Deciphering the plasma membrane hallmarks of apoptotic cells: Phosphatidylserine transverse redistribution and calcium entry

Background

Atherosclerosis and coronary heart disease (CHD) are significant contributors to morbidity and mortality in developed countries. A noted exception is the low mortality of CHD in France, particularly the southwest region. This phenomenon, commonly referred to as the French paradox, may be associated with high consumption of red wine. We investigate whether the cardioprotective activity of red wine may involve the grape skin-derived polyphenol, resveratrol. We further test the possibility that resveratrol acts by modulating structural and functional changes in endothelial cells lining the blood vessel wall.

Results

Bovine pulmonary artery endothelial cells (BPAEC) were incubated with resveratrol, with and without concurrent exposure to simulated arterial shear stress. Resveratrol significantly affected proliferation and shape of BPAEC; growth was suppressed and cells became elongated, based on morphologic analysis of rhodamine-conjugated phalloidin stained F-actin by confocal microscopy. Using selective signaling inhibitors, we showed that the resveratrol-induced cellular phenotype was dependent on intracellular calcium and tyrosine kinase activities, and assembly of actin microfilaments and microtubules, but was unrelated to PKC activity. Exposure to simulated arterial flow revealed that, whereas controls cells easily detached from the culture support in a time-dependent manner, resulting in total cell loss after a 5 min challenge with simulated arterial flow conditions, a significant percentage of the treated cells remained attached to the cultured plastic coverslips under identical experimental conditions, suggesting that they adhered more strongly to the surface. Western blot analysis shows that whereas cells treated with 25 μM and 100 μM resveratrol had no change in total ERK1/2, treatment did result in an increase in phosphorylated ERK1/2, which probably involved stabilization of the active enzyme. An increase in nitric oxide synthase expression was detected as early as 6 h and persisted for up to 4 days of treatment.

Conclusions

Results of our studies show that resveratrol interacts with endothelial cells in vitro to elicit morphological and structural changes; the observed changes support the interpretation that resveratrol acts as a cardioprotective agent.     

Phosphatidylserine decarboxylase governs plasma membrane fluidity and impacts drug susceptibilities of Candida albicans cells

Plasma membrane (PM) lipid composition imbalances affect drug susceptibilities of the human pathogen Candida albicans. The PM fundamental structure is made up of phospholipid bilayer where phosphatidylethanolamine (PE) contributes as second major phospholipid moieties, which is asymmetrically distributed between the two leaflets of the bilayer. PSD1 and PSD2 genes encode phosphatidylserine decarboxylase which converts phosphatidylserine (PS) into PE in C. albicans cells. Genetic manipulation of PSD1 and PSD2 genes is known to impact virulence, cell wall thickness and mitochondrial function in C. albicans. In the present study, we have examined the impact of PSD1 and PSD2 deletion on physiochemical properties of PM. Our fluorescence recovery after photobleaching (FRAP) experiments point that the PM of psd1Δ/Δ psd2Δ/Δ mutant strain displays increased membrane fluidity and reduced PM dipole potential. Further, the result of PSD1 and PSD2 deletion on the thermotropic phase behavior monitored by differential scanning calorimetry (DSC) showed that in comparison to WT, the apparent phase transition temperature is reduced by ~3 °C in the mutant strain. The functional consequence of altered physical state of PM of psd1Δ/Δ psd2Δ/Δ mutant strain was evident from observed high diffusion of fluorescent dye rhodamine 6G and radiolabelled fluconazole (FLC). The higher diffusion of FLC resulted in an increased drug accumulation in psd1Δ/Δ psd2Δ/Δ mutant cells, which was manifested in an increased susceptibility to azoles. To the best of our knowledge, these results constitute the first report on the effect of the levels of phospholipid biosynthesis enzyme on physiochemical properties of membranes and drug susceptibilities of Candida cells.     

Cyclic GMP mediates the agonist-stimulated increase in plasma membrane calcium entry in the pancreatic acinar cell

The present studies were performed to determine the role of cyclic GMP in regulating agonist mediated calcium entry in the pancreatic acinar cell. In guinea pig-dispersed pancreatic acini the findings demonstrated that carbachol stimulated a transient 20-40-fold rise in cellular cyclic GMP followed by a sustained 3-4-fold rise in cellular cyclic GMP. The guanylate cyclase inhibitor, 6-anilino-5,8-quinolinedione (LY83583), caused a dose-dependent inhibition of carbachol-stimulated increases in cellular cyclic GMP both during the initial transient large increase in cyclic GMP and the sustained increase in cyclic GMP. LY83583 also inhibited cellular Ca2+ influx during carbachol stimulation and reloading of the agonist-sensitive pool of Ca2+ at the termination of carbachol stimulation with atropine. The effect of the inhibition on reloading of the agonist-sensitive pool was secondary to its effects on the plasma membrane C2+ entry. The addition of dibutyryl cyclic GMP to LY83583-treated acini restored Ca2+ influx across the plasma membrane. Nitroprusside increased both cellular cyclic GMP and the rate of Ca2+ influx. During periods when plasma membrane Ca2+ entry was activated, cellular cyclic GMP levels were increased. These results suggest that agonist-induced increases in cellular cyclic GMP are necessary and sufficient to mediate the effects of the agonist on the plasma membrane Ca2+ entry mechanism.     

Independence of plasma membrane blebbing from other biochemical and biological characteristics of apoptotic cells

Plasma membrane blebs are observed in many types of apoptotic cells, but their physiological roles remain to be clarified. We examined whether there is a causative connection between membrane blebbing and other apoptotic changes in Jurkat cells induced to undergo apoptosis by doxorubicin in the presence or absence of Y-27632, an inhibitor of the Rho kinase ROCK-I. The inclusion of the drug made most membrane blebs disappear, while other changes, such as chromatin condensation, inactivation of mitochondrial enzymes, externalization of the membrane phospholipid phosphatidylserine, and removal of cell surface sialic acid, remained unaffected. Furthermore, these apoptotic cells were phagocytosed by macrophages as efficiently as normally apoptosing cells. These results indicate that blebbing of the plasma membrane occurs independently from other apoptotic changes and is not involved in the recognition and engulfment of apoptotic cells by macrophages.     

Phosphatidylserine (PS) induces PS receptor-mediated macropinocytosis and promotes clearance of apoptotic cells.

Efficient phagocytosis of apoptotic cells is important for normal tissue development, homeostasis, and the resolution of inflammation. Although many receptors have been implicated in the clearance of apoptotic cells, the roles of these receptors in the engulfment process have not been well defined. We developed a novel system to distinguish between receptors involved in tethering of apoptotic cells versus those inducing their uptake. Our results suggest that regardless of the receptors engaged on the phagocyte, ingestion does not occur in the absence of phosphatidylserine (PS). Further, recognition of PS was found to be dependent on the presence of the PS receptor (PSR). Both PS and anti-PSR antibodies stimulated membrane ruffling, vesicle formation, and "bystander" uptake of cells bound to the surface of the phagocyte. We propose that the phagocytosis of apoptotic cells requires two events: tethering followed by PS-stimulated, PSR-mediated macropinocytosis.     

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.     

ABC1 promotes engulfment of apoptotic cells and transbilayer redistribution of phosphatidylserine

ATP-binding-cassette transporter 1 (ABC1) has been implicated in processes related to membrane-lipid turnover. Here, using in vivo loss-of-function and in vitro gain-of-function models, we show that ABC1 promotes Ca2+-induced exposure of phosphatidylserine at the membrane, as determined by a prothrombinase assay, membrane microvesiculation and measurement of transbilayer redistribution of spin-labelled phospholipids. That ABC1 promotes engulfment of dead cells is shown by the impaired ability of ABC1-deficient macrophages to engulf apoptotic preys and by the acquisition of phagocytic behaviour by ABC1 transfectants. Release of membrane phospholipids and cholesterol to apo-AI, the protein core of the cholesterol-shuttling high-density lipoprotein (HDL) particle, is also ABC1-dependent. We propose that both the efficiency of apoptotic-cell engulfment and the efflux of cellular lipids depend on ABC1-induced perturbation of membrane phosphatidylserine turnover. Transient local exposure of anionic phospholipids in the outer membrane leaflet may be sufficient to alter the general properties of the membrane and thus influence discrete physiological functions.     

Increase in cytosolic calcium maintains plasma membrane integrity through the formation of microtubule ring structure in apoptotic cervical cancer cells induced by trichosanthin

This study investigates the role of dysregulated cytosolic free calcium ([Ca²⁺]c) homeostasis on microtubule (MT) ring structure in apoptotic cervical cancer (HeLa) cells induced by trichosanthin (TCS), a type I ribosome inactivating protein (RIP). The TCS-induced decrease in cell viability was significantly enhanced in combination with the specific calcium chelator, EGTA-AM. Sequestration of [Ca²⁺]c markedly disrupted the special MT ring structure. Furthermore, TCS tended to increase LDH release, whereas no significant differences were observed until 48 h of the treatment. In contrast, combined addition of EGTA-AM or colchicine (an inhibitor of tubulin polymerization) significantly reinforced LDH release. The data suggest that TCS-elevated [Ca²⁺]c maintains plasma membrane integrity via the formation of the MT ring structure in apoptotic HeLa cells.     

Activation of calcium entry by the tumor promoter thapsigargin in parotid acinar cells. Evidence that an intracellular calcium pool and not an inositol phosphate regulates calcium fluxes at the plasma membrane

The depletion of an inositol 1, 4,5-trisphosphate-sensitive intracellular Ca2+ pool has been proposed to be the signal for Ca2+ entry in agonist-activated cells. Consistent with this idea, thapsigargin, which releases intracellular Ca2+ without inositol phosphate formation, has been reported to activate Ca2+ entry in certain cells. We now report the effects of thapsigargin on Ca2+ entry in parotid acinar cells. In fura-2-loaded parotid acinar cells, thapsigargin caused a sustained elevation of [Ca2+], but did not increase inositol phosphate formation. In the absence of extracellular Ca2+, the increase in [Ca2+], was transient, suggesting that thapsigargin activates both the release of Ca2+ from intracellular stores and the entry of Ca2+ from the extracellular space. In the absence of extracellular Ca2+, pretreatment with methacholine, an agonist believed to mobilize Ca2+ through the production of inositol 1,4,5-trisphosphate, inhibited but did not completely block the response to thapsigargin; likewise, pretreatment with thapsigargin inhibited the response to methacholine. In permeabilized cells, thapsigargin gradually released Ca2+, whereas inositol 1,4,5-trisphosphate caused a rapid and transient discharge of Ca2+. The simultaneous addition of thapsigargin with inositol 1,4,5-trisphosphate evoked a maximum Ca2+ release similar to that for inositol 1,4,5-trisphosphate alone, but the reuptake seen with inositol 1,4,5-trisphosphate alone was abolished. In intact cells, methacholine and thapsigargin together produced a greater initial release of Ca2+ than either alone, but they were not additive in the sustained phase of Ca2+ mobilization. These results demonstrate that the mechanisms for activation of Ca2+ entry by thapsigargin and methacholine are the same and are consistent with the idea that entry is initiated by the depletion of the intracellular inositol 1,4,5-trisphosphate-sensitive Ca2+ pool. The results also indicate that, in contrast to previously proposed models, Ca2+ entry into agonist-activated cells occurs directly across the plasma membrane to the cytoplasm rather than through a cycle of uptake and release by the intracellular Ca2+ pool.     

Spermine oxidation leads to necrosis with plasma membrane phosphatidylserine redistribution in mouse leukemia cells

Oxidation by copper/quinone-containing serum amine oxidases (SAO) is a well-known cause of polyamine cytotoxicity. Spermine oxidation exerts potent immunosuppressive effects in animal cells, but the cell death mechanism involved remains unclear. We compared biochemical and morphological parameters of SAO-mediated cell death in L1210 mouse leukemia cells with normal or amplified ornithine decarboxylase gene expression with those observed during apoptosis induced by deregulated polyamine uptake or by okadaic acid. None of the characteristic features of apoptotic cell death (e.g., chromatin condensation, nuclear fragmentation, internucleosomal DNA cleavage, poly(ADP-ribose) polymerase cleavage) were observed during spermine oxidation-mediated cell death, which was clearly necrotic by morphological criteria. Inhibition of a wide spectrum of caspases did not prevent SAO-dependent cell death, whereas N-acetylcysteine completely abolished the cytotoxic effects of spermine oxidation. Catalase only delayed spermine oxidation-induced cell death without affecting its modality or preventing depletion of intracellular glutathione, suggesting that both H(2)O(2) and aminoaldehydes generated by SAO-mediated spermine oxidation contribute to SAO-induced necrosis. Interestingly, redistribution of phosphatidylserine to the outer leaflet of the plasma membrane, usually a diagnostic feature of apoptosis, preceded necrotic cytolysis triggered by spermine oxidation. Thus, L1210 cell death caused by SAO-mediated spermine oxidation has all the attributes of primary necrosis, but is also accompanied by loss of phospholipid asymmetry, indicating that the latter phenomenon may not be unique to apoptosis. Phosphatidylserine exposure, a potent engulfment signal for phagocytes, might contribute to the immunosuppressive effects of plasma polyamines through a controlled and rapid necrotic process involving SAO.     

Passive transport of calcium into plasma membrane fractions of myometrium cells

The apparent values of intravesicular volume (45 microliter/mg of protein), maximal capacity of adsorbed calcium binding on the inner surface of the vesicles (4.5 nmol/mg of protein) and dissociation constants for the Ca2+-binding site complexes (36 microM) were determined from the analysis of peculiarities of passive transport of 45Ca2+ into cow myometrium sarcolemmal vesicles. The kinetics of passive efflux of ionized Ca2+ from the vesicles is described by a two-phase exponential curve. Dilution of the vesicles with a dilution medium is associated with a rapid efflux of ionized Ca2+ from the intravesicular space resulting in dissociation of the Ca2+-binding site complexes on the inner surface of the vesicles and, correspondingly, in the passage from a rapid to the slow phase of Ca2+ efflux from the vesicles which is limited by the dissociation of the Ca2+-binding site complexes. The values of the apparent rate constants for the transmembrane transfer of Ca2+ and dissociation of the Ca2+-binding site complexes (0.73 and 0.02 min-1, respectively) and the permeability of sarcolemmal vesicles for the cation (10(-15) mol of Ca2+/cm2.s) were determined. Alkalinization of the dilution medium stimulates 45Ca2+ release from the vesicles. The blockers of passive Co2+ and Mn2+ transport injected into the vesicles inhibit the efflux of 45Ca2+ from the vesicles. The data obtained were used to analyze the role of sarcolemma in the Ca2+ control of myometrium contraction.     

Adenovirus adsorption and sterol redistribution in KB cell plasma membrane

Filipin was used as a chemical probe for localization of sterols in freeze-fractured plasma membrane of KB cells. After adenovirus particle adsorption, marked changes occurred in the number and planar distribution of sterols and of intramembranous particles (IMPs). Filipin-sterol complexes became more abundant and both sterols and IMPs aggregated in a network pattern. It was suggested that redistribution of sterols and rearrangement of IMPs were interconnected phenomena, which represented an early cellular response to adenovirus attachment.     

Electrogenic calcium transport in plasma membrane of rat pancreatic acinar cells

Summary ATP-dependent⁴⁵Ca²⁺ uptake was investigated in purified plasma membranes from rat pancreatic acinar cells. Plasma membranes were purified by four subsequent precipitations with MgCl₂ and characterized by marker enzyme distribution. When compared to the total homogenate, typical marker enzymes for the plasma membrane, (Na⁺,K⁺)-ATPase, basal adenylate cyclase and CCK-OP-stimulated adenylate cyclase were enriched by 43-fold, 44-fold, and 45-fold, respectively. The marker for the rough endoplasmic reticulum was decreased by fourfold compared to the total homogenate. Comparing plasma membranes with rough endoplasmic reticulum, Ca²⁺ uptake was maximal with 10 and 2 mol/liter free Ca²⁺, and half-maximal with 0.9 and 0.5 mol/liter free Ca²⁺. It was maximal at 3 and 0.2 mmol/liter free Mg²⁺ concentration, at an ATP concentration of 5 and 1 mmol/liter, respectively, and at pH 7 for both preparations. When Mg²⁺ was replaced by Mn²⁺ or Zn²⁺ ATP-dependent Ca²⁺ uptake was 63 and 11%, respectively, in plasma membranes; in rough endoplasmic reticulum only Mn²⁺ could replace Mg²⁺ for Ca²⁺ uptake by 20%. Other divalent cations such as Ba²⁺ and Sr²⁺ could not replace Mg²⁺ in Ca²⁺ uptake. Ca²⁺ uptake into plasma membranes was not enhanced by oxalate in contrast to Ca²⁺ uptake in rough endoplasmic reticulum which was stimulated by 7.3-fold. Both plasma membranes and rough endoplasmic reticulum showed cation and anion dependencies of Ca²⁺ uptake. The sequence was K⁺>Rb⁺>Na⁺>Li⁺>choline⁺ in plasma membranes and Rb⁺K⁺Na⁺>Li⁺>choline⁺ for rough endoplasmic reticulum. The anion sequence was Cl^–Br^–I^–>SCN^–>NO ₃ ^– >isethionate^– >cyclamate^–>gluconate^–>SO ₄ ^2– glutarate^– and Cl^–>Br>gluconate>SO ₄ ^2– >NO ₃ ^– >I^–>cyclamate^–SCN^–, respectively. Ca²⁺ uptake into plasma membranes appeared to be electrogenic since it was stimulated by an inside-negative K⁺ and SCN diffusion potential and inhibited by an inside-positive diffusion potential. Ca²⁺ uptake into rough endoplasmic reticulum was not affected by diffusion potentials. We assume that the Ca²⁺ transport mechanism in plasma membranes as characterized in this study represents the extrusion system for Ca²⁺ from the cell that might be involved in the regulation of the cytosolic Ca²⁺ level.     

Amino acids modulate calcium permeability of the plasma membrane of human neuroblastoma cells

Four different amino acids (kainate, N-methyl-D-aspartate, L-cysteine sulfinate and D,L-2-amino-5-phosphonovalerate) have been observed to stimulate uptake of 45Ca2+ into human neuroblastoma cells. This stimulation of uptake is specific and many amino acids which are structural analogs of the above compounds are without activity. The calcium movement is not inhibited by compounds which block voltage-dependent calcium channels. Biological specificity is observed in which some cell lines respond to the amino acids and others do not. It is concluded that these amino acids are acting on a class of receptors whose physiological role is modulation of neuronal metabolism by modulating the calcium permeability of the plasma membrane. The amino acids can substitute for the, as yet, unidentified natural agonists, albeit with low affinity.     

Insulin protects pancreatic acinar cells from cytosolic calcium overload and inhibition of plasma membrane calcium pump

Acute pancreatitis is a serious and sometimes fatal inflammatory disease of the pancreas without any reliable treatment or imminent cure. In recent years, impaired metabolism and cytosolic Ca(2+) ([Ca(2+)](i)) overload in pancreatic acinar cells have been implicated as the cardinal pathological events common to most forms of pancreatitis, regardless of the precise causative factor. Therefore, restoration of metabolism and protection against cytosolic Ca(2+) overload likely represent key therapeutic untapped strategies for the treatment of this disease. The plasma membrane Ca(2+)-ATPase (PMCA) provides a final common path for cells to "defend" [Ca(2+)](i) during cellular injury. In this paper, we use fluorescence imaging to show for the first time that insulin treatment, which is protective in animal models and clinical studies of human pancreatitis, directly protects pancreatic acinar cells from oxidant-induced cytosolic Ca(2+) overload and inhibition of the PMCA. This protection was independent of oxidative stress or mitochondrial membrane potential but appeared to involve the activation of Akt and an acute metabolic switch from mitochondrial to predominantly glycolytic metabolism. This switch to glycolysis appeared to be sufficient to maintain cellular ATP and thus PMCA activity, thereby preventing Ca(2+) overload, even in the face of impaired mitochondrial function.     

Capacitative calcium entry: sensing the calcium stores

A long-standing mystery in the cell biology of calcium channel regulation is the nature of the signal linking intracellular calcium stores to plasma membrane capacitative calcium entry channels. An RNAi-based screen of selected Drosophila genes has revealed that a calcium-binding protein, stromal interaction molecule (STIM), plays an essential role in the activation of these channels and may be the long sought sensor of calcium store content.     

Homocysteine inhibits store-mediated calcium entry in human endothelial cells: evidence for involvement of membrane potential and actin cytoskeleton

The role of homocysteine for store-operated calcium influx was investigated in human umbilical cord endothelial cell line. Homocysteine significantly decreased thapsigargin-evoked Ca2+ entry, membrane hyperpolarization and actin polymerization. GSH and DTT prevented homocysteine-induced inhibition of thapsigargin-evoked Ca2+ entry, membrane hyperpolarization and actin polymerization; while GSSG had the opposite effect. Homocysteine blocked large conductance Ca2+-activated K+ (BK(Ca)) channels in a concentration-dependent manner and related to the redox status of the endothelial cells. BK(Ca) channels opener NS1619 reversed thapsigargin-evoked Ca2+ entry, membrane hyperpolarization and actin polymerization; BK(Ca) channels inhibitor iberiotoxin had the opposite effect. The findings suggest that homocysteine is involved in store-regulated Ca2+ entry through membrane potential-dependent and actin cytoskeleton-dependent mechanisms, redox status of homocysteine and BK(Ca) channels may play a regulatory role in it.     

Homocysteine inhibits store-mediated calcium entry in human endothelial cells: Evidence for involvement of membrane potential and actin cytoskeleton

The role of homocysteine for store-operated calcium influx was investigated in human umbilical cord endothelial cell line. Homocysteine significantly decreased thapsigargin-evoked Ca²⁺ entry, membrane hyperpolarization and actin polymerization. GSH and DTT prevented homocysteine-induced inhibition of thapsigargin-evoked Ca²⁺ entry, membrane hyperpolarization and actin polymerization; while GSSG had the opposite effect. Homocysteine blocked large conductance Ca²⁺-activated K⁺ (BK_Ca) channels in a concentration-dependent manner and related to the redox status of the endothelial cells. BK_Ca channels opener NS1619 reversed thapsigargin-evoked Ca²⁺ entry, membrane hyperpolarization and actin polymerization; BK_Ca channels inhibitor iberiotoxin had the opposite effect. The findings suggest that homocysteine is involved in store-regulated Ca²⁺ entry through membrane potential-dependent and actin cytoskeleton-dependent mechanisms, redox status of homocysteine and BK_Ca channels may play a regulatory role in it. (Mol Cell Biochem 269: 37–47, 2005)