Aminophospholipid Translocation Research Articles

Caspase-mediated cleavage of phospholipid flippase for apoptotic phosphatidylserine exposure.

Phospholipids are asymmetrically distributed in the plasma membrane. This asymmetrical distribution is disrupted during apoptosis, exposing phosphatidylserine (PtdSer) on the cell surface. Using a haploid genetic screen in human cells, we found that ATP11C (adenosine triphosphatase type 11C) and CDC50A (cell division cycle protein 50A) are required for aminophospholipid translocation from the outer to the inner plasma membrane leaflet; that is, they display flippase activity. ATP11C contained caspase recognition sites, and mutations at these sites generated caspase-resistant ATP11C without affecting its flippase activity. Cells expressing caspase-resistant ATP11C did not expose PtdSer during apoptosis and were not engulfed by macrophages, which suggests that inactivation of the flippase activity is required for apoptotic PtdSer exposure. CDC50A-deficient cells displayed PtdSer on their surface and were engulfed by macrophages, indicating that PtdSer is sufficient as an "eat me" signal.

Localization, Purification, and Functional Reconstitution of the P4-ATPase Atp8a2, a Phosphatidylserine Flippase in Photoreceptor Disc Membranes

P(4)-ATPases comprise a relatively new subfamily of P-type ATPases implicated in the energy-dependent translocation of aminophospholipids across cell membranes. In this study, we report on the localization and functional properties of Atp8a2, a member of the P(4)-ATPase subfamily that has not been studied previously. Reverse transcription-PCR revealed high expression of atp8a2 mRNA in the retina and testis. Within the retina, immunofluorescence microscopy and subcellular fractionation studies localized Atp8a2 to outer segment disc membranes of rod and cone photoreceptor cells. Atp8a2 purified from photoreceptor outer segments by immunoaffinity chromatography exhibited ATPase activity that was stimulated by phosphatidylserine and to a lesser degree phosphatidylethanolamine but not by phosphatidylcholine or other membrane lipids. Purified Atp8a2 was reconstituted into liposomes containing fluorescent-labeled phosphatidylserine to measure the ability of Atp8a2 to flip phosphatidylserine across the lipid bilayer. Fluorescence measurements showed that Atp8a2 flipped fluorescent-labeled phosphatidylserine from the inner leaflet of liposomes (equivalent to the exocytoplasmic leaflet of cell membranes) to the outer leaflet (equivalent to cytoplasmic leaflet) in an ATP-dependent manner. Our studies provide the first direct biochemical evidence that purified P(4)-ATPases can translocate aminophospholipids across membranes and further implicates Atp8a2 in the generation and maintenance of phosphatidylserine asymmetry in photoreceptor disc membranes.

Phospholipid Scramblase Isoform Expression in Pregnant Rat Uterus

Phospholipid scramblases (PLSCRs), a family of novel membrane proteins, facilitate the translocation of aminophospholipids from the inner to the exterior leaf of the cell membrane. Four isoforms of PLSCR (PLSCR1-4) have been reported in mouse and human. The studies described in this report sought to characterize the uterine expression of the PLSCR isoforms in the near-term pregnant rat. Uterine tissue was obtained from timed-pregnant Sprague-Dawley rats. Total RNA was isolated, treated with DNase, and used in qualitative and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) studies utilizing PLCSR isoform PCR primers. A rat spleen cDNA bacteriophage library was used as a template for PCR-based sequencing to determine the cDNA and translated amino acid sequences for the PLSCR3 and PLSCR4 homologs expressed in rats. The 5' and 3' untranslated regions were obtained using 5' and 3' Rapid Amplification of cDNA Ends (RACE) techniques. RT-PCR studies confirmed expression of PLSCR3 and PLSCR4 in the endometrial and myometrial layers of the pregnant rat uterus; in contrast, PLSCR1 and PLSCR2 were not found in uterine tissues. The cDNA sequence for the rat PLSCR3 homolog was found to be 1642 nucleotides, having 92% identity with mouse and 80% with human PLSCR3. The rat PLSCR4 homolog has a cDNA sequence of 1879 nucleotides, having an 89% identity with mouse and 72% identity with human PLSCR4 homologs. The intrauterine expression of PLSCR3 and PLSCR4 provides a dynamic mechanism by which aminophospholipid translocation can be regulated, thereby modulating the activity of various membrane proteins that are involved in inflammation and coagulation-related events.

Mechanisms by Which Intracellular Calcium Induces Susceptibility to Secretory Phospholipase A2 in Human Erythrocytes

Exposure of human erythrocytes to the calcium ionophore ionomycin rendered them susceptible to the action of secretory phospholipase A(2) (sPLA(2)). Analysis of erythrocyte phospholipid metabolism by thin-layer chromatography revealed significant hydrolysis of both phosphatidylcholine and phosphatidylethanolamine during incubation with ionomycin and sPLA(2). Several possible mechanisms for the effect of ionomycin were considered. Involvement of intracellular phospholipases A(2) was excluded since inhibitors of these enzymes had no effect. Assessment of membrane oxidation by cis-parinaric acid fluorescence and comparison to the oxidants diamide and phenylhydrazine revealed that oxidation does not participate in the effect of ionomycin. Incubation with ionomycin caused classical physical changes to the erythrocyte membrane such as morphological alterations (spherocytosis), translocation of aminophospholipids to the outer leaflet of the membrane, and release of microvesicles. Experiments with phenylhydrazine, KCl, quinine, merocyanine 540, the calpain inhibitor E-64d, and the scramblase inhibitor R5421 revealed that neither phospholipid translocation nor vesicle release was required to induce susceptibility. Results from fluorescence spectroscopy and two-photon excitation scanning microscopy using the membrane probe laurdan argued that susceptibility to sPLA(2) is a consequence of increased order of membrane lipids.

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.

Identification and Functional Expression of Four Isoforms of ATPase II, the Putative Aminophospholipid Translocase: EFFECT OF ISOFORM VARIATION ON THE ATPase ACTIVITY AND PHOSPHOLIPID SPECIFICITY

ATPase II, a vanadate-sensitive and phosphatidylserine-dependent Mg(2+)-ATPase, is a member of a subfamily of P-type ATPase and is presumably responsible for aminophospholipid translocation activity in eukaryotic cells. The aminophospholipid translocation activity plays an important physiological role in the maintenance of membrane phospholipid asymmetry that is observed in the plasma membrane as well as the membranes of certain cellular organelles. While the preparations of ATPase II from different sources share common fundamental properties, such as substrate specificity, inhibitor spectrum, and phospholipid dependence, they are divergent in several characteristics. These include specific ATPase activity and phospholipid selectivity. We report here the identification of four isoforms of ATPase II in bovine brain. These isoforms are formed by a combination of two major variations in their primary sequences and show that the structural variation of these isoforms has functional significance in both ATPase activity and phosholipid selectivity. Furthermore, studies with the phosphoenzyme intermediate of ATPase II and its recombinant isoforms revealed that phosphatidylserine is essential for the dephosphorylation of the intermediate. Without phosphatidylserine, ATPase II would be accumulated as phosphoenzyme in the presence of ATP, resulting in the interruption of its catalytic cycle.

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 Mg2+-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 Kdof 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 μM free intracellular Ca2+. The wider applicability of the method and the physiological implications of the calcium sensitivity determined is discussed.

Asymmetric distribution of phosphatidylethanolamine in C. albicans: possible mediation by CDR1, a multidrug transporter belonging to ATP binding cassette (ABC) superfamily.

By using two molecular probes, we demonstrate that only 4% of total phosphatidylethanolamine (PtdEtn) in the plasma membrane (PM) of a human pathogenic yeast, Candida albicans, is present in its external half. Evidence is presented to show that the availability of PtdEtn could be related to the expression of a multidrug transporter CDR1 of C. albicans, and the process is energy-dependent. A homozygous CDR1 disruptant strain of C. albicans shows almost 23% reduction in the external labelling of PtdEtn. This report shows that, similar to human MDRs, yeast multidrug transporter could also be involved in aminophospholipid translocation.

Shear-induced platelet activation and platelet microparticle formation in native human blood

Shear-induced platelet activation and platelet microparticle formation are triggered in native human blood by high arterial shear or by a sudden increase in shear as introduced by a stenosis with potential consequences for collagen-induced platelet thrombus formation. Blood was drawn from healthy volunteers and directly perfused ex vivo over various well-defined eccentric stenoses. Shear-induced platelet activation was determined by using flow cytometry to assess: 1) GPIIb-IIIa activation by fluorescein isothiocyanate (FITC)-labeled Mab PAC-1; and 2) translocation of membrane aminophospholipids (procoagulant activity) by FITC-labeled Annexin V. Microparticle formation was measured by flow cytometry and FITC-labeled Mab Y2/51 directed against GPIIIa. Significant platelet activation and platelet microparticle formation were elicited when the wall shear rate reached 10,500 sec −1 for a period of 0.075 sec. Prolonged exposure to or a rapid increase in shear further enhanced activation and microparticle formation. Shear-induced platelet activation was associated with significantly increased collagen-induced platelet thrombus formation that was insensitive to aspirin ingestion. Exposure of native blood to very high shear thus activates platelets to express GPIIb-IIIa, renders the platelet membrane procoagulant and stimulates microparticle formation. These responses are associated with enhanced collagen-induced thrombus formation by prostaglandin-independent mechanisms.

Shear-induced platelet activation and platelet microparticle formation at blood flow conditions as in arteries with a severe stenosis.

In the present study, we investigated whether high arterial shear stresses at various exposure times or a sudden increase in shear stress introduced by a stenosis affect platelet activation and platelet microparticle formation in native human blood. We used a parallel-plate perfusion chamber device through which nonanticoagulated human blood was drawn (10 mL/min) by a pump directly from an antecubital vein through the flow channel of a perfusion chamber at wall shear rates of 420, 2600, and 10500 s-1. In another set of experiments, an eccentric stenosis was introduced into the flow channel. Wall shear rates of 2600 or 10500 s-1 at the stenosis apex were maintained at the same flow rate. The wall shear rate upstream and downstream of these stenoses was 420 s-1. A shear rate of 420 s-1 is within the range of those encountered in healthy small coronary arteries, whereas those of 2600 and 10500 s-1 are representative for vessels with various degrees of stenotic lesions. The blood was exposed to these shear rates for periods varying from 0.075 to 3.045 seconds. Platelet activation was assessed as activated glycoprotein (GP) IIb/IIIa by FITC-labeled monoclonal antibody (MAb) PAC-1 and aminophospholipid translocation by FITC-labeled annexin V. Microparticle formation was quantified by FITC-labeled MAb Y2/51 directed against GP IIIa. Significant platelet activation and formation of microparticles were observed at 10500 s-1 only (P < .008). This shear-induced platelet activation and microparticle formation were enhanced by introduction of a thrombus-promoting surface consisting of type III human collagen fibrils. Introduction of the most severe stenosis at 10500 s-1 further increased platelet activation (P < .017). The collagen-induced thrombus formation increased the platelet thrombus volume at 10500 s-1 from 16.5 to 33.8 microns3/microns2 (P < .003) on the stenosis apex when the most severe stenosis was used. A correlation (P < .0001) between platelet thrombus volume and platelet microparticle formation was observed in the presence of the eccentric stenoses. Apparently, high shear stress (315 dynes/cm2 at 10500 s-1), as encountered in severe atherosclerotic arteries, activated platelets and triggered platelet microparticle formation. In contrast, no significant platelet activation or formation of platelet microparticles was observed at physiological shear (420 s-1) or at the shear condition simulating shear in arteries with a less severe stenosis (2600 s-1). The data imply that platelets are activated and form microparticles in native blood at very high shear stresses. These events are potentiated by prolonged exposure to the high shear or by a sudden change of increasing shear due to the stenosis. The latter situation apparently enhances platelet thrombus formation at the stenosis.

Characterization of the correlation between ATP-dependent aminophospholipid translocation and Mg2+-ATPase activity in red blood cell membranes.

Pseudosubstrates and inhibitors of ATPases were studied with respect to their capability to modulate the kinetic behavior of Mg2+-ATPase and aminophospholipid translocation in red blood cell ghosts. ATP was substituted by the pseudosubstrates of P-type ATPases acetyl phosphate and p-nitrophenyl phosphate. With both pseudosubstrates, aminophospholipid translocation from the outer to the inner leaflets of resealed erythrocyte ghosts could be observed, although with a significantly decreased velocity compared to that in presence of ATP, both with respect to phosphate hydrolysis and translocation. Similarly, the apparent affinities for the pseudosubstrates were much lower than for ATP. Among the inhibitors studied, suramin acted as a competitive inhibitor of ATP towards both Mg2+-ATPase activity and aminophospholipid translocation. However, the inhibition of translocation occurred at a higher inhibitor concentration than the inhibition of Mg2+-ATPase activity. With elaiophylin, only a partial inhibition of Mg2+-ATPase activity could be detected, but translocation of labeled phosphatidylserine was almost completely abolished. With eosin Y, an almost complete inhibition of both Mg2+-ATPase activity and translocation could be achieved. The observed responses of aminophospholipid translocation to ATPase inhibitors strongly suggest that a P-type ATPase, part of which displays a Mg2+-ATPase activity, is involved in aminophospholipid translocation.

A Subfamily of P-Type ATPases with Aminophospholipid Transporting Activity

The appearance of phosphatidylserine on the surface of animal cells triggers phagocytosis and blood coagulation. Normally, phosphatidylserine is confined to the inner leaflet of the plasma membrane by an aminophospholipid translocase, which has now been cloned and sequenced. The bovine enzyme is a member of a previously unrecognized subfamily of P-type adenosine triphosphatases (ATPases) that may have diverged from the primordial enzyme before the separation of the known families of ion-translocating ATPases. Studies in Saccharomyces cerevisiae suggest that aminophospholipid translocation is a general function of members of this family.

Transbilayer movement of fluorescent and spin-labeled phospholipids in the plasma membrane of human fibroblasts: a quantitative approach.

All phospholipids in the plasma membrane of eukaryotic cells are subject to a slow passive transbilayer movement. In addition, aminophospholipids are recognized by the so-called aminophospholipid translocase, and are rapidly moved from the exoplasmic to the cytoplasmic leaflet of the plasma membrane at the expense of ATP hydrolysis. Though these principal pathways of transbilayer movement of phospholipids probably apply to all eukaryotic plasma membranes, studies of the actual kinetics of phospholipid redistribution have been largely confined to non-nucleated cells (erythrocytes). Experiments on nucleated cells are complicated by endocytosis and metabolism of the lipid probes inserted into the plasma membrane. Taking these complicating factors into account, we performed a detailed kinetic study of the transbilayer movement of short-chain fluorescent (N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl); NBD) and, for the first time, spin-labeled analogues of phosphatidylcholine (PC), -ethanolamine (PE), -serine (PS), and sphingomyelin (SM) in the plasma membrane of cultured human gingival fibroblasts. At 20 degrees C, the passive transbilayer diffusion of NBD analogues was very slow, and the choline-containing NBD analogues were internalized predominantly by endocytosis. Spin-labeled analogues of PC and SM showed higher passive transbilayer diffusion rates, and probably entered the cell by both passive transbilayer movement and endocytosis. In contrast, the rapid uptake of NBD- and spin-labeled aminophospholipid analogues could be mainly ascribed to the action of the aminophospholipid translocase, since it was inhibited by ATP depletion and N-ethylmaleimide pretreatment. The initial velocity of NBD-aminophospholipid translocation was eight to ten times slower than that of the corresponding spin-labeled lipid, and the half-times of redistribution of NBD-PS and spin-labeled PS were 7.2 and 3.6 minutes, respectively. Our data indicate that in human fibroblasts the initial velocity of aminophospholipid translocation is at least one order of magnitude higher than that in human erythrocytes, which should be sufficient to maintain the phospholipid asymmetry in the plasma membrane.

Protein-dependent translocation of aminophospholipids and asymmetric transbilayer distribution of phospholipids in the plasma membrane of ram sperm cells.

We have investigated the transbilayer movement of phospholipids in the plasma membrane of ram sperm cells using spin- and fluorescence-labeled lipid analogues. After incorporation into the outer leaflet, phosphatidylcholine (PC) and sphingomyelin (SM) moved slowly to the inner cytoplasmic leaflet, whereas phosphatidylserine (PS) and phosphatidylethanolamine (PE) rapidly disappeared from the exoplasmic monolayer. Variation of the initial velocity of the relocation kinetics vs the amount of analogue incorporated into the membrane suggests a saturability of the transbilayer movement of aminophospholipids. ATP depletion or pretreatment with N-ethylmaleimide of ram sperm cells reduced the fast inward motion of PS and PE, indicating a protein-mediated aminophospholipid translocation. The results suggest for the plasma membrane of ram sperm cells the presence of an aminophospholipid translocase and an asymmetric transversal lipid distribution with aminophospholipids preferentially located in the inner leaflet and choline-containing phospholipids in the outer leaflet. The relevance of the transversal segregation of phospholipids for membrane fusion processes occurring during fertilization is discussed.

Relationship of phospholipid distribution to shape change in Ca(2+)-crenated and recovered human erythrocytes.

Echinocytosis induced by elevation of intracellular Ca2+ in human erythrocytes can be reversed by removal of the cation. Using back-extraction of radiolabeled dilauroyl phospholipid analogs which had been incorporated into the cell membrane, we examined the relationship between this reversible shape transformation and phospholipid distribution. Upon Ca2+ crenation of cells, surface exposure of phosphatidylserine and phosphatidylethanolamine was observed simultaneously with inward diffusion of phosphatidylcholine. Removal of Ca2+ allowed resequestration of exposed phosphatidylserine to the membrane inner monolayer, but randomized phosphatidylethanolamine and phosphatidylcholine were not redistributed to their original states. Both shape reversion and retranslocation of phosphatidylserine were reversibly inhibited vanadate. On the other hand, the cell shape recovery was found to be independent of membrane skeleton and phosphoinositide metabolism and was supported by ATP resynthesis only under conditions where the aminophospholipid translocator is active. Other Ca(2+)-mediated biochemical changes, such as generation of diacylglycerol and fatty acids, were found to have no effect on Ca2+ crenation or its reversal, or upon transbilayer distribution of any phospholipid. These findings suggest that Ca2+ induces phospholipid redistribution, possibly by direct interaction with the lipid bilayer and, further, that metabolic recovery from Ca2+ crenation reflects selective retransport of phosphatidylserine to the membrane inner monolayer.

Hydrogen Peroxide Oxidation Induces the Transfer of Phospholipids from the Membrane into the Cytosol of Human Erythrocytes

The effects of oxidative damage on membrane phospholipid organization were examined in human erythrocytes. Exposure to H2O2 induced shape changes in these cells; normal discocytes became echinocytic, and stomatocytes generated by foreign phosphatidylserine incorporation reverted to discoid morphology. H2O2 treatment also inhibited phosphatidylserine transport from the outer to inner membrane monolayer, consistent with earlier reports on oxidative sensitivity of the aminophospholipid translocator. The morphological changes are consistent with movement of inner monolayer lipids to the outer monolayer, as might be expected if aminophospholipid sequestration is compromised. However, lipid extraction and prothrombinase activation assays showed no increased exposure of phosphatidylserine on the cell surface. Instead, phosphatidylserine was found associated with the cytosolic fraction of H2O2-treated cells. These observations suggest that oxidative damage alters the lipid organization of erythrocyte membranes, not by randomizing the lipid classes within the bilayer, but by inducing extraction of inner monolayer components into the cytosol.

Phosphatidylserine headgroup diastereomers translocate equivalently across human erythrocyte membranes

The natural chiral phospholipid substrates for the plasma membrane aminophospholipid translocator are L-α-phosphatidyl- L-serine and L-α-phosphatidylethanolamine. The glyceric D-stereoisomers of these lipids, D-α-phosphatidyl- L-serine and D-α-phosphatidylethanolamine, are not translocated (Martin, O.C. and Pagano, R.E. (1987) J. Biol. Chem. 262, 5890–5898). We have synthesized a diastereomer of phosphatidylserine, L-α-phosphatidyl- D-serine, to study the effects of headgroup stereochemistry on translocation. The diastereomer was synthesized as the dilauroyl (12:0) species, and the translocation was monitored by human erythrocyte morphology changes at 25°C and 37°C. Unlike other phosphatidylserine stereoisomers, L-α-phosphatidyl- Dserine is translocated to the same degree as the natural L,L-isomer. Incorporation of apparently equal amounts of the L,D- and L,L-diastereomers does produce minor quantitative differences in the cell morphological response, possibly as a result of differences in lipid packing of the two isomers.

Effects of exogenous phospholipids on platelet activation

Intercalation of amphipaths into the plasma membrane of platelets has a marked effect on their morphology. Incubation of platelets with phosphatidylcholines (PC) results in rounding of the platelet body and spiculation, while incubation with aminophospholipids such as dilauroylphosphatidylserine (DLPS) results in a biphasic shape change consistent with the bilayer couple model (Sheetz, M.P. and Singer, S.J. (1982) Proc. Natl. Acad. Sci. USA 71, 4457–4461) and with the activity of an aminophospholipid translocator facilitating transverse bilayer diffusion (Daleke, D.L. and Huestis, W.H. (1985) Biochemistry 24, 5406–5416). The present study extends this work to investigate the effects of PC and PS on platelet responses to a natural agonist, thrombin. PC incorporation produces a concentration-dependent progression of shape changes, beginning with surface ruffling and development of fine spicules, followed by sphering of the cell body, and ending with the apparent loss of spicules. PC reduces platelet responses to thrombin only under conditions that promote membrane vesiculation, seen morphologically as a loss of spicules and biochemically as a loss of 14C-PC labeled membrane. PS homologues of varying acyl chain composition induce concentration- and time-dependent platelet sphering. Incorporation of PS inhibits thrombin-induced platelet shape change, granule secretion, and protein phosphorylation. Inhibition of these responses requires transit of the exogenous PS to the cytofacial leaflet of the membrane bilayer.

Dithiothreitol stimulates the activity of the plasma membrane aminophospholipid translocator

Metabolic depletion induces human erythrocytes to crenate, a shape change that is reversed when ATP is regenerated by nutrient supplementation. In the presence of the sulfhydryl reducing agent dithiothreitol (DTT), this shape reversal is exaggerated, proceeding beyond normal discoid morphology to stomatocytic forms. DTT-induced stomatocytosis does not correlate consistently with alterations in cell ATP, spectrin phosphorylation, or phosphoinositide metabolism (Truong, H.-T.N., Ferrell, J.E., Jr. and Huestis, W.H. (1986) Blood 67, 214–221). The effect of DTT on outer-to-inner-monolayer transport of aminophospholipids was examined by monitoring shape changes induced by dilauroylphosphatidylserine (DLPS). Stomatocytosis induced by transport of this exogenous lipid to the membrane inner monolayer is accelerated and exaggerated by DTT. The effect of DTT on DLPS translocation is reversible and temperature dependent, consistent with the intervention of reducing agents in the activity of the aminophospholipid translocator. These findings bear on the relationship between cell redox status and shape regulation.

Transbilayer mobility and distribution of red cell phospholipids during storage.

We studied phospholipid topology and transbilayer mobility in red cells during blood storage. The distribution of phospholipids was determined by measuring the reactivity of phosphatidylethanolamine with fluorescamine and the degradation of phospholipids by phospholipase A2 and sphingomyelinase C. Phospholipid mobility was measured by determining transbilayer movements of spin-labeled phospholipids. We were unable to detect a change in the distribution of endogenous membrane phospholipids in stored red cells even after 2-mo storage. The rate of inward movement of spin-labeled phosphatidylethanolamine and phosphatidylserine was progressively reduced, whereas that for phosphatidylcholine was increased. These changes in phospholipid translocation correlated with a fall in cellular ATP. However, following restoration of ATP, neither the rate of aminophospholipid translocation nor the transbilayer movement of phosphatidylcholine were completely corrected. Taken together, our findings demonstrate that red cell storage alters the kinetics of transbilayer mobility of phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine, the activity of the aminophospholipid translocase, but not the asymmetric distribution of endogenous membrane phospholipids, at least at a level detectable with phospholipases. Thus, if phosphatidylserine appearance on the outer monolayer is a signal for red cell elimination, the amount that triggers macrophage recognition is below the level of detection upon using the phospholipase technique.