ATP-dependent asymmetric distribution of spin-labeled phospholipids in the erythrocyte membrane: relation to shape changes.

Spin-labeled analogs of phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine have been used to study phospholipid transverse diffusion and asymmetry in the human erythrocyte membrane. Ascorbate reduction was used to assess the transbilayer distribution of the labels. All three spin-labeled phospholipids initially incorporated into the outer leaflet of the membrane. On fresh erythrocytes at 5 degrees C, the phosphatidylcholine label remained mainly in the outer leaflet. In contrast, the phosphatidylserine and phosphatidylethanolamine labels underwent rapid transverse diffusion that led to their asymmetric distribution in favor of the inner leaflet. The latter effect was reversibly inhibited after ATP depletion of the erythrocytes and could be reproduced on resealed erythrocyte ghosts only if hydrolyzable Mg-ATP was included in the internal medium. It is suggested that an ATP-driven transport of amino phospholipids toward the inner leaflet could be the major cause of the phospholipid asymmetry in the erythrocyte membrane. It is also proposed that the same mechanism could explain the ATP requirement of the maintenance of the erythrocyte membrane discoid shape.     

Identification of a functional role for lipid asymmetry in biological membranes: Phosphatidylserine-skeletal protein interactions modulate membrane stability

Asymmetric distribution of phospholipids is ubiquitous in the plasma membranes of many eukaryotic cells. The majority of the aminophospholipids are located in the inner leaflet whereas the cholinephospholipids are localized predominantly in the outer leaflet. Several functional roles for asymmetric phospholipid distribution in plasma membranes have been suggested. Disruption of lipid asymmetry creates a procoagulant surface on platelets and serves as a trigger for macrophage recognition of apoptotic cells. Furthermore, the dynamic process of phospholipid translocation regulates important cellular events such as membrane budding and endocytosis. In the present study, we used the red cell membrane as the model system to explore the contribution of phospholipid asymmetry to the maintenance of membrane mechanical properties. We prepared two different types of membranes in terms of their phospholipid distribution, one in which phospholipids were scrambled and the other in which the asymmetric distribution of phospholipids was maintained and quantitated their mechanical properties. We documented that maintenance of asymmetric distribution of phospholipids resulted in improved membrane mechanical stability. The greater difficulty in extracting the spectrin-actin complex at low-ionic strength from the membranes with asymmetric phospholipid distribution further suggested the involvement of interactions between aminophospholipids in the inner leaflet and skeletal proteins in modulating mechanical stability of the red cell membrane. These findings have enabled us to document a functional role of lipid asymmetry in regulating membrane material properties.     

The distribution of erythrocyte phospholipids in hereditary spherocytosis demonstrates a minimal role for erythrocyte spectrin on phospholipid diffusion and asymmetry

In the human erythrocyte membrane phosphatidylcholine and sphingomyelin reside mainly in the outer leaflet, whereas the aminophospholipids, phosphatidylethanolamine and phosphatidylserine, are mainly found in the inner leaflet. Maintenance of phospholipid asymmetry has been assumed to involve interactions between the aminophospholipids and the membrane skeleton, in particular spectrin. To investigate whether spectrin contributes to maintaining the phospholipid transbilayer distribution and kinetics of redistribution, we studied erythrocytes from hereditary spherocytosis patients whose spectrin levels ranged from 34% to 82% of normal. The phospholipid composition and the accessibility of membrane phospholipids to hydrolysis by phospholipases were in the normal range. Spin-labeled phosphatidylserine and phosphatidylethanolamine analogues that had been introduced into the outer leaflet were rapidly transported at 37 degrees C to the inner leaflet, whereas the redistribution of spin-labeled phosphatidylcholine was slower. The kinetics of transbilayer movement of these spin-labeled phospholipid in all samples was in the normal range and was not affected by the level of spectrin. Although these erythrocyte membranes contained as little as 34% of the normal level of spectrin and were characterized by several physical abnormalities, the composition, distribution, and transbilayer kinetics of the phospholipids were found to be normal. We therefore conclude that spectrin plays, at best, only a minor role in maintaining the distribution of erythrocyte membrane phospholipid.     

Characterization of lipid domains in erythrocyte membranes.

Fluorescence digital imaging microscopy was used to study the lateral distribution of the lipid components in erythrocyte membranes. Intact erythrocytes labeled with phospholipids containing a fluorophore attached to one fatty acid chain showed an uneven distribution of the phospholipids in the membrane thereby demonstrating the presence of membrane domains. The enrichment of the lipotropic compound chlor-promazine in domains in intact erythrocytes also suggested that the domains are lipid-enriched regions. Similar membrane domains were present in erythrocyte ghosts. The phospholipid enrichment was increased in the domains by inducing membrane protein aggregation. Double-labeling experiments were done to determine the relative distributions of different phospholipids in the membrane. Vesicles made from extracted lipids did not show the presence of domains consistent with the conclusion that membrane proteins were responsible for creating the domains. Overall, it was found that large domains exist in the red blood cell membrane with unequal enrichment of the different phospholipid species.     

The neonatal erythrocyte and its oxidative susceptibility

Erythrocytes from newborns have altered lipid composition and organization across the membrane bilayer. There does not appear to be any major difference in the protein profile of the membrane in neonatal and adult erythrocytes. The erythrocyte membrane of newborns is particularly susceptible to oxidative damage presumably due to low vitamin E and reduced activities of antioxidative enzymes. Neonatal erythrocytes have reduced membrane fluidity and shortened survival even though cord blood may have a relatively young population of erythrocytes. Shortened survival can be caused by a combination of factors, such as differences in cell geometry, membrane lipid composition, immunoglobulin binding to the surface, and membrane oxidative damage. Some of these factors may be developmental whereas others may be related to the vulnerability of neonatal erythrocytes to in vivo oxidative stress. Vitamin E deficiency in the newborn period is well documented; however, this conclusion is based on plasma vitamin E levels. Whether or not erythrocytes of newborns also exhibit vitamin E deficiency needs investigation. It is not known if the most dense, presumably senescent, erythrocytes are the ones most susceptible to membrane oxidation and are therefore major contributors to altered membrane lipid organization across the membrane bilayer and to hypercoagulability in the newborn period. Whether or not changes in the phospholipid organization in neonatal erythrocytes are developmental or are related to the vitamin E deficiency of newborns is not known.     

Evidence against phospholipid asymmetry in intracellular membranes from liver.

We have studied the distribution of phospholipids across the membrane of microsomal vesicles and Golgi-derived secretory vesicles from rat liver by the use of phospholipases. Model studies on single-bilayer phospholipid vesicles showed that phospholipase A2 (phosphatide 2-acyl-hydrolase, EC 3.1.1.4) cleaved at least 80% of the lipids on the outer surface of such vesicles without significant attack on the inner surface. In microsomal vesicles approximately 40% of the outer surface phospholipids were cleaved before the enzyme gained access to the interior of the vesicles. The same conclusion was reached for Golgi vesicles. By following the degradation of the three major phospholipids in intact microsomes and in extracted lipids we found that the same fraction of each of these phospholipids was exposed on the outer surface of the microsomal vesicles. Corresponding experiments with Golgi vesicles showed that distinctly different fractions of phosphatidylcholine and phosphatidylethanolamine were present on the surface of these vesicles. However, the difference was accounted for by enrichment of phosphatidylcholine in intravesicular particles rather than by asymmetry across the vesicle membrane. The results from specific hydrolysis of phosphatidylinositol confirmed an essentially symmetric distribution of this phospholipid across the microsomal and the Golgi vesicle membranes.     

Phospholipid composition and organization in model β-thalassemic erythrocytes

The membrane phospholipid organization in human red blood cells (RBC) is rigidly maintained by a complex system of enzymes. However, several elements of this system are sensitive to oxidative damage. An important component in the destruction of β-thalassemic RBC is the generation of reactive oxygen species and the release of redox-active iron by the unpaired α-hemoglobin chains. Consequently, we hypothesized that the presence of this oxidative stress to the RBC membrane could lead to alterations in membrane lipid organization. Model β thalassemic RBC, prepared by the introduction of excess α-globin in the cell, have previously been shown to exhibit structural and functional changes almost identical to those observed in β-thalassemic cells. After 24 hr at 37°C, the model β thalassemic cells exhibited a significant loss of deformability, as measured by ektacytometric analysis, indicative of extensive membrane damage. However, a normal steady-state distribution of endogenous phospholipids was found, as evidenced by the accessibility of membrane phospholipids to hydrolysis by phospholipases. Similarly, the kinetics of transbilayer movement of spin-labeled phosphatidylserine (PS) and phosphatidylethanolamine (PE) in all samples was in the normal range and was not affected by the presence of excess α-globin chains. In contrast, a faster rate of spin-labeled phosphatidylcholine (PC) transbilayer movement was observed in these cells. While control RBC exhibited a complete loss of their initial (2 mol%) lysophosphatidylcholine (LPC) levels following 24 hr of incubation at 37°C, 1.5 mol% LPC was still present in model β-thalassemic cells, suggesting an altered phospholipid molecular species turnover, possibly as a result of an increased repair of oxidatively damaged phospholipids. © 1996 Wiley-Liss, Inc.     

Plasmalogen phospholipids - facts and theses to their antioxidative qualities

Fatty aldehyde dimethyl acetals (DMA) derived from plasma and erythrocyte membrane plasmalogen phospholipids of 109 donors, aged 25-91 years, were measured as weight percent of total phospholipid fatty acids and DMA. The age range from 70 to 90 years (n = 82) was divided into age groups of five years each. Cumulative distributions of the DMA values of these age groups, when compared with those of 17 younger persons (aged 25-41 years), revealed a tendency to higher DMA values in the youngest age group, and to lower values in the oldest one. Linear regressions were computed between age and hexadecanaldimethylacetal (16:0 DMA) or octadecanaldimethylactal (18:0 DMA) of erythrocyte membrane and plasma phospholipids. Statistically significant negative correlations with age were obtained. Because of their sensitivity to oxidation reactions, a role of plasmalogens as a natural antioxidant in oxidative defense mechanisms appears to be convincing. However, it will possibly be difficult to separate the effects of normal aging on the decline of plasmalogen phospholipid levels in some tissues from those of certain pathological conditions - including hyperlipidemia and atherosclerosis.     

Electrospray ionization mass spectroscopic analysis of human erythrocyte plasma membrane phospholipids.

Electrospray ionization mass spectrometry (ESI-MS) was utilized for the structural determination and quantitative analysis of individual phospholipid molecular species from subpicomole amounts of human erythrocyte plasma membrane phospholipids. The sensitivity of ESI-MS was 2-3 orders of magnitude greater than that achievable with fast-atom bombardment mass spectrometry (FAB-MS). Phospholipid structure determination and quantitative analysis with ESI-MS can be performed directly from chloroform extracts of biologic samples, obviating the need for prior chromatographic separation of phospholipid classes which has been necessary in FAB-MS phospholipid analyses. Furthermore, ESI-MS is uncomplicated by differential fragmentation of molecular ions and idiosyncratic surface desorption, allowing the quantitation of phospholipids with coefficients of determination (r2) > 0.99 and accuracies > 95%. More than 50 human erythrocyte plasma membrane phospholipid constituents were identified by direct ESI-MS analysis of chloroform extracts of plasma membranes derived from the equivalent of < 1 microliter of whole blood. The major ethanolamine glycerophospholipid subclass in erythrocyte plasma membranes was plasmenylethanolamine that was highly enriched in polyunsaturated fatty acids at the sn-2 position. Collectively, these results demonstrate that ESI-MS of phospholipids is an enabling strategy for the direct structural determination and quantitative analysis of subpicomole amounts of phospholipids from biologic samples.     

The composition of plasma lipoproteins and erythrocyte membranes in cholesterol-fed pigs with partial ileal bypass

The composition of plasma lipoproteins and erythrocyte membranes was studied in cholesterol-fed pigs with a partial ileal bypass. Cholesterol feeding caused marked increases in the plasma concentrations of cholesterol and phospholipids. In spite of continuation of cholesterol feeding, PIB reduced plasma concentrations of cholesterol and phospholipids towards basal values. PIB completely counteracted the dietary cholesterol induced alterations in the lipid composition of the apoprotein B containing plasma lipoproteins, but not in the HDL2 fraction. It is suggested that PIB specifically influences the metabolism of the atherogenic, apoprotein B containing lipoproteins. Dietary cholesterol caused significant increases in the ratios of cholesterol:phospholipids and phosphatidylcholine: sphingomyelin in erythrocytes. The high-cholesterol diet also increased the content of linoleic acid in erythrocyte phosphatidylcholine. PIB completely nullified the cholesterol-induced increase in the cholesterol:phospholipid ratio, but not the increase in the phosphatidylcholine:sphingomyelin ratio. The percentage of linoleic acid in erythrocyte phosphatidylcholine was unaffected by PIB. Neither cholesterol feeding nor PIB had an effect on the lipid fluidity of erythrocyte membranes, as measured by fluorescence polarization, using the probe diphenylhexatriene. Possible compensatory mechanisms operating to control homeostasis of lipid fluidity of erythrocyte membranes are discussed.     

Membrane tolerance to ethanol is rapidly lost after withdrawal: a model for studies of membrane adaptation.

The structural properties of liver microsomes and erythrocytes obtained from rats that had been chronically administered ethanol were examined by electron spin resonance (ESR) following ethanol withdrawal for 1-10 days. Membranes obtained from control animals exhibited considerable molecular disordering upon the addition of ethanol in vitro (50-100 mM). Conversely, microsomal and erythrocyte membranes from alcoholic animals were resistant to this disordering by ethanol (membrane tolerance). These membrane properties were also apparent in lipid bilayers comprised of either total lipids or phospholipids isolated from the control and alcoholic animals. While several weeks of ethanol administration were required for both erythrocytes and microsomes to develop membrane tolerance, erythrocytes from alcoholic animals were disordered by ethanol in vitro after the animals had been withdrawn from ethanol for only 1 day. The same rapid loss of tolerance was observed in microsomes after 2 days of withdrawal. The same time course for the loss of tolerance was observed in lipid bilayers prepared from the total lipid and phospholipid extracts. No significant differences in the cholesterol/phospholipid ratio were observed between the microsomal or erythrocyte membranes isolated before and after withdrawal. Thus, alterations in the microsomal and erythrocyte phospholipids, and not cholesterol content, were responsible for conveying membrane tolerance. Membrane structural properties can be rapidly adjusted in a mammalian system in response to the withdrawal of the external membrane perturbant ethanol. The withdrawal model, which begins with established membrane tolerance and leads to rapid and complete loss of tolerance, provides a model to analyze the compositional changes responsible for this tolerance to disordering by ethanol.     

Mechanisms of myocardial membrane alterations in endotoxin shock: roles of phospholipase and phosphorylation

Current progress in the studies of myocardial membrane alterations during endotoxin shock indicates that endotoxin administration impairs (Na(+) + K(+)-ATPase enzyme system by disrupting the coordination of the ouabain receptor subunit and the catalytic subunit of the enzyme system, and that the disruption is due to an alteration in the lipid microenvironment and a decrease in the phosphorylated intermediate of the enzyme cycle. Studies of the membrane lipid profile provide evidence that endotoxin administration modifies the molecular structure of cardiac membrane lipids in association with the activation of phospholipases A1 and A2 and with the inhibition of phospholipid methylating enzymes. Using liposomes as a membrane model for investigation, endotoxin was found to be capable of modifying the physical property of membrane phospholipids by altering the molecular packing and the phase transition temperature of lipid bilayers. Further studies with Na(+)-Ca2+ exchange system in cardiac sarcolemma have established the roles of phospholipase A2 and protein phosphorylation on the endotoxin-induced derangement in myocardial Na(+)-Ca2+ exchange. Based on these studies, it is concluded that endotoxin administration exerts multiple injuries in different membrane-associated enzyme/receptor systems and that the mechanisms responsible for the endotoxin-induced membrane damage can be categorized into two conceptual frameworks: namely, changes in membrane lipid microenvironment in response to phospholipase A activation and alterations in the phosphorylation of the enzyme/receptor proteins.     

Preservation of cardiolipin content during aging in rat heart interfibrillar mitochondria.

Aging selectively decreases the rate of oxidative phosphorylation in the interfibrillar population of cardiac mitochondria (IFM) located between the myofibers. In contrast, subsarcolemmal mitochondria (SSM), located below the plasma membrane, remain unaffected. IFM from elderly (24-month-old) Fischer 344 rats have a decreased specific activity of complexes III and IV. Complexes III and IV require an inner mitochondrial membrane lipid environment enriched in the oxidatively sensitive phospholipid cardiolipin for maximal activity. We asked if aging decreases the content or alters the composition of cardiolipin as a potential mechanism of the aging defect in IFM. The content and composition of mitochondrial phospholipids were measured in SSM and IFM from adult and aging rat hearts. Aging did not alter the content of mitochondrial phospholipids, including cardiolipin, in either population of mitochondria. The composition of cardiolipin based on characterization of both acyl group and the individual molecular species of cardiolipin was also unaltered by aging. Lipid-mediated oxidative modification of complex III subunits was not detected, making cardiolipin-derived oxidative damage to complex III unlikely. Thus, alterations in cardiolipin are not the mechanism for the aging defect in IFM in Fischer 344 rats.     

Physical effects underlying the transition from primitive to modern cell membranes

To understand the emergence of Darwinian evolution, it is necessary to identify physical mechanisms that enabled primitive cells to compete with one another. Whereas all modern cell membranes are composed primarily of diacyl or dialkyl glycerol phospholipids, the first cell membranes are thought to have self-assembled from simple, single-chain lipids synthesized in the environment. We asked what selective advantage could have driven the transition from primitive to modern membranes, especially during early stages characterized by low levels of membrane phospholipid. Here we demonstrate that surprisingly low levels of phospholipids can drive protocell membrane growth during competition for single-chain lipids. Growth results from the decreasing fatty acid efflux from membranes with increasing phospholipid content. The ability to synthesize phospholipids from single-chain substrates would have therefore been highly advantageous for early cells competing for a limited supply of lipids. We show that the resulting increase in membrane phospholipid content would have led to a cascade of new selective pressures for the evolution of metabolic and transport machinery to overcome the reduced membrane permeability of diacyl lipid membranes. The evolution of phospholipid membranes could thus have been a deterministic outcome of intrinsic physical processes and a key driving force for early cellular evolution.     

Hydrophilic bile salts enhance differential distribution of sphingomyelin and phosphatidylcholine between micellar and vesicular phases: potential implications for their effects in vivo

BACKGROUND/AIMS: The hepatocyte canalicular membrane outer leaflet contains both phosphatidylcholine (PC) and sphingomyelin (SM). Normally, PC is the exclusive phospholipid in bile. We examined effects of bile salt hydrophobicity on cytotoxicity and on differential SM and PC distribution between detergent-resistant aggregated vesicles (model for detergent-resistant canalicular membrane) and mixed micelles or small unilamellar vesicles (representing lipid phases in bile). METHODS: Aggregated vesicles were obtained by ultracentrifugation of cholesterol-supersaturated model systems containing SM, PC and various bile salts, micelles by ultrafiltration and unilamellar vesicles by dialysis of the supernatant. Erythrocyte hemolysis and lactate dehydrogenase release from CaCo-2 cells upon incubation with various micelles were quantified. RESULTS: Preferential SM distribution and lipid solubilization in aggregated vesicles increased in rank order taurodeoxycholate < taurocholate < tauroursodeoxycholate < taurohyodeoxycholate, with reciprocal PC enrichment in micelles and small unilamellar vesicles. Including small amounts of PC within taurohyodeoxycholate micelles increased cytotoxicity with more erythrocyte hemolysis and LDH release from CaCo-2 cells upon incubation, but decreased cytotoxicity in case of tauroursodeoxycholate micelles. CONCLUSIONS: Hydrophilic but not hydrophobic bile salts preserve integrity of pathophysiologically relevant phosphatidylcholine plus sphingomyelin-containing bilayers. Enhanced biliary phospholipid secretion during taurohyodeoxycholate but not during tauroursodeoxycholate therapy (Hepatology 25 (1997) 1306) may relate to different interactions of these bile salts with phospholipids.     

Molecular alterations in hepatocyte transport mechanisms in acquired cholestatic liver disorders

Recent advances in the molecular cloning of membrane transport systems that determine bile formation have facilitated studies of the molecular mechanisms of cholestatic liver disease. The present review summarizes what has been learned about the molecular alterations of these membrane transporters in hepatocytes and cholangiocytes in acquired cholestatic liver disorders. Much of this information has been obtained from the study of animal models of cholestasis and from more limited studies in clinical cholestatic liver diseases. Many of these responses may be interpreted as adaptations that serve to diminish cholestatic liver injury.     

Effects of oxidative damage of membrane protein thiol groups on erythrocyte membrane viscoelasticities

In three oxidative damaging systems: the diamide-mercaptoethanol redox modification system (DM), the pyrogallol oxygen free radicals system (PG) and the hypoxanthine-xanthine oxidase oxygen free radical system (HXO), the effect of erythrocyte membrane oxidative damage on membrane viscoelasticities was investigated with micropipette aspiration method. The experimental results indicated that erythrocyte membrane oxidative damage has a great influence upon the membrane mechanical properties. The oxidative damage led to decrease of contents of membrane protein thiol radical. The scanning of SDS-PAGE presented that membrane proteins form the higher molecular weight component (HMP) by the cross-linking of membrane protein thiol radicals that might hinder the conformational change of membrane protein. This might be the reason for the increased membrane elastic modulus and viscous coefficient upon treating erythrocytes with the oxidative damaging systems. A significant negative logarithm regression relation was found between the membrane elastic modulus, mu, or viscoefficient, eta, and the contents of membrane protein thiol radicals. These experimental results suggested that thiol radicals oxidative damage reaction due to the superoxides anions (*O2-) may be an important molecular mechanism inducing changes of membrane viscoelasticities or whole cell deformability of erythrocyte under physiological and pathological oxidative stress.     

Acceleration of Coagulation by Spectrin-Free Erythrocytic Vesicles: Evidence for Lipid Flip-Flop during Vesicle Formation

Abstract. Spectrin-free erythrocytic vesicles isolated from outdated liquid-preserved blood samples show a distinct increase of the clot-promoting activity compared with membrane phospholipid equivalents of intact erythrocytes. We suppose that, among other remodelling processes, local spectrin detachment from the inner membrane surface may trigger a flip-flop-mediated disturbance of the membrane lipid asymmetry. The interactions of spectrin with the cytoplasmic membrane surface seem to be essential for the structural membrane integrity including the lipid asymmetry.     

Lipid abnormalities in myocardial cell injury

Considerable evidence indicates that abnormalities in fatty acid and phospholipid metabolism are important in the pathogenesis of the membrane dysfunction that leads to irreversible myocardial cell injury during myocardial ischemia and related conditions. Membrane dysfunction is mediated by phospholipid degradation and by the accumulation of amphipathic lipid species, including free fatty acids, long-chain acyl-coenzyme A esters, long-chain acylcarnitines, and lipid peroxides. Accumulation of free arachidonic acid, a fatty acid normally stored in membrane phospholipids, is a sensitive indicator of phospholipid degradation. Ongoing work is aimed at defining mechanisms of the phospholipid alterations that appear to involve phospholipase-mediated phospholipid catabolism and impaired phospholipid synthesis.