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ABSTRACT: P-glycoprotein (Pgp) and multidrug resistance-associated proteins (MRPs) are ATP-dependent transporters involved in efflux of toxins and xenobiotics from cells. When overexpressed, these transporters can mediate multidrug resistance (MDR) in mammalian cells, and changes in Pgp expression and sequence are associated with drug resistance in helminths. In addition to the role they play in drug efflux, MDR transporters are essential components of normal cellular physiology, and targeting them may prove a useful strategy for development of new therapeutics or of compounds that enhance the efficacy of current anthelmintics. We previously showed that expression of Schistosoma mansoni MDR transporters increases in response to praziquantel (PZQ), the current drug of choice against schistosomiasis, and that reduced PZQ sensitivity correlates with higher levels of these parasite transporters. We have also shown that PZQ inhibits transport by SMDR2, a Pgp orthologue from S. mansoni, and that PZQ is a likely substrate of SMDR2. Here, we examine the physiological roles of SMDR2 and SmMRP1 (the S. mansoni orthologue of MRP1) in S. mansoni adults, using RNAi to knock down expression, and pharmacological agents to inhibit transporter function. We find that both types of treatments disrupt parasite egg deposition by worms in culture. Furthermore, administration of different MDR inhibitors to S. mansoni-infected mice results in a reduction in egg burden in host liver. These schistosome MDR transporters therefore appear to play essential roles in parasite egg production, and can be targeted genetically and pharmacologically. Since eggs are responsible for the major pathophysiological consequences of schistosomiasis, and since they are also the agents for transmission of the disease, these results suggest a potential strategy for reducing disease pathology and spread.
PLoS Neglected Tropical Diseases 12/2011; 5(12):e1425. · 4.69 Impact Factor
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ABSTRACT: Schistosomes are the causative agents of schistosomiasis, a neglected tropical disease affecting hundreds of millions worldwide and a major global health burden. Current control of schistosomiasis depends largely on a single drug, praziquantel (PZQ). One potential physiological target for new antischistosomal drugs is the parasite's excretory system, which removes wastes and xenobiotics. Multidrug resistance (MDR) transporters that are members of the ATP-binding cassette (ABC) superfamily of proteins are ATP-dependent efflux pumps involved in removal of toxins and xenobiotics from cells. They mediate the phenomenon of multidrug resistance, in which cells resistant to one drug show cross-resistance to a broad range of other agents, and are also associated with reduced drug susceptibility in parasitic helminths. In this review, we survey the different types of ABC transporter genes present within the schistosome genome, and examine recent evidence indicating that at least some of these transporters may play a role in fine-tuning susceptibility of schistosomes to PZQ. Disruption of their function may therefore provide a strategy for enhancing drug action or overcoming or attenuating drug resistance. Furthermore, dissection of the roles these transporters may play in normal schistosome physiology could potentially lead to identification of highly "druggable" targets for new antischistosomals.
Experimental Parasitology 03/2011; 132(1):2-6. · 2.12 Impact Factor
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Michael Föller,
Diwakar Bobbala,
Saisudha Koka,
Krishna M Boini,
Hasan Mahmud, Ravi S Kasinathan,
Ekaterina Shumilina,
Kerstin Amann,
Golo Beranek,
Ulrike Sausbier,
Peter Ruth,
Matthias Sausbier,
Florian Lang,
Stephan M Huber
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ABSTRACT: Increased cytosolic Ca(2+) concentrations activate Gardos K(+) channels in human erythrocytes with membrane hyperpolarization, efflux of K(+), Cl⁻, and osmotically obliged H₂O resulting in cell shrinkage, a phenomenon referred to as Gardos effect. We tested whether the Gardos effect delays colloid osmotic hemolysis of injured erythrocytes from mice lacking the Ca(2+)-activated K(+) channel K(Ca)3.1. To this end, we applied patch clamp and flow cytometry and determined in vitro as well as in vivo hemolysis. As a result, erythrocytes from K(Ca)3.1-deficient (K(Ca)3.1(-/-)) mice lacked Gardos channel activity and the Gardos effect. Blood parameters, reticulocyte count, or osmotic erythrocyte resistance, however, did not differ between K(Ca)3.1(-/-) mice and their wild-type littermates, suggesting low or absent Gardos channel activity in unstressed erythrocytes. Oxidative stress-induced Ca(2+) entry and phospholipid scrambling were significantly less pronounced in K(Ca)3.1(-/-) than in wild-type erythrocytes. Moreover, in vitro treatment with α-toxin from Staphylococcus aureus, which forms pores in the cellular membrane, resulted in significantly stronger hemolysis of K(Ca)3.1(-/-) than of wild-type erythrocytes. Intravenous injection of α-toxin induced more profound hemolysis in K(Ca)3.1(-/-) than in wild-type mice. Similarly, intra-peritoneal application of the redox-active substance phenylhydrazine, an agent for the induction of hemolytic anemia, was followed by a significantly stronger decrease of hematocrit in K(Ca)3.1(-/-) than in wild-type mice. Finally, malaria infection triggered the activation of K(Ca)3.1 and transient shrinkage of the infected erythrocytes. In conclusion, K(Ca)3.1 channel activity and Gardos effect counteract hemolysis of injured erythrocytes, thus decreasing hemoglobin release into circulating blood.
Pflügers Archiv - European Journal of Physiology 11/2010; 460(6):1029-44. · 4.46 Impact Factor
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ABSTRACT: The ATP-binding cassette (ABC) superfamily of proteins comprises several ATP-dependent efflux pumps involved in transport of toxins and xenobiotics from cells. These transporters are essential components of normal physiology, and a subset is associated with development of multidrug resistance. P-glycoprotein (Pgp) and the multidrug resistance-associated proteins (MRPs) represent two classes of these multidrug resistance (MDR) transporters. MRP1 is one type of mammalian MRP, which preferentially transports anionic compounds and compounds detoxified by cellular enzymes such as glutathione-S-transferase. It also transports signaling molecules, including immunomodulators. In schistosomes, both Pgp and MRP substrates localize to the excretory system, a potentially attractive target for new antischistosomals. We have previously shown that expression of schistosome Pgp (SMDR2) is altered in worms exposed to praziquantel (PZQ), the current drug of choice against schistosomiasis, and is expressed at higher levels in worms from isolates with reduced PZQ susceptibility. We have also shown that PZQ interacts directly with SMDR2. Here, we examine the relationship between PZQ and SmMRP1, a Schistosoma mansoni homolog of mammalian MRP1. SmMRP1 RNA is differentially expressed in adult males and females, and levels increase transiently following exposure of adult worms to sub-lethal concentrations of PZQ. A corresponding, though delayed, increase in anti-MRP1-immunoreactive protein also occurs following exposure to PZQ. PZQ-insensitive juvenile worms express higher levels of both SmMRP1 and SMDR2 RNA than mature adults, consistent with the hypothesis that increases in levels of schistosome multidrug transporters may be involved in development or maintenance of reduced susceptibility to PZQ.
Molecular and Biochemical Parasitology 09/2010; 173(1):25-31. · 2.55 Impact Factor
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ABSTRACT: Estimated to affect nearly 300 million people worldwide, schistosomiasis is caused by parasitic flatworms of the genus Schistosoma. The major pathological consequences of chronic schistosomiasis are associated with soluble egg antigens (SEA) secreted from schistosome egg deposits in liver and other organs. The vigorous immune responses induced by egg antigens result in granuloma formation and other pathophysiological symptoms such as hepatosplenomegaly and anemia. Risk of anemia correlates with infection intensity and the level of host hemoglobin is inversely proportional to egg count. Schistosomiasis-associated anemia could be multifactorial, but the potential link and molecular underpinnings are unclear. Here, we evaluate whether S. mansoni SEA affects survival of mouse erythrocytes. Erythrocytes incubated with different concentrations of SEA were tested for various markers of erythrocyte cell death. Erythrocytes exposed to SEA exhibit elevated intracellular Ca(2+) levels as measured by Fluo-3 AM fluorescence in flow cytometry, and they also display concentration-dependent, Ca(2+)-dependent, and heat-sensitive increases in phosphatidyl serine exposure. Further, SEA-exposed erythrocytes show increased fluorescence using the in situ apoptosis marker CaspACE FITC, indicating the involvement of caspase-mediated cell deformation. Taken together, these results offer several lines of experimental evidence for SEA-induced erythrocyte cell death and may provide new insights into factors contributing to schistosomiasis-associated anemia.
Cellular Physiology and Biochemistry 01/2010; 26(4-5):767-74. · 2.86 Impact Factor
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ABSTRACT: P-glycoprotein (Pgp) is an ATP-dependent efflux pump involved in transport of xenobiotics from cells that, when overexpressed, can mediate multidrug resistance in mammalian cells. Pgp may be a candidate target for new anthelmintics, as it plays critical roles in normal cell physiology, in removal of drugs from cells, and potentially in the development of drug resistance. Schistosomes are parasitic flatworms that cause schistosomiasis, which affects hundreds of millions of people worldwide. Here, we express SMDR2, a Pgp homologue from Schistosoma mansoni (Platyhelminthes), in Chinese hamster ovary (CHO) cells and use fluorescence-based assays to examine the functional and pharmacological properties of this transporter. Membrane vesicles from stably transfected CHO cells expressing recombinant SMDR2 show significant increases in rhodamine transport and ATP hydrolysis compared with those from control cells or cells transfected with empty vector. SMDR2-mediated transport is inhibited by the Pgp modulators verapamil (IC(50)=12.1 muM) and nifedipine, and also by praziquantel, the current drug of choice against schisotosomiasis (IC(50)=17.4 muM). Efflux measurements of a fluorescent analog of praziquantel indicate that it is also a substrate for SMDR2. The interaction of praziquantel with SMDR2 may offer new strategies for potentiating the action of praziquantel and possibly overcoming drug resistance.
The FASEB Journal 10/2009; 24(1):128-35. · 5.71 Impact Factor
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ABSTRACT: One potential physiological target for new antischistosomals is the parasite's system for excretion of wastes and xenobiotics. P-glycoprotein (Pgp), a member of the ATP-binding-cassette superfamily of proteins, is an ATP-dependent efflux pump involved in transport of toxins and xenobiotics from cells. In vertebrates, increased expression of Pgp is associated with multidrug resistance in tumor cells. Pgp may also play a role in drug resistance in helminths. In this report, we examine the relationship between praziquantel (PZQ), the current drug of choice against schistosomiasis, and Pgp expression in Schistosoma mansoni. We show that levels of RNA for SMDR2, a Pgp homolog from S. mansoni, increase transiently in adult male worms following exposure to sub-lethal concentrations (100-500 nM) of PZQ. A corresponding, though delayed, increase in anti-Pgp immunoreactive protein expression occurs in adult males following exposure to PZQ. The level of anti-Pgp immunoreactivity in particular regions of adult worms also increases in response to PZQ. Adult worms from an Egyptian S. mansoni isolate with reduced sensitivity to PZQ express increased levels of SMDR2 RNA and anti-Pgp-immunoreactive protein, perhaps indicating a role for multidrug resistance proteins in development or maintenance of PZQ resistance.
Molecular and Biochemical Parasitology 06/2009; 167(1):54-9. · 2.55 Impact Factor
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Philipp A Lang, Ravi S Kasinathan,
Verena B Brand,
Christophe Duranton,
Camelia Lang,
Saisudha Koka,
Ekaterina Shumilina,
Daniela S Kempe,
Valerie Tanneur,
Ahmad Akel, [......],
Peter G Kremsner,
Sebastian Wesselborg,
Stefan Laufer,
Christoph S Clemen,
Claudia Herr,
Angelika A Noegel,
Thomas Wieder,
Erich Gulbins,
Florian Lang,
Stephan M Huber
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ABSTRACT: 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 PGE(2) formation, Ca(2+) 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 PGE(2)-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.
Cellular Physiology and Biochemistry 01/2009; 24(5-6):415-28. · 2.86 Impact Factor
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Michael Föller,
Susanne Feil,
Kamran Ghoreschi,
Saisudha Koka,
Andrea Gerling,
Martin Thunemann,
Franz Hofmann,
Beat Schuler,
Johannes Vogel,
Bernd Pichler, Ravi S Kasinathan,
Jan P Nicolay,
Stephan M Huber,
Florian Lang,
Robert Feil
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ABSTRACT: To explore the functional significance of cGMP-dependent protein kinase type I (cGKI) in the regulation of erythrocyte survival, gene-targeted mice lacking cGKI were compared with their control littermates. By the age of 10 weeks, cGKI-deficient mice exhibited pronounced anemia and splenomegaly. Compared with control mice, the cGKI mutants had significantly lower red blood cell count, packed cell volume, and hemoglobin concentration. Anemia was associated with a higher reticulocyte number and an increase of plasma erythropoietin concentration. The spleens of cGKI mutant mice were massively enlarged and contained a higher fraction of Ter119(+) erythroid cells, whereas the relative proportion of leukocyte subpopulations was not changed. The Ter119(+) cGKI-deficient splenocytes showed a marked increase in annexin V binding, pointing to phosphatidylserine (PS) exposure at the outer membrane leaflet, a hallmark of suicidal erythrocyte death or eryptosis. Compared with control erythrocytes, cGKI-deficient erythrocytes exhibited in vitro a higher cytosolic Ca(2+) concentration, a known trigger of eryptosis, and showed increased PS exposure, which was paralleled by a faster clearance in vivo. Together, these results identify a role of cGKI as mediator of erythrocyte survival and extend the emerging concept that cGMP/cGKI signaling has an antiapoptotic/prosurvival function in a number of cell types in vivo.
Proceedings of the National Academy of Sciences 06/2008; 105(18):6771-6. · 9.68 Impact Factor
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ABSTRACT: Human erythrocytes express cation channels which contribute to the background leak of Ca(2+), Na(+) and K(+). Excessive activation of these channels upon energy depletion, osmotic shock, Cl(-) depletion, or oxidative stress triggers suicidal death of erythrocytes (eryptosis), characterized by cell-shrinkage and exposure of phosphatidylserine at the cell surface. Eryptotic cells are supposed to be cleared from circulating blood. The present study aimed to identify the cation channels. RT-PCR revealed mRNA encoding the non-selective cation channel TRPC6 in erythroid progenitor cells. Western blotting indicated expression of TRPC6 protein in erythrocytes from man and wildtype mice but not from TRPC6(-/-) mice. According to flow-cytometry, Ca(2+) entry into human ghosts prepared by hemolysis in EGTA-buffered solution containing the Ca(2+) indicator Fluo3/AM was inhibited by the reducing agent dithiothreitol and the erythrocyte cation channel blockers ethylisopropylamiloride and amiloride. Loading of the ghosts with antibodies against TRPC6 or TRPC3/6/7 but neither with antibodies against TRPM2 or TRPC3 nor antibodies pre-adsorbed with the immunizing peptides inhibited ghost Ca(2+) entry. Moreover, free Ca(2+) concentration, cell-shrinkage, and phospholipid scrambling were significantly lower in Cl(-)-depleted TRPC6(-/-) erythrocytes than in wildtype mouse erythrocytes. In conclusion, human and mouse erythrocytes express TRPC6 cation channels which participate in cation leak and Ca(2+)-induced suicidal death.
Cellular Physiology and Biochemistry 02/2008; 21(1-3):183-92. · 2.86 Impact Factor
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Christophe Duranton,
Valerie Tanneur,
Camelia Lang,
Verena B Brand,
Saisudha Koka, Ravi S Kasinathan,
Martina Dorsch,
Hans J Hedrich,
Stefan Baumeister,
Klaus Lingelbach,
Florian Lang,
Stephan M Huber
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ABSTRACT: The intraerythrocytic development of P. falciparum induces New Permeability Pathways (NPP) in the membrane of the parasitized erythrocyte which provide the parasite with nutrients, adjust the erythrocyte electrolyte composition to the needs of the parasite, and dispose of metabolic waste products and osmolytes. Patch-clamp recordings identified inwardly and outwardly rectifying (OR) anion conductances in the host erythrocyte membrane as electrophysiological correlate of the NPP. The OR conductance is regulated by serum. Here we show that serum albumin (SA) stimulated OR-generated Cl(-) and lactate outward currents with an EC(50) of approximately 100 nM while other proteins such as ovalbumin or casein did not. The stimulatory efficacy did not differ between fatty acid free bovine SA and recombinant human SA and disruption of the SA tertiary structure abolished the effect suggesting that intact SA protein and not other bound factors interact with the erythrocyte membrane. Taken together, the data indicate a high affinity and specificity interaction of native SA with the parasitized erythrocytes which might underlie the observed dependence of P. falciparum growth in vitro on SA.
Cellular Physiology and Biochemistry 02/2008; 22(5-6):395-404. · 2.86 Impact Factor
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ABSTRACT: To test for redox regulation of anion channels in erythroid cells, we exposed K562 cells to oxidants and measured changes in transmembrane Cl(-) currents using patch-clamp, and in intracellular Cl(-) content using the Cl(-) selective dye MQAE. Oxidation with tert-butylhydroperoxide or H(2)O(2) produced a plasma membrane anion permeability with a permselectivity of NO(3)(-)>lactate(-)>gluconate(-). The permeability increase was paralleled by insertion of ClC-3 protein into the plasma membrane as evident from immunofluorescence microscopy and surface biotinylation. Down-regulation of ClC-3 protein by RNA interference as assessed by immunoblotting decreased the oxidation-stimulated permeability. In conclusion, oxidation induces surface expression of ClC-3 and activation of a ClC-3-dependent anion permeability in K562 cells.
FEBS Letters 11/2007; 581(28):5407-12. · 3.54 Impact Factor
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ABSTRACT: Eryptosis, a suicidal death of mature erythrocytes, is characterized by decrease of cell volume, cell membrane blebbing, and breakdown of cell membrane asymmetry with phosphatidylserine exposure at the cell surface. Triggers of eryptosis include increased cytosolic Ca(2+) activity, which could result from activation of Ca(2+)-permeable cation channels. Ca(2+) triggers phosphatidylserine exposure and activates Ca(2+)-sensitive K(+) channels, leading to cellular K(+) loss and cell shrinkage. The cation channels and thus eryptosis are stimulated by Cl(-) removal and inhibited by erythropoietin. The present experiments explored eryptosis in transgenic mice overexpressing erythropoietin (tg6). Erythrocytes were drawn from tg6 mice and their wild-type littermates (WT). Phosphatidylserine exposure was estimated from annexin binding and cell volume from forward scatter in fluorescence-activated cell sorting (FACS) analysis. The percentage of annexin binding was significantly larger and forward scatter significantly smaller in tg6 than in WT erythrocytes. Transgenic erythrocytes were significantly more resistant to osmotic lysis than WT erythrocytes. Cl(-) removal and exposure to the Ca(2+) ionophore ionomycin (1 microM) increased annexin binding and decreased forward scatter, effects larger in tg6 than in WT erythrocytes. The K(+) ionophore valinomycin (10 nM) triggered eryptosis in both tg6 and WT erythrocytes and abrogated differences between genotypes. An increase of extracellular K(+) concentration to 125 mM blunted the difference between tg6 and WT erythrocytes. Fluo-3 fluorescence reflecting cytosolic Ca(2+) activity was larger in tg6 than in WT erythrocytes. In conclusion, circulating erythrocytes from tg6 mice are sensitized to triggers of eryptosis but more resistant to osmotic lysis, properties at least partially due to enhanced Ca(2+) entry and increased K(+) channel activity.
AJP Regulatory Integrative and Comparative Physiology 10/2007; 293(3):R1127-34. · 3.34 Impact Factor
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Ahmad Akel,
Carsten A Wagner,
Jana Kovacikova, Ravi S Kasinathan,
Valentin Kiedaisch,
Saisudha Koka,
Seth L Alper,
Ingolf Bernhardt,
Thomas Wieder,
Stephan M Huber,
Florian Lang
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ABSTRACT: Genetic defects of anion exchanger 1 (AE1) may lead to spherocytic erythrocyte morphology, severe hemolytic anemia, and/or cation leak. In normal erythrocytes, osmotic shock, Cl(-) removal, and energy depletion activate Ca(2+)-permeable cation channels with Ca(2+)-induced suicidal erythrocyte death, i.e., surface exposure of phosphatidylserine, cell shrinkage, and membrane blebbing, all features typical for apoptosis of nucleated cells. The present experiments explored whether AE1 deficiency favors suicidal erythrocyte death. Peripheral blood erythrocyte numbers were significantly smaller in gene-targeted mice lacking AE1 (AE1(-/-) mice) than in their wild-type littermates (AE1(+/+) mice) despite increased percentages of reticulocytes (AE1(-/-): 49%, AE1(+/+): 2%), an indicator of enhanced erythropoiesis. Annexin binding, reflecting phosphatidylserine exposure, was significantly larger in AE1(-/-)erythrocytes/reticulocytes ( approximately 10%) than in AE1(+/+) erythrocytes ( approximately 1%). Osmotic shock (addition of 400 mM sucrose), Cl(-) removal (replacement with gluconate), or energy depletion (removal of glucose) led to significantly stronger annexin binding in AE1(-/-) erythrocytes/reticulocytes than in AE1(+/+) erythrocytes. The increase of annexin binding following exposure to the Ca(2+) ionophore ionomycin (1 muM) was, however, similar in AE1(-/-) and in AE1(+/+) erythrocytes. Fluo3 fluorescence revealed markedly increased cytosolic Ca(2+) permeability in AE1(-/-) erythrocytes/reticulocytes. Clearance of carboxyfluorescein diacetate succinimidyl ester-labeled erythrocytes/reticulocytes from circulating blood was more rapid in AE1(-/-) mice than in AE1(+/+) mice and was accelerated by ionomycin treatment in both genotypes. In conclusion, lack of AE1 is associated with enhanced Ca(2+) entry and subsequent scrambling of cell membrane phospholipids.
AJP Cell Physiology 06/2007; 292(5):C1759-67. · 3.54 Impact Factor
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ABSTRACT: Non-selective (NSC) cation channels participate in the Ca(2+) leak of human erythrocytes. Sustained activity of these channels triggers suicidal erythrocyte death (eryptosis), which is characterized by Ca(2+)-stimulated cell shrinkage and phosphatidylserine (PS) exposure. PS-exposing erythrocytes are rapidly cleared from circulating blood. PGE(2) activates the NSC channels, and erythrocyte PGE(2) formation is stimulated by a decrease in intra- or extracellular Cl(-) concentration. In addition, the intraerythrocytic malaria parasite Plasmodium falciparum activates the NSC channels, most probably to accomplish Na(+) and Ca(2+) entry into the erythrocyte cytosol required for parasite development. By Ca(2+) uptake the parasite maintains a low Ca(2+) concentration in the erythrocyte cytosol and thus delays the suicidal death of the host erythrocyte. Flufenamic acid has previously been shown to inhibit NSC channels. The present study thus explored the effect of flufenamic acid on erythrocyte Ca(2+) entry, on suicidal erythrocyte death and on intraerythrocytic growth of P. falciparum. Within 48 h, replacement of extracellular Cl(-) with gluconate or application of PGE(2) (50 microM) increased Fluo3 fluorescence reflecting cytosolic Ca(2+) activity, decreased forward scatter reflecting cell volume and increased annexin V binding reflecting PS exposure in FACS analysis. All those effects were significantly blunted in the presence of flufenamic acid (10 microM). Flufenamic acid (25 microM) further significantly delayed the intraerythrocytic growth of P. falciparum and the PS exposure of the infected erythrocytes. The present observations disclose a novel effect of flufenamic acid, which may allow the pharmacological manipulation of erythrocyte survival and the course of malaria.
Archiv für Experimentelle Pathologie und Pharmakologie 02/2007; 374(4):255-64. · 2.65 Impact Factor
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ABSTRACT: Prostaglandin-E2 (PGE2) is known to trigger suicidal death of nucleated cells (apoptosis) and enucleated erythrocytes (eryptosis). In erythrocytes PGE2 induced suicidal cell death involves activation of nonselective cation channels leading to Ca2+ entry followed by cell shrinkage and triggering of Ca2+ sensitive cell membrane scrambling with phosphatidylserine (PS) exposure at the cell surface. The present study was performed to explore whether PGE2 induces apoptosis of nucleated cells similarly through cation channel activation and to possibly disclose the molecular identity of the cation channels involved. To this end, Ca2+ activity was estimated from Fluo3 fluorescence, mitochondrial potential from DePsipher fluorescence, phosphatidylserine exposure from annexin binding, caspase activation from caspAce fluorescence, cell volume from FACS forward scatter, and DNA fragmentation utilizing a photometric enzyme immunoassay. Stimulation of K562 human leukaemia cells with PGE2 (50 microM) increased cytosolic Ca2+ activity, decreased forward scatter, depolarized the mitochondrial potential, increased annexin binding, led to caspase activation and resulted in DNA fragmentation. Gene silencing of the Ca2+-permeable transient receptor potential cation channel TRPC7 significantly blunted PGE2-induced triggering of PS exposure and DNA fragmentation. In conclusion, K562 cells express Ca2+-permeable TRPC7 channels, which are activated by PGE2 and participate in the triggering of apoptosis.
Cellular Physiology and Biochemistry 02/2006; 17(5-6):201-10. · 2.86 Impact Factor
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Valérie Tanneur,
Christophe Duranton,
Verena B Brand,
Ciprian D Sandu,
Canan Akkaya, Ravi S Kasinathan,
Christian Gachet,
Ronald Sluyter,
Julian A Barden,
James S Wiley,
Florian Lang,
Stephan M Huber
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ABSTRACT: 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.
The FASEB Journal 02/2006; 20(1):133-5. · 5.71 Impact Factor
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Stephan M Huber,
Christophe Duranton,
Guido Henke,
Claudia Van De Sand,
Volker Heussler,
Ekaterina Shumilina,
Ciprian D Sandu,
Valerie Tanneur,
Verena Brand, Ravi S Kasinathan,
Karl S Lang,
Peter G Kremsner,
Christian A Hübner,
Marco B Rust,
Karin Dedek,
Thomas J Jentsch,
Florian Lang
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ABSTRACT: Intraerythrocytic growth of the human malaria parasite Plasmodium falciparum depends on delivery of nutrients. Moreover, infection challenges cell volume constancy of the host erythrocyte requiring enhanced activity of cell volume regulatory mechanisms. Patch clamp recording demonstrated inwardly and outwardly rectifying anion channels in infected but not in control erythrocytes. The molecular identity of those channels remained elusive. We show here for one channel type that voltage dependence, cell volume sensitivity, and activation by oxidation are identical to ClC-2. Moreover, Western blots and FACS analysis showed protein and functional ClC-2 expression in human erythrocytes and erythrocytes from wild type (Clcn2(+/+)) but not from Clcn2(-/-) mice. Finally, patch clamp recording revealed activation of volume-sensitive inwardly rectifying channels in Plasmodium berghei-infected Clcn2(+/+) but not Clcn2(-/-) erythrocytes. Erythrocytes from infected mice of both genotypes differed in cell volume and inhibition of ClC-2 by ZnCl(2) (1 mm) induced an increase of cell volume only in parasitized Clcn2(+/+) erythrocytes. Lack of ClC-2 did not inhibit P. berghei development in vivo nor substantially affect the mortality of infected mice. In conclusion, activation of host ClC-2 channels participates in the altered permeability of Plasmodium-infected erythrocytes but is not required for intraerythrocytic parasite survival.
Journal of Biological Chemistry 11/2004; 279(40):41444-52. · 4.77 Impact Factor
