Loss of membrane phospholipid asymmetry in platelets and red cells may be associated with calcium-induced shedding of plasma membrane and inhibition of aminophospholipid translocase

Influx of calcium in platelets and red cells produces formation of vesicles shed from the plasma membrane. The time course of the shedding process closely correlates with the ability of both cells to stimulate prothrombinase activity when used as a source of phospholipid in the prothrombinase assay. This reflects increased surface exposure of phosphatidylserine, presumably resulting from a loss in membrane asymmetry. Evidence is presented that the shed vesicles have a random phospholipid distribution, while the remnant cells show a progressive loss of membrane phospholipid asymmetry when more shedding occurs. Removal of intracellular calcium produces a decrease of procoagulant activity of the remnant cells but not of that of the shed vesicles. This is consistent with reactivation of aminophospholipid translocase activity, being first inhibited by intracellular calcium and subsequently reactivated upon calcium removal. Involvement of aminophospholipid translocase is further supported by the observation that reversibility of procoagulant activity is also dependent on metabolic ATP and reduced sulfhydryl groups. The finding that this reversibility process is not apparent in shed vesicles may be ascribed to the absence of translocase or to a lack of ATP. These data support and extend the suggestion made by Sims et al. [1989) J. Biol. Chem. 264, 17049-17057) that membrane fusion, which is required for shedding to occur, produces transient flip-flop sites for membrane phospholipids. Furthermore, the present results indicate that scrambling of membrane phospholipids can only occur provided that aminophospholipid translocase is inactive.     

Regulated externalization of phosphatidylserine at the cell surface: implications for apoptosis

The regulated loss of plasma membrane phosphatidylserine (PS) asymmetry is critical to many biological processes. In particular, the appearance of PS at the cell surface, a hallmark of apoptosis, prepares the dying cell for engulfment and elimination by phagocytes. While it is well established that PS externalization is regulated by activation of a calcium-dependent phospholipid scramblase activity in concert with inactivation of the aminophospholipid translocase, there is no evidence indicating that these processes are triggered and regulated by apoptotic regulatory mechanisms. Using a novel model system, we show that PS externalization is inducible, reversible, and independent of cytochrome c release, caspase activation, and DNA fragmentation. Additional evidence is presented indicating that the outward movement of plasma membrane PS requires sustained elevation in cytosolic Ca2+ in concert with inactivation of the aminophospholipid translocase and is inhibited by calcium channel blockers.     

Electric field pulses induce reversible shape transformation of human erythrocytes

Electric field pulses >2-3 kV cm¹ long known to induce membrane poration and fusion of erythrocytes as well as to enhance the transbilayer mobility of phospholipids and to perturb aminophospholipid asymmetry, are shown to induce, at 0 C., transformation of the discocytic cells into echinocytes and spheroechinocytes. The extent of transformation increases with strength, duration and number of pulses. Its time course is biphasic., a major rapid phase (t/2 ～ 5 s) being followed by a minor one, lasting for 2-3 h. Shape transformation goes along with the exofacial exposure of phosphatidylserine (PS), detected by FITC-annexin V binding and quantified by a calibration curve established via externally inserted dilauroylphosphatldylserine. Incubation of these echinocytes at 37 C leads to a rapid recovery of the discocytic shape followed by slower formation of stomatocytes. Shape recovery is temperature dependent (E_a ～100 kJ/mol), and can be impaired by depletion of ATP or Mg⁺⁺ and by addition of vanadate or fluoride. Shape recovery and stomatocyte formation go along with a rapid loss of annexin binding in about 45% of the cells while the rest maintains its binding capacity. In the presence of vanadate, annexin binding increases in all cells. The results are discussed in the light of the bilayer couple concept of erythrocyte shape and the enhanced transverse mobility of phospholipids. Echinocyte formation is most likely caused by the reorientation of endofacial aminopho-spholipids to the outer leaflet of the bilayer. Shape recovery and stomatocyte formation probably result from a continuous reinternalization of PS via the ATP dependent aminophospholipid translocase, but may also be supported by downhill movement of PC to the inner leaflet and by other yet unidentified processes.     

Ca2+ sensitivity of phospholipid scrambling in human red cell ghosts.

The phospholipids in plasma membranes of erythrocytes, as well as platelets, lymphocytes and other cells are asymmetrically distributed, with sphingomyelin and phosphatidylcholine residing predominantly in the outer leaflet of the bilayer, and phosphatidylserine and phosphatidylethanolamine in the inner leaflet. It is known that Ca2+ can disrupt the phospholipid asymmetry by activation of a protein known as phospholipid scramblase, which affects bidirectional phospholipid movement in a largely non-selective manner. As Ca2+ also inhibits aminophospholipid translocase, whose Mg(2+)-ATPase activity is responsible for active translocation of aminophospholipids from the outer to the inner leaflet, it is important to accurately determine the sensitivity of scramblase to intracellular free Ca2+. In the present study we have utilized the favourable Kd of Mag-fura-2 for calcium in the high micromolar range to determine free Ca2+ levels associated with lipid scrambling in resealed human red cell ghosts. The Ca2+ sensitivity was measured in parallel to the translocation of a fluorescent-labelled lipid incorporated into the ghost bilayer. The phospholipid scrambling was found to be half-maximally activated at 63-88 microM free intracellular Ca2+. The wider applicability of the method and the physiological implications of the calcium sensitivity determined is discussed.     

Phospholipid transverse mobility modifications in plasma membranes of activated platelets: an ESR study.

Spin labeled phospholipid analogs were used to directly study changes in aminophospholipid translocase activity in activated platelets. In thrombin-activated platelets, the translocase activity was slightly stimulated, whereas no vesicle formation or proteolysis of cytoskeletal protein occurred. Ca2+ ionophore A23187-mediated activation produced vesiculation and proteolysis. Additionally, the translocase activity was completely inhibited, probably due to a sharp rise the intracellular Ca2+ concentration, as shown when platelets were activated in the presence of various A23187 and Ca2+ concentrations and by the recovery of the translocase activity when Ca2+ was complexed with EGTA. No translocase activity was found in vesicles. Whereas vesiculation and translocase inhibition can occur independently of proteolysis, this later accentuated the shedding phenomenon.     

Transbilayer phospholipid movement and the clearance of apoptotic cells

When lymphocytes (and other cells) die by apoptosis, they orchestrate their own orderly removal by macrophages, and thereby prevent the inflammation that would otherwise attend cell lysis. As part of their demise, apoptotic cells disrupt the normal asymmetric distribution of phospholipids across their plasma membranes, an asymmetry normally maintained by an aminophospholipid translocase. This disruption of asymmetry, mediated by an activity known as the scramblase, generates ligands on the cell surface that trigger phagocytosis of the dying cell before lysis can occur. This crucial alteration of the plasma membrane is not dependent on caspase-mediated proteolysis, but quite unexpectedly, it is required both on the apoptotic target cell and on the phagocyte that engulfs it. At least in the phagocyte, this rearrangement may depend on the activity of an ABC ATPase, termed ABC1 in mammals and ced-7 in C. elegans.     

Asymmetric distribution of phospholipids in spectrin-poor erythrocyte vesicles

We have investigated by electron spin resonance, at 37 degrees C, the outside-inside passage and the equilibrium distribution of spin-labeled phospholipids, respectively, in ATP-containing ghosts, in heat-treated erythrocytes, and in heat-induced vesicles. The heat-treated vesicles were spectrin depleted to approximately 25% of the original content and had lost almost 100% of the other cytoskeletal proteins. Yet the vesicles, as long as they contained ATP, were capable of translocating the aminophospholipids with the same efficiency as the heat-treated erythrocytes, and almost with the same efficiency as ATP-containing ghosts. In the vesicles, sphingomyelin and phosphatidylcholine analogues underwent a very slow transverse diffusion as in native cells. We conclude that spectrin and other cytoskeleton proteins are not major factors for the establishment and maintenance of phospholipid asymmetry in human erythrocytes, which may be chiefly due to the aminophospholipid translocase activity.     

Involvement of ATP-dependent aminophospholipid translocation in maintaining phospholipid asymmetry in diamide-treated human erythrocytes

Crosslinking of membrane skeletal proteins such as spectrin by oxidation of their SH-groups can be provoked by treatment of intact erythrocytes with diamide. Shortly after exposure of human erythrocytes to diamide and despite the transverse destabilization of the lipid bilayer that was observed in these cells (Franck, P.F.H., Op den Kamp, J.A.F., Roelofsen, B. and Van Deenen, L.L.M. (1986) Biochim. Biophys. Acta 857, 127-130), no abnormalities could be detected regarding the asymmetric distribution of the phospholipids when probed by either the prothrombinase assay or brief exposure of the cells to a modified phospholipase A2 with enhanced membrane penetrating capacity. This asymmetry appeared to undergo dramatic changes however, when the ATP content of the cytosol had decreased to less than 10% of its original level during prolonged incubation of the treated cells. These observations indicate that the initial maintenance of phospholipid asymmetry in diamide-treated erythrocytes can be solely ascribed to the action of the ATP-dependent aminophospholipid translocase. This view is supported by experiments involving radiolabeled phospholipids of which trace amounts had been inserted into the outer membrane leaflet of diamide-treated red cells and which still showed a preferential translocation of both aminophospholipids in favour of the inner monolayer, be it that the efficiency of the translocase was found to be impaired when compared to control cells.     

Back and forth

Summary

That some membranes restrict certain lipid species to one side of the bilayer and others to the opposite side has been known for two decades. However, how this asymmetric transbilayer distribution is generated and controlled, how many and what type of membranes are so structured, and even the reason for its existence is just now beginning to be understood. It has been a decade since the discovery of an activity which transports in an ATP-dependent manner only the aminophospholipids from the outer to the inner leaflet of the plasma membrane. This aminophospholipid translocase has yet to be isolated, reconstituted, and identified molecularly. Elevating intracellular Ca²⁺ allows all the major classes of phospholipids to move freely across the bilayer, scrambling lipids and dissipating asymmetry. The nature of this pathway and its mode of activation by Ca²⁺ remain to be determined. Though loss of transbilayer asymmetry by blood cells clearly produces a procoagulant surface and increases interactions with the reticuloendothelial system, it remains to be elucidated whether maintenance of blood homeostasis is just one expression of a more general raison d'ětre for lipid asymmetry. It is these persisting uncertainties and gaps in our knowledge which make the field such an interesting and exciting challenge at the present time.     

Lipid translocation across the plasma membrane of mammalian cells.

The plasma membrane, which forms the physical barrier between the intra- and extracellular milieu, plays a pivotal role in the communication of cells with their environment. Exchanging metabolites, transferring signals and providing a platform for the assembly of multi-protein complexes are a few of the major functions of the plasma membrane, each of which requires participation of specific membrane proteins and/or lipids. It is therefore not surprising that the two leaflets of the membrane bilayer each have their specific lipid composition. Although membrane lipid asymmetry has been known for many years, the mechanisms for maintaining or regulating the transbilayer lipid distribution are still not completely understood. Three major players have been presented over the past years: (1) an inward-directed pump specific for phosphatidylserine and phosphatidylethanolamine, known as aminophospholipid translocase; (2) an outward-directed pump referred to as 'floppase' with little selectivity for the polar headgroup of the phospholipid, but whose actual participation in transport of endogenous lipids has not been well established; and (3) a lipid scramblase, which facilitates bi-directional migration across the bilayer of all phospholipid classes, independent of the polar headgroup. Whereas a concerted action of aminophospholipid translocase and floppase could, in principle, account for the maintenance of lipid asymmetry in quiescent cells, activation of the scramblase and concomitant inhibition of the aminophospholipid translocase causes a collapse of lipid asymmetry, manifested by exposure of phosphatidylserine on the cell surface. In this article, each of these transporters will be discussed, and their physiological importance will be illustrated by the Scott syndrome, a bleeding disorder caused by impaired lipid scrambling. Finally, phosphatidylserine exposure during apoptosis will be briefly discussed in relation to inhibition of translocase and simultaneous activation of scramblase.     

Merocyanine 540 as a fluorescent probe of altered membrane phospholipid asymmetry in activated whole blood platelets

BACKGROUND: Platelet activation leads to the loss of a natural asymmetry of membrane phospholipids (PL) and the subsequent exposure of negatively charged PL in platelets with procoagulant activity that can be monitored routinely with annexin V (AN-V). METHODS: Flow cytometric analysis of merocyanine 540 (MC540) binding may be the alternate choice for the monitoring of platelet procoagulant activity. Due to the increased partition of negatively charged phosphatidylserine (PS) in the membrane outer leaflet of activated platelets, the interaction with MC540 is reduced. RESULTS: Collagen, which facilitated platelet PL bilayer symmetrization, vastly reduced MC540 fluorescence and augmented AN-V binding to platelets. Such a collagen-induced symmetrization was further augmented in the presence of thrombin receptor-activating peptide (TRAP, SFLLRNPNDKYEPF). In the presence of VO(4) ((-3)) (the inhibitor of aminophospholipid translocase), the rebuilt of membrane asymmetry was attenuated, which resulted in further reduced MC540 fluorescence and enhanced AN-V binding in activated cells. In platelets incubated with thapsigargin, the inhibitor of platelet tubular system Ca(2+) ATP-ase, which elevates intraplatelet Ca(2+) concentration, TRAP increased AN-V and reduced MC540 binding. The chelating of Ca(2+) with EGTA outside of activated platelets reduced AN-V binding, but did not affect MC540-positive platelets. The fluctuations in reduced staining with MC540 paralleled enhanced AN-V binding (r = -0.481, P < 0.01), especially for strong "procoagulant" activating agents. CONCLUSIONS: (1) MC540 may be used in whole blood flow cytometry for the monitoring of platelet membrane symmetrization as an alternate or compounding method to AN-V. (2) Platelet staining with MC540 is sensitive to the fluctuations in the intraplatelet [Ca(2+)] during platelet activation. (3) Use of MC540 is characterized by improved diagnostic precision and reliability compared with AN-V.     

Compensating lipid fluxes generated by the aminophospholipid translocase

By a combined kinetic and thermodynamic model on the transbilayer dynamics and asymmetric distribution of lipids in the red blood cell, compensating lipid fluxes to the exoplasmic leaflet have been analysed, counterbalancing the active transport of aminophospholipids to the cytoplasmic monolayer by the aminophospholipid translocase. The compensating fluxes are assumed to be of passive nature generated by forces of lateral mechanical stress and of lipid concentration differences between the two monolayers. These forces are shown to be caused and maintained by the operation of the aminophospholipid translocase. Simulations reveal that a reduction of the compensating fluxes upon ATP-depletion can be attributed to the inhibition of the aminophospholipid translocase. Thus, a Mg(2+)- and ATP-dependence of the outward movement of phospholipid analogues in the plasma membrane of red blood cells can be expected independent of the existence and operation of an ATP-dependent 'floppase' activity.     

Regulation of phosphatidylserine exposure and phagocytosis of apoptotic T lymphocytes

In lymphocytes, an asymmetric distribution of phospholipids across the plasma membrane is maintained by an ATP-dependent translocase which specifically transports aminophospholipids from the outer to the inner leaflet of the bilayer. During apoptosis, this enzyme is down-regulated and a lipid flipsite, termed the scramblase, is activated. Together, these events lead to the appearance of phosphatidylserine (PS) on the cell surface. In DO11.10 T lymphocyte hybridoma cells undergoing apoptosis, the kinetics of PS externalization are paralleled by the development of PS-sensitive phagocytosis by macrophages. This parallel is also observed when PS externalization is effected directly by application of a Ca2+ ionophore, suggesting that PS externalization is not only necessary, but sufficient, to generate a recognition signal. The broad spectrum aspartate-directed cysteine protease (caspase) inhibitor zVAD-fmk blocks externalization of PS and terminal cell lysis after induction of apoptosis by anti-CD3 antibody, but is ineffective when apoptosis is induced in the same cells by treatment with glucocorticoid. These results suggest that apoptosis induced by glucocorticoid does not require the same zVAD-sensitive caspase steps which are required for Fas/FasL-dependent death induced by anti-CD3 antibody, and that the action of these proteases is also not required for PS externalization. Extracellular Ca2+ is required to complete the later stages of apoptosis in DO11.10 cells, and its removal restores normal transport of PS, suggesting that down-regulation of the aminophospholipid translocase and up-regulation of the scramblase are not effected by irreversible protease cleavage.     

Bilayer/cytoskeleton interactions in lipid-symmetric erythrocytes assessed by a photoactivable phospholipid analogue

Two mechanisms have been proposed for maintenance of transbilayer phospholipid asymmetry in the erythrocyte plasma membrane, one involving specific interactions between the aminophospholipids of the inner leaflet of the bilayer and the cytoskeleton, particularly spectrin, and the other involving the aminophospholipid translocase. If the former mechanism is correct, then erythrocytes which have lost their asymmetric distribution of phospholipids should display altered bilayer/cytoskeleton interactions. To test this possibility, normal erythrocytes, erythrocytes from patients with chronic myelogenous leukemia or sickle disease, and lipid-symmetric and -asymmetric erythrocyte ghosts were labeled with the radioactive photoactivable analogue of phosphatidylethanolamine, 2-(2-azido-4-nitrobenzoyl)-1-acyl-sn-glycero-3-phospho[14C]ethanolamine ([14C]AzPE), previously shown to label cytoskeletal proteins from the bilayer. The labeling pattern of cytoskeletal proteins in pathologic erythrocytes and lipid-asymmetric erythrocyte ghosts was indistinguishable from normal erythrocytes, indicating that the probe detects no differences in bilayer/cytoskeleton interactions in these cells. In contrast, in lipid-symmetric erythrocyte ghosts, labeling of bands 4.1 and 4.2 and actin, and to a lesser extent ankyrin, by [14C]AzPE was considerably reduced. Significantly, however, labeling of spectrin was unaltered in the lipid-symmetric ghosts, suggesting that its relationship with the bilayer is normal in these lipid-symmetric cells. These results do not support a model in which spectrin is involved in the maintenance of an asymmetric distribution of phospholipids in erythrocytes.     

Phosphatidylserine plasma membrane asymmetry in vivo: a pancellular phenomenon which alters during apoptosis

The distribution of phospholipids across the two leaflets of the plasma membrane is important for many cellular processes including phagocytosis and hemostasis. In the present study we investigated the in vivo plasma membrane distribution of the aminophospholipid phosphatidylserine in mouse embryos with a novel technique employing Annexin V, a Ca2+ dependent phosphatidylserine binding protein, conjugated to fluorescein isothiocyanate and biotin. Annexin V directly applied to cryostat sections labeled the plasma membrane of all cells at the interface. In contrast, Annexin V injected intracardially into viable mouse embryos labeled almost exclusively apoptotic cells. These apoptotic cells were visible in all tissues and derived from all germ layers. Our experiments demonstrate that phosphatidylserine is asymmetrically distributed between the two leaflets of the plasma membrane in virtually all cell types in vivo and that this asymmetry is lost early during apoptosis.     

Regulation of aminophospholipid asymmetry in murine fibroblast plasma membranes by choline and ethanolamine analogues

The regulation of the asymmetric distribution of aminophospholipids in mammalian cell plasma membranes is not understood at this time. One approach to determine the nature of such regulatory mechanisms is to attempt alteration of the plasma membrane phospholipid composition. Choline analogues such as N,N'-dimethylethanolamine and N-monomethylethanolamine lowered the quantity of phosphatidylethanolamine in the plasma membrane of LM fibroblasts grown in defined medium without serum. Ethanolamine supplementation increased the phosphatidylethanolamine content while ethanolamine analogues such as 2-amino-2-methyl-1-propanol, 2-amino-1-butanol, 1-aminopropanol, and 3-aminopropanol did not alter the aminophospholipid content significantly. The transverse distribution of aminophospholipids in the plasma membrane was determined by use of a chemical labelling reagent trinitrobenzenesulfonic acid. The percent phosphatidylethanolamine trinitrophenylated by trinitrobenzenesulfonate in the outer plasma membrane monolayer of LM cells supplemented with choline analogues was not altered. In contrast, ethanolamine analogue supplementation increased the percentage of aminophospholipid in the outer monolayer 2--3-fold. Ethanolamine analogue-containing phospholipids were distributed asymmetrically across the plasma membrane with 85 to 91% being located in the inner monolayer of the plasma membrane, a distribution similar to that of phosphatidylethanolamine. The fatty acyl composition of aminophospholipids in the outer monolayer was in all cases more saturated than in the corresponding phospholipids of the inner monolayer. However, choline analogues and especially the ethanolamine analogues reduced this difference. Thus, base analogues of choline and ethanolamine may alter the aminophospholipid asymmetry, the surface charge, and the acyl chain asymmetry of LM cell plasma membranes.     

Chilling tolerance in Arabidopsis involves ALA1, a member of a new family of putative aminophospholipid translocases

The lipid composition of membranes is a key determinant for cold tolerance, and enzymes that modify membrane structure seem to be important for low-temperature acclimation. We have characterized ALA1 (for aminophospholipid ATPase1), a novel P-type ATPase in Arabidopsis that belongs to the gene family ALA1 to ALA11. The deduced amino acid sequence of ALA1 is homologous with those of yeast DRS2 and bovine ATPase II, both of which are putative aminophospholipid translocases. ALA1 complements the deficiency in phosphatidylserine internalization into intact cells that is exhibited by the drs2 yeast mutant, and expression of ALA1 results in increased translocation of aminophospholipids in reconstituted yeast membrane vesicles. These lines of evidence suggest that ALA1 is involved in generating membrane lipid asymmetry and probably encodes an aminophospholipid translocase. ALA1 complements the cold sensitivity of the drs2 yeast mutant. Downregulation of ALA1 in Arabidopsis results in cold-affected plants that are much smaller than those of the wild type. These data suggest a link between regulation of transmembrane bilayer lipid asymmetry and the adaptation of plants to cold.     

Membrane cholesterol in the regulation of aminophospholipid asymmetry and phagocytosis in oxidized erythrocytes

Cholesterol is known to affect several membrane functions, including membrane susceptibility to oxidative stress. In order to gain a better understanding of the relationship between cholesterol contents, structural integrity, and degree of survival in oxidatively stressed erythrocytes, here we analyzed the transbilayer phospholipid distribution, the morphology, and the degree of clearance observed in cholesterol-modified (enriched or depleted) and unmodified (control) erythrocytes exposed to tert-butylhydroperoxide. We report that the modification of cholesterol contents in erythrocytes promotes the externalization of phosphatidylserine (PS) to the membrane surface, which is consistent with a concomitant inhibition of aminophospholipid translocase (APLT) and an increased uptake of modified erythrocytes by macrophages. Moreover, cholesterol depletion modifies the transbilayer aminophospholipid distribution induced by oxidative stress to a great extent, significantly increasing PS externalization, which is associated with the strongest decrease in APLT activity. The loss of normal PS asymmetry is positively correlated with enhanced phagocytosis, and an increase in echinocyte forms is observed in all oxidized erythrocytes. We envisage that PS externalization could be due, at least in part, to the decrease in APLT activity induced by oxidative stress, the activity of which is also dependent on membrane cholesterol contents.     

Polyamine regulation of plasma membrane phospholipid flip-flop during apoptosis.

During apoptosis, phosphatidylserine (PS) is moved from the plasma membrane inner leaflet to the outer leaflet where it triggers recognition and phagocytosis of the apoptotic cell. Although the mechanisms of PS appearance during apoptosis are not well understood, it is thought that declining activity of the aminophospholipid translocase and calcium-mediated, nonspecific flip-flop of phospholipids play a role. As previous studies in the erythrocyte ghost have shown that polyamines can alter flip-flop of phospholipids, we asked whether alterations in cellular polyamines in intact cells undergoing apoptosis would affect PS appearance, either by altering aminophospholipid translocase activity or phospholipid flip-flop. Cells of the human leukemic cell line, HL-60, were incubated with or without the ornithine decarboxylase inhibitor, difluoromethylornithine (DFMO), and induced to undergo apoptosis by ultraviolet irradiation. Whereas DFMO treatment resulted in profound depletion of putrescine and spermidine (but not spermine), it had no effect on caspase activity, DNA fragmentation, or plasma membrane vesiculation, typical characteristics of apoptosis. Notably, DFMO treatment prior to ultraviolet irradiation did not alter the decline in PS inward movement by the aminophospholipid translocase as measured by the uptake of 6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminocaproyl] (NBD)-labeled PS detected in the flow cytometer. Conversely, the appearance of endogenous PS in the plasma membrane outer leaflet detected with fluorescein isothiocyanate-labeled annexin V and enhanced phospholipid flip-flop detected by the uptake of 1-palmitoyl-1-[6-[(7-nitro-2-1, 3-benzoxadiazol-4-yl)aminocaproyl]-sn-glycero-3-phosphocholine (NBD-PC) seen during apoptosis were significantly inhibited by prior DFMO treatment. Importantly, replenishment of spermidine, by treatment with exogenous putrescine to bypass the metabolic blockade by DFMO, restored both enhanced phospholipid flip-flop and appearance of PS during apoptosis. Such restoration was seen even in the presence of cycloheximide but was not seen when polyamines were added externally just prior to assay. Taken together, these data show that intracellular polyamines can modulate PS appearance resulting from nonspecific flip-flop of phospholipids across the plasma membrane during apoptosis.     

Mathematical modelling of lipid transbilayer movement in the human erythrocyte plasma membrane

A model is presented to simulate transverse lipid movement in the human erythrocyte membrane. The model is based on a system of differential equations describing the time-dependence of phospholipid redistribution and the steady state distribution between the inner and outer membrane monolayer. It takes into account several mechanisms of translocation: (i) ATP-dependent transport via the aminophospholipid translocase; (ii) protein-mediated facilitated and (iii) carrier independent transbilayer diffusion. A reasonable modelling of the known lipid asymmetry could only be achieved by introducing mechanism (iii). We have called this pathway the compensatory flux, which is proportional to the gradient of phospholipids between both membrane leaflets. Using realistic model parameters, the model allows the calculation of the transbilayer motion and distribution of endogenous phospholipids of the human erythrocyte membrane for several biologically relevant conditions. Moreover, the model can also be applied to experiments usually performed to assess phospholipid redistribution in biological membranes. Thus, it is possible to simulate transbilayer motion of exogenously added phospholipid analogues in erythrocyte membranes. Those experiments have been carried out here in parallel using spin labeled lipid analogues. The general application of this model to other membrane systems is outlined.Abbreviations PBS phosphate buffered saline - DFP diisopropyl fluorophosphate - ESR electron spin resonance - RBC red blood cells - PC phosphatidylcholine - PE phosphatidylethanolamine - PS phosphatidylserine - SM sphingomyelin - (0,2)PC 1-palmitoyl-2(4doxylpentanoyl)-PC - (0,2)PE 1-palmitoyl-2(4-doxylpentanoyl)-PE - (0,2) PS 1-palmitoyl-2(4-doxylpentanoyl)-PS