[Current insights into anemia in old age : Summary of the symposium "Anemia in old age" on the occasion of the annual congress of the German Society for Geriatrics (DGG) 2016 in Stuttgart]. / Aktuelle Einblicke in die Anämie im Alter : Zusammenfassung zum Symposium "Anämie im Alter" anlässlich der DGG-Jahrestagung 2016 in Stuttgart.

Anemia in advanced age is often a multifactorial condition requiring an interdisciplinary approach. The contributions to the opening interdisciplinary symposium on anemia in older subjects focused on physiological and histopathological as well as on nephrological and neurogeriatric aspects and on the therapeutic implications of this underdiagnosed, yet highly frequent disease. The symposium was the kick-off event for the founding of the German Geriatric Society special interest group on anemia in advanced age.

Impact of cyclin-dependent kinase CDK4 inhibition on eryptosis.

BACKGROUND/AIMS: The cyclin-dependent kinase 4 (CDK4) participates in the regulation of apoptosis of nucleated cells by altering transcriptional regulation of genes governing cell proliferation and cell death. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine (PS) exposure at the cell surface. As mature erythrocytes lack nuclei, acute stimulation of eryptosis cannot result from altered gene expression. Eryptosis is triggered by isotonic cell shrinkage following Cl- removal (replacement with the impermeant organic anion gluconate) or by oxidative stress (exposure to 0.3 mM tertbutyl-hydroperoxide [tBOOH]). The present study explored whether CDK4 is expressed in erythrocytes and whether the CDK4 inhibitors II (NSC625987) and III (ryuvidine) influence eryptosis. METHODS: Western blotting was utilized for determination of the presence of CDK4 protein in human erythrocytes, and FACS analysis to determine Fluo3 fluorescence (reflecting cytosolic Ca2+), annexin-V-binding (reflecting PS-exposure) and forward scatter (reflecting cell volume). RESULTS: CDK4 protein was present in human erythrocytes. Cl- removal was followed by decrease of forward scatter and increase of both annexin-V-binding and Fluo3 fluorescence, an effect significantly curtailed by CDK4 inhibitors II and III. Furthermore, CDK4 inhibition blunted enhanced PS-exposure elicited by tBOOH treatment. CONCLUSIONS: The present observations disclose the presence of CDK4 protein in human erythrocytes and the suppression of suicidal erythrocyte death by pharmacological inhibition of CDK4.

Enhanced suicidal erythrocyte death contributing to anemia in the elderly.

BACKGROUND/AIMS: Anemia, a common condition in the elderly, could result from impaired formation and/or from accelerated loss of circulating erythrocytes. The latter could result from premature suicidal erythrocyte death or eryptosis characterized by phosphatidylserine (PS) 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 eryptosis is altered in elderly individuals and, if so, to identify underlying mechanisms. METHODS: Blood was drawn from healthy young (n=11, age 31.3 ± 1.7 years) and elderly (n=16, age 88.6 ± 0.9 years) individuals. PS exposure was estimated from annexin V-binding, cell volume from forward scatter, [Ca(2+)]i from Fluo3-fluorescence, reactive oxygen species (ROS) from 2',7'dichlorodihydrofluorescein fluorescence, reduced glutathione (GSH) from mercury orange fluorescence and ceramide from FITC-conjugated antibody binding in flow cytometry. Measurements were made in erythrocytes from freshly drawn blood and in erythrocytes exposed in vitro for 24 h to plasma from young or elderly individuals. RESULTS: Elderly individuals suffered from severe anemia (hemoglobin 10.5 ± 0.3 g/100 ml) despite enhanced number of reticulocytes (2.3 ± 0.2%). The percentage of PS-exposing erythrocytes was significantly higher in the elderly (2.5 ± 0.2%) than in the young volunteers (1.3 ± 0.1%). The increase in PS exposure was paralleled by significant increase of ROS and significantly decreased levels of reduced GSH. Erythrocyte [Ca(2+)]i, and ceramide abundance tended to be higher in the elderly, differences, however, not reaching statistical significance. CONCLUSIONS: The anemia of elderly individuals is mainly if not exclusively due to enhanced eryptosis, resulting at least in part from GSH deficiency and increased oxidative stress.

Vitamin D-Rich Diet in Mice Modulates Erythrocyte Survival.

BACKGROUND/AIMS: Epidemiological evidence suggests that vitamin D deficiency is associated with anemia. The potent metabolite 1,25(OH)2 vitamin D3 [1,25(OH)2D3] activates various signaling cascades regulating a myriad of cellular functions including suicidal cell death or apoptosis. Suicidal death of erythrocytes or eryptosis is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine (PS) externalization. Stimulation of eryptosis may limit lifespan of circulating erythrocytes and thus cause anemia. In the present study, we explored the effect of a high vitamin D diet (10,000 I.U. vitamin D for 14 days) in mice on eryptosis. METHODS: Plasma concentrations of erythropoietin were estimated using an immunoassay kit, blood count using an electronic hematology particle counter, relative reticulocyte numbers using Retic-COUNT® reagent, PS exposure at the cell surface from annexin V binding, cell volume from forward scatter, and cytosolic Ca(2+) ([Ca(2+)]i) from Fluo3-fluorescence in FACS analysis. RESULTS: Vitamin D treatment decreased mean corpuscular volume, reticulocyte count, and plasma erythropoietin levels. Vitamin D treatment slightly but significantly decreased forward scatter but did not significantly modify spontaneous PS exposure and [Ca(2+)]i of freshly drawn erythrocytes. Vitamin D treatment augmented the stimulation of PS exposure and cell shrinkage following exposure to hyperosmotic shock (addition of 550 mM sucrose) or energy depletion (glucose removal) without significantly modifying [Ca(2+)]i. CONCLUSIONS: The present observations point to a subtle effect of exogenous vitamin D supplementation on erythrocyte survival.

Stimulation of eryptosis by cryptotanshinone.

BACKGROUND/AIMS: Cryptotanshinone, a component of Salvia miltiorrhiza Bunge roots, may trigger suicidal death or apoptosis of tumor cells and has thus been recommended for the prevention and treatment of malignancy. On the other hand, Cryptotanshinone has been shown to counteract apoptosis of neurons and hepatocytes. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, a suicidal death characterized by cell shrinkage and phosphatidylserine translocation to the erythrocyte surface. Eryptosis may be triggered by increase of cytosolic Ca(2+)-activity ([Ca(2+)]i). The present study explored whether Cryptotanshinone stimulates eryptosis. METHODS: Forward scatter was taken as measure of cell volume, annexin V binding for identification of phosphatidylserine-exposing erythrocytes and Fluo3-fluorescence for determination of [Ca(2+)]i. RESULTS: A 48 h exposure of human erythrocytes to Cryptotanshinone (10 µM) was followed by significant decrease of forward scatter, significant increase of the percentage annexin-V-binding cells and significant increase of [Ca(2+)]i. The effect of Cryptotanshinone (1 µM) on annexin-V-binding was virtually abrogated by removal of extracellular Ca(2+). CONCLUSION: Cryptotanshinone is a powerful stimulator of suicidal erythrocyte death or eryptosis, which is effective mainly, if not exclusively, by stimulation of Ca(2+) entry.

Stimulation of erythrocyte cell membrane scrambling by gedunin.

BACKGROUND/AIMS: Gedunin, an inhibitor of heat shock protein HSP90, triggers apoptosis of tumor cells and is thus effective against malignancy. Moreover, the drug has antimalarial potency. 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 may be triggered by increase of cytosolic Ca(2+)-activity ([Ca(2+)]i). The present study explored whether gedunin stimulates eryptosis. METHODS: Forward scatter was determined to estimate cell volume, annexin V binding to identify phosphatidylserine-exposing erythrocytes, hemoglobin release to depict hemolysis, and Fluo3-fluorescence to quantify [Ca(2+)]i. RESULTS: A 48 h exposure of human erythrocytes to gedunin significantly increased [Ca(2+)]i (12 µM), significantly decreased forward scatter (24 µM) and significantly increased annexin-V-binding (12 µM). The effect of gedunin (24 µM) on annexin-V-binding was virtually abrogated by removal of extracellular Ca(2+). CONCLUSION: Gedunin stimulates suicidal erythrocyte death or eryptosis, an effect mainly if not exclusively due to stimulation of Ca(2+) entry.

Induction of suicidal erythrocyte death by novobiocin.

BACKGROUND: Novobiocin, an aminocoumarin antibiotic, interferes with heat shock protein 90 and hypoxia inducible factor dependent gene expression and thus compromises cell survival. Similar to survival of nucleated cells, erythrocyte survival could be disrupted by eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and by phospholipd scrambling of the cell membrane with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca(2+)-activity ([Ca(2+)]i). The Ca(2+) sensitivity of phospholipid scrambling is enhanced by ceramide. The present study explored, whether novobiocin elicits eryptosis. METHODS: [Ca(2+)]i was estimated from Fluo3-fluorescence, ceramide abundance utilizing fluorescent antibodies, cell volume from forward scatter, phosphatidylserine-exposure from annexin V binding. RESULTS: A 48 hours exposure to novobiocin (500 µM) was followed by a significant increase of [Ca(2+)]i, decrease of forward scatter, increase of annexin-V-binding and enhanced ceramide formation. Removal of extracellular Ca(2+) virtually abrogated the increase of annexin-V-binding following novobiocin exposure. CONCLUSIONS: Novobiocin stimulates eryptosis, an effect at least in part due to entry of extracellular Ca(2+) and formation of ceramide.

Liver cell death and anemia in Wilson disease involve acid sphingomyelinase and ceramide.

Wilson disease is caused by accumulation of Cu(2+) in cells, which results in liver cirrhosis and, occasionally, anemia. Here, we show that Cu(2+) triggers hepatocyte apoptosis through activation of acid sphingomyelinase (Asm) and release of ceramide. Genetic deficiency or pharmacological inhibition of Asm prevented Cu(2+)-induced hepatocyte apoptosis and protected rats, genetically prone to develop Wilson disease, from acute hepatocyte death, liver failure and early death. Cu(2+) induced the secretion of activated Asm from leukocytes, leading to ceramide release in and phosphatidylserine exposure on erythrocytes, events also prevented by inhibition of Asm. Phosphatidylserine exposure resulted in immediate clearance of affected erythrocytes from the blood in mice. Accordingly, individuals with Wilson disease showed elevated plasma levels of Asm, and displayed a constitutive increase of ceramide- and phosphatidylserine-positive erythrocytes. Our data suggest a previously unidentified mechanism for liver cirrhosis and anemia in Wilson disease.

Patulin-induced suicidal erythrocyte death.

BACKGROUND: Patulin, the most common mycotoxin in apples and apple-derived products, triggers apoptosis and has thus been considered for the treatment of cancer. Similar to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and by cell membrane scrambling leading to phosphatidylserine-exposure at the erythrocyte surface. Stimulators of eryptosis include increase of cytosolic Ca(2+)-activity ([Ca(2+)]i). The present study explored, whether exposure of human erythrocytes to patulin is followed by eryptosis. METHODS: Forward scatter was measured to estimate cell volume, annexin V binding to detect phosphatidylserine-exposure, hemoglobin release to quantify hemolysis, and Fluo3-fuorescence to determine [Ca(2+)]i. RESULTS: A 48 h exposure to patulin significantly increased [Ca(2+)]I (5 µM), significantly decreased forward scatter (5 µM) and significantly increased annexin-V-binding (2.5 µM). Patulin (10 µM) induced annexin-V-binding was virtually abrogated by removal of extracellular Ca(2+). CONCLUSION: Patulin stimulates Ca(2+) entry into erythrocytes, an effect triggering suicidal erythrocyte death or eryptosis.

Inhibition of suicidal erythrocyte death by probucol.

Probucol, an antioxidant and anti-inflammatory agent counteracting atherosclerosis and restenosis, is partially effective by influencing suicidal cell death or apoptosis. In analogy to apoptosis of nucleated cells, suicidal death of erythrocytes or eryptosis is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the erythrocyte surface. Eryptosis is stimulated by increase in cytosolic Ca(2+) activity, for example, after energy depletion or oxidative stress. The present study explored whether probucol influences eryptosis. Phosphatidylserine exposure was estimated from annexin-V-binding, cell volume from forward scatter (FSC), and cytosolic Ca(2+) concentration from fluo-3 fluorescence in flow cytometry. As a result, energy depletion (48-hour glucose removal) increased annexin-V-binding, decreased FSC, and increased fluo-3 fluorescence. Probucol (≤30 µM) did not significantly modify annexin-V-binding, FSC, or fluo-3 fluorescence in the presence of glucose but (at ≥5 µM) blunted the effect of glucose depletion on annexin-V-binding. Probucol (≥20 µM) only slightly blunted the effects of glucose depletion on FSC and fluo-3 fluorescence. Ca(2+) ionophore ionomycin (1 µM) and oxidative stress (30-minute exposure to 0.3 mM of tert-butylhydroperoxide) increased annexin-V-binding, effects again blunted by 30 µM of probucol. In conclusion, probucol blunts cell membrane scrambling after energy depletion and oxidative stress, effects primarily because of interference with the scrambling effects of increased cytosolic Ca(2+) concentration.

Sunitinib-sensitive suicidal erythrocyte death.

Sunitinib, a multikinase inhibitor, stimulates apoptosis and is thus utilized for the treatment of malignancy. Even though lacking mitochondria and nuclei, critical elements in apoptosis of nucleated cells, erythrocytes may undergo eryptosis, an apoptosis-like suicidal death, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine-exposure at the cell surface. Triggers of eryptosis include activation of Ca(2+) permeable cation channels with subsequent increase of cytosolic Ca(2+)-activity ([Ca(2+)](i)), ceramide formation, ATP-depletion, stimulation of p38 kinase and caspase activation. The present study explored, whether sunitinib stimulates eryptosis. [Ca(2+)](i )was estimated from Fluo-3-fluorescence, cell volume from forward scatter, phosphatidylserine-exposure from annexin-V-binding, hemolysis from hemoglobin release, ceramide abundance from anti-ceramide antibody binding, and cytosolic ATP from luciferin-luciferase activity. A 48 h exposure to sunitinib (10 µM) significantly decreased forward scatter and increased annexin-V-binding, effects paralleled by significant increase of [Ca(2+)](i). Sunitinib exposure was followed by a slight but significant increase of hemolysis. Sunitinib induced annexin-V-binding was slightly, but significantly blunted by removal of extracellular Ca(2+), by p38 kinase inhibitor SB203580 (10 µM) and by the pancaspase inhibitor zVAD (10 µM). Sunitinib, however, did not significantly modify cytosolic ATP and ceramide abundance. The present observations reveal that sunitinib is able to trigger suicidal death in erythrocytes even in the absence of nuclei and mitochondria.

Induction of programmed erythrocyte death by gambogic acid.

Gambogic acid, a xanthone from Garcinia hanburyi, stimulates apoptosis and has thus anticancer potency. Similar to apoptosis of nucleated cells, erythrocytes may undergo apoptosis-like suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine-exposure at the cell surface. Eryptosis could be triggered by increase of cytosolic Ca(2+)-activity ([Ca(2+)](i)), ceramide formation, ATP-depletion and caspase activation. The present study explored, whether gambogic acid triggers eryptosis of human erythrocytes. [Ca(2+)](i )was estimated utilizing Fluo-3 fluorescence, cell volume from forward scatter, phosphatidylserine-exposure from annexin-V-binding, hemolysis from hemoglobin release, ceramide abundance utilizing antibodies, and cytosolic ATP with luciferin- luciferase. A 48 h exposure to gambogic acid (500 nM) significantly increased [Ca(2+)](i), stimulated ceramide formation, decreased forward scatter and increased annexin-V-binding. Gambogic acid exposure was followed by a slight but significant increase of hemolysis. Gambogic acid did not significantly modify cytosolic ATP-concentration. Removal of extracellular Ca(2+) slightly, but significantly blunted the effect of gambogic acid (500 nM) on annexin-V-binding. The present observations disclose a novel effect of gambogic acid, i.e. stimulation of suicidal death of human erythrocytes or eryptosis, paralleled by Ca(2+)-entry, ceramide formation, cell shrinkage and phosphatidylserine-exposure.

Stimulation of suicidal erythrocyte death by ipratropium bromide.

BACKGROUND/AIMS: Ipratropium bromide, an anticholinergic agent widely used in obstructive lung disease, has previously been shown to trigger suicidal death of nucleated cells or apoptosis. Despite their lack of mitochondria and nuclei, key organelles in the execution of apoptosis, erythrocytes may similarly undergo suicidal cell death, which is characterized by cell shrinkage and by cell membrane scrambling with phosphatidylserine-exposure at the cell surface. Triggers of eryptosis include increase of cytosolic Ca(2+)-activity ([Ca(2+)](i)). The present study explored whether ipratropium bromide triggers eryptosis. METHODS: [Ca Ca(2+)](i) was estimated utilizing Fluo3 fluorescence, cell volume from forward scatter, phosphatidylserine-exposure from annexin-V-binding, and hemolysis from hemoglobin release. RESULTS: A 48 h exposure to ipratropium bromide (1 nM) significantly increased [Ca(2+)](i), decreased forward scatter and increased annexin-V-binding. Ipratropium bromide treatment was followed by slight but significant increase of hemolysis. Removal of extracellular Ca(2+) or inhibition of Ca(2+) permeable cation channels with amiloride (1 mM) virtually abolished cell membrane scrambling. Ca(2+) ionophore ionomycin (1 µM, 30 min) increased the percentage of phosphatidylserine exposing erythrocytes to similarly high levels in the absence and presence of ipratropium bromide (1 nM). CONCLUSIONS: Ipratropium bromide triggers suicidal erythrocyte death or eryptosis, an effect mainly due to stimulation of Ca(2+)-entry.

Sulindac sulfide--induced stimulation of eryptosis.

BACKGROUND: Sulindac sulfide, a non-steroidal anti-inflammatory drug (NSAID), stimulates apoptosis of tumor cells and is thus effective against malignancy. In analogy to apoptosis of nucleated cells, erythrocytes may undergo eryptosis, an apoptosis-like suicidal erythrocyte death, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine-exposure at the cell surface. Stimulators of eryptosis include increase of cytosolic Ca(2+)-activity ([Ca(2+)](i)) and ceramide formation. The present study explored, whether sulindac sulfide stimulates eryptosis. METHODS: [Ca(2+)](i) was estimated from Fluo-3 fluorescence, cell volume from forward scatter, phosphatidylserine-exposure from binding of fluorescent annexin-V, hemolysis from hemoglobin release, and ceramide abundance utilizing fluorescent antibodies. RESULTS: A 48 h exposure to sulindac sulfide (≤ 20 µM) was followed by significant increase of [Ca(2+)](i), enhanced ceramide abundance, decreased forward scatter and increased percentage of annexin-V-binding erythrocytes. Sulindac sulfide triggered slight but significant hemolysis. Removal of extracellular Ca(2+) significantly blunted, but did not abrogate the effect of sulindac sulfide (20 µM) on annexin-V-binding. CONCLUSION: Sulindac sulfide stimulates the suicidal death of erythrocytes or eryptosis, an effect paralleled by Ca(2+)-entry, ceramide formation, cell shrinkage and phosphatidylserine-exposure.

Tanshinone IIA stimulates erythrocyte phosphatidylserine exposure.

Tanshinone IIA, an antimicrobial, antioxidant, antianaphylactic, antifibrotic, vasodilating, antiatherosclerotic, organo-protective and antineoplastic component from the rhizome of Salvia miltiorrhiza, is known to trigger apoptosis of tumor cells. Tanshinone IIA is effective in part through mitochondrial depolarization and altered gene expression. Erythrocytes lack mitochondria and nuclei but may undergo eryptosis, an apoptosis-like suicidal cell death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Eryptosis is triggered by increase of cytosolic Ca(2+) activity, ATP depletion and ceramide formation. The present study explored, whether tanshinone IIA elicits eryptosis. Cytosolic Ca(2+)-concentration was determined from Fluo3-fluorescence, cell volume from forward scatter, phosphatidylserine exposure from binding of fluorescent annexin V, hemolysis from hemoglobin concentration in the supernatant, ATP concentration utilizing luciferin-luciferase and ceramide formation utilizing fluorescent anticeramide antibodies. Clearance of circulating erythrocytes was estimated by CFSE-labeling. A 48 h exposure to tanshinone IIA (≥10 µM) significantly increased cytosolic Ca(2+)-concentration, decreased ATP concentration (25 µM), increased lactate concentration (25 µM), increased ceramide formation (25 µM), decreased forward scatter, increased annexin-V-binding and increased (albeit to a much smaller extent) hemolysis. The effect of 25 µM tanshinone IIA on annexin-V binding was partially reversed in the nominal absence of Ca(2+). Labelled tanshinone IIA-treated erythrocytes were more rapidly cleared from the circulating blood in comparison to untreated erythrocytes. The present observations reveal a completely novel effect of tanshinone IIA, i.e. triggering of Ca(2+) entry, ATP depletion and ceramide formation in erythrocytes, events eventually leading to eryptosis with cell shrinkage and cell membrane scrambling.

Enhanced erythrocyte membrane exposure of phosphatidylserine following sorafenib treatment: an in vivo and in vitro study.

BACKGROUND: Sorafenib (Nexavar(®)), a polytyrosine kinase inhibitor, stimulates apoptosis and is thus widely used for chemotherapy in hepatocellular carcinoma (HCC). Hematological side effects of Nexavar(®) chemotherapy include anemia. Erythrocytes may undergo apoptosis-like suicidal death or eryptosis, which is characterized by cell shrinkage and phosphatidylserine-exposure at the cell surface. Signaling leading to eryptosis include increase in cytosolic Ca(2+)activity ([Ca(2+)](i)), formation of ceramide, ATP-depletion and oxidative stress. The present study explored, whether sorafenib triggers eryptosis in vitro and in vivo. METHODS: [Ca(2+)](i )was estimated from Fluo3-fluorescence, cell volume from forward scatter, phosphatidylserine-exposure from annexin-V-binding, hemolysis from hemoglobin release, ceramide with antibody binding-dependent fluorescence, cytosolic ATP with a luciferin-luciferase-based assay, and oxidative stress from 2',7' dichlorodihydrofluorescein diacetate (DCFDA) fluorescence. RESULTS: A 48 h exposure of erythrocytes to sorafenib (≥0.5 µM) significantly increased Fluo 3 fluorescence, decreased forward scatter, increased annexin-V-binding and triggered slight hemolysis (≥5 µM), but did not significantly modify ceramide abundance and cytosolic ATP. Sorafenib treatment significantly enhanced DCFDA-fluorescence and the reducing agents N-acetyl-L-cysteine and tiron significantly blunted sorafenib-induced phosphatidylserine exposure. Nexavar(®) chemotherapy in HCC patients significantly enhanced the number of phosphatidylserine-exposing erythrocytes. CONCLUSIONS: The present observations disclose novel effects of sorafenib, i.e. stimulation of suicidal erythrocyte death or eryptosis, which may contribute to the pathogenesis of anemia in Nexavar(®)-based chemotherapy.

Hexavalent chromium-induced erythrocyte membrane phospholipid asymmetry.

Hexavalent (VI) chromium is a global contaminant with cytotoxic activity. Chromium (VI) induces oxidative stress, inflammation, cell proliferation, malignant transformation and may trigger carcinogenesis and at the same time apoptosis. The toxic effects of chromium (VI) at least partially result from mitochondrial injury and DNA damage. Erythrocytes lack mitochondria and nuclei but may experience an apoptosis-like suicidal cell death, i.e. eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Eryptosis may result from increase of cytosolic Ca(2+) activity, ATP depletion and/or ceramide formation. The present study explored, whether chromium (VI) triggers eryptosis. Fluo-3-fluorescence was employed to determine cytosolic Ca(2+)-concentration, forward scatter to estimate cell volume, binding of fluorescent annexin V to detect phosphatidylserine exposure, hemoglobin concentration in the supernatant to quantify hemolysis, luciferin-luciferase to determine cytosolic ATP concentration and fluorescent anti-ceramide antibodies to uncover ceramide formation. A 48 h exposure to chromium (VI) (≥10 µM) significantly increased cytosolic Ca(2+)-concentration, decreased ATP concentration (20 µM), decreased forward scatter, increased annexin V-binding and increased (albeit to a much smaller extent) hemolysis. Chromium (VI) did not significantly modify ceramide formation. The effect of 20 µM chromium (VI) on annexin V binding was partially reversed in the nominal absence of Ca(2+). The present observations disclose a novel effect of chromium (VI), i.e. Ca(2+) entry and cytosolic ATP depletion in erythrocytes, effects resulting in eryptosis with cell shrinkage and cell membrane scrambling.

Enhanced Ca2+ entry, ceramide formation, and apoptotic death of erythrocytes triggered by plumbagin.

Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone, 1), a natural product from plants with potential anticancer potency, induces apoptosis. Mechanisms involved in 1-induced apoptosis include mitochondrial depolarization, inactivation of NF-κB, and altered expression of anti- and proapoptotic Bcl proteins. Similar to nucleated cells, erythrocytes may undergo suicidal death or eryptosis, which, like apoptosis, results in cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Triggers of eryptosis include increase of cytosolic Ca(2+) activity ([Ca(2+)]i) and ceramide formation. The present study explored whether 1 stimulates eryptosis. Cell volume was estimated from forward scatter, phosphatidylserine exposure from annexin-V-binding, hemolysis from hemoglobin release, [Ca(2+)]i from Fluo-3 fluorescence, and ceramide abundance utilizing antibodies. A 48 h exposure to 1 (2 µM) decreased forward scatter and increased annexin-V-binding significantly, events paralleled by increased [Ca(2+)]i and ceramide formation. Exposure to 1 was followed by a slight but significant increase of hemolysis. Removal of extracellular Ca(2+) slightly, but significantly blunted the effect of 1 (2 µM) on annexin-V-binding. The present observations demonstrate that 1 may trigger suicidal death of erythrocytes, cells devoid of mitochondria and nuclei.

Enhanced apoptotic death of erythrocytes induced by the mycotoxin ochratoxin A.

BACKGROUND: The mycotoxin ochratoxin A, an agent responsible for endemic Balkan nephropathy is known to trigger apoptosis and thus being toxic to several organs including the kidney. The mechanisms involved in ochratoxin A induced apoptosis include oxidative stress. Sequelae of ochratoxin intoxication include anemia. Similar to apoptosis of nucleated cells, erythrocytes may undergo suicidal cell death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling resulting in phosphatidylserine-exposure at the cell surface. Eryptosis could be triggered by Ca2+ -entry through oxidant sensitive unspecificcation channels increasing cytosolic Ca2+ activity ([Ca2+]i). The Ca2+ -sensitivity of cell membrane scrambling could be enhanced and eryptosis thus triggered by ceramide. The removal of suicidal erythrocytes may lead to anemia. Moreover, eryptotic erythrocytes could adhere to the vascular wall thus impeding microcirculation. The present study explored, whether ochratoxin A stimulates eryptosis. METHODS: Fluo3-fluorescence was utilized to determine [Ca2+]i, forward scatter to estimate cell volume, annexin-V-binding to identify phosphatidylserine-exposing cells, fluorescent antibodies to detect ceramide formation and hemoglobin release to quantify hemolysis. Moreover, adhesion to human vascular endothelial cells (HUVEC) was determined utilizing a flow chamber. RESULTS: A 48 h exposure to ochratoxin A was followed by significant increase of Fluo3-fluorescencei (≥ 2.5 µM), increase of ceramide abundance (10 µM), decrease of forward scatter (≥ 5 µM) and increase of annexin-V-binding (≥ 2.5 µM). Ochratoxin A exposure slightly but significantly enhanced hemolysis (10 µM). Ochratoxin (10 µM) enhanced erythrocyte adhesion to HUVEC. Removal of extracellular Ca2+ significantly blunted, but did not abrogate ochratoxin A-induced annexin V binding. CONCLUSIONS: Ochratoxin A triggers suicidal erythrocyte death or eryptosis, an effect partially but not fully due to stimulation of Ca2+ -entry.

Impaired mast cell activation in gene-targeted mice lacking the serum- and glucocorticoid-inducible kinase SGK1.

The PI3K pathway plays a pivotal role in the stimulation of mast cells. PI3K-dependent kinases include the serum- and glucocorticoid-inducible kinase 1 (SGK1). The present study explored the role of SGK1 in mast cell function. Mast cells were isolated from bone marrow (BMMC) of SGK1 knockout mice (sgk1(-/-)) and their wild-type littermates (sgk1(+/+)). The BMMC number as well as CD117, CD34, and FcepsilonRI expression in BMCCs were similar in both genotypes. Upon Ag stimulation of the FcepsilonRI receptor, Ca(2+) entry but not Ca(2+) release from intracellular stores was markedly impaired in sgk1(-/-) BMMCs. The currents through Ca(2+)-activated K+ channels induced by Ag were significantly higher in sgk1(+/+) BMMCs than in sgk1(-/-) BMMCs. Treatment with the Ca(2+) ionophore ionomycin (1 microM) led to activation of the K+ channels in both genotypes, indicating that the Ca(2+)-activated K+ channels are similarly expressed and sensitive to activation by Ca(2+) in sgk1(+/+) and sgk1(-/-) BMMCs, and that blunted stimulation of Ca(2+)-activated K+ channels was secondary to decreased Ca(2+) entry. Ag-IgE-induced degranulation and early IL-6 secretion were also significantly blunted in sgk1(-/-) BMMCs. The decrease in body temperature following Ag treatment, which reflects an anaphylactic reaction, was substantially reduced in sgk1(-/-) mice, pointing to impaired mast cell function in vivo. Serum histamine levels measured 30 min after induction of an anaphylactic reaction were significantly lower in sgk1(-/-) than in sgk1(+/+)mice. The observations reveal a critical role for SGK1 in ion channel regulation and the function of mast cells, and thus disclose a completely novel player in the regulation of allergic reaction.