Christophe Duranton

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 850 mOsm for 24 h, to tert-butyl-hydroperoxide (1 mM) for 15 min, or to glucose-free medium for 48 h, all elicit erythrocyte shrinkage and annexin binding, both sequelae being blunted by removal of extracellular Ca2+ and mimicked by ionomycin (1 μM). Osmotic shrinkage and oxidative stress activate Ca2+-permeable cation channels and increase cytosolic Ca2+ concentration. The channels are inhibited by amiloride (1 mM), 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.

Oxidation induces a Cl−-dependent cation conductance in human red blood cells

Oxidative stress induces complex alterations of membrane proteins in red blood cells (RBCs) eventually leading to haemolysis. To study changes of membrane ion permeability induced by oxidative stress, whole‐cell patch‐clamp recordings and haemolysis experiments were performed in control and oxidised human RBCs. Control RBCs exhibited a small cation‐selective whole‐cell conductance (236 ± 38 pS; n= 8) which was highly sensitive to the external Cl− concentration: replacement of NaCl in the bath by sodium gluconate induced an increase of this cation conductance by about 85 %. Exposing RBCs to t‐butylhydroxyperoxide (1 mm for 10 min) induced a twofold increase in this cation conductance which was further stimulated after replacement of extracellular Cl− by gluconate, Br−, I− or SCN−. In addition, lowering the ionic strength of the bath solution by isosmotic substitution of NaCl by sorbitol activated the cation conductance. The Cl−‐sensitive and oxidation‐induced cation conductance was Ca2+ permeable, exhibited a permselectivity of Cs+ > K+ > Na+= Li+ >> NMDG+, and was partially inhibited by amiloride (1 mm) and almost completely inhibited by GdCl3 (150 μm), but was insensitive to TEA, BaCl2, NPPB, flufenamic acid or quinidine. DIDS (100 μm) reversibly inhibited the activation of the cation conductance by removal of external Cl−. Oxidation induced haemolysis in NaCl‐bathed human RBCs. This haemolysis was attenuated by amiloride (1 mm) and inhibited by replacement of bath Na+ by the impermeant cation NMDG+. The Na+‐ and Ca2+‐permeable conductance might be involved in haemolytic diseases induced by elevated oxidative stress, such as glucose‐6‐phosphate dehydrogenase deficiency.

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.

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.

Plasmodium falciparum activates endogenous Cl− channels of human erythrocytes by membrane oxidation

Intraerythrocytic survival of the malaria parasite Plasmodium falciparum requires that host cells supply nutrients and dispose of waste products. This solute transport is accomplished by infection‐induced new permeability pathways (NPP) in the erythrocyte membrane. Here, whole‐cell patch–clamp and hemolysis experiments were performed to define properties of the NPP. Parasitized but not control erythrocytes constitutively expressed two types of anion conductances, differing in voltage dependence and sensitivity to inhibitors. In addition, infected but not control cells hemolyzed in isosmotic sorbitol solution. Both conduct ances and hemolysis of infected cells were inhibited by reducing agents. Conversely, oxidation induced identical conductances and hemolysis in non‐infected erythrocytes. In conclusion, P.falciparum activates endogenous erythrocyte channels by applying oxidative stress to the host cell 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.

Accelerated Clearance of Plasmodium-infected Erythrocytes in Sickle Cell Trait and Annexin-A7 Deficiency

The course of malaria does not only depend on the virulence of the parasite Plasmodium but also on properties of host erythrocytes. Here, we show that infection of erythrocytes from human sickle cell trait (HbA/S) carriers with ring stages of P. falciparum led to significantly enhanced PGE2 formation, Ca2+ permeability, annexin-A7 degradation, phosphatidylserine (PS) exposure at the cell surface, and clearance by macrophages. P. berghei-infected erythrocytes from annexin-A7-deficient (annexin-A7-/-) mice were more rapidly cleared than infected wildtype cells. Accordingly, P. berghei-infected annexin-A7-/- mice developed less parasitemia than wildtype mice. The cyclooxygenase inhibitor aspirin decreased erythrocyte PS exposure in infected annexin-A7-/- mice and abolished the differences of parasitemia and survival between the genotypes. Conversely, the PGE2-agonist sulprostone decreased parasitemia and increased survival of wild type mice. In conclusion, PS exposure on erythrocytes results in accelerated clearance of Plasmodium ring stage-infected HbA/S or annexin-A7-/- erythrocytes and thus confers partial protection against malaria in vivo.

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.

Cadmium-Induced Autophagy in Rat Kidney: An Early Biomarker of Subtoxic Exposure

Environmental exposures to cadmium (Cd) are a major cause of human toxicity. The kidney is the most sensitive organ; however, the natures of injuries and of adaptive responses have not been adequately investigated, particularly in response to environmental relevant Cd concentrations. In this study, rats received a daily ip injection of low CdCl₂ dose (0.3 mg Cd/kg body mass) and killed at 1, 3, and 5 days of intoxication. Functional, ultrastructural, and biochemical observations were used to evaluate Cd effects. We show that Cd at such subtoxic doses does not affect the tubular functions nor does it induce apoptosis. Meanwhile, Cd accumulates within lysosomes of proximal convoluted tubule (PCT) cells where it triggers cell proliferation and autophagy. By developing an immunohistochemical assay, a punctate staining of light chain 3-II is prominent in Cd-intoxicated kidneys, as compared with control. We provide the evidence of a direct upregulation of autophagy by Cd using a PCT cell line. Compared with the other heavy metals, Cd is the most powerful inducer of endoplasmic reticulum stress and autophagy in PCT cells, in relation to the hypersensitivity of PCT cells. Altogether, these findings suggest that kidney cortex adapts to subtoxic Cd dose by activating autophagy, a housekeeping process that ensures the degradation of damaged proteins. Given that Cd is persistent within cytosol, it might damage proteins continuously and impair at long-term autophagy efficiency. We therefore propose the autophagy pathway as a new sensitive biomarker for renal injury even after exposure to subtoxic Cd doses.