Plasma Membrane Phospholipid Asymmetry Research Articles

Effect of age on plasma membrane asymmetry and membrane fluidity in human leukocytes and platelets.

We determined whether ageing changes in plasma membrane phospholipid asymmetry were related to changes in membrane physical characteristics. Plasma membrane asymmetry was determined in polymorphonuclear leukocytes (PMN), lymphocytes, and platelets from 45 healthy young (mean 29 years, 26 male) and 28 healthy elderly (mean 70 years, 15 male) subjects by flow cytometric measurement of annexin V binding to cell surface phosphatidylserine. Membrane fluidity in lymphocytes and platelets from young and elderly subjects was determined by fluorescence polarization of 1,6-diphenyl- 1,3,5-hexatriene (DPH) and (4-trimethylammonium)-DPH (TMA). In elderly subjects, a higher proportion of lymphocytes had specific annexin V binding to phosphatidylserine (PS) than in young subjects (young: median percentage of cells with specific annexin V binding to PS 5.3 [second to fourth quintiles range 3.8-8.7]; elderly: 8.5 [5.2-17.2]; p = .028). No ageing change in annexin V binding to PMN was observed (young: 35.0% [21.8-53.5]; elderly: 39.6% [27.4-69.8]; p = .42). Platelets had no specific annexin V binding (young: median molecules of annexin V specific binding 3.8 [0.4-11.3]; elderly: -1.4 [-4.8-1.7]; p = .23). Superficial membrane fluidity was increased in lymphocytes (TMA anisotropy, young: 0.271 [0.259-0.289]; elderly: 0.262 [0.242-0.279];p = .004), but not in platelets (young: 0.273 [0.259-0.293]; elderly: 0.269 [0.248-0.284]; p = .12). Lymphocyte annexin V binding correlated with TMA (r = -.65, p = .022), but not DPH anisotropy (r = -.39, p = .18). Plasma membrane asymmetry is decreased with age in human lymphocytes, but not in human PMN or platelets. The increased proportion of lymphocytes with loss of plasma membrane asymmetry corresponds to the ageing changes in superficial membrane fluidity observed in lymphocytes. Such alterations in lymphocyte plasma membrane structure with age could account for changes in membrane-bound receptor function described with ageing, and may contribute to alterations in immune responsiveness and vascular thrombosis seen in older humans.

“Apoptotic” biochemical cascades in synaptic compartments: Roles in adaptive plasticity and neurodegenerative disorders

Apoptosis is a form of cell death historically defined by morphological and biochemical changes that occur in the cell body and nucleus. However, in contrast to nonneuronal cells in which apoptosis has been most intensively studied, neurons exhibit elaborate morphologies with synaptic connections often located at sites a great distance from the cell body. Signaling events occurring in synaptic terminals are believed to play important roles in either promoting (e.g., activation of glutamate receptors in postsynaptic spines) or preventing (e.g., activation of neurotrophic factors in presynaptic terminals) neuronal cell death in various physiological and pathological settings. We have found that apoptotic biochemical cascades can be activated locally in synaptic terminals and neurites and have shown that such cascades can result in local functional and morphological alterations and can also propagate to the cell body resulting in neuronal death. Prostate apoptosis response-4 production, caspase activation, loss of plasma membrane phospholipid asymmetry, mitochondrial dysfunction, and production of factors capable of inducing nuclear chromatin condensation and fragmentation can all occur locally in synaptic terminals in response to various stimuli. Activation of receptors for neurotrophic factors (e.g., basic fibroblast growth factor, secreted form of amyloid precursor protein α, and activity-dependent neurotrophic factor) and cytokines (e.g., tumor necrosis factor-α) in synaptic terminals can exert synaptoprotective actions that either can be transduced locally or may require signals to the nucleus and back. In addition to their roles in synaptic degeneration and neuron death, apoptotic cascades may play roles in synaptic plasticity. For example, we found that caspase activation can lead to proteolysis of certain glutamate receptor subunits and that this action of capases is correlated with reduced calcium responses to glutamate. We propose that apoptotic cascades function in a continuum in which low levels of activation play roles in adaptive responses to “stressors,” whereas higher levels of activation mediate synaptic degeneration and cell death. J. Neurosci. Res. 58:152–166, 1999. © 1999 Wiley-Liss, Inc.

“Apoptotic” biochemical cascades in synaptic compartments: Roles in adaptive plasticity and neurodegenerative disorders

Apoptosis is a form of cell death historically defined by morphological and biochemical changes that occur in the cell body and nucleus. However, in contrast to nonneuronal cells in which apoptosis has been most intensively studied, neurons exhibit elaborate morphologies with synaptic connections often located at sites a great distance from the cell body. Signaling events occurring in synaptic terminals are believed to play important roles in either promoting (e.g., activation of glutamate receptors in postsynaptic spines) or preventing (e.g., activation of neurotrophic factors in presynaptic terminals) neuronal cell death in various physiological and pathological settings. We have found that apoptotic biochemical cascades can be activated locally in synaptic terminals and neurites and have shown that such cascades can result in local functional and morphological alterations and can also propagate to the cell body resulting in neuronal death. Prostate apoptosis response-4 production, caspase activation, loss of plasma membrane phospholipid asymmetry, mitochondrial dysfunction, and production of factors capable of inducing nuclear chromatin condensation and fragmentation can all occur locally in synaptic terminals in response to various stimuli. Activation of receptors for neurotrophic factors (e.g., basic fibroblast growth factor, secreted form of amyloid precursor protein alpha, and activity-dependent neurotrophic factor) and cytokines (e.g., tumor necrosis factor-alpha) in synaptic terminals can exert synaptoprotective actions that either can be transduced locally or may require signals to the nucleus and back. In addition to their roles in synaptic degeneration and neuron death, apoptotic cascades may play roles in synaptic plasticity. For example, we found that caspase activation can lead to proteolysis of certain glutamate receptor subunits and that this action of capases is correlated with reduced calcium responses to glutamate. We propose that apoptotic cascades function in a continuum in which low levels of activation play roles in adaptive responses to "stressors," whereas higher levels of activation mediate synaptic degeneration and cell death.

Plasma membrane phospholipid integrity and orientation during hypoxic and toxic proximal tubular attack

Acute cell injury can activate intracellular phospholipase A2 (PLA2) and can inhibit plasma membrane aminophospholipid translocase(s). The latter maintains inner/outer plasma membrane phospholipid (PL) asymmetry. The mechanistic importance of PLA2-mediated PL breakdown and possible PL redistribution ("flip flop") to lethal tubule injury has not been well defined. This study was performed to help clarify these issues. Proximal tubule segments (PTS) from normal CD-1 mice were subjected to either 30 minutes of hypoxia, Ca2+ ionophore (50 microM A23187), or oxidant attack (50 microM Fe). Lethal cell injury [the percentage of lactate dehydrogenase (LDH) release], plasma membrane PL expression [two-dimensional thin layer chromatography (TLC)], and free fatty acid (FFA) levels were then assessed. "Flip flop" was gauged by preferential decrements in phosphatidylserine (PS) versus phosphatidylcholine (PC; PS/PC ratios) in response to extracellular (Naja) PLA2 exposure. Hypoxia induced approximately 60% LDH release, but no PL losses were observed. FFA increments suggested, at most 3% or less PL hydrolysis. Naja PLA2 reduced PLs in hypoxic tubules, but paradoxically, mild cytoprotection resulted. In contrast to hypoxia, Ca2+ ionophore and Fe each induced significant PL losses (6 to 15%) despite minimal FFA accumulation or cell death (26 to 27% LDH release). Arachidonic acid markedly inhibited PLA2 activity, potentially explaining an inverse correlation (r = -0.91) between tubule FFA accumulation and PL decrements. No evidence for plasma membrane "flip flop" was observed. In vivo ischemia reperfusion and oxidant injury (myohemoglobinuria) induced 0 and 24% cortical PL depletion, respectively, validating these in vitro data. (a) Plasma membrane PLs are well preserved during acute hypoxic/ischemic injury, possibly because FFA accumulation (caused by mitochondrial inhibition) creates a negative feedback loop, inhibiting intracellular PLA2. (b) Exogenous PLA2 induces PL losses during hypoxia, but decreased cell injury can result. Together these findings suggest that PL loss may not be essential to hypoxic cell death. (c) Oxidant/Ca2+ overload injury induces early PL losses, perhaps facilitated by ongoing mitochondrial FFA metabolism, and (d) membrane "flip flop" does not appear to be an immediate mediator of acute necrotic tubular cell death.

Independent mechanisms for macrophage binding and macrophage phagocytosis of damaged erythrocytes. Evidence of receptor cooperativity.

The binding and phagocytosis of oxidatively damaged red blood cells (OxRBCs) by mouse peritoneal macrophages can be inhibited by oxidatively modified LDL (OxLDL), implying some commonality at their receptor-binding domains. Studies from many different laboratories support the view that OxRBC binding is due to the disruption of plasma membrane phospholipid asymmetry and the subsequent exposure of phosphatidylserine (PS) on the outer membrane leaflet. Presumably, oxidation of LDL creates a surface structure on it in some way homologous to the PS-rich domain on OxRBCs. Apoptotic cells in some instances are also recognized because of PS exposure on the outer leaflet of the membrane, and apoptotic cells are a common feature of atherosclerotic lesions. In the present studies, the mechanisms of binding and internalization of cells recognized by virtue of their membrane PS were studied using OxRBCs or vanadate-treated erythrocytes (VaRBCs) as models. Disruption of phospholipid asymmetry with vanadate produced cells that were bound by macrophages in the same divalent cation-dependent manner as OxRBCs. However, whereas OxRBCs were rapidly phagocytosed, VaRBCs were not. Stimulation of mouse macrophages with phorbol myristate acetate resulted in a concentration-dependent induction of phagocytosis of bound VaRBCs, an effect that could be prevented by the protein kinase C inhibitor staurosporine. Because phagocytosis of OxRBCs occurred unassisted, we speculated that there must be additional membrane changes induced by oxidation (over and above the disruption of phospholipid asymmetry) that contribute to phagocytosis of OxRBCs, possibly resulting in the ligation of a distinct receptor that does not necessarily contribute to adherence. This proposal is supported by the finding that ligation of macrophage Fc gamma receptors by the anti-Fc gamma RII/RIII antibody 2.4G2 triggers the phagocytosis of bound VaRBCs. Phagocytosis is also triggered by subthreshold opsonization of VaRBC, i.e., by antibody concentrations that do not by themselves cause binding and phagocytosis of native RBCs. Finally, treatment with low concentrations of glutaraldehyde, which causes membrane protein cross-linking, promotes the phagocytosis of VaRBCs, but, at the low concentration used, has little or no effect on binding and phagocytosis of native RBCs. We suggest that the internalization of damaged cells, bound because of PS exposure, requires the cooperation of a PS-binding receptor with at least one additional receptor to trigger an intracellular signaling pathway to initiate phagocytosis.

Reconstitution of phospholipid scramblase activity from human blood platelets.

Cellular activation, accompanied by elevation of cytoplasmic Ca2+ levels, can induce a progressive loss of plasma membrane phospholipid asymmetry, resulting from increased transbilayer movement (flip-flop) of phospholipids. While this process has been demonstrated in a variety of different cells, it is most active in blood platelets. In order to test whether this lipid scrambling process is mediated by a membrane protein, platelet membranes were solubilized in cholate and fractionated by anion exchange chromatography, and fractions were reconstituted into phospholipid vesicles by detergent dialysis in the presence of small amounts of fluorescent (NBD) phospholipids. Using dithionite reduction to monitor the transbilayer location of NBD phospholipids, it was shown that addition of Ca2+ and ionomycin to vesicles reconstituted with a particular fraction results in transbilayer movement of the fluorescent phospholipid analogs from the vesicle's inner to outer leaflet. Lipid vesicles reconstituted in the absence of membrane protein, or reconstituted with another platelet membrane protein fraction, were devoid of this activity. Heating the active fraction or incubating it with pronase or the SH reagent pyridyldithioethylamine markedly diminished the ability of the vesicles to translocate fluorescent phospholipid analogs across the bilayer in response to Ca2+ and ionophore. These results argue that a membrane protein (or proteins) from blood platelets is required to catalyze Ca2+-induced transbilayer movement of phospholipids, suggesting its (or their) involvement in the loss of lipid asymmetry that can occur during cellular activation.

Recognition of oxidatively damaged and apoptotic cells by an oxidized low density lipoprotein receptor on mouse peritoneal macrophages: role of membrane phosphatidylserine.

We recently reported that oxidized low density lipoprotein (OxLDL), but not acetyl LDL (AcLDL), inhibited the binding and phagocytosis of nonopsonized, oxidatively damaged red blood cells (OxRBCs) by mouse peritoneal macrophages, implying the involvement of a "scavenger receptor" other than the AcLDL receptor. Numerous studies establish that loss of plasma membrane phospholipid asymmetry, which increases phosphatidylserine expression on the outer leaflet of the membrane, can play a key role in macrophage recognition of damaged and apoptotic cells. We report here that this recognition is in part attributable to the same mouse macrophage receptor that recognizes OxLDL. As described in an accompanying paper, this is a plasma membrane protein of 94-97 kDa. Phosphatidylserine liposomes show strong ligand binding to the same 94- to 97-kDa protein and this binding is inhibited by OxLDL but not by AcLDL. Inhibition of the RBC membrane phospholipid translocase by incubation with sodium vanadate caused a progressive increase in the appearance of phosphatidylserine on the cell surface and a parallel increase in the binding of these RBCs to macrophages, binding that was inhibited by OxLDL. Finally, OxLDL also inhibited the binding of sickled RBCs and apoptotic thymocytes to mouse macrophages. However, the latter was incomplete (approximately 50%), suggesting that other receptors are also involved. We suggest that the OxLDL receptor plays a significant role in recognition of damaged and apoptotic cells.

A model for the extracellular release of PAF: The influence of plasma membrane phospholipid asymmetry

Recent studies suggesting that cellular activation leads to enhanced transbilayer movement of phospholipids and loss of plasma membrane phospholipid asymmetry lead us to hypothesize that such events may govern the release of PAF, a potent, but variably released, lipid mediator synthesized by numerous inflammatory cells. To model these membrane events, we studied the transbilayer movement of PAF across the human erythrocyte and erythrocyte ghost plasma membrane, membranes with documented phospholipid asymmetry which can be deliberately manipulated. Utilizing albumin to extract outer leaflet PAF, transbilayer movement of PAF was shown to be significantly enhanced in erythrocytes and ghosts altered to lose membrane asymmetry when compared to movement in those with native menbrane asymmetry. Verification of membrane changes was demonstrated using merocyanine 540 (MC540), a dye which preferentially stains loosely packed or hydrophobic membranes, and acceleration of the modified Russell's viper venom clotting assay by externalized anionic phospholipids. Utilizing the erythrocyte ghost loaded with PAF in either the outer or the inner leaflet, enhanced transbilayer movement to the opposite leaflet was seen to accompany loss of membrane asymmetry. Studies utilizing ghosts loaded with albumin intracellularly demonstrated that ‘acceptor’ molecules binding PAF further influence the disposition of PAF across the plasma membrane. Taken together, these findings suggest that the net release of PAF from activated inflammatory cells will depend on localization of PAF to the plasma membrane, transbilayer movement, which is facilitated by alteration of membrane phospholipid asymmetry, and removal from the membrane by extracellular and intracellular ‘acceptor’ molecules.

Differentiation-related differences in the plasma membrane phospholipid asymmetry of myogenic and fibrogenic cells

We have determined the asymmetric distribution of two aminophospholipids phosphatidylethanolamine and phosphatidylserine in the plasma membrane of chick embryo fibroblast and myoblasts. Right-side-out membrane preparations were incubated with two different amidating reagents, trinitrobenzenesulfonate and isethionylacetimidate, under nonpenetrating conditions. Inside-out membranes were incubated with trinitrobenzenesulfonate. In fibroblasts, the similar plateau values suggested that 35% of the phosphatidylethanolamine and 20% of the phosphatidylserine is externally disposed. These values agree with previous measurements on fibroblast plasma membranes. In myoblasts, however, labelling plateaux were achieved which suggested that 65% of the phosphatidylethanolamine and 45% of the phosphatidylserine is externally disposed. This represents a 2–3-fold increase in potentially fusogenic lipids on the external leaflet of the plasma membrane. This unique distribution of aminophospholipids in myoblasts extends through the stage of development during which myoblasts become competent to fuse and form myotubes in culture. Two inferences may be drawn from these results. First, the external concentration of aminophospholipids in myoblasts is enriched significantly over that of fibroblasts or erythrocytes. This orientation may contribute to its fusion competence. Second, although large amounts of externally disposed aminophospholipid may be necessary for myoblast fusion, they do not confer fusion competence.