sq-23377 and Hemolysis

The high potency antipsychotic drug trifluoperazine (10-[3-(4-methyl-1-piperazinyl)-propyl]-2-(trifluoromethyl)-(10)H-phenothiazine dihydrochloride; TFP) may either counteract or promote suicidal cell death or apoptosis. Similar to apoptosis, erythrocytes may enter eryptosis, characterized by phosphatidylserine exposure at the cell surface and cell shrinkage. Eryptosis can be stimulated by an increase in cytoplasmic Ca2+ concentration ([Ca2+]i) and inhibited by nitric oxide (NO). We explored whether TFP treatment of erythrocytes induces phosphatidylserine exposure, cell shrinkage, and calcium influx, whether it impairs S-nitrosylation and whether these effects are inhibited by NO.. Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, and protein nitrosylation from fluorescence switch of the Bodipy-TMR/Sypro Ruby signal.. Exposure of human erythrocytes to TFP significantly enhanced the percentage of annexin-V-binding cells, raised [Ca2+]i, and decreased S-nitrosylation. The effect of TFP on annexin-V-binding was not affected by removal of extracellular Ca2+ alone, but was significantly inhibited by pre-treatment with sodium nitroprusside (SNP), an effect significantly augmented by additional removal of extracellular Ca2+. A 3 hours treatment with 0.1 µM Ca2+ ionophore ionomycin triggered annexin-V-binding and cell shrinkage, effects fully reversed by removal of extracellular Ca2+.. TFP induces eryptosis and decreases protein S-nitrosylation, effects blunted by nitroprusside. The effect of nitroprusside is attenuated in the presence of extracellular Ca2+.

Topics: Action Potentials; Calcium; Cell Size; Eryptosis; Erythrocyte Membrane; Erythrocytes; Hemolysis; Humans; Ionomycin; Microscopy, Fluorescence; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Patch-Clamp Techniques; Phosphatidylserines; Protein Processing, Post-Translational; Trifluoperazine

The antihistaminic drug Terfenadine may trigger apoptosis of tumor cells, an effect unrelated to its effect on histamine receptors. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal death of erythrocytes characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling triggering eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, and ceramide. The present study explored, whether Terfenadine is capable to trigger eryptosis.. Flow cytometry was employed to estimate phosphatidylserine abundance at the erythrocyte surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, abundance of reactive oxygen species (ROS) from 2',7'-dichlorodihydrofluorescein (DCF) diacetate dependent fluorescence, and ceramide abundance at the human erythrocyte surface utilizing specific antibodies. Hemolysis was quantified from haemoglobin concentration in the supernatant.. A 48 hours exposure of human erythrocytes to Terfenadine (≥ 5 µM) significantly increased the percentage of annexin-V-binding cells and triggered hemolysis without significantly modifying the average forward scatter. Terfenadine (7.5 µM) significantly increased Fluo3-fluorescence, but did not significantly modify DCF fluorescence or ceramide abundance. The effect of Terfenadine on annexin-V-binding was significantly blunted but not abolished by removal of extracellular Ca2+. Exposure of human erythrocytes to Ca2+ ionophore ionomycin (1 µM, 15 min) triggered annexin-V-binding, an effect augmented by Terfenadine pretreatment (10 µM, 48 hours).. Terfenadine triggers phospholipid scrambling of the human erythrocyte cell membrane, an effect in part due to entry of extracellular Ca2+ and in part due to sensitizing human erythrocyte cell membrane scrambling to Ca2+.

Topics: Calcium; Calcium Ionophores; Ceramides; Eryptosis; Erythrocyte Membrane; Erythrocytes; Flow Cytometry; Hemolysis; Histamine H1 Antagonists, Non-Sedating; Humans; Ionomycin; Phosphatidylserines; Reactive Oxygen Species; Terfenadine

Ribavirin in combination with interferon-α is the standard treatment for chronic hepatitis C, but often induces severe anemia forcing discontinuation of the therapy. Whereas suppression of bone marrow by interferon may impact on the production of erythrocytes, it has been suggested that accumulation of ribavirin in erythrocytes induces alterations causing an early removal of these cells by the mononuclear phagocytic system. Externalization of phosphatidylserine, which is exclusively present in the cytoplasmic leaflet of the plasma membrane, is a recognition signal for phagocytosis in particular of apoptotic cells. Here, we demonstrate that surface exposure of phosphatidylserine upon prolonged treatment of erythrocytes with ribavirin results mainly from inactivation of the aminophospholipid translocase, an ATP-dependent lipid pump, which specifically transports phosphatidylserine from the outer to the inner leaflet of the plasma membrane. Inactivation is due to severe ATP depletion, although competitive inhibition by ribavirin or its phosphorylated derivatives cannot be excluded. Phospholipid scramblase, responsible for collapse of lipid asymmetry, appears to be of minor importance as erythrocytes of patients with the Scott syndrome, lacking Ca(2+)-induced lipid scrambling, are equally sensitive to ribavirin treatment. Neither the antioxidant N-acetylcysteine nor the pan-caspase inhibitor Q-VD-OPH did affect ribavirin-induced phosphatidylserine exposure, suggesting that oxidative stress or apoptotic-related mechanisms are not involved in this process. In conclusion, we propose that spontaneous loss of lipid asymmetry, not corrected by aminophospholipid translocase activity, is the mechanism for ribavirin-induced phosphatidylserine exposure that may contribute to ribavirin-induced anemia.

Topics: Adenosine Triphosphate; Antiviral Agents; Cells, Cultured; Erythrocytes; Hemolysis; Humans; Ionomycin; Phosphatidylserines; Phospholipid Transfer Proteins; Ribavirin

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.

Topics: Anemia, Hemolytic; Animals; Anion Exchange Protein 1, Erythrocyte; Annexin A5; Apoptosis; Calcium; Chlorides; Erythrocyte Membrane; Erythrocytes; Glucose; Hemolysis; Hypertonic Solutions; Ionomycin; Ionophores; Mice; Mice, Knockout; Osmotic Pressure; Phosphatidylserines; Platelet Count; Reticulocyte Count; Reticulocytes; Reticulocytosis; Sucrose; Time Factors

Haemolysin Kanagawa, a toxin from Vibrio parahaemolyticus, is known to trigger haemolysis. Flux studies indicated that haemolysin forms a cation channel. In the present study, channel properties were elucidated by patch clamp and functional significance of ion fluxes by fluorescence-activated cell sorting (FACS) analysis. Treatment of human erythrocytes with 1 U ml-1 haemolysin within minutes induces a non-selective cation permeability. Moreover, haemolysin activates clotrimazole-sensitive K+ channels, pointing to stimulation of Ca2+-sensitive Gardos channels. Haemolysin (1 U ml-1) leads within 5 min to slight cell shrinkage, which is reversed in Ca2+-free saline. Erythrocytes treated with haemolysin (0.1 U ml-1) do not undergo significant haemolysis within the first 60 min. Replacement of extracellular Na+ with NMDG+ leads to slight cell shrinkage, which is potentiated by 0.1 U ml-1 haemolysin. According to annexin binding, treatment of erythrocytes with 0.1 U ml-1 haemolysin leads within 30 min to breakdown of phosphatidylserine asymmetry of the cell membrane, a typical feature of erythrocyte apoptosis. The annexin binding is significantly blunted at increased extracellular K+ concentrations and by K+ channel blocker clotrimazole. In conclusion, haemolysin Kanagawa induces cation permeability and activates endogenous Gardos K+ channels. Consequences include breakdown of phosphatidylserine asymmetry, which depends at least partially on cellular loss of K+.

Topics: Annexins; Apoptosis; Calcium; Cations; Cell Membrane Permeability; Cell Size; Clotrimazole; Erythrocyte Membrane; Erythrocytes; Flow Cytometry; Hemolysin Proteins; Hemolysis; Humans; In Vitro Techniques; Ionomycin; Patch-Clamp Techniques; Potassium; Potassium Channels; Vibrio parahaemolyticus

Erythrocyte Ca2+ overload is known to occur in several different disease states, and to affect the erythrocyte membrane deformability. We show here that an increase in intracellular Ca2+ concentration in erythrocytes, induced by ionomycin, caused a reduction in ATP levels. Concomitant to the fall in ATP, a marked activation of phosphofructokinase (PFK) (EC 2.7.1.11), the rate-limiting enzyme in glycolysis, in the membrane skeleton fraction occurred. The increase in the membrane skeleton-bound PFK activity was most probably mediated by Ca2+, as direct addition of Ca2+ to the membrane skeleton fraction from the erythrocyte induced an enhancement of the bound PFK activity. Time-response curves revealed that erythrocyte hemolysis did not occur during the first 30 min of incubation with ionomycin, when the membrane skeleton-bound PFK was activated. Longer incubation time resulted in solubilization of the membrane skeleton-bound PFK and a concomitant hemolysis of the erythrocytes. These results suggest that the Ca2+-induced activation of membrane skeleton-bound PFK, and thereby glycolysis, the sole source of energy in erythrocytes, may be a defense mechanism to surmount the damage induced by high Ca2+ levels.

Topics: Adenosine Triphosphate; Animals; Calcium; Dose-Response Relationship, Drug; Erythrocyte Membrane; Erythrocytes; Hemolysis; Ionomycin; Ionophores; Male; Phosphofructokinase-1; Rats; Time Factors

Calcium in millimolar concentrations protected mouse erythrocytes from hemolysis caused by Vibrio vulnificus cytolysin without affecting the release of intracellular K+ from the cells. This effect was maximal at 25 mM CaCl2. The protection was not absolute and could be partially overcome by increased concentrations of cytolysin. Calcium failed to block both the binding and oligomer formation of cytolysins on the erythrocyte membrane. After pore formation, the continued presence of calcium is required for the prevention of hemolysis. There was hardly any inflow of calcium into the erythrocytes through pores as measured by 45Ca2+ uptake. The presence of calcium after the abolition of Ca2+ gradient by ionomycin cannot inhibit the hemolysis caused by cytolysin. These results suggest that calcium exerts its major inhibitory effect on V. vulnificus cytolysin-induced hemolysis as an osmotic protectant, and that cytolysin may become an useful tool for permeabilizing cells selectively for small ions such as potassium or sodium while preventing the Ca2+ flow.

Topics: Animals; Calcium; Cytotoxins; Erythrocyte Membrane; Hemolysis; In Vitro Techniques; Ionomycin; Mice; Potassium; Raffinose; Vibrio