Stimulation of Phospholipid Scrambling of the Erythrocyte Membrane by 9-Cis-Retinoic Acid.

BACKGROUND/AIMS: The endogenous retinoid 9-cis-retinoic acid has previously been shown to trigger apoptosis in a wide variety of cells including several tumor cells and has thus been suggested for the treatment of malignancy. Similar to apoptosis of nucleated cells, erythrocytes may enter suicidal erythrocyte death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Cellular mechanisms participating in the accomplishment of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i) and formation of ceramide. The present study explored, whether 9-cis-retinoic acid induces eryptosis and whether the effect involves Ca2+ and/or ceramide. METHODS: Flow cytometry was employed to estimate erythrocyte volume from forward scatter, phosphatidylserine exposure at the cell surface from annexin-V-binding, [Ca2+]i from Fluo3-fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was quantified from hemoglobin concentration in the supernatant. RESULTS: A 48 hours exposure of human erythrocytes to 9-cis-retinoic acid (≥ 0.5 µg/ml) significantly increased the percentage of annexin-V-binding cells and significantly decreased forward scatter. Exposure to 9-cis-retinoic acid (≥ 0.5 µg/ml) significantly increased Fluo3-fluorescence, and the effect of 9-cis-retinoic acid on annexin-V-binding was significantly blunted by removal of extracellular Ca2+. Exposure to 9-cis-retinoic acid (1 µg/ml) further significantly increased the ceramide abundance at the erythrocyte surface and significantly increased hemolysis. CONCLUSIONS: 9-cis-retinoic acid triggers phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part downstream of Ca2+ and ceramide.

Stimulation of Erythrocyte Cell Membrane Scrambling by C-Reactive Protein.

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.

Bromfenvinphos induced suicidal death of human erythrocytes.

The organophosphorus pesticide bromfenvinphos ((E,Z)-O,O-diethyl-O-[1-(2,4-dichlorophenyl)-2-bromovinyl] phosphate) has been shown to decrease hematocrit and hemoglobin levels in blood presumably by triggering oxidative stress of erythrocytes. Oxidative stress is known to activate erythrocytic Ca(2+) permeable unselective cation channels leading to Ca(2+) entry and increase of cytosolic Ca(2+) activity ([Ca(2+)]i), which in turn triggers eryptosis, the suicidal death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. The present study explored, whether and how bromfenvinphos induces eryptosis. To this end, phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, hemolysis from hemoglobin release, [Ca(2+)]i from Fluo3-fluorescence, and ROS formation from DCFDA dependent fluorescence. As a result, a 48hour exposure of human erythrocytes to bromfenvinphos (≥100µM) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter, significantly increased Fluo3-fluorescence, and significantly increased DCFDA fluorescence. The effect of bromfenvinphos on annexin-V-binding and forward scatter was significantly blunted, but not abolished by removal of extracellular Ca(2+). In conclusion, bromfenvinphos triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to stimulation of ROS formation and Ca(2+) entry.

Stimulation of Suicidal Erythrocyte Death by Garcinol.

BACKGROUND/AIMS: The benzophenone garcinol from dried fruit rind of Garcinia indica counteracts malignancy, an effect at least in part due to stimulation of apoptosis. The proapototic effect of garcinol is attributed in part to inhibition of histone acetyltransferases and thus modification of gene expression. Moreover, garcinol triggers mitochondrial depolarisation. Erythrocytes lack gene expression and mitochondria but are nevertheless able to enter apoptosis-like suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include oxidative stress, energy depletion and Ca2+ entry with increase of cytosolic Ca2+ activity ([Ca2+]i). The present study explored, whether and how garcinol induces eryptosis. METHODS: To this end, phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, hemolysis from hemoglobin release, [Ca2+]i from Fluo3-fluorescence, ROS formation from DCFDA dependent fluorescence and cytosolic ATP levels utilizing a luciferin-luciferase-based assay. RESULTS: A 24 hours exposure of human erythrocytes to garcinol (2.5 or 5 µM) significantly increased the percentage of annexin-V-binding cells. Garcinol decreased (at 1 µM and 2.5 µM) or increased (at 5 µM) forward scatter. Garcinol (5 µM) further increased Fluo3-fluorescence, increased DCFDA fluorescence, and decreased cytosolic ATP levels. The effect of garcinol on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+. CONCLUSIONS: Garcinol triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to stimulation of ROS formation, energy depletion and Ca2+ entry.

Oxaliplatin Induced Suicidal Death of Human Erythrocytes.

BACKGROUND/AIMS: The alkylating drug oxaliplatin is widely used for chemotherapy of malignancy. Oxaliplatin is effective by inducing both, necrosis and apoptosis. Similar to necrosis or apoptosis of nucleated cells, erythrocytes may enter hemolysis, which is apparent from hemoglobin release or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include oxidative stress and/or Ca2+ entry with increase of cytosolic Ca2+ activity ([Ca2+]i). The present study explored, whether and how oxaliplatin induces eryptosis. METHODS: Phosphatidylserine exposure at the cell surface was quantified utilizing annexin-V-binding, cell volume estimated from forward scatter, hemolysis deduced from hemoglobin release, [Ca2+]i determined utilizing Fluo-3 fluorescence, and reactive oxygen species (ROS) abundance visualized using 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) dependent fluorescence. RESULTS: A 48 hours exposure of human erythrocytes to oxaliplatin (10 µg/ml) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter, significantly increased Fluo-3 fluorescence, and significantly increased DCFDA fluorescence. The effect of oxaliplatin on annexin-V-binding and forward scatter was rather augmented by removal of extracellular Ca2+, but was significantly blunted in the presence of the antioxidant N-acetyl-cysteine (1 mM). CONCLUSIONS: Oxaliplatin triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect partially dependent on ROS formation.

Triggering of Suicidal Erythrocyte Death Following Boswellic Acid Exposure.

BACKGROUND/AIMS: The antinflammatory natural product boswellic acid is effective against cancer at least in part by inducing tumor cell apoptosis. Similar to apoptosis of nucleated cells erythrocytes may enter eryptosis, a suicidal death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include oxidative stress, increase of cytosolic Ca(2+)-activity ([Ca(2+)]i), energy depletion, ceramide formation and p38 kinase activation. The present study tested, whether and how boswellic acid induces eryptosis. METHODS: Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, hemolysis from hemoglobin release, [Ca(2+)]i from Fluo3-fluorescence, ceramide abundance utilizing specific antibodies, reactive oxygen species (ROS) from 2',7'-dichlorodihydrofuorescein diacetate (DCFDA) fluorescence, and cytosolic ATP concentration utilizing a luciferin-luciferase assay kit. RESULTS: A 24 hours exposure of human erythrocytes to boswellic acid (5 µg/ml) significantly increased the percentage of annexin-V-binding cells (to 9.3 ± 0.9 %) and significantly decreased forward scatter. Boswellic acid did not significantly modify [Ca(2+)]i, cytosolic ATP, ROS, or ceramide abundance. The effect of boswellic acid on annexin-V-binding was significantly blunted, but not abolished by p38 kinase inhibitors skepinone (2 µM) and SB203580 (2 µM). CONCLUSIONS: Boswellic acid stimulates cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part dependent on p38 protein kinase activity.

Clofazimine Induced Suicidal Death of Human Erythrocytes.

BACKGROUND/AIMS: The antimycobacterial riminophenazine clofazimine has previously been shown to up-regulate cellular phospholipase A2 and to induce apoptosis. In erythrocytes phospholipase A2 stimulates eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Phospholipase A2 is in part effective by fostering formation of prostaglandin E2, which triggers Ca(2+) entry. Stimulators of Ca(2+) entry and eryptosis further include oxidative stress and energy depletion. The present study tested, whether and how clofazimine induces eryptosis. METHODS: Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, hemolysis from hemoglobin release, cytosolic Ca(2+) activity ([Ca(2+)]i) from Fluo3-fluorescence, reactive oxygen species (ROS) from 2', 7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, and cytosolic ATP level utilizing a luciferin-luciferase assay kit. RESULTS: A 24-48 hours exposure of human erythrocytes to clofazimine (≥ 1.5 µg/ml) significantly increased the percentage of annexin-V-binding cells without appreciably modifying forward scatter. Clofazimine significantly increased [Ca(2+)]i, significantly decreased cytosolic ATP, but did not significantly modify ROS. The effect of clofazimine on annexin-V-binding was significantly blunted, but not fully abolished by removal of extracellular Ca(2+), and by phospholipase A2 inhibitor quinacrine (25 µM). Clofazimine further augmented the effect of Ca(2+) ionophore ionomycin (0.1 µM) on eryptosis. The clofazimine induced annexin-V-binding was, however, completely abrogated by combined Ca(2+) removal and addition of quinacrine. CONCLUSION: Clofazimine stimulates phospholipid scrambling of the erythrocyte cell membrane, an effect in part dependent on entry of extracellular Ca(2+), paralleled by cellular energy depletion and sensitive to phospholipase A2 inhibitor quinacrine.

Enhanced eryptosis following auranofin exposure.

BACKGROUND/AIMS: The antiinflammatory, antimicrobial and anticancer drug auranofin has previously been shown to trigger apoptosis, the suicidal death of nucleated cells. Side effects of the drug include anaemia. At least in theory the anaemia could result from stimulation of suicidal death of erythrocytes or eryptosis, which involves cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. METHODS: Stimulators of eryptosis include oxidative stress and increase of cytosolic Ca2+-activity ([Ca2+]i). In the present study, phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, hemolysis from hemoglobin release, reactive oxygen species (ROS) from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, and [Ca2+]i from Fluo3-fluorescence. RESULTS: A 24 hours exposure of human erythrocytes to auranofin (≥5 µg/ml) significantly increased the percentage of annexin-V-binding cells (from 2.2 ± 0.5 to 17.4 ± 1.5%), significantly decreased forward scatter and significantly enhanced ROS. At higher concentrations (10 µg/ml) auranofin triggered slight hemolysis (from 2.1 ± 0.2 to 3.2 ± 0.3%). CONCLUSIONS: Auranofin stimulates cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least partially due to induction of oxidative stress.

Stimulation of suicidal erythrocyte death by PRIMA-1.

BACKGROUND: The anticarcinogenic drug PRIMA-1 (p53 reactivation and induction of massive apoptosis 1) induces suicidal death of tumor cells, an effect in large part attributed to the up-regulation of the proapoptotic transcription factor p53. Erythrocytes are lacking gene transcription but are nevertheless able to enter eryptosis, a 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 Ca(2+)-activity ([Ca(2+)]i) and ceramide formation. The present study tested whether PRIMA-1 stimulates eryptosis. METHODS: Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, [Ca(2+)]i from Fluo3-fluorescence, ceramide abundance from binding of specific antibodies, and ROS formation from DCFDA fluorescence. RESULTS: A 48 h exposure of human erythrocytes to PRIMA-1 (25 µM) significantly increased the percentage of annexin-V-binding cells without significantly influencing [Ca(2+)]i or forward scatter. PRIMA-1 (100 µM) induced annexin-V-binding was not significantly blunted by removal of extracellular Ca(2+) or by the caspase-3 inhibitor zVAD. PRIMA-1 (100 µM) further increased the ceramide abundance at the cell surface and ROS formation. CONCLUSIONS: PRIMA-1 stimulates phosphatidylserine translocation at the erythrocyte cell membrane, an effect at least partially due to up-regulation of ceramide abundance and ROS formation.

Triggering of Suicidal Erythrocyte Death by Ruxolitinib.

BACKGROUND/AIMS: The JAK1/JAK2 tyrosine kinase inhibitor ruxolitinib is widely used for the treatment of myeloproliferative neoplasm-associated myelofibrosis and other malignancies. Most important side effects include anemia. A common cause of anemia is accelerated suicidal death of erythrocytes or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Mechanisms contributing to the triggering of eryptosis include oxidative stress, Ca2+ entry with increase of cytosolic Ca2+ activity ([Ca2+]i), and activation of distinct kinases, such as p38 mitogen activated protein (MAP) kinase. The present study explored whether and how ruxolitinib induces eryptosis. METHODS: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, hemolysis from hemoglobin release, [Ca2+]i from Fluo3-fluorescence, and ROS formation from DCFDA dependent fluorescence. RESULTS: A 48 hours exposure of human erythrocytes to ruxolitinib (25 µM) significantly increased the percentage of annexin-V-binding cells and significantly decreased forward scatter. Ruxolitinib did not significantly modify Fluo3-fluorescence and DCFDA fluorescence and the effect of ruxolitinib on annexin-V-binding was not significantly modified by removal of extracellular Ca2+. The effect of ruxolitinib on annexin-V-binding was, however, significantly blunted by the p38 MAP kinase inhibitor SB203580 and virtually abolished by the p38 MAP kinase inhibitor skepinone. CONCLUSION: Ruxolitinib triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part requiring p38 MAP kinase activity.

Stimulation of Suicidal Erythrocyte Death by the Antimalarial Drug Mefloquine.

BACKGROUND: The antimalarial drug mefloquine has previously been shown to stimulate apoptosis of nucleated cells. Similar to apoptosis, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include oxidative stress, increase of cytosolic Ca2+-activity ([Ca2+]i), and ceramide. METHODS: Phosphatidylserine abundance at the cell surface was estimated from annexin V binding, cell volume from forward scatter, reactive oxidant species (ROS) from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, [Ca2+]i from Fluo3- fluorescence, and ceramide abundance from specific antibody binding. RESULTS: A 48 h treatment of human erythrocytes with mefloquine significantly increased the percentage of annexin-V-binding cells (≥5 µg/ml), significantly decreased forward scatter (≥5 µg/ml), significantly increased ROS abundance (5 µg/ml), significantly increased [Ca2+]i (7.5 µg/ml) and significantly increased ceramide abundance (10 µg/ml). The up-regulation of annexin- V-binding following mefloquine treatment was significantly blunted but not abolished by removal of extracellular Ca2+. Even in the absence of extracellular Ca2+, mefloquine significantly increased annexin-V-binding. CONCLUSIONS: Mefloquine treatment leads to erythrocyte shrinkage and erythrocyte membrane scrambling, effects at least partially due to induction of oxidative stress, increase of [Ca2+]i and up-regulation of ceramide abundance.

Programmed erythrocyte death following in vitro Treosulfan treatment.

BACKGROUND/AIMS: The cytotoxic drug Treosulfan is clinically used for the treatment of malignancy. A common side effect of Treosulfan treatment is anemia. Treosulfan is at least partially effective by triggering apoptosis of tumor cells. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, a suicidal death characterized by cell shrinkage and translocation of phosphatidylserine from the inner to the outer leaflet of the plasma membrane. Triggers of eryptosis include oxidative stress, Ca(2+)-entry and increase of cytosolic Ca(2+)-activity ([Ca(2+)]i). The present study explored whether Treosulfan stimulates eryptosis, which may contribute to development of anemia. METHODS: Erythrocyte volume was estimated from forward scatter, phosphatidylserine abundance at the erythrocyte surface from Fluorescein isothiocyanate (FITC)-annexin-V-binding, [Ca(2+)]i from Fluo3 fluorescence and reactive oxygen species (ROS) from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA)-fluorescence. RESULTS: A 48 hours exposure of human erythrocytes to Treosulfan (800 µg/ml) significantly decreased erythrocyte forward scatter, increased the percentage of annexin-V-binding cells, increased [Ca(2+)]i, and increased ROS. The effect of Treosulfan on annexin-V-binding was virtually abrogated by removal of extracellular Ca(2+). CONCLUSION: Treosulfan stimulates suicidal erythrocyte death or eryptosis at least in part by inducing oxidative stress and stimulating Ca(2+) entry.

Rapid Upregulation of Orai1 Abundance in the Plasma Membrane of Platelets Following Activation with Thrombin and Collagen Related Peptide.

BACKGROUND: Blood platelets accomplish primary hemostasis following vascular injury and contribute to the orchestration of occlusive vascular disease. Platelets are activated by an increase of cytosolic Ca2+-activity ([Ca2+]i), which is accomplished by Ca2+-release from intracellular stores and subsequent store operated Ca2+ entry (SOCE) through Ca2+ release activated Ca2+ channel moiety Orai1. Powerful activators of platelets include thrombin and collagen related peptide (CRP), which are in part effective by activation of small G- protein Rac1. The present study explored the influence of thrombin and CRP on Orai1 protein abundance and cytosolic Ca2+-activity ([Ca2+]i) in platelets drawn from wild type mice. METHODS: Orai1 protein surface abundance was quantified utilizing CF™488A conjugated antibodies, and [Ca2+]i was determined with Fluo3-fluorescence. RESULTS: In resting platelets, Orai1 protein abundance and [Ca2+]i were low. Thrombin (0.02 U/ml) and CRP (5ug/ml) within 2 min increased [Ca2+]i and Orai1 protein abundance at the platelet surface. [Ca2+]i was further increased by Ca2+ ionophore ionomycin (1 µM) and by store depletion with the sarcoendoplasmatic Ca2+ ATPase inhibitor thapsigargin (1 µM). However, Orai1 protein abundance at the platelet surface was not significantly affected by ionomycin and only slightly increased by thapsigargin. The effect of thrombin and CRP on Orai1 abundance and [Ca2+]i was significantly blunted by Rac1 inhibitor NSC23766 (50 µM). CONCLUSION: The increase of [Ca2+]i following stimulation of platelets with thrombin and collagen related peptide is potentiated by ultrarapid Rac1 sensitive translocation of Orai1 into the cell membrane.

Impact of Na+/Ca2+ Exchangers on Therapy Resistance of Ovary Carcinoma Cells.

BACKGROUND/AIMS: According to previous observations, enhanced store-operated Ca2+-entry (SOCE) accomplished by the pore forming ion channel unit Orai1 and its regulator STIM1 contribute to therapy resistance of ovary carcinoma cells. Ca2+ signaling is further shaped by Ca2+ extrusion through K+-independent (NCX) and/or K+-dependent (NCKX) Na+/Ca2+-exchangers. The present study thus explored whether therapy resistance is further paralleled by altered expression and/or function of Na+/Ca2+-exchangers. METHODS: In therapy resistant (A2780cis) and therapy sensitive (A2780sens) ovary carcinoma cells transcript levels were estimated from RT-PCR, cytosolic Ca2+-activity ([Ca2+]i) from Fura-2-fluorescence, Na+/Ca2+-exchanger activity from the increase of [Ca2+]i (x0394;[Ca2+]i) and from whole cell current (Ica) following abrupt replacement of Na+ containing (130 mM) and Ca2+ free extracellular perfusate by Na+ free and Ca2+ containing (2 mM) extracellular perfusate, as well as cell death from PI -staining in flow cytometry. RESULTS: The transcript levels of NCX3, NCKX4, NCKX5, and NCKX6, slope and peak of x0394;[Ca2+]i as well as Ica were significantly higher in therapy resistant than in therapy sensitive ovary carcinoma cells. The Na+/Ca2+-exchanger inhibitor KB-R7943 (10 µM) significantly blunted x0394;[Ca2+]i and significantly augmented the cisplatin-induced cell death of therapy resistant ovary carcinoma cells without significantly modifying cisplatin-induced cell death of therapy sensitive ovary carcinoma cells. CONCLUSION: Enhanced Na+/Ca2+-exchanger activity may contribute to the therapy sensitivity of ovary carcinoma cells.

Fucoxanthin Induced Suicidal Death of Human Erythrocytes.

BACKGROUND/AIMS: Fucoxanthin, a carotenoid isolated from brown seaweeds, induces suicidal death or apoptosis of tumor cells and is thus considered for the treatment or prevention of malignancy. In analogy to apoptosis of nucleated cell, erythrocytes may enter eryptosis, the suicidal death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include Ca2+ entry with increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress and activation of p38 kinase or protein kinase C. The present study explored, whether and how fucoxanthin induces eryptosis. METHODS: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, hemolysis from hemoglobin release, [Ca2+]i from Fluo3-fluorescence, and abundance of reactive oxygen species (ROS) from DCFDA dependent fluorescence and lipid peroxidation using BODIPY fluoresence. RESULTS: A 48 hours exposure of human erythrocytes to fucoxanthin significantly increased the percentage of annexin-V-binding cells (≥ 50 µM), significantly decreased average forward scatter (≥ 25 µM), significantly increased hemolysis (≥ 25 µM), significantly increased Fluo3-fluorescence (≥ 50 µM), significantly increased lipid peroxidation, but did not significantly modify DCFDA fluorescence. The effect of fucoxanthin on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+, and was insensitive to p38 kinase inhibitor skepinone (2 µM) and to protein kinase C inhibitor calphostin (100 nM). CONCLUSION: Fucoxanthin triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to stimulation of Ca2+ entry.

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.

Inhibition of Phosphate-Induced Vascular Smooth Muscle Cell Osteo-/Chondrogenic Signaling and Calcification by Bafilomycin A1 and Methylamine.

BACKGROUND/AIMS: Excessive phosphate concentrations trigger vascular calcification, an active process promoted by osteoinduction of vascular smooth muscle cells (VSMCs) with increased expression and activity of transcription factor RUNX2 (Core-binding factor α1, CBFA1), alkaline phosphatase (ALPL), TGFß1, transcription factor NFAT5, and NFAT5-sensitive transcription factor SOX9. The osteoinductive signaling and vascular calcification of hyperphosphatemic klotho-hypomorphic mice could be reversed by treatment with NH4Cl, effects involving decrease of TGFß1 and inhibition of NFAT5-dependent osteoinductive signaling. Known effects of NH4Cl include alkalinization of acidic cellular compartments. The present study explored whether osteo-/chondrogenic signaling could be influenced by alkalinization of acidic cellular compartments following inhibition of the vacuolar H+ ATPase with bafilomycin A1 or following dissipation of the pH gradient across the membranes of acidic cellular compartments with methylamine. METHODS: Primary human aortic smooth muscle cells (HAoSMCs) were treated with high phosphate to trigger osteo-/chondrogenic signaling and calcification in the absence or presence of bafilomycin A1 or methylamine. Calcium content was determined using a QuantiChrom Calcium assay, ALP activity by a colorimetric assay and transcript levels by quantitative RT-PCR. RESULTS: High phosphate increased significantly the calcium deposition, CBFA1 and ALPL mRNA expression as well as alkaline phosphatase activity in HAoSMCs, all effects ameliorated by both, bafilomycin A1 and methylamine. High phosphate further significantly up-regulated the mRNA levels of TGFB1, NFAT5 and SOX9, effects significantly blunted by additional treatment with bafilomycin A1 or methylamine. Treatment of HAoSMCs with human TGFß1 protein or high phosphate up-regulated NFAT5, SOX9, CBFA1 and ALPL mRNA expression to similarly high levels which could not be further increased by combined treatment with high phosphate and TGFß1. Bafilomycin A1 failed to reverse the osteo-/chondrogenic signaling triggered by high phosphate together with TGFß1. CONCLUSIONS: Inhibition of the vacuolar H+ ATPase or dissipation of the pH gradient across the membranes of acidic cellular compartments both disrupt osteo-/chondrogenic signaling and calcium deposition in VSMCs, observations supporting the hypothesis that vascular calcification requires acidic cellular compartments.

Stimulation of suicidal erythrocyte death by artesunate.

BACKGROUND: The artemisinin derivative artesunate is effective in the treatment of severe malaria and is considered for the treatment of malignancy. Artesunate triggers tumor cell apoptosis, an effect at least in part mediated by mitochondria. Even though lacking mitochondria, erythrocytes may similarly enter suicidal death or eryptosis, which is characterized by cell shrinkage and breakdown of the phospholipid asymmetry of the cell membrane with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca(2+)-activity ([Ca(2+)]i), ceramide formation, and oxidative stress. The present study explored whether artesunate stimulates eryptosis. METHODS: Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, [Ca(2+)]i from Fluo3-fluorescence, ceramide abundance from binding of specific antibodies, and oxidative stress from 2',7'-dichlorodihydrofluorescein-diacetate fluorescence. RESULTS: A 48 h exposure of human erythrocytes to artesunate significantly increased the percentage of annexin-V-binding cells (≥ 9 µg/ml) without significantly influencing forward scatter. Artesunate significantly increased [Ca(2+)]i. The stimulation of annexin-V-binding by artesunate (15 µg/ml) was significantly blunted but not abolished by removal of extracellular Ca(2+). Artesunate increased the ceramide abundance at the cell surface and the 2',7'-dichlorodihydrofluorescein-diacetate fluorescence. CONCLUSIONS: Artesunate stimulates phosphatidylserine translocation at the erythrocyte cell membrane, an effect at least partially due to increase of [Ca(2+)]i, stimulation of ceramide formation and generation of oxidative stress.

Tannic acid induced suicidal erythrocyte death.

BACKGROUND: The polyphenol tannic acid with antioxidant and antimicrobial potency may trigger suicidal death of nucleated cells or apoptosis and thus may counteract tumor growth. In analogy to apoptosis of nucleated cells, erythrocytes may undergo eryptosis, a suicidal death characterized by cell shrinkage and cell membrane scrambling with appearance of phosphatidylserine at the erythrocyte surface. A major trigger of eryptosis is increase of cytosolic Ca(2+)-activity ([Ca(2+)]i). Erythrocytes could be sensitized to the eryptotic effect of cytosolic Ca(2+) by ceramide. METHODS: Cell volume has been estimated from forward scatter, phosphatidylserine abundance at the erythrocyte surface from annexin V binding, hemolysis from hemoglobin release, [Ca(2+)]i from Fluo3-fuorescence and ceramide utilizing fluorescent antibodies. RESULTS: A 48 h treatment with tannic acid was followed by significant decrease of forward scatter (≥ 1 µg/ml) and significant increase of annexin-V-binding (≥ 10 µg/ml). Tannic acid did not significantly modify [Ca(2+)]i (up to 50 µM) but significantly increased ceramide formation (50 µM). The annexin-V-binding following tannic acid treatment (50 µM) was significantly blunted in the nominal absence of extracellular Ca(2+). CONCLUSIONS: Tannic acid stimulates eryptosis, an effect at least partially due to ceramide formation with subsequent sensitization of erythrocytes to cytosolic Ca(2+).

Stimulation of suicidal erythrocyte death by increased extracellular phosphate concentrations.

BACKGROUND/AIM: Anemia in renal insufficiency results in part from impaired erythrocyte formation due to erythropoietin and iron deficiency. Beyond that, renal insufficiency enhances eryptosis, the suicidal erythrocyte death characterized by phosphatidylserine-exposure at the erythrocyte surface. Eryptosis may be stimulated by increase of cytosolic Ca(2+)-activity ([Ca(2+)]i). Several uremic toxins have previously been shown to stimulate eryptosis. Renal insufficiency is further paralleled by increase of plasma phosphate concentration. The present study thus explored the effect of phosphate on erythrocyte death. METHODS: Cell volume was estimated from forward scatter, phosphatidylserine-exposure from annexin V binding, and [Ca(2+)]i from Fluo3-fluorescence. RESULTS: Following a 48 hours incubation, the percentage of phosphatidylserine exposing erythrocytes markedly increased as a function of extracellular phosphate concentration (from 0-5 mM). The exposure to 2 mM or 5 mM phosphate was followed by slight but significant hemolysis. [Ca(2+)]i did not change significantly up to 2 mM phosphate but significantly decreased at 5 mM phosphate. The effect of 2 mM phosphate on phosphatidylserine exposure was significantly augmented by increase of extracellular Ca(2+) to 1.7 mM, and significantly blunted by nominal absence of extracellular Ca(2+), by additional presence of pyrophosphate as well as by presence of p38 inhibitor SB203580. CONCLUSION: Increasing phosphate concentration stimulates erythrocyte membrane scrambling, an effect depending on extracellular but not intracellular Ca(2+) concentration. It is hypothesized that suicidal erythrocyte death is triggered by complexed CaHPO4.