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Putative functions of the heterotrimeric G-protein subunit Gαi2-dependent signaling include ion channel regulation, cell differentiation, proliferation and apoptosis. Erythrocytes may, similar to apoptosis of nucleated cells, undergo eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine (PS) exposure. Eryptosis may be triggered by increased cytosolic Ca(2+) activity and ceramide. In the present study, we show that Gαi2 is expressed in both murine and human erythrocytes and further examined the survival of erythrocytes drawn from Gαi2-deficient mice (Gαi2(-/-)) and corresponding wild-type mice (Gαi2(+/+)). Our data show that plasma erythropoietin levels, erythrocyte maturation markers, erythrocyte counts, hematocrit and hemoglobin concentration were similar in Gαi2(-/-) and Gαi2(+/+) mice but the mean corpuscular volume was significantly larger in Gαi2(-/-) mice. Spontaneous PS exposure of circulating Gαi2(-/-) erythrocytes was significantly lower than that of circulating Gαi2(+/+) erythrocytes. PS exposure was significantly lower in Gαi2(-/-) than in Gαi2(+/+) erythrocytes following ex vivo exposure to hyperosmotic shock, bacterial sphingomyelinase or C6 ceramide. Erythrocyte Gαi2 deficiency further attenuated hyperosmotic shock-induced increase of cytosolic Ca(2+) activity and cell shrinkage. Moreover, Gαi2(-/-) erythrocytes were more resistant to osmosensitive hemolysis as compared to Gαi2(+/+) erythrocytes. In conclusion, Gαi2 deficiency in erythrocytes confers partial protection against suicidal cell death.

Background: The microtubule assembly inhibitor nocodazole has been shown to trigger caspase-independent mitotic death and caspase dependent apoptosis. Similar to apoptosis of nucleated cells, erythrocytes may undergo eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress and ceramide. The present study explored, whether and how nocodazole induces eryptosis., Methods: Flow cytometry was employed to determine phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, the abundance of reactive oxygen species (ROS) from 2',7'-dichlorodihydrofluorescein (DCF) diacetate dependent fluorescence as well as ceramide surface abundance utilizing specific antibodies. Tubulin abundance was quantified by TubulinTracker™ Green reagent and visualized by confocal microscopy., Results: A 48 hours exposure of human erythrocytes to nocodazole (≥ 30 µg/ml) significantly increased the percentage of annexin-V-binding cells without significantly modifying average forward scatter. Nocodazole significantly increased Fluo3-fluorescence, significantly increased DCF fluorescence and significantly increased ceramide surface abundance. The effect of nocodazole on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+ and was not modified in the presence of Caspase 3 inhibitor zVAD (1 µM). Nocodazole treatment reduced the content of total tubulin., Conclusions: Nocodazole triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to stimulation of Ca2+ entry, oxidative stress and ceramide., (© 2016 The Author(s) Published by S. Karger AG, Basel.)

The polyphenolic flavonoid Baicalein has been shown to trigger suicidal death or apoptosis of tumor cells and is thus considered for the prevention and treatment of malignancy. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i) and ceramide. The present study explored whether Baicalein stimulates eryptosis. To this end, forward scatter was taken for measurement of cell volume, annexin-V-binding for phosphatidylserine-exposure, Fluo3 fluorescence for [Ca2+]i and fluorescent antibodies for ceramide abundance. As a result, a 48 h exposure of human erythrocytes to Baicalein was followed by significant decrease of forward scatter (≥10 µM), significant increase of the percentage of annexin-V-binding cells (≥25 µM), significant increase of [Ca2+]i (50 µM) and significant increase of ceramide abundance (50 µM). The effect of Baicalein (50 µM) on annexin-V-binding was significantly blunted but not abrogated by removal of extracellular Ca2+. In conclusion, at the concentrations employed, Baicalein stimulates suicidal erythrocyte death or eryptosis, an effect at least in part due to the combined effects of Ca2+ entry and ceramide formation.

Unlabelled: Anemia in end-stage renal disease (ESRD) results mainly from erythropoietin and iron deficiency. Anemia could be confounded, however, by accelerated clearance of circulating erythrocytes because of premature suicidal erythrocyte death or eryptosis characterized by phosphatidylserine exposure at the erythrocyte surface. Triggers of eryptosis include increased cytosolic Ca(2+) concentration ([Ca(2+)]i), oxidative stress, and ceramide. The present study explored whether and how ESRD influences eryptosis. Blood was drawn from healthy volunteers (n = 20) as well as ESRD patients (n = 20) prior to and after hemodialysis. Phosphatidylserine exposure was estimated from annexin V binding, [Ca(2+)]i from Fluo3-fluorescence, reactive oxygen species (ROS) from 2',7'dichlorodihydrofluorescein fluorescence, and ceramide from fluorescein-isothiocyanate-conjugated antibody binding in flow cytometry. Measurements were made in erythrocytes from freshly drawn blood and in erythrocytes from healthy volunteers exposed in vitro for 24 h to plasma from healthy volunteers or ESRD patients prior to and following dialysis. The patients suffered from anemia (hemoglobin 10.1 ± 0.5 g/100 ml) despite 1.96 ± 0.34 % reticulocytes. The percentage of phosphatidylserine-exposing erythrocytes was significantly higher in ESRD patients (0.84 ± 0.09 %) than in healthy volunteers (0.43 ± 0.04 %) and was significantly increased immediately after dialysis (1.35 ± 0.13 %). The increase in phosphatidylserine exposure was paralleled by increase in [Ca(2+)]i, oxidative stress, and ceramide abundance. As compared to addition of plasma from healthy individuals, addition of predialytic but not of postdialytic plasma from ESRD patients increased phosphatidylserine exposure, [Ca(2+)]i, ROS, and ceramide abundance. In conclusion, both, dialyzable components of uremic plasma and dialysis procedure, trigger eryptosis at least in part by increasing erythrocyte [Ca(2+)]i, ROS, and ceramide formation., Key Messages: Anemia in uremia results in part from eryptosis, the suicidal erythrocyte death. Eryptosis in uremia is triggered in part by a dialyzable plasma component. Eryptosis in uremia is further triggered by dialysis procedure. Eryptosis in uremia is in part due to increased cytosolic Ca(2+) concentration. Eryptosis in uremia is further due to oxidative stress and ceramide formation.

Saponins, naturally occurring glycosides and triterpene glycosides in plants, are considered useful in the prophylaxis and treatment of several disorders, including malignancy. The effect of these substances is partly attributable to induction of both apoptosis and necrosis. Saponin has previously been shown to trigger hemolysis. Erythrocytes may avoid hemolysis by entering programmed cell death or eryptosis, characterized by cell shrinkage and cell membrane scrambling, leading to phosphatidylserine exposure at the erythrocyte surface. Eryptosis is triggered by increase of cytosolic Ca(2+) activity ([Ca(2+)](i)). The present study explored, whether exposure of human erythrocytes to saponin modifies [Ca(2+)](i), ceramide formation, hemolysis, and eryptosis. Cell volume was estimated from forward scatter, phosphatidylserine exposure from annexin V binding, hemolysis from hemoglobin release, [Ca(2+)](i) from Fluo3-fluorescence, and ceramide utilizing specific antibodies. A 24 h exposure to saponin (15 µg/ml) resulted in a significant increase of annexin V binding and a significant stimulation of hemolysis. Saponin (15 µg/ml) further increased [Ca(2+)](i) and ceramide formation. Annexin V binding was significantly blunted but not abrogated in the nominal absence of extracellular Ca(2+). Saponin thus triggers cell membrane scrambling, an effect partially due to entry of extracellular Ca(2+) and ceramide formation.

The polyphenolic 1,2,3,4,6-penta-O-galloyl-beta-D-glucose from several medicinal herbs triggers apoptosis and has, thus, been proposed for treatment of malignancy. The substance is at least partially effective through caspase activation. In analogy to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and by phosphatidylserine translocation to the erythrocyte surface. Eryptosis is triggered by increase of cytosolic Ca2+-activity ([Ca2+]i). The sensitivity to [Ca2+]i is enhanced by ceramide. The present study explored whether penta-O-galloyl-β-D-glucose stimulates eryptosis. Cell volume was estimated from forward scatter, phosphatidylserine exposure from annexin V binding, hemolysis from hemoglobin-release, [Ca2+]i from Fluo3-fluorescence and ceramide abundance from fluorescent antibodies. A 48-h exposure of human erythrocytes to penta-O-galloyl-β-D-glucose significantly decreased forward scatter (50 µM) and significantly increased annexin V binding (10 µM). Up to 50 µM penta-O-galloyl-β-D-glucose did not significantly modify [Ca2+]i. However, the effect of penta-O-galloyl-β-D-glucose (25 µM) induced annexin V binding was slightly, but significantly, blunted by removal of extracellular Ca2+, pointing to sensitization of erythrocytes to the scrambling effect of Ca2+. Penta-O-galloyl-β-D-glucose (25 µM) further increased ceramide formation. In conclusion, penta-O-galloyl-β-D-glucose stimulates suicidal erythrocyte death or eryptosis, an effect partially due to stimulation of ceramide formation with subsequent sensitization of erythrocytes to Ca2+.

Background: Eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and phosphatidylserine-translocation, is triggered by fever and inflammation. Signaling includes increased cytosolic Ca2+-activity ([Ca2++]i), caspase activation, and ceramide. Inflammation is associated with increased plasma concentration of C-reactive protein (CRP). The present study explored whether CRP triggers eryptosis. Methods: Phosphatidylserine abundance at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ceramide abundance and caspase-3-activity utilizing FITC-conjugated antibodies. Moreover, blood was drawn from patients with acute appendicitis (9♀,11♂) and healthy volunteers (10♀,10♂) for determination of CRP, blood count and phosphatidylserine. Results: A 48h CRP treatment significantly increased the percentage of annexin-V-binding cells (⩾5μg/ml), [Ca2+]i (⩾5μg/ml), ceramide (20μg/ml) and caspase-activity (20μg/ml). Annexin- V-binding was significantly blunted by caspase inhibitor zVAD (10μM). The percentage of phosphatidylserine-exposing erythrocytes in freshly drawn blood was significantly higher in appendicitis patients (1.83±0.21%) than healthy volunteers (0.81±0.09%), and significantly higher following a 24h incubation of erythrocytes from healthy volunteers to patient plasma than to plasma from healthy volunteers. The percentage of phosphatidylserine-exposing erythrocytes correlated with CRP plasma concentration. Conclusion: C-reactive protein triggers eryptosis, an effect at least partially due to increase of [Ca2+]i, increase of ceramide abundance and caspase activation. [ABSTRACT FROM AUTHOR]

Background/Aims: Arteritis is an inflammatory disease of the vascular wall leading to ischemia and vascular occlusion. Complications of arteritis include anemia, which could, at least in theory, result from suicidal erythrocyte death or eryptosis, which is characterized by erythrocyte shrinkage and phosphatidylserine (PS) exposure at the erythrocyte surface. Cellular mechanisms involved in the stimulation of eryptosis include increased cytosolic Ca2+-concentration ([Ca2+]i), oxidative stress and ceramide formation. The present study explored whether and how arteritis influences eryptosis. Methods: Blood was drawn from patients suffering from arteritis (n=17) and from healthy volunteers (n=21). PS exposure was estimated from annexin V-binding, erythrocyte volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, reactive oxygen species (ROS) from DCFDA fluorescence and ceramide abundance from FITC-conjugated antibody binding in flow cytometry. The patients suffered from anemia despite 2.8±0.4% reticulocytes. Results: The percentage of PS-exposing erythrocytes was significantly higher in patients (1.1±0.1%) than in healthy volunteers (0.3±0.1%). The increase in PS exposure was paralleled by increase in oxidative stress and [Ca2+]i but not by significant changes of ceramide abundance. Erythrocyte PS exposure and ROS production were significantly enhanced in erythrocytes exposed to patient plasma as compared to exposure to plasma from healthy volunteers. Conclusion: Arteritis is associated with enhanced eryptosis due to increased [Ca2+]i and oxidative stress. The eryptosis contributes to or even accounts for the anemia in those patients. As eryptotic erythrocytes adhere to endothelial cells of the vascular wall, they could impede microcirculation and thus contribute to vascular occlusion. [ABSTRACT FROM AUTHOR]

Background/Aims: Chorein is a protein expressed in various cell types. Loss of function mutations of the chorein encoding gene VPS13A lead to chorea-acanthocytosis, an autosomal recessive genetic disease characterized by movement disorder and behavioral abnormalities. Recent observations revealed that chorein is a powerful regulator of actin cytoskeleton in erythrocytes, platelets, K562 and endothelial HUVEC cells. Methods: In the present study we have used Western blotting to study actin polymerization dynamics, laser scanning microscopy to evaluate in detail the role of chorein in microfilaments, microtubules and intermediate filaments cytoskeleton architecture and RT-PCR to assess gene transcription of the cytoskeletal proteins. Results: We report here powerful depolymerization of actin microfilaments both, in erythrocytes and fibroblasts isolated from chorea-acanthocytosis patients. Along those lines, morphological analysis of fibroblasts from chorea-acanthocytosis patients showed disarranged microtubular network, when compared to fibroblasts from healthy donors. Similarly, the intermediate filament networks of desmin and cytokeratins showed significantly disordered organization with clearly diminished staining in patient's fibroblasts. In line with this, RT-PCR analysis revealed significant downregulation of desmin and cytokeratin gene transcripts. Conclusion: Our results provide for the first time evidence that defective chorein is accompanied by significant structural disorganization of all cytoskeletal structures in human fibroblasts from chorea-acanthocytosis patients. © 2015 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The polyphenolic 1,2,3,4,6-penta-O-galloyl-beta-D-glucose from several medicinal herbs triggers apoptosis and has, thus, been proposed for treatment of malignancy. The substance is at least partially effective through caspase activation. In analogy to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and by phosphatidylserine translocation to the erythrocyte surface. Eryptosis is triggered by increase of cytosolic Ca2+-activity ([Ca2+]i). The sensitivity to [Ca2+]i is enhanced by ceramide. The present study explored whether penta-O-galloyl-β-D-glucose stimulates eryptosis. Cell volume was estimated from forward scatter, phosphatidylserine exposure from annexin V binding, hemolysis from hemoglobin-release, [Ca2+]i from Fluo3-fluorescence and ceramide abundance from fluorescent antibodies. A 48-h exposure of human erythrocytes to penta-O-galloyl-β-D-glucose significantly decreased forward scatter (50 μM) and significantly increased annexin V binding (10 μM). Up to 50 μM penta-O-galloyl-β-D-glucose did not significantly modify [Ca2+]i. However, the effect of penta-O-galloyl-β-D-glucose (25 μM) induced annexin V binding was slightly, but significantly, blunted by removal of extracellular Ca2+, pointing to sensitization of erythrocytes to the scrambling effect of Ca2+. Penta-O-galloyl-β-D-glucose (25 μM) further increased ceramide formation. In conclusion, penta-O-galloyl-β-D-glucose stimulates suicidal erythrocyte death or eryptosis, an effect partially due to stimulation of ceramide formation with subsequent sensitization of erythrocytes to Ca2+. [ABSTRACT FROM AUTHOR]