid interfacial region of membranes can be extracted from fluorescence decay heterogeneity properties.

The isolation and analysis of the luminal plasma membrane of calf urinary bladder epithelium.

The luminal plasma membrane of calf urinary bladder epithelium (urothelium) has been isolated by a method designed to preserve enzymic activity as well as structural integrity. The yield was about 80 micrograms per calf bladder. Low levels of 5' nucleotidase, Mg2+-ATPase and (Na+ + K+)-ATPase activities were found in the luminal membrane fraction. Cerebroside was the major lipid present and dodecyl sulphate gel electrophoresis revealed a complex protein and glycoprotein composition in the whole membrane. A membrane fraction consisting of only the plaque areas was shown to have a simpler protein composition with major polypeptides of apparent Mr 12 000 and 22 000. These may associate to form a 30 000 apparent Mr complex which could represent the individual 'particles' of the dodecameric subunits seen by electron microscopy in the plaque regions.

The dynamics of lipid motion in sarcoplasmic reticulum membranes determined by steady-state and time-resolved fluorescence measurements on 1,6-diphenyl-1,3,5-hexatriene and related molecules.

Steady-state and time-resolved fluorescence anisotropy measurements were made on 1,6-diphenyl-1,3,5-hexatriene (DPH), 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) and 1-acyl-2-(DPH)-phosphatidylcholine (DPH-PC) incorporated into sarcoplasmic reticulum membranes. The results were analysed in terms of the 'wobbling-in-cone' model. Considerable differences in the fluorescence parameters were found. In particular TMA-DPH and DPH-PC showed a smaller cone angle, relating to the range of acyl chain motion, compared to DPH, taken to be a reflection of a difference in probe locations. The influence of the protein component was also found to restrict DPH motion more than TMA-DPH and DPH-PC. Effectiveness in assessment of perturbation of the membrane by the non-esterified fatty acid, oleic acid again revealed differences. The steady-state anisotropy decreased on addition of oleic acid; a recovery to control values was observed with DPH but not with the other probes. Time-resolved parameters followed the same pattern. The results of this work demonstrated the effectiveness of these three probes in revealing differences in membrane properties, such as protein and fatty acid perturbation of membrane lipid structure and dynamics.

Solvent relaxation in lipid bilayers with dansyl probes.

The solvent relaxation properties of the dansyl group attached to two lipids (dansylphosphatidylethanolamine and dansylphosphatidylserine), a fatty acid (dansylundecanoic acid), and two drugs (dansylbenzocaine and dansylpropranolol) were compared in a variety of different lipid systems. Several methods for characterising solvent relaxation were compared in detail for dansylpropranolol in bilayer vesicles of egg phosphatidylcholine. It was shown that the relaxation process is non-monoexponential; nevertheless, for comparative purposes, a model was adopted in which the lifetime associated with the negative exponent in a two exponential decay analysis, obtained at a particular energy on the red edge of emission, was taken as an approximation to a 'solvent relaxation' rate. A negative exponent, indicative of solvent relaxation processes, occurring in the nanosecond time-scale, was found only for dansylpropranolol, dansylPE and dansylundecanoic acid. On addition of the spin probe, 5-doxylstearate, the negative exponent was unaffected in liquid-crystalline phase lipids but was no longer found in gel-phase lipid in the case of dansylpropranolol, while for dansylPE the relaxation time was reduced. On the basis of these types of measurement it was possible to distinguish between different lipid environments using the same probe or between different dansyl environments of the different probes in the same lipid in cases where this would have been difficult or impossible solely on the basis of steady-state or fluorescence lifetime measurements.

Chronic ethanol intoxication induces adaptive changes at the membrane protein/lipid interface.

Modifications were found to occur at the membrane protein/lipid interface of liver microsomes in animals that had been subjected to chronic ethanol ingestion. The effects were revealed by probing this region with 1,6-diphenyl-1,3,5-hexatriene (DPH), trimethylammonium-DPH (TMA-DPH) and DPH attached to the sn-2 chain of phosphatidylcholine (1-palmitoyl-2-[[2-[4-(6-phenyl-trans-1,3,5-hexatrienyl) phenyl]ethyl]carbonyl]-3-sn-phosphatidylcholine, DPH-PC). In intact membranes, it was found that the decay of the excited state was heterogeneous, this being modeled by fitting the data to a fluorescence lifetime distribution. The full-width of the distribution at half-maximum, which relates to the degree of excited state environmental heterogeneity, increased for each fluorophore, as a result of chronic ethanol treatment. For TMA-DPH and DPH the excited state heterogeneity could have arisen from, (i) the protein/lipid interface and (ii) varied degrees of water penetration into the lipid, due to the ability of these fluorophores to sample along the bilayer normal. By contrast, the DPH in DPH-PC, due to its tethering, was only able to sample the heterogeneity at the protein/lipid interface, as confirmed by a homogeneous decay in vesicles of microsomal lipid extracts. The increased degree of DPH-PC fluorescence decay heterogeneity in microsomes from chronic ethanol-treated animals as compared to controls, was found to persist in vesicles of extracted lipids, when apocytochrome C was included in the vesicle preparations as a model protein. This effectively eliminated a protein modification from being responsible and indicated that a chronic-ethanol induced alteration in the lipids was being expressed in the form of a physico-chemical modification at the protein/lipid interface. The degree of DPH-PC environmental heterogeneity was also directly increased by ethanol, however, membranes from chronic ethanol-treated animals were resistant to this effect, showing that the phenomenon of 'membrane tolerance' extends to the membrane protein/lipid interface.

On the use of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine in the study of lipid polymorphism.

The change in the fluorescence properties of dioleoyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanola mine (N-NBD-PE) as an indicator of the (liquid-crystalline) bilayer-to-non-bilayer hexagonalII (HII) phase transition has been investigated. Lipid bilayer systems which are known to undergo the bilayer-to-HII phase transition on addition of Ca2+ were compared with systems which can undergo aggregation and fusion but not HII phase formation. The former included Ca2+-triggered non-bilayer transitions in cardiolipin and in phosphatidylethanolamine mixed with phosphatidylserine. The latter type of system investigated included the addition of polylysine to cardiolipin and Ca2+ to phosphatidylserine. Freeze-fracture electron microscopy was used to confirm that under the experimental conditions used, the formation of HII phase was occurring in the first type of system, but not in the second, which was stable in the bilayer state. It was found that the fluorescence intensity of N-NBD-PE (at 1 mol% of the phospholipids) increased in both types of system, irrespective of the formation of the HII phase. A dehydration at the phospholipid head group is a common feature of the formation of the HII phase, the interaction of divalent cations with phosphatidylserine and the interaction of polylysine with lipid bilayers, suggesting that this may be the feature which affects the fluorescence properties of the NBD. The finding of a fluorescence intensity increase in systems lacking HII phase involvement clearly indicates that the effect is not unique to the formation of the HII phase. Thus, while offering high sensitivity and the opportunity to follow kinetics of lipid structural changes, changes in the N-NBD-PE fluorescence properties should be interpreted with caution in the study of the bilayer-to-HII phase transition.

The effects of dietary (n-3) fatty acid supplementation on lipid dynamics and composition in rat lymphocytes and liver microsomes.

Rats were fed diets devoid of (n-3) fatty acids (olive oil supplementation) or high in (n-3) fatty acids (fish oil supplementation) for a period of 10 days. In spleen lymphocytes and liver microsomes derived from animals fed fish oil diets, relatively high levels of (n-3) eicosapentaenoic (20:5), docosapentaenoic (22:5) and docosahexaenoic acids (22:6) were obtained compared to minimal levels when fed the olive oil diet. When the average lipid motional properties were examined by measuring the fluorescence anisotropy of diphenylhexatriene, no significant different was found between intact liver microsomes from animals fed the two diets. However, when lipid motion was examined in vesicles of phosphatidylcholine, isolated from the microsomes from fish oil fed animals (21.4% (n-3) fatty acids), the fluorescence anisotropy was significantly less than the corresponding phosphatidylcholine from olive oil fed animals (5.6% (n-3) fatty acids), indicating a more disordered or fluid bilayer in the presence of higher levels of (n-3) fatty acids. Phosphatidylethanolamine (n-3) fatty acids were also elevated after fish oil supplementation (41.3% of total fatty acids), compared to the level after olive oil supplementation (21.4%). The major effect of the fish oil supplementation was a replacement of (n-6) arachidonic acid by the (n-3) fatty acids and when this was 'modeled', using liposomes of synthetic lipids, 1-palmitoyl-2-arachidonyl(n-6) or docosahexaenoyl(n-3)-phosphatidylcholine, significant differences in lipid motional properties were found, with the docosahexaenoate conferring a more disordered or fluid lipid environment. Thus it appears that although lipid order/fluidity can be significantly decreased by increases in the highly unsaturated (n-3) fatty acid levels, alterations in membrane domain organization and/or phospholipid molecular species composition effectively compensated for the changes, at least as far as average lipid motional properties in the intact membranes was concerned.

Effects of ethanol on protein kinase C activity induced by filamentous actin.

Protein kinase C (PKC) can be activated by interaction with filamentous actin (F-actin) in the absence of membrane lipids (S.J. Slater, S.K. Milano, B.A. Stagliano, K.J. Gergich, J.P. Curry, F.J. Taddeo and C.D. Stubbs, Biochemistry 39 (2000) 271-280). Here, the effects of ethanol on the F-actin-induced activities of a panel of PKC isoforms consisting of 'conventional' (cPKC) alpha, betaI, gamma, 'novel' (nPKC) delta, epsilon and 'atypical' (aPKC) zeta were investigated using purified PKC and F-actin. Ethanol was found to inhibit the Ca2+- and phorbol ester-dependent activities of cPKCalpha and betaI, and the Ca2+- and phorbol ester-independent activity of cPKCgamma, whereas the activities of nPKCdelta, epsilon and aPKCzeta were unaffected. Although the activities of cPKCalpha and betaI induced by saturating levels of phorbol ester were inhibited by ethanol, the binding of these isozymes to F-actin was unaffected within the same phorbol ester concentration range. Conversely, within submaximal levels of phorbol ester, cPKCalpha and betaI activities were unaffected by ethanol whereas binding to F-actin was inhibited. The potency of the inhibition of F-actin-induced cPKCbetaI activity increased with n-alkanol chain length up to n-hexanol, after which it declined. The results indicate that PKC activities associated with F-actin, and therefore cellular processes involving the actin cytoskeleton, are potential targets for ethanol action. The effects of ethanol on these processes may differ according to the particular regulating PKC isoform, its intracellular localization and the presence of activators and cofactors.

The effects of non-lamellar forming lipids on membrane protein-lipid interactions.

The role of lipid polymorphism in the regulation of membrane-associated protein function is examined, based on recent studies which showed that changes in the levels of phosphatidylethanolamine (PE), cholesterol and phospholipid unsaturation, modulate the activity of the key signal transduction enzyme, protein kinase C (PKC). It is shown that effects of membrane compositional changes on PKC activity involve a perturbation of protein-lipid interactions with the head group region rather than with the hydrophobic interior of the bilayer. A key determinant in the perturbation of these interactions is suggested to be an elastic curvature energy, termed curvature stress, which results from the unfavorable packing of non-lamellar forming lipids in a planar bilayer. PKC activity is shown to be a biphasic function of curvature stress, with an optimum value of this parameter corresponding to an optimally active PKC conformation. Thus, it is shown that the maximal activity of conformationally distinct PKC isoforms may require a different optimum value of curvature stress. Furthermore, it is hypothesized that curvature stress may have differing effects on the conformation of membrane-associated PKC activity induced by diacylglycerols, phorbol esters or other activators, based on recent studies showing that these agents induce the formation of disparate active conformers of the enzyme.

Application of flow microcalorimetry to analytical problems-I. Determination of organophosphorus pesticides by inhibition of cholinesterase.

Flow microcalorimetry has been applied to the determination of organophosphorus pesticides by inhibition of cholinesterase. One direct inhibitor (TEPP) and one latent inhibitor (parathion) were investigated. The former is determinable at concentrations of about 10(-6)M and the latter at about 10(-4)M. The inhibitory power of parathion is increased if methanol is used as solvent.

Effect of ethanol on platelet phospholipase A2.

Platelet aggregation is known to be inhibited by ethanol, and this has been suggested to be one of the attenuating effects of ethanol in cardiovascular disease. Recent studies have implicated an inhibition of phospholipase A2 induced arachidonic acid release, since the production of prostanoids that are formed from arachidonic acid and are involved in the aggregation process has been shown to be diminished by ethanol. Phospholipase A2 is found in platelets in both a cytosolic form, from where it may translocate to the plasma membrane to release arachidonic acid, and in a secretory form which is released extracellularly upon activation. In the present study, the effect of ethanol on the secretion of phospholipase A2 and on its activity was determined. It was found that ethanol inhibited phospholipase A2 secretion but not its activity. By contrast, the activity of the cytosolic form of phospholipase A2 was inhibited by ethanol.

Effect of increasing the level of omega-3 fatty acids on rat skeletal muscle sarcoplasmic reticulum.

The effect of dietary supplementation with fish oil as compared to corn oil on the lipid dynamics and calcium ATPase activity of rat skeletal sarcoplasmic reticulum was examined. After four-week supplementation with fish oil, the levels of eicosapentaenoic (20:5 omega 3), docosapentaenoic (22:5 omega 3) and docosahexaenoic (22:6 omega 3) acids in the total lipids were 5.3, 5.5 and 28.1% of the total fatty acids, respectively. In contrast, with corn oil only 22:6 was found (8.9%). The level of these fatty acids in phosphatidylethanolamine from the membranes of animals fed fish oil was 4.2 (20:5), 5.4 (22:5) and 49.1% (22:6); and for phosphatidylcholine it was 5.4 (20:5), 4.6 (22:5) and 17.4% (22:6). Again, in corn oil fed animals, only 22:6 was found in appreciable amounts, namely 28.3% in phosphatidylethanolamine and 1.8% in phosphatidylcholine. The steady state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to assess lipid order and was found to be only slightly less for membranes from animals supplemented with fish oil (0.120) as compared to those supplemented with corn oil (0.124). The calcium ATPase was found to be unaffected by supplementation consistent with the observed modest changes in lipid order as well as with suggestions that the enzyme is relatively insensitive to the level of unsaturation. It could be argued that if large increases in fatty acyl polyunsaturation in mammalian cell membranes would lead to marked alterations in bulk membrane lipid motional properties, this may not be in the interest of preserving physiological function.(ABSTRACT TRUNCATED AT 250 WORDS)

Polyunsaturation in cell membranes and lipid bilayers and its effects on membrane proteins.

The effect of variation of the degree of cis-unsaturation on cell membrane protein functioning was investigated using a model lipid bilayer system and protein kinase C (PKC). This protein is a key element of signal transduction. Furthermore it is representative of a class of extrinsic membrane proteins that show lipid dependent interactions with cell membranes. To test for dependence of activity on the phospholipid unsaturation, experiments were devised using a vesicle assay system consisting of phosphatidylcholine (PC) and phosphatidylserine (PS) in which the unsaturation was systematically varied. Highly purified PKC alpha and epsilon were obtained using the baculovirus-insect cell expression system. It was shown that increased PC unsaturation elevated the activity of PKC alpha. By contrast, increasing the unsaturation of PS decreased the activity of PKC alpha, and to a lesser extent PKC epsilon. This result immediately rules out any single lipid bilayer physical parameter, such as lipid order, underlying the effect. It is proposed that while PC unsaturation effects are explainable on the basis of a contribution to membrane surface curvature stress, the effects of PS unsaturation may be due to specific protein-lipid interactions. Overall, the results indicate that altered phospholipid unsaturation in cell membranes that occurs in certain disease states such as chronic alcoholism, or by dietary manipulations, are likely to have profound effects on signal transduction pathways involving PKC and similar proteins.

The effect of concanavalin A on the incorporation of fatty acids into lymphocyte phospholipids.

The incorporation of radioactively labeled palmitic, stearic, oleic, linoleic and arachidonic acid into the phospholipids of lymphocytes was studied. When concanavalin A (Con A) was added a gradual increase in the incorporation was found over the period investigated (0-4 h). Changes were not detected over the first 15 min. The increased incorporation at 4 h was shown to be Con A dose dependent and the Con A concentrations for the maximum effect on fatty acid incorporation (at 4 h) and on [3H]thymidine incorporation (at 72 h) were the same. With the exception of stearic acid all the other fatty acids showed an increased incorporation into phosphatidyl choline on addition of Con A, linoleic and oleic acids also had an increased incorporation into phosphatidyl ethanolamine.