Pharmacological modification of protein kinase CK1 (casein kinase 1) has previously been shown to influence suicidal erythrocyte death or eryptosis, which is triggered by activation of Cl--sensitive Ca2+-permeable cation channels. Ca2+ entering through those channels stimulates cell membrane scrambling and opens Ca2+-activated K+-channels resulting in KCl exit and thus cell shrinkage. The specific CK1-inhibitor D4476 (1 µM) blunted, whereas the specific CK1 αactivator pyrvinium pamoate (10 µM) enhanced cell membrane scrambling. The substances were at least partially effective through modification of cytosolic Ca2+-activity. The present study explored, whether pyrvinium pamoate indeed influences Cl--sensitive cation-channels in erythrocytes. As a result, removal of Cl-increased Fluo3-fluorescence (reflecting cytosolic Ca2+-activity), triggered cell membrane scrambling (apparent from annexin-V-binding), and decreased forward scatter (pointing to cell shrinkage). Pyrvinium pamoate significantly augmented the effect of Cl--removal on Fluo3 fluorescence and annexin-V-binding, but blunted the effect on forward scatter. According to whole cell patch clamp recording, Cl-removal activated a cation current, which was significantly enhanced by pyrvinium pamoate. Pyrvinium pamoate inhibited Ca2+-activated K+-channels. Ca2+-ionophore ionomycin (1 µM) decreased forward scatter, an effect significantly blunted by pyrvinium pamoate. In conclusion, pyrvinium pamoate activates Cl--sensitive Ca2+-permeable cation channels with subsequent Ca2+-entry and inhibits Ca2+-activated K+-channels thus blunting the stimulating effect of Ca2+ on those channels, K+-exit and thus cell shrinkage.
Sphingosine kinase 1 phosphorylates sphingosine, which is converted to ceramide by ceramide synthetase. Ceramide triggers eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and phosphatidylserine (PS) exposure at the erythrocyte surface. Erythrocytes lack sphingosine phosphate-degrading enzymes and thus store large quantities of sphingosine phosphate. The present study explored the influence of sphingosine and sphingosine phosphate on eryptosis. [Ca2+]i, was estimated from Fluo3 fluorescence, cell volume from forward scatter and PS exposure from annexin V-binding in FACS analysis. Sphingosine (0.1 – 10 µM) but not sphingosine-1- phosphate (0.1 – 10 µM) increased [Ca2+]i, decreased cell volume and increased PS-exposure. The observations disclose sphingosine, but not sphingosine-1-phosphate, as a strong inducer of eryptosis.
Background: Furosemide, a loop diuretic inhibiting the renal tubular Na+,K+,2Cl- cotransporter, has been shown to decrease cytosolic Ca2+ concentration ([Ca2+]i) in platelets and erythrocytes. [Ca2+]i in erythrocytes is a function of Ca2+ permeable cation channels. Activation of those channels e.g. by energy depletion or oxidative stress leads to increase of [Ca2+]i, which in turn triggers eryptosis, a suicidal erythrocyte death characterized by cell membrane scrambling. The present study was performed to explore whether furosemide influences the cation channels and thus influences eryptosis. Methods: Cation channel activity was determined by whole-cell patch clamp, [Ca2+]i utilizing Fluo3 fluorescence and annexin V binding to estimate cell membrane scrambling with phosphatidylserine exposure. Results: A 45 min exposure to furosemide (10 and 100 µM) slightly, but significantly decreased cation channel activity and [Ca2+]i in human erythrocytes drawn from healthy individuals. ATP-depletion (> 3 hours, +37°C, 6 mM ionosine and 6 mM iodoacetic acid) enhanced the non-selective cation channel activity, increased [Ca2+]i and triggered cell membrane scrambling, effects significantly blunted by furosemide (10 – 100 µM). Oxidative stress by exposure to tert-butylhydroperoxide (0.1 –1 mM) similarly enhanced the non-selective cation channels activity, increased [Ca2+]i and triggered cell membrane scrambling, effects again significantly blunted by furosemide (10 – 100 µM). Conclusions: The present study shows for the first time that the loop diuretic furosemide applied at micromolar concentrations (10 – 100 µM) inhibits non-selective cation channel activity in and eryptosis of human erythrocytes.
Dicoumarol, a widely used anticoagulant, may cause anemia, which may result from enhanced erythrocyte loss due to bleeding or due to accelerated erythrocyte death. Erythrocytes may undergo suicidal death or eryptosis, characterized by cell shrinkage and phospholipid scrambling of the cell membrane. Eryptosis may be triggered by increase of cytosolic 2+-activity ([Ca2+]i). The present study explored, whether dicoumarol induces eryptosis. [Ca2+]i was estimated from Fluo3-fluorescence, cation channel activity utilizing whole cell patch clamp, cell volume from forward scatter, phospholipid scrambling from annexin-V-binding, and hemolysis from haemoglobin release. Exposure of erythrocytes for 48 hours to dicoumarol (=10 µM) significantly increased [Ca2+]i, enhanced cation channel activity, decreased forward scatter, triggered annexin-V-binding and elicited hemolysis. Following exposure to 30 µM dicoumarol, annexin-V-binding affected approximately 15%, and hemolysis 2% of treated erythrocytes. The stimulation of annexin-V-binding by dicoumarol was abrogated in the nominal absence of 2+. In conclusion, dicoumarol stimulates suicidal death of erythrocytes by stimulating Ca2+ entry and subsequent triggering of 2+ dependent cell membrane scrambling.
Annexin A7 is a ubiquitously expressed Ca(2+)- and phospholipid-binding protein. Erythrocytes from mice lacking annexin A7 (anxA7(-/-)) are deformed and relatively resistant to osmotic swelling. In normal erythrocytes, hyperosmotic shock, Cl(-) removal, and energy depletion (glucose removal) trigger PGE(2) formation, which stimulates Ca(2+)-permeable cation channels, increases cytosolic Ca(2+) activity ([Ca(2+)](i)), and thus triggers suicidal death of erythrocytes or eryptosis, characterized by scrambling of the cell membrane with phosphatidylserine exposure at the cell surface. The present experiments explored the influence of annexin A7 deficiency on eryptosis. In erythrocytes from annexin A7-deficient mice (anxA7(-/-)) and wild-type mice (anxA7(+/+)), PGE(2) formation was determined utilizing an immunoassay, ion channel activity by whole-cell patch clamp recording, [Ca(2+)](i) by fluo3 fluorescence, and phosphatidylserine exposure by binding of annexin A5 in fluorescence activated cell sorter (FACS) analysis. Erythrocyte number and hematocrit were significantly smaller in blood from anx7(-/-) than in anx7(+/+) mice. Cl(-)-removal (replacement with gluconate) stimulated PGE(2)-formation, activated cation currents, increased [Ca(2+)](i), and triggered phosphatidylserine exposure, effects significantly more pronounced in anx7(-/-) than in anx7(+/+) erythrocytes. Hyperosmotic shock (addition of 400 mM sucrose) and glucose depletion (removal of glucose) similarly increased cytosolic Ca(2+) activity and triggered phosphatidylserine exposure, effects again significantly more pronounced in anx7(-/-) than in anx7(+/+) erythrocytes. The effects of Cl(-) removal on PGE(2) formation and the cation current, as well as the effect of hypertonic cell shrinkage on [Ca(2+)](i) and cell membrane scrambling, were blunted following inhibition of cyclooxygenase by aspirin or diclofenac. In conclusion, lack of annexin A7 sensitizes the erythrocytes for "proapoptotic" Ca(2+) overload, an effect shortening the life span of the affected erythrocytes and, thus, leading to anemia.
Excessive glucose concentrations foster glycation and thus premature aging of erythrocytes. The present study explored whether glycation-induced erythrocyte aging is paralleled by features of suicidal erythrocyte death or eryptosis, which is characterized by cell membrane scrambling with subsequent phosphatidylserine exposure at the cell surface and cell shrinkage. Both are triggered by increases of cytosolic Ca(2+) concentration ([Ca(2+)](i)), which may result from activation of Ca(2+) permeable cation channels. Glycation was accomplished by exposure to high glucose concentrations (40 and 100 mM), phosphatidylserine exposure estimated from annexin binding, cell shrinkage from decrease of forward scatter, and [Ca(2+)](i) from Fluo3-fluorescence in analysis via fluorescence-activated cell sorter. Cation channel activity was determined by means of whole-cell patch clamp. Glycation of total membrane proteins, immunoprecipitated TRPC3/6/7, and immunoprecipitated L-type Ca(2+) channel proteins was estimated by Western blot testing with polyclonal antibodies used against advanced glycation end products. A 30-48-h exposure of the cells to 40 or 100 mM glucose in Ringer solution (at 37 degrees C) significantly increased glycation of membrane proteins, hemoglobin (HbA(1c)), TRPC3/6/7, and L-type Ca(2+) channel proteins, enhanced amiloride-sensitive, voltage-independent cation conductance, [Ca(2+)](i), and phosphatidylserine exposure, and led to significant cell shrinkage. Ca(2+) removal and addition of Ca(2+) chelator EGTA prevented the glycation-induced phosphatidylserine exposure and cell shrinkage after glycation. Glycation-induced erythrocyte aging leads to eryptosis, an effect requiring Ca(2+) entry from extracellular space.
Survival of the malaria pathogen Plasmodium falciparum in host erythrocytes requires the opening of new permeability pathways (NPPs) in the host cell membrane, accomplishing entry of nutrients, exit of metabolic waste products such as lactate and movement of inorganic ions such as Cl⁻, Na⁺ and Ca²⁺. The molecular identity of NPPs has remained largely elusive but presumably involves several channels, which partially can be activated by oxidative stress in uninfected erythrocytes. One NPP candidate is aquaporin 9 (AQP9), a glycerol-permeable water channel expressed in erythrocytes. Gene-targeted mice lacking functional AQP9 (aqp⁻/⁻) survive infection with the malaria pathogen Plasmodium berghei better than their wild-type littermates (aqp9⁺/⁺). In the present study whole-cell patch-clamp recordings were performed to explore whether ion channel activity is different in erythrocytes from aqp⁻/⁻ and aqp9⁺/⁺ mice. As a result, the cation conductance (K⁺ > Na⁺ > Ca²⁺ ≫ NMDG⁺) was significantly lower in erythrocytes from aqp⁻/⁻ than in erythrocytes from aqp9⁺/⁺ mice. Oxidative stress by exposure for 15-30 min to 1 mM H₂O₂ or 1 mM tert-butyl-hydroperoxide enhanced the cation conductance and increased cytosolic Ca²⁺ concentration, effects significantly less pronounced in erythrocytes from aqp⁻/⁻ than in erythrocytes from aqp9⁺/⁺ mice. In conclusion, lack of AQP9 decreases the cation conductance of erythrocytes, an effect that possibly participates in the altered susceptibility of AQP9-deficient mice to infection with P. berghei.
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