Aminophospholipid translocase activity in JEG-3; a choriocarcinoma model of cytotrophoblast differentiation

The plasma membrane is characterized by a non-symmetrical distribution of phospholipids; the outer monolayer of the plasma membrane consists primarily of phosphatidylcholine (PC), and the aminophospholipids, phosphatidylserine (PS) and phosphatidylethanolamine (PE), preferentially reside in the inner monolayer. Asymmetry is maintained by a membrane associated ATP-dependent aminophospholipid translocase that preferentially relocates PS and PE from the outer to the inner monolayer. Although in most cells the translocase minimizes expression of PS on the outer surface, differentiating trophoblasts express increasing levels of surface PS. One possible explanation of prolonged PS externalization is that trophoblasts lack an effective aminophospholipid translocase. To test this hypothesis, fluorescent PC and PS analogues, NBD-PC and NBD-PS, were introduced into the plasma membrane of a choriocarcinoma model of trophoblast, JEG-3 cells. After incubation, the fluorescent lipid remaining on the outer monolayer was removed by incubation with fetal bovine serum. JEG-3 cells selectively translocated 80 per cent of the NBD-PS without significant translocation of NBD-PC. The process was significantly inhibited by N-ethylmaleimide (NEM) and vanadate. It is concluded that this model of trophoblast contains an active aminophospholipid translocase.     

Safety and immunogenicity of capsular polysaccharide-tetanus toxoid conjugate vaccines for group B streptococcal types Ia and Ib

The translocation of spin-labeled analogues of phosphatidylcholine (4-doxylpentanoyl-PC, SL-PC), phosphatidylethanolamine (SL-PE), phosphatidylserine (SL-PS), and sphingomyelin (SL-SM) from the outer to the inner leaflet of the plasma membrane bilayer was investigated in dog kidney MDCK II and human colon Caco-2 cells. Disappearance from the outer leaflet was assayed using back-exchange to serum albumin. Experiments with cells in suspension as well as with polarized cells on filters were performed at reduced temperatures (10 and 20 degreesC) to suppress endocytosis and hydrolysis of spin-labeled lipids. For both epithelial cell lines, a fast ATP-dependent inward movement of the aminophospholipids SL-PS and SL-PE was found, while SL-SM was only slowly internalized without any effect of ATP depletion. The kinetics of redistribution of SL-PC were clearly different between the two cell lines. In MDCK II cells, SL-PC was rapidly internalized in an ATP-dependent and N-ethylmaleimide-sensitive manner and at a rate similar to that of the aminophospholipids. In contrast, in Caco-2 cells the inward movement of SL-PC was much slower than that of the aminophospholipids, did not depend on ATP, and was not N-ethylmaleimide-sensitive. Inhibitor studies indicated that the outward-translocating multidrug resistance P-glycoprotein present in these cells did not affect the kinetics of inward translocation. Internalization was always similar on the apical and basolateral cell surface, suggesting the presence of the same phospholipid translocator(s) on both surface domains of epithelial cells. We propose that Caco-2 cells contain the well-known aminophospholipid translocase, while MDCK II cells contain either two translocases, namely, the aminophospholipid translocase and a phosphatidylcholine-specific translocase, or one translocase of a new type, translocating aminophospholipids as well as phosphatidylcholine.     

Transmembrane phospholipid distribution in blood cells: control mechanisms and pathophysiological significance

This review deals with current concepts on the regulation and function of phospholipid asymmetry in biological membranes. This ubiquitous phenomenon is characterized by a distinctly different lipid composition between the inner and outer leaflet of the membrane bilayer. Transbilayer asymmetry is controlled by different membrane proteins that function as lipid transporters, catalyzing uni- or bi-directional transbilayer movement of lipids. Under normal conditions, an ATP-dependent protein (aminophospholipid translocase) generates and maintains phospholipid asymmetry by promoting unidirectional transport of aminophospholipids from the outer- to the inner leaflet. The membrane lipid asymmetry may be compromised during cellular activation by a Ca2+-dependent transporter (lipid scramblase) that facilitates rapid bi-directional movement of all major phospholipid classes. A major consequence of this collapse of lipid asymmetry is the exposure of phosphatidylserine (PS) at the outer membrane surface. Surface exposure of PS has important physiological and pathological implications for blood coagulation, apoptosis, and cell-cell recognition.     

Transverse redistribution of phospholipids during human platelet activation: evidence for a vectorial outflux specific to aminophospholipids

The redistribution kinetics of phospholipids during human platelet activation by calcium ionophore were investigated to determine the specificity of the observed phospholipid outflux [Bassé et al. (1993) Biochemistry 32, 2337]. (1) Two double-labeling experiments were performed with a combination of equal amounts of spin- and fluorescently-labeled phosphatidylserine and phosphatidylcholine. During A23187-induced activation, 50% of the internal phosphatidylserine analogs were rapidly (t1/2 < 1 min) reexposed on the platelet surface while no reciprocal influx of the external phosphatidylcholine analogs was observed. (2) Treatment with chlorpromazine allowed the internalization of 20% of external spin-labeled sphingomyelin or spin-labeled phosphatidylcholine, without either inducing platelet activation or interfering with aminophospholipid translocase activity or with A23187-induced activation (dense granule secretion and vesicle shedding). During A23187-induced activation, none of the previously internalized choline head phospholipids were exposed externally, while spin-labeled phosphatidylserine outward movements were similar irrespective of whether platelets were pretreated or not pretreated with chlorpromazine. Our results demonstrated that during strong platelet activation (1) the PL excess in the internal leaflet, due to the probe addition, is not responsible for their outflux; (2) the rapid aminophospholipid outflux is definitely a vectorial outflux not counterbalanced by a rapid reciprocal influx of choline head phospholipids (i.e., not scrambling); and (3) the vectorial outflux is specific for aminophospholipids since previously internalized sphingomyelin and phosphatidylcholine did not move outward. This suggests that the specific vectorial outflux of aminophospholipids could be catalyzed by a "reverse aminophospholipid translocase" activity.     

Regulatory mechanisms in maintenance and modulation of transmembrane lipid asymmetry: pathophysiological implications

The two leaflets of the plasma membrane of eukaryotic cells differ in lipid composition: the outer leaflet comprises mainly neutral choline containing phospholipids, whereas the aminophospholipids reside almost exclusively in the cytoplasmic leaflet. The importance of transmembrane lipid asymmetry may be judged from the fact that the cell invests energy to maintain this situation for which at least two regulatory mechanisms are held responsible. A translocase, selective for aminophospholipids, acts as an ATP-dependent pump for rapid inward movement of phosphatidylserine (PS) and phosphatidylethanolamine; in addition, a non-selective, but also ATP-dependent pump causes outward movement of phospholipids, be it at a much lower rate compared to the inward transport by the aminophospholipid translocase. These two systems, acting in concert, are thought to be the main players in the maintenance of a dynamic equilibrium of the phospholipids over both membrane leaflets. Dissipation of membrane lipid asymmetry can be elicited in different cell types under a variety of conditions; in particular, platelets upon activation rapidly lose their normal plasma membrane lipid distribution, but also in other blood cells, lipid asymmetry can be lost, be it at a much lower rate and extent than in platelets. A putative protein, referred to as "scramblase' has been described, which requires the continuous presence of elevated intracellular Ca(2+)-levels, to allow a rapid, non-selective and bidirectional transbilayer movement of phospholipids. Although scrambling of lipids does not require ATP as such, preliminary studies suggest the possible involvement of one or more phosphorylated proteins. The most prominent consequence of the loss of phospholipid asymmetry is exposure of PS in the outer leaflet of the plasma membrane. Surface-exposed PS serves several important physiological functions: it promotes assembly of enzyme complexes of the coagulation cascade, it forms a signal for cell-cell recognition, which is important for cell scavenging processes. Surface-exposure of PS is an early phenomenon of apoptosis and appears to be involved in efficient removal of these cells. In addition, PS in the outer leaflet of cells is thought to play a role in cell fusion processes. It may be clear from the foregoing, that the amount of PS present at the cell surface needs to be tightly controlled, and that an impairment of this process leads to either excessive- or diminished exposition of PS which may have several pathophysiological consequences.     

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.     

Transbilayer movement of NBD-labeled phospholipids in red blood cell membranes: outward-directed transport by the multidrug resistance protein 1 (MRP1)

The outward movement (flop) of fluorescently labeled analogues of phosphatidylserine (PS) and phosphatidylcholine (PC) in human and murine red blood cells (RBC) was examined. 1-Oleoyl-2-[6(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl (C6-NBD) analogues of PS and PC were incorporated in the inner leaflet of the plasma membrane through the action of aminophospholipid translocase or through equilibration upon prolonged incubation, respectively. After removal of noninternalized probe, externalization of C6-NBD-PS or C6-NBD-PC from the inner to outer leaflet was monitored by continuous incubation of the cells in the presence of bovine serum albumin. Flop rates for both probes in intact human RBC were virtually identical (t1/2 approximately 1.5 h), confirming earlier findings by Bitbol et al. [Bitbol, M., et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 6783-6787] and Connor et al. [Connor, J., et al. (1992) J. Biol. Chem. 267, 19412-19417]. Flop activity in resealed RBC ghosts could only be found upon coinclusion of both ATP and oxidized glutathione (GSSG). Furthermore, flop in intact cells was sensitive to verapamil (IC50 = 5-7 microM), vincristine (IC50 = 20 microM), and indomethacin (IC50 = 50 microM), suggesting the involvement of proteins conferring multidrug resistance (MDR). Experiments with RBC from knock-out mice for multidrug resistance P-glycoproteins (Mdr1a/1b-/- and Mdr2-/-) and multidrug resistance protein 1 (Mrp1-/-) revealed that Mrp1 is responsible for the observed flop of the fluorescent lipid analogues. We found no indications for outward transport of endogenous PS by any of these drug-transporting proteins as measured by a sensitive prothrombinase assay. Neither aminophospholipid translocase nor Ca2+-induced lipid scramblase activities were affected in RBC of these knock-out mice. We conclude that lipid floppase activity, as detected with lipid probes, reflects the activity of MRP1 recognizing the modified lipid analogues as xenobiotics to be expelled from the cell.     

Oxidative damage does not alter membrane phospholipid asymmetry in human erythrocytes

Oxidant-induced damage has been proposed to be the underlying mechanism for loss of membrane phospholipid asymmetry in the erythrocyte membrane. In sickle cell disease, thalassemia, and diabetes as well as in senescent erythrocytes, an apparent correlation between oxidative damage and loss of phosphatidylserine asymmetry has been reported. In the present study, erythrocytes were subjected to various levels of oxidative stress and/or sulfhydryl modifying agents. The transmembrane location of phosphatidylserine (PS) was assessed by FITC-conjugated annexin V labeling and the PS-dependent prothrombinase assay. Transbilayer movement of spin-labeled PS was used to determine aminophospholipid translocase activity. Our data show that cells did not expose PS as the result of oxidative stress induced by phenylhydrazine, hydrogen peroxide, tert-butyl hydroperoxide, cumene hydroperoxide, or sulfhydryl modification by N-ethylmaleimide (NEM) and diamide, even under conditions that led to severe cellular damage and impairment of aminophospholipid translocase activity. In contrast, the increase of intracellular calcium induced by treatment with calcium and ionophore A23187 leads to a rapid scrambling of the lipid bilayer and the exposure of PS, which can be exacerbated by the inhibition of aminophospholipid translocase activity. Oxidation of the cells with hydrogen peroxide or phenylhydrazine did not affect A23187-induced uptake of calcium, but partly inhibited calcium-induced membrane scrambling. In conclusion, oxidative damage of erythrocytes does not induce exposure of phosphatidylserine on the membrane surface, but can interfere with both aminophospholipid translocase activity and calcium-induced randomization of membrane phospholipids.     

Phospholipid translocation from the outer to the inner leaflet of synaptic vesicle membranes isolated from the electric organ of Japanese electric ray Narke japonica

The phospholipid translocation from the outer to the inner leaflet of synaptic vesicles isolated from the electric organ of the Japanese electric ray, Narke japonica, was measured using fluorescent phospholipid probes. Phosphatidylcholine (PC), phosphatidylethanolamine (PE), or phosphatidylserine (PS) with a fluorescent NBD-labeled short acyl chain at the sn-2 position was mixed with purified synaptic vesicles and the probe in the outer leaflet of the membranes was reduced with dithionite to quench the fluorescence from time to time. The percentage of fluorescence remaining after the dithionite treatment served as an index for the phospholipid translocation. The results obtained indicated that about 30, 13, and 9% of NBD-PE, NBD-PS, and NBD-PC, respectively, were translocated from the outer to the inner leaflet in 3 h. Thus, the translocation activity in synaptic vesicle membranes was much higher for PE than for PS, in contrast to the previous results obtained with plasma membranes, including synaptosomal membranes. The percentages of the phospholipid in the inner leaflet at equilibrium were estimated to be 41, 31, and 14% for PE, PS, and PC, respectively. The translocation was inhibited by pretreatment with an SH reagent, iodoacetamide, indicating the involvement of a proteinaceous translocator. These data may provide a biochemical basis for elucidating the mechanisms of membrane fusion and exocytosis at nerve endings.     

Rapid kinetics of insertion and accessibility of spin-labeled phospholipid analogs in lipid membranes: a stopped-flow electron paramagnetic resonance approach

Spin-labeled phospholipid analogs have been employed to probe the transbilayer distribution of endogenous phospholipids in various membrane systems. To determine the transmembrane distribution of the spin-labeled analogs, the analogs are usually inserted into the membrane of interest and subsequently the amount of analog in the outer membrane leaflet is determined either by chemical reduction with ascorbate or by back-exchange to bovine serum albumin (BSA). For accurate determination of the transbilayer distribution of analogs, both the kinetics of incorporation and those of accessibility of analogs to ascorbate or BSA have to be fast in comparison to their transbilayer movement. By means of stopped-flow electron paramagnetic resonance (EPR) spectroscopy, we have studied the kinetics of incorporation of the spin-labeled phosphatidylcholine (PC) analog 1-palmitoyl-2-(4-doxylpentanoyl)-sn-glycero-3-phosphocholine (SL-PC) and of its accessibility to chemical reduction and to back-exchange at room temperature. Incorporation of SL-PC into the outer leaflet of egg phosphatidylcholine (EPC) and red cell ghost membranes was essentially completed within 5 s. Ninety percent of the SL-PC molecules located in the outer membrane leaflet of those membranes were extracted by BSA within 15 s. All exterior-facing SL-PC molecules were reduced by ascorbate in a pseudo-first-order reaction within 60 s in EPC membranes and within 90 s in red cell ghost membranes. The rate of the reduction process could be enhanced by approximately 30-fold when 6-O-phenyl-ascorbic acid was used instead of ascorbate as the reducing agent. The results are discussed in light of assaying rapid transbilayer movement of spin-labeled analogs in biological membranes.     

Release of phospholipids from erythrocyte membranes by taurocholate is determined by their transbilayer orientation and hydrophobic backbone

Bile salts mediate a specific release of phosphatidylcholine (PC) from the canalicular membrane into the bile fluid. We utilized human red blood cells (RBC) as a model system to study the release of endogenous phospholipids as well as phospholipid analogues from plasma membranes in the presence of the bile salt taurocholate (TC). Short- and long-chain fluorescent as well as spin-labeled analogues with various headgroups were chosen. RBC were labeled either on the exoplasmic or on the cytoplasmic leaflet with the analogues and incubated with various concentrations of TC. Analogues on the exoplasmic layer could be readily released by TC. Release was most efficient above the critical micellar concentration (CMC) of TC. Release was independent of the headgroup, but depended on the fatty acid chain length of the analogues; i.e., it was lower for long-chain than for short-chain labeled phospholipids. Analogues on the cytoplasmic leaflet were efficiently shielded from TC-mediated release. The preferential release of endogenous PC and sphingomyelin (SM) from the erythrocyte membrane above the CMC supports the conclusion that TC-mediated release of phospholipids occurs preferentially from the exoplasmic leaflet independent of their headgroup. However, the extent of release of endogenous phospholipids was significantly lower in comparison to that of analogues, endorsing the relevance of the hydrophobic backbone for bile salt mediated release of phospholipids. Implications for the mechanism of the release of PC from the canalicular membrane into the bile fluid are discussed.     

Studies of the mechanism for enhanced cell surface factor VIIa/tissue factor activation of factor X on fibroblast monolayers after their exposure to N-ethylmaleimide

Fibroblast monolayers constitutively expressing surface membrane tissue factor (TF) were treated with 0.1 mM N-ethylmaleimide (NEM) for 1 min to inhibit aminophospholipid translocase activity without inducing general cell damage. This resulted in increased anionic phospholipid in the outer leaflet of the cell surface membrane as measured by the binding of 125I-annexin V and by the ability of the monolayers to support the generation of prothrombinase. Specific binding of 125I-rVIIa to TF on NEM-treated monolayers was increased 3- to 4-fold over control monolayers after only brief exposure to 125I-rVIIa, but this difference progressively diminished with longer exposure times. A brief exposure of NEM-treated monolayers to rVIIa led to a maximum 3- to 4-fold enhancement of VIIa/TF catalytic activity towards factor X over control monolayers, but, in contrast to the binding studies, this 3- to 4-fold difference persisted despite increasing time of exposure to rVIIa. Adding prothrombin fragment 1 failed to diminish the enhanced VIIa/TF activation of factor X of NEM-treated monolayers. Moreover, adding annexin V, which was shown to abolish the ability of NEM to enhance factor X binding to the fibroblast monolayers, also failed to diminish the enhanced VIIa/TF activation of factor X. These data provide new evidence for a possible mechanism by which availability of anionic phospholipid in the outer layer of the cell membrane limits formation of functional VIIa/TF complexes on cell surfaces.     

Nonmediated flip-flop of anionic phospholipids and long-chain amphiphiles in the erythrocyte membrane depends on membrane potential

The nonmediated inward translocation (flip) of the anionic fluorescent N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)- (NBD-)labeled phospholipid phosphatidylmethanol (PM) from the outer to the inner membrane leaflet of human erythrocytes and vice versa depends on membrane potential. Interestingly, inside-positive potentials due to chloride gradients and the native chloride conductance of the cells resulted in an increase of the flip rates. This flip enhancement could be suppressed by addition of gramicidin D, which increases cation conductance, or 4,4'-diisothiocyanatostilbene-2,2'-disufonate (DIDS), which inhibits anion conductance. Conversely, inside negative potentials established by an outward-directed K+ gradient in the presence of gramicidin on DIDS-treated cells resulted in a decrease of flip rate. Flip rate exhibited an exponential dependence on membrane potential. The opposite effects of the positive and negative potentials were obtained for the outward translocation (flop) from the inner to the outer membrane leaflet. Similar potential dependencies were found for the nonmediated flip of anionic NBD-labeled phosphatidic acid (PA) and 2-(N-decyl)aminonaphthalene-6-sulfonic acid (2,6-DENSA) following blockage of the band-3-mediated component of flip. The membrane potential also influences the stationary distribution of the anionic lipids between the inner and outer leaflets. The distribution is shifted to the inner leaflet by increasingly positive potentials and to the outer leaflet by increasingly negative potentials. It is concluded that nonmediated flip-flop of the anionic phospholipids and the long-chain sulfonate represents electrogenic translocation of the unprotonated charged lipids across the hydrophobic barrier.     

The lantibiotic nisin induces transmembrane movement of a fluorescent phospholipid

Nisin is a pore-forming antimicrobial peptide. The capacity of nisin to induce transmembrane movement of a fluorescent phospholipid in lipid vesicles was investigated. Unilamellar phospholipid vesicles that contained a fluorescent phospholipid (1-acyl-2-(6-[(7-nitro-2-1, 3-benzoxadiazol-4-yl)amino]caproyl)-sn-glycero-3-phosphocholine) in the inner leaflet of the bilayer were used. Nisin-induced movement of the fluorescent phospholipid from the inner leaflet to the outer leaflet of the membrane reached stable levels, which were dependent on the concentration of nisin added. The rate constant k of this nisin-induced transmembrane movement increased with the nisin concentration but was not dependent on temperature within the range of 5 to 30 degrees C. In contrast, the rate constant of movement of fluorescent phospholipid from vesicle to vesicle strongly depended on temperature. The data indicate that nisin transiently disturbs the phospholipid organization of the target membrane.     

Some relationships between membrane phospholipid domains, conformational order, and cell shape in intact human erythrocytes

A novel method developed in this laboratory [D.J. Moore et al., Biochemistry 35 (1996) 229-235; D.J. Moore et al., Biochemistry 36 (1997) 660-664] to study the conformational order and the propensity for domain formation of specific phospholipids in intact human erythrocytes is extended to two additional species. Acyl chain perdeuterated 1,2-dilauroylphosphatidylethanolamine (diC12PE-d46) was incorporated preferentially (in separate experiments) into the inner leaflet of stomatocytic erythrocytes and into the outer leaflet of echinocytic erythrocytes, while acyl chain perdeuterated 1,2-dipentadecanoylphosphatidylcholine (diC15PC-d58) was incorporated into the outer leaflet of echinocytic erythrocytes. The conformational order and phase behavior of the incorporated molecules were monitored through FT-IR studies of the temperature dependence of the CD2 stretching vibrations. For both diC12PE-d46 and diC15PC-d58, the gel-->liquid crystal phase transition persisted when these lipids were located in the outer leaflet of echinocytic cells, a result indicative of the persistence of phospholipid domains. In each case, the transition widths were broadened compared to the pure lipids, suggestive of either small domains or the presence of additional molecular components within the domains. The conformational order of diC12PE-d46 differed markedly depending on its location and the morphology of the cells. When located predominantly in the inner membrane of stomatocytes, the phase transition of this species was abolished and the conformational order compared with pure lipid vesicles at the same temperature was much lower. The current results along with our previous studies provide a sufficient experimental basis to deduce some general principles of phospholipid conformational order and organization in both normal and shape-altered erythrocytes.     

Contribution of platelet microparticle formation and granule secretion to the transmembrane migration of phosphatidylserine

Activation of human platelets by complement proteins, C5b-9, thrombin plus collagen, or a Ca2+ ionophore results in surface exposure of phosphatidylserine (PS), accompanied by the expression of membrane catalytic activity for the tenase (VIIaIXa) and prothrombinase (VaXa) coagulation enzyme complexes. The mechanism underlying this surface exposure of PS upon platelet activation remains unresolved. Using fluorescent derivatives of PS (NBD-PS), we have investigated how the transmembrane migration of PS is related to microvesiculation of the platelet plasma membrane and to fusion of storage granules with the plasma membrane. Gel-filtered platelets were incubated with NBD-PS, allowing 90 +/- 10% of the incorporated NBD-PS to accumulate into the inner leaflet of the plasma membrane. Migration of NBD-PS from the inner leaflet to the plasma membrane surface was monitored by time-based flow cytometry, and correlated with the appearance of platelet microparticles and alpha-granule secretion. Platelet activation by C5b-9 or the Ca2+ ionophore, A23187, increased surface exposure of NBD-PS, due to acceleration of the apparent rate of migration from inner to outer plasma membrane leaflets. The onset of this accelerated migration of NBD-PS to the surface coincided with the onset of plasma membrane vesiculation, and the NBD-PS that partitioned into the membrane of the shed microparticle was also rapidly exposed to the surface (t1/2 < 2 min). In addition to a temporal correlation, microparticle formation and the surface exposure of inner leaflet NBD-PS showed a similar requirement for Ca2+. These results demonstrate that agonist-induced microvesiculation of the platelet plasma membrane is accompanied by accelerated migration of a PS analogue from the inner leaflet to the surface of the shed microparticle membrane, suggesting the mechanism by which induction of platelet microparticle formation exposes catalytic surface for tenase and prothrombinase assembly.     

Isoflurane alters proximal tubular cell susceptibility to toxic and hypoxic forms of attack

BACKGROUND: Fluorinated anesthetics can profoundly alter plasma membrane structure and function, potentially impacting cell injury responses. Because major surgery often precipitates acute renal failure, this study assessed whether the most commonly used fluorinated anesthetic, isoflurane, alters tubular cell responses to toxic and hypoxic attack. METHODS: Mouse proximal tubule segments were incubated under control conditions or with a clinically relevant isoflurane dose. Cell viability (lactate dehydrogenase release), deacylation (fatty acid, such as C20:4 levels), and adenosine triphosphate (ATP) concentrations were assessed under one or more of the following conditions: (a) exogenous phospholipase A2 (PLA2) or C20:4 addition, (b) Ca2+ overload (A23187 ionophore), (c) increased metabolic work (Na ionophore), and (d) hypoxia- or antimycin A-induced attack. Isoflurane's effect on NBD phosphatidylserine uptake (an index of plasma membrane aminophospholipid translocase activity) was also assessed. RESULTS: Isoflurane alone caused trivial deacylation and no lactate dehydrogenase release. However, it strikingly sensitized to both PLA2- and A23187-induced deacylation and cell death. Isoflurane also exacerbated C20:4's direct membrane lytic effect. Under conditions of mild ATP depletion (Na ionophore-induced increased ATP consumption; PLA2-induced mitochondrial suppression), isoflurane provoked moderate/severe ATP reductions and cell death. Conversely, under conditions of maximal ATP depletion (hypoxia, antimycin), isoflurane conferred a modest cytoprotective effect. Isoflurane blocked aminophospholipid translocase activity, which normally maintains plasma membrane lipid asymmetry (that is, preventing its "flip flop"). CONCLUSIONS: Isoflurane profoundly and differentially affects tubular cell responses to toxic and hypoxic attack. Direct drug-induced alterations in lipid trafficking/plasma membrane orientation and in cell energy production are likely involved. Although the in vivo relevance of these findings remains unknown, they have potential implications for intraoperative renal tubular cell structure/function and how cells may respond to superimposed attack.     

Uptake of aluminum and gallium into tissues of the rat: influence of antibody against the transferrin receptor

A phosphatidylcholine-like phospholipid expressed in the outer leaflet of the cell membrane shortly after mitogenic activation of T-cells is described, based on the binding of monoclonal antibody 90. 60.3. Expression of the 90.60.3 phospholipid antigen in T-cells is activation-dependent. Once expressed, the 90.60.3 phospholipid is in direct physical association with the interleukin-2 (IL-2) binding domain of IL-2 receptor alpha subunits, but does not affect IL-2 binding. The association is specific, because the 90.60.3 phospholipid is not found in association with other domains of IL-2 receptor alpha subunits, or near IL-2 receptor beta or gamma subunits. Culturing cytokine-dependent cell lines in the presence of monoclonal antibody 90.60.3 potentiates IL-2-dependent cell survival and proliferation in a dose-dependent manner. In contrast, IL-4-dependent responses are not potentiated. Taken together, the data suggest that specific plasma membrane phospholipids expressed in the outer leaflet after T-cell activation associate with the IL-2 binding domain of IL-2 receptor alpha subunits (and perhaps other cytokine receptors), and may play a role in regulating receptor mobility or signal transduction.     

Effect of a glycerol-containing hypotonic medium on erythrocyte phospholipid asymmetry and aminophospholipid transport during storage

Previous studies from our laboratory have shown that under blood bank storage conditions red blood cell (RBC) ATP and lipid content were better maintained in a glycerol-containing hypotonic experimental additive solution (EAS 25) than in the conventional storage medium Adsol. The objective of this study was to determine the mechanism of the protective effect of EAS 25, by measuring transmembrane phospholipid asymmetry and the membrane integrity of stored RBCs. Split units of packed RBCs were stored in either EAS 25 or Adsol. RBCs were analyzed after 0, 42, and 84 days and vesicles shed from stored RBCs were analyzed after 84 days of storage. Phospholipid asymmetry was measured by phospholipase A2 digestion (RBCs) and activation of the prothrombinase complex (RBCs, vesicles). RBC membrane exhibited a significantly greater (P < 0.01) amount of phosphatidylethanolamine externalized after storage in Adsol than in EAS 25 (44.3% +/- 11.7 vs. 25.3% +/- 5.7, respectively). Prothrombin converting activities in RBCs were significantly lower than in shed vesicles (P < 0.001) suggesting the presence of phosphatidylserine in the outer monolayer of vesicle, but not in RBC membranes. The rates of inwardly-directed aminophospholipid transport in RBCs decreased by 50% and glutathione levels decreased by approximately 50% in both media. RBC cholesterol and phospholipid content of stored RBCs remained significantly greater (P < 0.01) in EAS 25 than in Adsol. The results indicate that despite comparable reduction in the rate of aminophospholipid transport and reduced GSH concentrations, RBC phospholipid asymmetry was better maintained during storage in EAS 25 than in Adsol. The data suggest that glycerol in the hypotonic EAS helps preserve RBC lipid organization and membrane integrity during storage.     

The interference effects of hexadecylphosphocholine on proliferation and membrane phospholipid metabolism in human myeloid leukemia cell lines

Membrane phospholipids are important regulators of cellular function. The phospholipid activities, such as lipid composition and transportation, contribute to cellular homeostasis in the lifespan of cells. Alterations in phospholipids result in the movement of bilayer lipids and the initiation of coagulation, recognition and internalization. Hexadecylphosphocholine (HePC) exerts antitumor potencies and represents a new class of antitumor agents targeted to the cellular membrane. Human myeloid leukemia cell lines HL-60 and K562 employed in this study were inhibited by HePC in vitro. The results indicate that the HL-60 cell line was sensitive, while K562 was resistant to HePC. Synthetic HePC is an alkyllysophospholipid analog which interacted with the cell membrane, thereby altering lipid composition and metabolism of membrane phospholipids and modulating intracellular calcium in human myeloid leukemia HL-60 and K562 cell lines. The contents of membrane phospholipids, including phosphatidylinositol (PI), phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidylethanolamine (PE), were determined quantitatively with high performance liquid chromatography. The sensitivity of myeloid leukemia HL-60 and K562 cell lines to HePC probably depends on the different distribution of these four phospholipids in the cellular membrane, or on the response of these phospholipids to HePC. The cytosolic free calcium ([Ca++]i) concentration increased by HePC confirmed that [Ca++]i was released from the intracellular calcium pool and is associated with cell differentiation and apoptosis. We investigated the hypothesis that the antiproliferative effect of HePC was mediated through the interference with cellular membrane phospholipids, including choline-containing phospholipids (PC), aminophospholipids (PE and PS) and PI, in eukaryotic cells.