Karl S. Lang

Mechanisms of Suicidal Erythrocyte Death

Erythrocyte injury such as osmotic shock, oxidative stress or energy depletion stimulates the formation of prostaglandin E2 through activation of cyclooxygenase which in turn activates a Ca2+ permeable cation channel. Increasing cytosolic Ca2+ concentrations activate Ca2+ sensitive K+ channels leading to hyperpolarization, subsequent loss of KCl and (further) cell shrinkage. Ca2+ further stimulates a scramblase shifting phosphatidylserine from the inner to the outer cell membrane. The scramblase is sensitized for the effects of Ca2+ by ceramide which is formed by a sphingomyelinase following several stressors including osmotic shock. The sphingomyelinase is activated by platelet activating factor PAF which is released by activation of phospholipase A2. Phosphatidylserine at the erythrocyte surface is recognised by macrophages which engulf and degrade the affected cells. Moreover, phosphatidylserine exposing erythrocytes may adhere to the vascular wall and thus interfere with microcirculation. Erythrocyte shrinkage and phosphatidylserine exposure (‘eryptosis’) mimic features of apoptosis in nucleated cells which however, involves several mechanisms lacking in erythrocytes. In kidney medulla, exposure time is usually too short to induce eryptosis despite high osmolarity. Beyond that high Cl- concentrations inhibit the cation channel and high urea concentrations the sphingomyelinase. Eryptosis is inhibited by erythropoietin which thus extends the life span of circulating erythrocytes. Several conditions trigger premature eryptosis thus favouring the development of anemia. On the other hand, eryptosis may be a mechanism of defective erythrocytes to escape hemolysis. Beyond their significance for erythrocyte survival and death the mechanisms involved in ‘eryptosis’ may similarly contribute to apoptosis of nucleated cells.

Cation channels trigger apoptotic death of erythrocytes

AbstractErythrocytes are devoid of mitochondria and nuclei and were considered unable to undergo apoptosis. As shown recently, however, the Ca2+-ionophore ionomycin triggers breakdown of phosphatidylserine asymmetry (leading to annexin binding), membrane blebbing and shrinkage of erythrocytes, features typical for apoptosis in nucleated cells. In the present study, the effects of osmotic shrinkage and oxidative stress, well-known triggers of apoptosis in nucleated cells, were studied. Exposure to 850u2009mOsm for 24u2009h, to tert-butyl-hydroperoxide (1u2009mM) for 15u2009min, or to glucose-free medium for 48u2009h, all elicit erythrocyte shrinkage and annexin binding, both sequelae being blunted by removal of extracellular Ca2+ and mimicked by ionomycin (1u2009μM). Osmotic shrinkage and oxidative stress activate Ca2+-permeable cation channels and increase cytosolic Ca2+ concentration. The channels are inhibited by amiloride (1u2009mM), which further blunts annexin binding following osmotic shock, oxidative stress and glucose depletion. In conclusion, osmotic and oxidative stress open Ca2+-permeable cation channels in erythrocytes, thus increasing cytosolic Ca2+ activity and triggering erythrocyte apoptosis.

Ion channels in cell proliferation and apoptotic cell death.

Cell proliferation and apoptosis are paralleled by altered regulation of ion channels that play an active part in the signaling of those fundamental cellular mechanisms. Cell proliferation must - at some time point - increase cell volume and apoptosis is typically paralleled by cell shrinkage. Cell volume changes require the participation of ion transport across the cell membrane, including appropriate activity of Cl− and K+ channels. Besides regulating cytosolic Cl− activity, osmolyte flux and, thus, cell volume, most Cl− channels allow HCO3− exit and cytosolic acidification, which inhibits cell proliferation and favors apoptosis. K+ exit through K+ channels may decrease intracellular K+ concentration, which in turn favors apoptotic cell death. K+ channel activity further maintains the cell membrane potential, a critical determinant of Ca2+ entry through Ca2+ channels. Cytosolic Ca2+ may trigger mechanisms required for cell proliferation and stimulate enzymes executing apoptosis. The switch between cell proliferation and apoptosis apparently depends on the magnitude and temporal organization of Ca2+ entry and on the functional state of the cell. Due to complex interaction with other signaling pathways, a given ion channel may play a dual role in both cell proliferation and apoptosis. Thus, specific ion channel blockers may abrogate both fundamental cellular mechanisms, depending on cell type, regulatory environment and condition of the cell. Clearly, considerable further experimental effort is required to fully understand the complex interplay between ion channels, cell proliferation and apoptosis.

Enhanced programmed cell death of iron-deficient erythrocytes

Exposure of erythrocytes to osmotic shock, oxidative stress, and energy depletion activates Cl–sensitive and Ca2+‐permeable cation channels. Subsequent Ca2+ entry triggers eryptosis, characterized by erythrocyte shrinkage, membrane blebbing, and phosphatidylserine exposure all features typical for apoptotic death of nucleated cells. Erythrocytes exposing phosphatidylserine are recognized, bound, engulfed, and degraded by macrophages. Eryptosis thus fosters clearance of affected erythrocytes from circulating blood. Iron deficiency leads to anemia, in part by decreasing erythrocyte life span. In this study, phosphatidylserine exposure, cell size, and cytosolic Ca2+ were measured by FACS analysis of annexin‐V binding, forward scatter, and Fluo‐3 fluorescence, respectively. Erythrocytes from mice on control diet were compared with erythrocytes from mice exposed 10 weeks to iron‐deficient diet. Iron deficiency significantly (P<0.001) enhanced erythrocyte annexin‐V binding (from 2.4 to 3.7%), decreased forward scatter (from 544 to 393), and increased cytosolic Ca2+ concentration. 45Ca2+ flux measurements and patch clamp experiments revealed enhanced Ca2+ uptake (by 2.3‐fold) and cation channel activity. The half‐life of fluorescence‐labeled, iron‐deficient, or Ca2+‐loaded erythrocytes was significantly reduced compared with control erythrocytes. Thus, the experiments reveal a novel mechanism triggered by iron deficiency, which presumably contributes to accelerated clearance of erythrocytes in iron deficiency anemia.

Involvement of ceramide in hyperosmotic shock- induced death of erythrocytes

AbstractErythrocytes lack nuclei and mitochondria, the organelles important for apoptosis of nucleated cells. However, following increase of cytosolic Ca2+ activity, erythrocytes undergo cell shrinkage, cell membrane blebbing and breakdown of phosphatidylserine asymmetry, all features typical for apoptosis in nucleated cells. The same events are observed following osmotic shock, an effect mediated in part by activation of Ca2+-permeable cation channels. However, erythrocyte death following osmotic shock is blunted but not prevented in the absence of extracellular Ca2+ pointing to additional mechanisms. As shown in this study, osmotic shock (950u2009mOsm) triggers sphingomyelin breakdown and formation of ceramide. The stimulation of annexin binding following osmotic shock is mimicked by addition of ceramide or purified sphingomyelinase and significantly blunted by genetic (aSM-deficient mice) or pharmacologic (50u2009μM 3,4-dichloroisocoumarin) knockout of sphingomyelinase. The effect of ceramide is blunted but not abolished in the absence of Ca2+. Conversely, osmotic shock-induced annexin binding is potentiated in the presence of sublethal concentrations of ceramide. In conclusion, ceramide and Ca2+ entry through cation channels concert to trigger erythrocyte death during osmotic shock.

Enhanced Erythrocyte Apoptosis in Sickle Cell Anemia, Thalassemia and Glucose-6-Phosphate Dehydrogenase Deficiency

Erythrocyte diseases such as sickle cell anemia, thalassemia and glucose-6-phosphate dehydrogenase deficiency decrease the erythrocyte life span, an effect contributing to anemia. Most recently, erythro-cytes have been shown to undergo apoptosis upon increase of cytosolic Ca2+ activity. The present study has been performed to explore whether sickle cell anemia, thalassemia and glucose-6-phosphate dehydrogenase deficiency enhance the sensitivity of erythrocytes to osmotic shock, oxidative stress or energy depletion, all maneuvers known to increase cytosolic Ca2+ activity. To this end, annexin binding as an indicator of apoptosis has been determined by FACS analysis. Erythrocytes from healthy individuals, from patients with sickle cell anemia, thalassemia or glucose-6-phosphate dehydrogenase deficiency all responded to osmotic shock (up to 950 mOsm by addition of sucrose for 24 hours), to oxidative stress (up to 1.0 mM tetra-butyl-hydroxyperoxide tBOOH) and to energy depletion (up to 48 hours glucose deprivation) with enhanced annexin binding. However, the sensitivity of sickle cells and of glucose-6-phosphate dehydrogenase deficient cells to osmotic shock and of sickle cells, thalassemic cells and glucose-6-phosphate dehydrogenase deficient cells to oxidative stress and to glucose depletion was significantly higher than that of control cells. Annexin binding was further stimulated by Ca2+ ionophore ionomycin with significantly higher sensitivity of sickle cells and glucose-6-phosphate dehydrogenase deficient cells as compared to intact cells. In conclusion, sickle cells, thalassemic cells and glucose-6-phosphate dehydrogenase deficient erythrocytes are more sensitive to osmotic shock, oxidative stress and/or energy depletion, thus leading to enhanced apoptosis of those cells. The accelerated apoptosis then contributes to the shortened life span of the defective erythrocytes.

Dependence of Plasmodium falciparum in vitro growth on the cation permeability of the human host erythrocyte.

Intraerythrocyte growth of the malaria parasite Plasmodium falciparum induces a Ca2+-permeable unselective cation conductance in the host cell membrane which is inhibited by ethylisopropylamiloride (EIPA) and is paralleled by an exchange of K+ by Na+ in the host cytosol. The present study has been performed to elucidate the functional significance of the electrolyte exchange. Whole-cell patch-clamp experiments confirmed the Ca2+ permeability and EIPA sensitivity of the Plasmodium falciparum induced cation channel. In further experiments, ring stage-synchronized parasites were grown in vitro for 48 h in different test media. Percentage of Plasmodium-infected and phosphatidylserine-exposing erythrocytes was measured with FACS analysis by staining with the DNA-dye Syto16 and annexin V, respectively. The increase of infected cells was not significantly affected by an 8 h replacement of NaCl in the culture medium with Na-gluconate but was significantly blunted by replacement of NaCl with KCl, NMDG-Cl or raffinose. Half maximal growth was observed at about 25 mM Na+. The increase of infected cells was further inhibited by EIPA (IC50< 10 µM) and at low extracellular free Ca2+. Infected cells displayed significantly stronger annexin binding, an effect mimicked by exposure of noninfected erythrocytes to oxidative stress (1 mM t-butylhydroperoxide for 15 min) or to Ca2+ ionophore ionomycin (1 µM for 60 min). The observations indicate that parasite growth requires the entry of both, Na+ and Ca2+ cations into the host erythrocyte probably through the EIPA sensitive cation channel. Ca2+ entry further induces break-down of the phospholipid asymmetry in the host membrane.

Electrophysiological Properties of the Plasmodium falciparum-Induced Cation Conductance of Human Erythrocytes

Intraerythrocyte survival of the malaria pathogen Plasmodium falciparumdepends on the induction of the new-permeability-pathways (NPPs) in the host cell membrane. NPPs are characterized as anion- and organic osmolyte-permeable channels which also exhibit a low but significant permeability for inorganic cations. To disclose the electrophyiologial properties of this infection-induced cation permeability whole-cell currents were recorded inPlasmodium falciparum-infected human erythrocytes (pRBC) using bath and pipette solutions with low Cl^- concentrations. The data disclose a nonselective cation conductance (G_cat) which activated upon removal of extracellular Cl^-. Upon activation, G_cat was 0.3 ± 0.05 nS (n=16) in control RBC and 2.0 ± 0.3 nS (n = 32) in pRBC indicating an induction of G_cat during the infection. G_cat of pRBC exibited a relative permselectivity for monovalent cations of Cs⁺ñK⁺>Na⁺>Li⁺ (P_Na/P_K ñ 0.5) with a significant permeability for Ca²⁺. G_cat of pRBC was inhibited by NPPs blockers (furosemide and NPPB) and cation channel blockers (amiloride, EIPA, GdCl₃) with the highest sensitivity to EIPA (IC₅₀ñ0.5µM). Most importantly, the blocker sensitivities differed between the infection-induced anion conductances and G_cat suggesting that G_cat and the anion conductances represent different channel proteins which in concert build up the NPPs.

Cell Volume Regulatory Ion Channels in Cell Proliferation and Cell Death

Alterations of cell volume are key events during both cell proliferation and apoptotic cell death. Cell proliferation eventually requires an increase of cell volume, and apoptosis is typically paralleled by cell shrinkage. Alterations of cell volume require the participation of ion transport across the cell membrane, including appropriate activity of Cl(-) and K(+) channels. Cl(-) channels modify cytosolic Cl(-) activity and mediate osmolyte flux, and thus influence cell volume. Most Cl(-) channels allow exit of HCO(3)(-), leading to cytosolic acidification, which in turn inhibits cell proliferation and favors apoptosis. K(+) exit through K(+) channels decreases cytosolic K(+) concentration, which may sensitize the cell for apoptotic cell death. K(+) channel activity further maintains the cell membrane potential, a critical determinant of Ca(2+) entry through Ca(2+) channels. Ca(2+) may, in addition, enter through Ca(2+)-permeable cation channels, which, in some cells, are activated by hyperosmotic shock. Increases of cytosolic Ca(2+) activity may trigger both mechanisms required for cell proliferation and mechanisms, leading to apoptosis. Thereby cell proliferation and apoptosis depend on magnitude and temporal organization of Ca(2+) entry, as well as activity of other signaling pathways. Accordingly, the same ion channels may participate in the stimulation of both cell proliferation and apoptosis. Specific ion channel blockers may thus abrogate both cellular mechanisms, depending on cell type and condition.

Channel-induced apoptosis of infected host cells—the case of malaria

Infection of erythrocytes by the malaria pathogen Plasmodium falciparum leads to activation of several distinct anion channels and a non-selective, Ca2+-permeable cation channel. All channel types are presumably activated by the oxidative stress generated by the pathogen. Similar or identical channels are activated by oxidation of non-infected erythrocytes. Activation of the non-selective cation channel allows entry of Ca2+ and Na+, both of which are required for intracellular growth of the pathogen. The entry of Ca2+ stimulates an intraerythrocytic scramblase that facilitates bi-directional phospholipid migration across the bilayer, resulting in breakdown of the phosphatidylserine asymmetry of the cell membrane. The exposure of phosphatidylserine at the outer surface of the cell membrane is presumably followed by binding to phosphatidylserine receptors on macrophages and subsequent phagocytosis of the affected erythrocyte. The lysosomal degradation may eventually eliminate the pathogen. The channel may thus play a dual role in pathogen survival. Absence of the channels is not compatible with pathogen growth, enhanced channel activity accelerates erythrocyte “apoptosis” that may represent a host defence mechanism serving to eliminate infected erythrocytes.