Valerie Tanneur

Cell volume in the regulation of cell proliferation and apoptotic cell death.

Cell proliferation must – at some time point – lead to increase of cell volume and one of the hallmarks of apoptosis is cell shrinkage. At constant extracellular osmolarity those alterations of cell volume must reflect respective changes of cellular osmolarity which are hardly possible without the participation of cell volume regulatory mechanisms. Indeed, as shown for ras oncogene expressing 3T3 fibroblasts, cell proliferation is paralleled by activation of Na+/H+ exchange and Na+,K+,2Cl- cotransport, the major transport systems accomplishing regulatory cell volume increase. Conversely, as evident from CD95-induced apoptotic cell death, apoptosis is paralleled by inhibition of Na+/H+ exchanger and by activation of Cl- channels and release of the organic osmolyte taurine, major components of regulatory cell volume decrease. However, ras oncogene activation leads to activation and CD95 receptor triggering to inhibition of K+ channels. The effects counteract the respective cell volume changes. Presumably, they serve to regulate cell membrane potential, which is decisive for Ca++ entry through ICRAC and the generation of cytosolic Ca++ oscillations in proliferating cells. As a matter of fact ICRAC is activated in ras oncogene expressing cells and inhibited in CD95-triggered cells. Activation of K+ channels and Na+/H+ exchanger as well as Ca++ oscillations have been observed in a wide variety of cells upon exposure to diverse mitogenic factors. Conversely, diverse apoptotic factors have been shown to activate Cl- channels and organic osmolyte release. Inhibition of K+ channels is apparently, however, not a constant phenomenon paralleling apoptosis which in some cells may even require the operation of K+ channels. Moreover, cell proliferation may at some point require activation of Cl- channels. In any case, the alterations of cell volume are obviously important for the outcome, as cell shrinkage impedes cell proliferation and apoptosis can be elicited by increase of extracellular osmolarity. At this stage little is known about the interplay of cell volume regulatory mechanisms and the cellular machinery leading to mitosis or death of the cell. Thus, considerable further experimental effort is required in this exciting area of cell physiology.

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.

Stimulation of erythrocyte ceramide formation by platelet-activating factor

Osmotic erythrocyte shrinkage leads to activation of cation channels with subsequent Ca2+ entry and stimulates a sphingomyelinase with subsequent formation of ceramide. Ca2+ and ceramide then activate a scramblase leading to breakdown of phosphatidylserine asymmetry of the cell membrane. The mediators accounting for activation of erythrocyte sphingomyelinase and phosphatidylserine exposure remained elusive. The study demonstrates that platelet-activating factor (PAF) is released from erythrocytes upon hyperosmotic cell shrinkage. The experiments further disclose the presence of PAF receptors in erythrocytes and show that PAF stimulates the breakdown of sphingomyelin and the release of ceramide from erythrocytes at isotonic conditions. PAF further triggers cell shrinkage (decrease of forward scatter) and phosphatidylserine exposure (annexin binding) of erythrocytes. The stimulation of annexin-binding is blunted by a genetic knockout of PAF receptors, by the PAF receptor antagonist ABT491 or by inhibition of sphingomyelinase with urea. In conclusion, PAF activates an erythrocyte sphingomyelinase and the then formed ceramide leads to the activation of scramblase with subsequent phosphatidylserine exposure.

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.

PGE(2) in the regulation of programmed erythrocyte death.

Hyperosmotic shock, energy depletion, or removal of extracellular Cl− activates Ca2+-permeable cation channels in erythrocyte membranes. Subsequent Ca2+ entry induces erythrocyte shrinkage and exposure of phosphatidylserine (PS) at the erythrocyte surface. PS-exposing cells are engulfed by macrophages. The present study explored the signalling involved. Hyperosmotic shock and Cl− removal triggered the release of prostaglandin E2 (PGE2). In whole-cell recording, activation of the cation channels by Cl− removal was abolished by the cyclooxygenase inhibitor diclophenac. In FACS analysis, phospholipase-A2 inhibitors quinacrine and palmitoyltrifluoromethyl-ketone, and cyclooxygenase inhibitors acetylsalicylic acid and diclophenac, blunted the increase of PS exposure following Cl− removal. PGE2 (but not thromboxane) induced cation channel activation, increase in cytosolic Ca2+ concentration, cell shrinkage, PS exposure, calpain activation, and ankyrin-R degradation. The latter was attenuated by calpain inhibitors-I/II, while PGE2-induced PS exposure was not. In conclusion, hyperosmotic shock or Cl− removal stimulates erythrocyte PS exposure through PGE2 formation and subsequent activation of Ca2+-permeable cation channels.

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.

Purinoceptors are involved in the induction of an osmolyte permeability in malaria-infected and oxidized human erythrocytes

In human erythrocytes, infection by the malaria parasite Plasmodium falciparum or oxidative stress induces a new organic osmolyte and anion permeability. To examine a role for autocrine purinoceptor signaling during this induction process, erythrocytic purinoceptor expression, and ATP release were determined. Furthermore, using pharmacological and genetic approaches the dependence on purinoceptor signaling of osmolyte permeability and Plasmodium development, both in vitro and in vivo, were assessed. Extracellular ATP did not induce an osmolyte permeability in non‐infected or non‐oxidized erythrocytes. ATP and other purinoceptor agonists increased the induction of osmolyte permeability during infection or oxidation as measured by isosmotic hemolysis and patch‐clamp recording. Purinoceptor antagonists and apyrase decreased the induced permeability. The observed pharmacology suggested the involvement of P2Y purinoceptors. Accordingly, human erythrocytes expressed P2Y1 protein. Moreover, P2Y1 deficient mouse erythrocytes exhibited a delayed appearance of the osmolyte permeability during P. berghei infection‐ or oxidation compared with wild‐type erythrocytes. Furthermore, the nonspecific purinoceptor antagonist suramin decreased in vitro growth and DNA/RNA amplification of P. falciparum in human erythrocytes and decreased in vivo growth of P. berghei. P. berghei developed slower in P2Y1‐deficient mice in vivo compared with wild‐type animals. In conclusion, induction of the osmolyte permeability in Plasmodium‐infected erythrocytes involves autocrine purinoceptor signaling.

Expression of the Serine/Threonine Kinase hSGK1 in Chronic Viral Hepatitis

The human serine/threonine kinase hSGK1 is expressed ubiquitously with highest transcript levels in pancreas and liver. This study has been performed to determine the hSGK1 distribution in normal liver and its putative role in fibrosing liver disease. HSGK1-localization was determined by in situr hybridization, regulation of hSGK1-transcription by Northern blotting, fibronectin synthesis and hSGK1 phosphorylation by Western blotting. In normal liver hSGK1 was mainly transcribed by Kupffer cells. In liver tissue from patients with chronic viral hepatitis, hSGK1 transcript levels were excessively high in numerous activated Kupffer cells and inflammatory cells localized within fibrous septum formations. HSGK1 transcripts were also detected in activated hepatic stellate cells. Accordingly, Western blotting revealed that tissue from fibrotic liver expresses excessive hSGK1 protein as compared to normal liver. TGF-β1 (2 ng/ml) increases hSGK1 transcription in both human U937 macro-phages and HepG2 hepatoma cells. H2O2 (0.3 mM) activated hSGK1 and increased fibronectin formation in HepG2 cells overexpressing hSGK1 but not in HepG2 cells expressing the inactive mutant hSGK1K127R. In conclusion hSGK1 is upregulated by TGF-β1 during hepatitis and may contribute to enhanced matrix formation during fibrosing liver disease.

Organic Osmolyte Permeabilities of the Malaria-induced Anion Conductances in Human Erythrocytes

Infection of human erythrocytes with the malaria parasite Plasmodium falciparum induces new permeability pathways (NPPs) in the host cell membrane. Isotopic flux measurements demonstrated that the NPP are permeable to a wide variety of molecules, thus allowing uptake of nutrients and release of waste products. Recent patch-clamp recordings demonstrated the infection-induced up-regulation of an inwardly and an outwardly rectifying Cl− conductance. The present experiments have been performed to explore the sensitivity to cell volume and the organic osmolyte permeability of the two conductances. It is shown that the outward rectifier has a high relative lactate permeability (Plactate/PCl = 0.4). Sucrose inhibited the outward-rectifier and abolished the infection-induced hemolysis in isosmotic sorbitol solution but had no or little effect on the inward-rectifier. Furosemide and NPPB blocked the outward-rectifying lactate current and the sorbitol hemolysis with IC50s in the range of 0.1 and 1 μM, respectively. In contrast, the IC50s of NPPB and furosemide for the inward-rectifying current were >10 μM. Osmotic cell-shrinkage inhibited the inwardly but not the outwardly rectifying conductance. In conclusion, the parasite-induced outwardly-rectifying anion conductance allows permeation of lactate and neutral carbohydrates, whereas the inward rectifier seems largely impermeable to organic solutes. All together, these data should help to resolve ongoing controversy regarding the number of unique channels that exist in P. falciparum–infected erythrocytes.