Inhibitory effect of cholesterol on interaction between cytoplasmic actin and liposomes, and restorative effect of high osmotic pressure

Although cholesterol is one of the major components of plasma membranes in eukaryotic cells, very little is known about its role in biological membranes. We reported previously (Okimasu et al., Cell Struct. Funct. 11, 273-283, 1986) that introduction of cholesterol into the liposomal membrane caused a decrease in membrane permeability, especially by the binding of cytoplasmic proteins to the liposomal membrane. The present study was carried out to further clarify the biochemical function of cholesterol in the membrane-protein interactions, especially under high osmotic pressure. The association of membranes with cytoplasmic proteins and their permeability were decreased by the introduction of cholesterol, but its effects were diminished in a hypertonic medium. The protein species associated with cholesterol-containing liposomes vary depending on the sort of hypertonic condition. It was suggested that since the degree of lipid packing by the cholesterol was reduced by the locally increased curvature in the lipid bilayer under high osmotic pressure, some cytoplasmic proteins can penetrate into the liposomal membrane.     

Lipid peroxidation induces cholesterol domain formation in model membranes

Numerous reports have established that lipid peroxidation contributes to cell injury by altering the basic physical properties and structural organization of membrane components. Oxidative modification of polyunsaturated phospholipids has been shown, in particular, to alter the intermolecular packing, thermodynamic, and phase parameters of the membrane bilayer. In this study, the effects of oxidative stress on membrane phospholipid and sterol organization were measured using small angle x-ray diffraction approaches. Model membranes enriched in dilinoleoylphosphatidylcholine were prepared at various concentrations of cholesterol and subjected to lipid peroxidation at physiologic conditions. At cholesterol-to-phospholipid mole ratios (C/P) as low as 0.4, lipid peroxidation induced the formation of discrete, membrane-restricted cholesterol domains having a unit cell periodicity or d-space value of 34 A. The formation of cholesterol domains correlated directly with lipid hydroperoxide levels and was inhibited by treatment with vitamin E. In the absence of oxidative stress, similar cholesterol domains were observed only at C/P ratios of 1.0 or higher. In addition to changes in sterol organization, lipid peroxidation also caused reproducible changes in overall membrane structure, including a 10 A reduction in the width of the surrounding, sterol-poor membrane bilayer. These data provided direct evidence that lipid peroxidation alters the essential organization and structure of membrane lipids in a manner that may contribute to changes in membrane function during aging and oxidative stress-related disorders.     

Some studies on lipid peroxidation in monomolecular and bimolecular lipid films

Summary Hydrogen peroxide generated from dissolved oxygen through the alloxandialuric acid cycle affected both the permeability and the stability of lipid bilayer membranes. The permeability of the artificial membranes varied directly with the hydrogen peroxide concentration. Membrane stability varied inversely with the hydrogen peroxide concentration. Bilayers formed from solutions containing both phospholipid and the antioxidant vitamin E were less permeable and more stable in the presence of hydrogen peroxide than bilayers generated from solutions containing phospholipid alone. Peroxidation of phospholipid monolayers caused first an expansion of the films presumably through the introduction of peroxide groups. Further oxidation of phospholipid monolayers led to contraction of the films presumably through the formation of water-soluble products. The results of the monolayer studies and a consideration of the possible kinetics for the peroxidation reaction sequence have been used to explain the changes in the permeability and the stability of lipid bilayer membranes. Our data suggest that oxidation of lipid in biological membranes may first increase membrane permeability and then decrease membrane stability.     

A spin-label study of the effect of gamma radiation on erythrocyte membrane. Influence of lipid peroxidation on membrane structure.

Gamma-irradiation of bovine erythrocyte membranes (0.1-4 Mrad) resulted in a decrease in the degree of order of membrane lipids, as measured by spin-labelled fatty acid esters, at the depth of C12 but not at the depth of C5. Dose dependence of this phenomenon corresponded to dose dependence of malondialdehyde formation in the membranes. On this basis a mechanism for the effect of lipid peroxidation on the membrance structure is proposed. Membrane proteins underwent radiation-induced conformational transitions revealed by maleimide spin label which could be also connected with lipid peroxidation.     

Lipid peroxidation increases the molecular order of microsomal membranes

The effect of enzymatic lipid peroxidation on the molecular order of microsomal membranes was evaluated by ESR spectroscopy using the spin probes 5-, 12-, and 16-doxyl-stearic acid. Rat liver microsomal membranes were peroxidized by the NADPH-dependent reaction in the presence of the chelate ADP-Fe3+. Peroxidation resulted in a preferential depletion of polyenoic fatty acids and an increase in the percentage composition of shorter fatty acyl chains. There was no change in the cholesterol/phospholipid ratio of the peroxidized microsomes. The molecular order of both control and peroxidized membranes decreased toward the central region of the bilayer, and the order parameter (S) of each probe was temperature dependent. Peroxidation of the microsomal membrane lipids resulted in an increase in the order parameter determined with the three stearic acid spin probes. Of the three probes, 12-doxylstearic acid was the most sensitive to the changes in membrane organization caused by peroxidation. These data indicate that ESR spectroscopy is a sensitive method of detecting changes in membrane order accompanying peroxidation of membrane lipids.     

Lipid peroxidation contributes to age-related membrane rigidity

The aim of this work was to assess the relative contributions of lipid peroxidation and cholesterol content to the increase in membrane rigidity observed during senescence. Membrane fluidity was manipulated through exposure to peroxidized or cholesterol-loaded liposomes. Small unilamella liposomes were prepared and either peroxidized by Fe⁺⁺-ADP-ascorbic acid or loaded with cholesterol. After incorporation of the liposomes into rat liver microsomal membranes, membrane fluidity was quantitated by measuring changes in polarization. Membranes exhibited a greater sensitivity to peroxidation than cholesterol in that incorporation of peroxidized liposomes induced microsomal membrane rigidity substantially more than did cholesterol-loaded liposomes. Thus it is proposed, based on data from the present and earlier studies, that membrane fluidity can be modulated readily by lipid peroxidation of membrane phospholipids, irrespective of the influences of cholesterol. These results support the proposal that alterations of lipid structure are more potent and effective than compositional changes in cholesterol in inducing age-related increases in membrane rigidity.     

Proton permeability and electric breakdown of phospholipid membranes after UV-irradiation

Lipid peroxidation induced in bilayer lipid membranes (BLM) by UV-irradiation leads to two types of effects: selective in proton permeability and electric breakdown of the membranes. Both phenomena are always observed but the contribution of each in the membrane conductivity increase depends on the lipid nature (degree of unsaturation of fatty acids) and the value of transmembrane applied to BLM or generated by the membrane itself.     

Cholesterol prevents interaction of the cell‐penetrating peptide transportan with model lipid membranes

Interaction of the cell‐penetrating peptide (CPP) cysteine‐transportan (Cys‐TP) with model lipid membranes was examined by spin‐label electron paramagnetic resonance (EPR). Membranes were labeled with lipophilic spin probes and the influence of Cys‐TP on membrane structure was studied. The influence of Cys‐TP on membrane permeability was monitored by the reduction of a liposome‐trapped water‐soluble spin probe. Cys‐TP caused lipid ordering in membranes prepared from pure dimyristoylphosphatidylcholine (DMPC) and in DMPC membranes with moderate cholesterol concentration. In addition, Cys‐TP caused a large increase in permeation of DMPC membranes. In contrast, with high cholesterol content, at which model lipid membranes are in the so‐called liquid‐ordered phase, no effect of Cys‐TP was observed, either on the membrane structure or on the membrane permeability. The interaction between Cys‐TP and the lipid membrane therefore depends on the lipid phase. This could be of great importance for understanding of the CPP–lipid interaction in laterally heterogeneous membranes, while it implies that the CPP–lipid interaction can be different at different points along the membrane. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Glucose promotes membrane cholesterol crystalline domain formation by lipid peroxidation

Oxidative damage to vascular cell membrane phospholipids causes physicochemical changes in membrane structure and lipid organization, contributing to atherogenesis. Oxidative stress combined with hyperglycemia has been shown to further increase the risk of vascular and metabolic diseases. In this study, the effects of glucose on oxidative stress-induced cholesterol domain formation were tested in model membranes containing polyunsaturated fatty acids and physiologic levels of cholesterol. Membrane structural changes, including cholesterol domain formation, were characterized by small angle X-ray scattering (SAXS) analysis and correlated with spectrophotometrically-determined lipid hydroperoxide levels. Glucose treatment resulted in a concentration-dependent increase in lipid hydroperoxide formation, which correlated with the formation of highly-ordered cholesterol crystalline domains (unit cell periodicity of 34 Å) as well as a decrease in overall membrane bilayer width. The effect of glucose on lipid peroxidation was further enhanced by increased levels of cholesterol. Treatment with free radical-scavenging agents inhibited the biochemical and structural effects of glucose, even at elevated cholesterol levels. These data demonstrate that glucose promotes changes in membrane organization, including cholesterol crystal formation, through lipid peroxidation.     

Decrease in beech (<Emphasis Type="Italic">Fagus sylvatica</Emphasis>) seed viability caused by temperature and humidity conditions as related to membrane damage and lipid composition

The aim of this study was to determine if the loss of germinability and viability of beech (Fagus sylvatica L.) seeds stored at different variants of temperature (4, 20, and 30 °C) and relative humidity (RH: 45 and 75 %) is associated with a loss of membrane integrity and changes in lipid composition. Beech seeds stored for 9 weeks gradually lost viability at a rate dependent on temperature and humidity. The harmful effect of temperature increased with growing humidity. The loss of seed viability was strongly correlated with an increase in membrane permeability and with production of lipid hydroxyperoxides (LHPO), which was regarded as an indicator of peroxidation of unsaturated fatty acids. The condition of membranes was assessed on the basis of their permeability and the state of lipid components: phospholipids and fatty acids. During seed storage we observed a decline in concentration of individual phospholipids and fatty acids, proportional to the loss of seeds viability. We also detected a decrease in concentrations of α-tocopherol and sterols, which play an important role in protection of membranes against the harmful influence of the environment. Our results show that the germinability of beech seeds declines rapidly at temperature above 0 °C and growing humidity. This is due mainly to the loss of membrane integrity, caused by peroxidation of unsaturated fatty acids.     

The mode of action of polyacetylene and thiophene photosensitizers on liposome permeability to glucose

The mode of action of the two photosensitizers 1-phenylhepta-1,3,5-triyne and alpha-terthienyl on membrane permeability was investigated using liposomes entrapped with glucose as a model membrane system. Upon exposure to UV-A light, alpha-terthienyl, and to a much lesser extent phenylheptatriyne, induced leakage of glucose via a photodynamic mechanism in liposomes which had a high degree of unsaturated fatty acid side chains. Enhanced permeability to glucose in these liposomes due to the action of alpha-terthienyl and phenylheptatriyne involved lipid peroxidation, but neither of the two assays used to monitor lipid peroxidation (malondialdehyde and peroxide formation) was directly correlated with the increase in liposome permeability. In liposomes with highly ordered lipid where the fatty acid side chains are saturated, alpha-terthienyl had no effect on glucose permeability. In contrast, phenylheptatriyne was very effective in increasing glucose permeability in these liposomes via a photodynamic mechanism. Addition of lysophosphatidylcholine, which perturbs the order of lipid packing, to these liposomes, completely inhibited the effect of phenylheptatriyne. Conversely, incorporation of cholesterol which increases lipid order, into egg PC liposomes, enhanced the action of phenylheptatriyne. These data suggest that under UV-A irradiation (a) alpha-terthienyl and phenylheptatriyne enhance permeability in liposomes with a high degree of unsaturation involving lipid peroxidation and (b) phenylheptatriyne enhances membrane permeability through some other mechanism when present in a bilayer with a highly ordered lipid environment.     

Alterations of human erythrocyte membrane fluidity by oxygen-derived free radicals and calcium

Two possible reasons for the structural alterations of cell membranes caused by free radicals are lipid peroxidation and an increase in the intracellular calcium ion concentration. To characterize the alterations in membrane molecular dynamics caused by oxygen-derived free radicals and calcium, human erythrocytes were spin-labeled with 5-doxyl stearic acid, and alterations in membrane fluidity were quantified by electron spin resonance oxidase (0.07 U/mL) decreased membrane fluidity, and the addition of superoxide dismutase and catalase inhibited the effect on membrane fluidity of the hypoxanthine-xanthine oxidase system. Hydrogen peroxide (0.1 and 1 nM) also decreased membrane fluidity and caused alterations to erythrocyte morphology. In addition, a decrease in membrane fluidity was observed in erythrocytes incubated with 2.8 mM CaCl₂. On the other hand, incubation of erythrocytes with calcium-free solution decreased the changes in membrane fluidity caused by hydrogen peroxide.

These results suggest that changes in membrane fluidity are directly due to lipid peroxidation and are indirectly the result of increased intracellular calcium concentration. We support the hypothesis that alterations of the biophysical properties of membranes caused by free radicals play an important role in cell injury, and that the accumulation of calcium amplifies the damge to membranes weakened by free radicals.     

The mode of action of polyacetylene and thiophene photosensitizers on liposome permeability to glucose

The mode of action of the two photosensitizers 1-phenylhepta-1,3,5-triyne and α-terthienyl on membrane permeability was investigated using liposomes entrapped with glucose as a model membrane system. Upon exposure to UV-A light, α-terthienyl, and to a much lesser extent phenylheptatriyne, induced leakage of glucose via a photodynamic mechanism in liposomes which had a high degree of unsaturated fatty acid side chains. Enhanced permeability to glucose in these liposomes due to the action of α-terthienyl and phenylheptatriyne involved lipid peroxidation, but neither of the two assays used to monitor lipid peroxidation (malondialdehyde and peroxide formation) was directly correlated with the increase in liposome permeability. In liposomes with highly ordered lipid where the fatty acid side chains are saturated, α-terthienyl had no effect on glucose permeability. In contrast, phenylheptatriyne was very effective in increasing glucose permeability in these liposomes via a photodynamic mechanism. Addition of lysophosphatidylcholine, which perturbs the order of lipid packing, to these liposomes, completely inhibited the effect of phenylheptatriyne. Conversely, incorporation of cholesterol which increases lipid order, into egg PC liposomes, enhanced the action of phenylheptatriyne. These data suggest that under UV-A irradiation (a) α-terthienyl and phenylheptatriyne enhance permeability in liposomes with a high degree of unsaturation involving lipid peroxidation and (b) phenylheptatriyne enhances membrane permeability through some other mechanism when present in a bilayer with a highly ordered lipid environment.     

Age-Related Differences in Synaptosomal Peroxidative Damage and Membrane Properties

Young, adult, and old rats were used to study the effect of age on the integrity and functioning of brain synaptosomes. An evaluation was made of the differences in lipid composition, membrane fluidity, Na+, K(+)-ATPase activity, and susceptibility to in vitro lipid peroxidation. There was an age-related increase in synaptosomal free fatty acids, with no modification in acyl chain composition, and a decrease in membrane phospholipids which increased the cholesterol/phospholipid mole ratio. With altered lipid composition, there was a corresponding age-dependent decrease in membrane fluidity, a reduction of Na+, K(+)-ATPase activity, and an overall greater susceptibility to in vitro lipid peroxidation. Furthermore, lipid peroxidation promoted strong modifications of the membrane fluidity, lipid composition, and Na+,K(+)-ATPase activity just as aging did, thus indicating a possible contribution of oxidative damage to ageing processes. The cases studied revealed that the greater responsiveness of old membranes to in vitro lipid peroxidation resulted in the highest degree of membrane alteration, indicating that all pathological states known to promote a peroxidative injury can have even more dramatic consequences when they take place in old brain.     

Radiation induced lipid peroxidation: factors which determine the oxidizability of lipids

Lipids are the essential components of cell membranes and lipoproteins. Their peroxidation plays an important role in numerous pathologies in which oxidative stress is involved. Lipid peroxidation occurs through a chain reaction that contributes to membrane damage in cells. It results in the conversion of fatty acids to polar hydroperoxides and leads to the breakdown or malfunction of the membrane. Lipids are amphiphilic molecules that aggregate in aqueous solutions into micelles and liposomes. The effect of this structural organization is significant in studies of radiation-induced peroxidation damage in highly ordered biological systems such as biological membranes. In this paper, a synthesis of the data concerning radioinduced lipid peroxidation is completed by an original review of the different parameters that determine lipid oxidizability. In addition, the influence of lipid aggregation and the effect of molecular packing are discussed.     

Inhibition of phospholipid hydrolysis by phospholipase A2 in microsomal and mitochondrial membranes subjected to lipid peroxidation

The rate of phospholipid hydrolysis in rat liver microsomal and mitochondrial membranes catalyzed by phospholipase A2 was shown to decrease after ascorbate + Fe2+-induced lipid peroxidation. The degree of inhibition was linearly dependent on the amount of lipid peroxidation products (malonyl dialdehyde) accumulated in the membrane. The decreased phospholipid hydrolysis rate in membranes after lipid peroxidation was registered using phospholipases A2 from two sources: porcine pancreas and bee venom. It was established that the inhibitory action of phospholipid peroxidation products was not linked with a direct effect on the enzyme and was not caused by depletion of phospholipase reaction substrates (as a result of lipid peroxidation). A possible role of lateral separation of oxidized and non-oxidized lipid phases in the mechanisms of inhibition of phospholipid hydrolysis by phospholipase A2 is discussed.     

Radiation-induced damage to mitochondrial D-beta-hydroxybutyrate dehydrogenase and lipid peroxidation

Radiation-induced damage to the reconstituted system of membrane-bound enzyme, D-beta-hydroxybutyrate dehydrogenase obtained from rat liver mitochondria, was investigated in relation to the lipid peroxidation of membranes. The activity of D-beta-hydroxybutyrate dehydrogenase in fresh mitochondria was very low in general and was not affected by irradiation because of little incorporation of substrates into mitochondria. However, the enzyme activity in one-day-aged mitochondria or submitochondrial particles was five times higher than that of fresh mitochondria and decreased with increasing radiation dose accompanying the increase in peroxidation of membrane lipids. The activity of D-beta-hydroxybutyrate dehydrogenase in the reconstituted system of the purified enzyme with irradiated liver microsomes or irradiated liposomes was decreased considerably in comparison with either unirradiated control or irradiated enzyme. Therefore, the radiation-induced decrease in the enzyme activity was thought to be caused mainly by peroxidation of membrane lipids and not to be due to direct damage by radiation to the enzyme molecule itself. Irradiation of microsomes, a component of the reconstituted system, caused decreases in phosphatidylcholine and phosphatidylethanolamine content and an increase in lysophosphatidylcholine content. In addition, arachidonic acid contents in phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine were also markedly decreased with increasing radiation dose. These results are discussed in terms of a mechanism involving radiation-induced damage to membrane function and structures.     

Lipids, curvature stress, and the action of lipid prodrugs: free fatty acids and lysolipid enhancement of drug transport across liposomal membranes

Molecular shape and its impact on bilayer curvature stress are powerful concepts for describing the effects of lipids and fatty acids on fundamental membrane properties, such as passive permeability and derived properties like drug transport across liposomal membranes. We illustrate these relationships by studying the effects of fatty acids and lysolipids on the permeation of a potent anti-cancer drug, doxorubicin, across the bilayer of a liposome in which the drug is encapsulated. Using a simple fluorescence assay, we have systematically studied the passive permeation of doxorubicin across liposomal membranes in different lipid phases: the solid-ordered phase (DPPC bilayers), the liquid-disordered phase (POPC lipid bilayers), and the liquid-ordered phase induced by high levels of cholesterol (DOPC + cholesterol lipid bilayers). The effect of different free fatty acids (FA) and lysolipids (LL), separately and in combination, on permeability was assessed to elucidate the possible mechanism of phospholipase A₂-triggered release in cancer tissue of liposomal doxorubicin formulations. In all cases, FAs applied separately lead to significant enhancement of permeability, most pronounced in liquid-disordered bilayers and less pronounced in solid and solid-ordered bilayers. LLs applied separately had only a marginal effect on permeability. FA and LL applied in combination lead to a synergistic enhancement of permeability in solid bilayers, whereas in liquid-disordered bilayers, the combined effect suppressed the otherwise strong permeability enhancement due to the FAs.     

The natural antioxidant rosmarinic acid spontaneously penetrates membranes to inhibit lipid peroxidation in situ

Exogenous molecules from dietary sources such as polyphenols are very efficient in preventing the alteration of lipid membranes by oxidative stress. Among the polyphenols, we have chosen to study rosmarinic acid (RA). We investigated the efficiency of RA in preventing lipid peroxidation and in interacting with lipids. We used liposomes of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC) to show that RA was an efficient antioxidant. By HPLC, we determined that the maximum amount of RA associated with the lipids was ~1 mol%. Moreover, by using Langmuir monolayers, we evidenced that cholesterol decreases the penetration of RA. The investigation of transferred lipid/RA monolayers by atomic force microscopy revealed that 1 mol% of RA in the membrane was not sufficient to alter the membrane structure at the nanoscale. By fluorescence, we observed no significant modification of membrane permeability and fluidity caused by the interaction with RA. We also deduced that RA molecules were mainly located among the polar headgroups of the lipids. Finally, we prepared DLPC/RA vesicles to evidence for the first time that up to 1 mol% of RA inserts spontaneously in the membrane, which is high enough to fully prevent lipid peroxidation without any noticeable alteration of the membrane structure due to RA insertion.     

Local Partition Coefficients Govern Solute Permeability of Cholesterol-Containing Membranes

The permeability of lipid membranes for metabolic molecules or drugs is routinely estimated from the solute’s oil/water partition coefficient. However, the molecular determinants that modulate the permeability in different lipid compositions have remained unclear. Here, we combine scanning electrochemical microscopy and molecular-dynamics simulations to study the effect of cholesterol on membrane permeability, because cholesterol is abundant in all animal membranes. The permeability of membranes from natural lipid mixtures to both hydrophilic and hydrophobic solutes monotonously decreases with cholesterol concentration [Chol]. The same is true for hydrophilic solutes and planar bilayers composed of dioleoyl-phosphatidylcholine or dioleoyl-phosphatidyl-ethanolamine. However, these synthetic lipids give rise to a bell-shaped dependence of membrane permeability on [Chol] for very hydrophobic solutes. The simulations indicate that cholesterol does not affect the diffusion constant inside the membrane. Instead, local partition coefficients at the lipid headgroups and at the lipid tails are modulated oppositely by cholesterol, explaining the experimental findings. Structurally, these modulations are induced by looser packing at the lipid headgroups and tighter packing at the tails upon the addition of cholesterol.