Christine Zelenak

University of Tuebingen, Tübingen, Baden-Wuerttemberg, Germany

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Publications (26)87.59 Total impact

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    ABSTRACT: 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. © 2014 S. Karger AG, Basel.
    07/2014; 34(2):432-442.
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    ABSTRACT: 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.
    Cellular Physiology and Biochemistry 08/2012; 30(4):876-888. · 3.42 Impact Factor
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    ABSTRACT: Carbon monoxide (CO) intoxication severely interferes with the oxygen-transporting function of haemoglobin. Beyond that, CO participates in the regulation of apoptosis. CO could be generated from CO-releasing molecules (CORM), such as the tricarbonyl-dichlororuthenium (II) dimer (CORM-2), which is presently considered for the treatment of vascular dysfunction, inflammation, tissue ischaemia and organ rejection. CORM-2 is at least partially effective by modifying gene expression and mitochondrial potential. Erythrocytes lack nuclei and mitochondria but may undergo suicidal cell death or eryptosis, characterized by cell shrinkage and phospholipid scrambling of the cell membrane. Eryptosis is triggered by the increase in cytosolic Ca(2+) activity ([ Ca(2+) ](i) ). The present study explored whether CORM-2 influences eryptosis. To this end, [ Ca(2+) ](i) was estimated from Fluo-3-fluorescence, cell volume from forward scatter, phospholipid scrambling from annexin-V-binding and haemolysis from haemoglobin release. CO-binding haemoglobin (COHb) was estimated utilizing a blood gas analyser. As a result, exposure of erythrocytes for 24 hr to CORM-2 (≥5 μM) significantly increased COHb, [ Ca(2+) ](i) , forward scatter, annexin-V-binding and haemolysis. Annexin-V-binding was significantly blunted by 100% oxygen and was virtually abolished in the nominal absence of Ca(2+) . In conclusion, CORM-2 stimulates cell membrane scrambling of erythrocytes, an effect largely due to Ca(2+) entry and partially reversed by O(2) .
    Basic & Clinical Pharmacology & Toxicology 06/2012; 111(5):348-55. · 2.18 Impact Factor
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    ABSTRACT: The oxidative stress-responsive kinase 1 (OSR1) is activated by WNK (with no K kinases) and in turn stimulates the thiazide-sensitive Na-Cl cotransporter (NCC) and the furosemide-sensitive Na-K-2Cl cotransporter (NKCC), thus contributing to transport and cell volume regulation. Little is known about extrarenal functions of OSR1. The present study analyzed the impact of decreased OSR1 activity on the function of dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity. DCs were cultured from bone marrow of heterozygous WNK-resistant OSR1 knockin mice (osr(KI)) and wild-type mice (osr(WT)). Cell volume was estimated from forward scatter in FACS analysis, ROS production from 2',7'-dichlorodihydrofluorescein-diacetate fluorescence, cytosolic pH (pH(i)) from 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein fluorescence, and Na(+)/H(+) exchanger activity from Na(+)-dependent realkalinization following ammonium pulse and migration utilizing transwell chambers. DCs expressed WNK1, WNK3, NCC, NKCC1, and OSR1. Phosphorylated NKCC1 was reduced in osr(KI) DCs. Cell volume and pH(i) were similar in osr(KI) and osr(WT) DCs, but Na(+)/H(+) exchanger activity and ROS production were higher in osr(KI) than in osr(WT) DCs. Before LPS treatment, migration was similar in osr(KI) and osr(WT) DCs. LPS (1 μg/ml), however, increased migration of osr(WT) DCs but not of osr(KI) DCs. Na(+)/H(+) exchanger 1 inhibitor cariporide (10 μM) decreased cell volume, intracellular reactive oxygen species (ROS) formation, Na(+)/H(+) exchanger activity, and pH(i) to a greater extent in osr(KI) than in osr(WT) DCs. LPS increased cell volume, Na(+)/H(+) exchanger activity, and ROS formation in osr(WT) DCs but not in osr(KI) DCs and blunted the difference between osr(KI) and osr(WT) DCs. Na(+)/H(+) exchanger activity in osr(WT) DCs was increased by the NKCC1 inhibitor furosemide (100 nM) to values similar to those in osr(KI) DCs. Oxidative stress (10 μM tert-butyl-hydroperoxide) increased Na(+)/H(+) exchanger activity in osr(WT) DCs but not in osr(KI) DCs and reversed the difference between genotypes. Cariporide virtually abrogated Na(+)/H(+) exchanger activity in both genotypes and blunted LPS-induced cell swelling and ROS formation in osr(WT) mice. In conclusion, partial OSR1 deficiency influences Na(+)/H(+) exchanger activity, ROS formation, and migration of dendritic cells.
    AJP Cell Physiology 05/2012; 303(4):C416-26. · 3.71 Impact Factor
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    ABSTRACT: 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 Ca(2+)-permeable cation channels. Ca(2+) entering through those channels stimulates cell membrane scrambling and opens Ca(2+)-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 Ca(2+)-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 Ca(2+)-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 Ca(2+)-activated K(+)-channels. Ca(2+)-ionophore ionomycin (1 µM) decreased forward scatter, an effect significantly blunted by pyrvinium pamoate. In conclusion, pyrvinium pamoate activates Cl(-)-sensitive Ca(2+)-permeable cation channels with subsequent Ca(2+)-entry and inhibits Ca(2+)-activated K(+)-channels thus blunting the stimulating effect of Ca(2+) on those channels, K(+)-exit and thus cell shrinkage.
    Cellular Physiology and Biochemistry 01/2012; 30(2):407-17. · 3.42 Impact Factor
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    ABSTRACT: 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.
    Cellular Physiology and Biochemistry 01/2012; 30(2):428-38. · 3.42 Impact Factor
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    ABSTRACT: 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.
    Cellular Physiology and Biochemistry 01/2012; 30(1):282-94. · 3.42 Impact Factor
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    ABSTRACT: The anti-inflammatory Nigella sativa component thymoquinone compromises the function of dendritic cells (DCs), key players in the regulation of innate and adaptive immunity. DC function is regulated by the Na(+)/H(+) exchanger (NHE), which is stimulated by lipopolysaccharides (LPS) and required for LPS-induced cell swelling, reactive oxygen species (ROS) production, TNF-α release and migration. Here we explored, whether thymoquinone influences NHE activity in DCs. To this end, bone marrow derived mouse DCs were treated with LPS in the absence and presence of thymoquinone (10 μM). Cytosolic pH (pH(i)) was determined from 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) fluorescence, NHE activity from the Na(+)-dependent realkalinization following an ammonium pulse, cell volume from forward scatter in FACS analysis, ROS production from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, TNF-α production utilizing ELISA and DC migration with transwell migration assays. As a result, exposure of DCs to LPS (1 μg/ml) led within 4 hours to transient increase of NHE activity. Thymoquinone did not significantly modify cytosolic pH or cellular NHE activity in the absence of LPS, but abrogated the effect of LPS on NHE activity. Accordingly, in the presence of thymoquinone LPS-treatment resulted in cytosolic acidification. LPS further increased forward scatter and ROS formation, effects similarly abrogated by thymoquinone. Again, in the absence of LPS, thymoquinone did not significantly modify ROS formation and cell volume. LPS further triggered TNF-α release and migration, effects again blunted in the presence of thymoquinone. NHE1 inhibitor cariporide (10 μM) blunted LPS induced TNF-α release and migration. The effects of thymoquinone on NHE activity and migration were reversed upon treatment of the cells with t-butyl hydroperoxide (TBOOH, 5 μM). In conclusion, thymoquinone blunts LPS induced NHE activity, cell swelling, oxidative burst, cytokine release and migration of bone marrow derived murine dendritic cells. NHE inhibition may thus contribute to the antiinflammatory action of thymoquinone.
    Cellular Physiology and Biochemistry 01/2012; 29(1-2):21-30. · 3.42 Impact Factor
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    ABSTRACT: Protein kinase CK1 (casein kinase 1) isoforms are involved in the regulation of various physiological functions including apoptosis. The specific CK1 inhibitor D4476 may either inhibit or foster apoptosis. Similar to apoptosis of nucleated cells, eryptosis, the suicidal death of erythrocytes, is paralleled by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Triggers of eryptosis include increase of cytosolic Ca(2+) activity following energy depletion (removal of glucose) or oxidative stress (exposure to the oxidant tert-butyl hydroperoxide [TBOOH]). Western blotting was utilized to verify that erythrocytes express the protein kinase CK1α, and FACS analysis to determine whether the CK1 inhibitor D4476 and CK1α activator pyrvinium pamoate modify forward scatter (reflecting cell volume), annexin V binding (reflecting phosphatidylserine exposure), and Fluo3 fluorescence (reflecting cytosolic Ca(2+) activity). As a result, both, human and murine erythrocytes express CK1 isoform α. Glucose depletion (48 hours) and exposure to 0.3 mM TBOOH (30 minutes) both decreased forward scatter, increased annexin V binding and increased Fluo3 fluorescence. CK1 inhibitor D4476 (10 μM) significantly blunted the decrease in forward scatter, the increase in annexin V binding and the increase in Fluo 3 fluorescence. (R)-DRF053, another CK1 inhibitor, similarly blunted the increase in annexin V binding upon glucose depletion. The CK1α specific activator pyrvinium pamoate (10 μM) significantly enhanced the increase in annexin V binding and Fluo3 fluorescence upon glucose depletion and TBOOH exposure. In the presence of glucose, pyrvinium pamoate slightly but significantly increased Fluo3 fluorescence. In conclusion, CK1 isoform α participates in the regulation of erythrocyte programmed cell death by modulating cytosolic Ca(2+) activity.
    Cellular Physiology and Biochemistry 01/2012; 29(1-2):171-80. · 3.42 Impact Factor
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    ABSTRACT: Rapamycin, a widely used immunosuppressive drug, has been shown to interfere with the function of dendritic cells (DCs), antigen-presenting cells contributing to the initiation of primary immune responses and the establishment of immunological memory. DC function is governed by the Na(+)/H(+) exchanger (NHE), which is activated by bacterial lipopolysaccharides (LPS) and is required for LPS-induced cell swelling, reactive oxygen species (ROS) production and TNF-α release. The present study explored, whether rapamycin influences NHE activity and/or ROS formation in DCs. Mouse DCs were treated with LPS in the absence and presence of rapamycin (100 nM). ROS production was determined from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, cytosolic pH (pH(i)) from 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) fluorescence, NHE activity from the Na(+)-dependent realkalinization following an ammonium pulse, cell volume from forward scatter in FACS analysis, and TNF-α production utilizing ELISA. In the absence of LPS, rapamycin did not significantly modify cytosolic pH, NHE activity or cell volume but significantly decreased ROS formation. LPS stimulated NHE activity, enhanced forward scatter, increased ROS formation, and triggered TNF-α release, effects all blunted in the presence of rapamycin. NADPH oxidase inhibitor Vas-2870 (10 μM) mimicked the effect of rapamycin on LPS induced stimulation of NHE activity and TNF-α release. The effect of rapamycin on TNF-α release was also mimicked by the antioxidant ROS scavenger Tempol (30 μM) and partially reversed by additional application of tert-butylhydroperoxide (10 μM). In conclusion, in DCs rapamycin disrupts LPS induced ROS formation with subsequent inhibition of NHE activity, cell swelling and TNF-α release.
    Cellular Physiology and Biochemistry 01/2012; 29(3-4):543-50. · 3.42 Impact Factor
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    ABSTRACT: 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.
    Biology of Metals 11/2011; 25(2):309-18. · 3.17 Impact Factor
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    ABSTRACT: Ursolic acid (1), a triterpenoid with pleotropic effects including inhibition of tumor growth, is well known to trigger apoptosis of nucleated cells. The effect is at least partially due to altered gene expression and mitochondrial dysfunction. Erythrocytes lack nuclei and mitochondria but, similar to nucleated cells, may undergo suicidal cell death or eryptosis, which is characterized by cell shrinkage and phospholipid scrambling of the cell membrane. Triggers of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), ceramide formation and/or ATP depletion. The present study has investigated whether or not 1 induces eryptosis. [Ca2+]i was estimated from Fluo-3 fluorescence, cell volume from forward scatter, phospholipid scrambling from annexin V binding, hemolysis from hemoglobin release, and cytosolic ATP concentration ([ATP]i) utilizing a luciferase assay and ceramide-utilizing fluorescent antibodies in FACS analysis. As a result, exposure of erythrocytes for 48 h to 1 (≥5 μM) did not significantly modify [ATP]i, but significantly increased [Ca2+]i, stimulated ceramide formation, decreased forward scatter, triggered annexin V binding, and elicited hemolysis. At 5 μM, 1 stimulated phospholipid scrambling in 10% and hemolysis in 2% of treated erythrocytes. Annexin V binding was blunted in the nominal absence of Ca2+. In conclusion, the food component ursolic acid stimulates suicidal death of erythrocytes, i.e., cells devoid of nuclei and mitochondria.
    Journal of Natural Products 09/2011; 74(10):2181-6. · 3.29 Impact Factor
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    ABSTRACT: Enniatin A, a peptide antibiotic and common food contaminant, triggers mitochondrial dysfunction and apoptosis. Even though lacking mitochondria, erythrocytes may similarly undergo suicidal cell death or eryptosis. Eryptosis is characterized by cell shrinkage and cell membrane phospholipid scrambling. Triggers of phospholipid scrambling include energy depletion and increase in cytosolic Ca(2+) activity ([Ca(2+) ](i) ). The present study explored whether enniatin A triggers phospholipid scrambling. Phospholipid scrambling was estimated from annexin-V-binding, cell volume from forward scatter (FSC), [Ca(2+) ](i) from Fluo3-fluorescence, cytosolic ATP-concentration ([ATP](i) ) using a luciferase assay and hemolysis from hemoglobin release. Exposure of erythrocytes for 48 h to enniatin A (≥ 2.5 μM) significantly increased [Ca(2+) ](i) , decreased [ATP](i) , decreased FSC, triggered annexin-V-binding and elicited hemolysis. Annexin-V-binding affected 25%, and hemolysis 2% of treated erythrocytes. Decreased [ATP](i) by glucose depletion for 48 h was similarly followed by increased [Ca(2+) ](i) , decreased FSC and annexin-V-binding. Enniatin A augmented the effect on [Ca(2+) ](i) and annexin-V-binding, but not on FSC. Annexin-V-binding was blunted by Ca(2+) removal, by the cation channel inhibitor amiloride (1 mM), by the protein kinase C inhibitor staurosporine (500 nM) but not by the pancaspase inhibitor zVAD (10 μM). The food contaminant enniatin A triggers ATP depletion and increases cytosolic Ca(2+) activity, effects resulting in suicidal erythrocyte death.
    Molecular Nutrition & Food Research 08/2011; 55 Suppl 2:S294-302. · 4.31 Impact Factor
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    ABSTRACT: Loss-of-function mutations in human adenomatous polyposis coli (APC) lead to multiple colonic adenomatous polyps eventually resulting in colonic carcinoma. Similarly, heterozygous mice carrying defective APC (apc(Min/+)) suffer from intestinal tumours. The animals further suffer from anaemia, which in theory could result from accelerated eryptosis, a suicidal erythrocyte death triggered by enhanced cytosolic Ca(2+) activity and characterized by cell membrane scrambling and cell shrinkage. To explore, whether APC-deficiency enhances eryptosis, we estimated cell membrane scrambling from annexin V binding, cell size from forward scatter and cytosolic ATP utilizing luciferin-luciferase in isolated erythrocytes from apc(Min/+) mice and wild-type mice (apc(+/+)). Clearance of circulating erythrocytes was estimated by carboxyfluorescein-diacetate-succinimidyl-ester labelling. As a result, apc(Min/+) mice were anaemic despite reticulocytosis. Cytosolic ATP was significantly lower and annexin V binding significantly higher in apc(Min/+) erythrocytes than in apc(+/+) erythrocytes. Glucose depletion enhanced annexin V binding, an effect significantly more pronounced in apc(Min/+) erythrocytes than in apc(+/+) erythrocytes. Extracellular Ca(2+) removal or inhibition of Ca(2+) entry with amiloride (1 mM) blunted the increase but did not abrogate the genotype differences of annexin V binding following glucose depletion. Stimulation of Ca(2+) -entry by treatment with Ca(2+) -ionophore ionomycin (10 μM) increased annexin V binding, an effect again significantly more pronounced in apc(Min/+) erythrocytes than in apc(+/+) erythrocytes. Following retrieval and injection into the circulation of the same mice, apc(Min/+) erythrocytes were more rapidly cleared from circulating blood than apc(+/+) erythrocytes. Most labelled erythrocytes were trapped in the spleen, which was significantly enlarged in apc(Min/+) mice. The observations point to accelerated eryptosis and subsequent clearance of apc(Min/+) erythrocytes, which contributes to or even accounts for the enhanced erythrocyte turnover, anaemia and splenomegaly in those mice.
    Journal of Cellular and Molecular Medicine 07/2011; 16(5):1085-93. · 4.75 Impact Factor
  • Kashif Jilani, Syed M Qadri, Christine Zelenak, Florian Lang
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    ABSTRACT: Oridonin triggers apoptosis of cancer cells and was suggested as anticancer agent. Oridonin is partially effective through mitochondrial depolarization and partially by modifying gene expression. Erythrocytes lack mitochondria and nuclei but may undergo eryptosis, a suicidal cell death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Triggers of eryptosis include increase of cytosolic Ca(2+)-activity, ATP depletion and ceramide formation. The present study explored, whether oridonin triggers eryptosis. Cytosolic Ca(2+)-concentration was estimated from Fluo3-fluorescence, cell volume from forward scatter in FACS analysis, phosphatidylserine exposure from binding of fluorescent annexin V, hemolysis from hemoglobin release, ATP concentration utilizing a luciferin-luciferase assay and ceramide abundance utilizing fluorescent anti-ceramide antibodies. A 48 h exposure to oridonin (≥25μM) significantly increased cytosolic Ca(2+)-concentration, increased ceramide formation, decreased forward scatter and triggered annexin V-binding (the latter in >20% of the erythrocytes). Oridonin didn't decrease ATP concentration and hemolysed <5% of erythrocytes. The effects of oridonin on annexin V binding were partially reversed in the nominal absence of Ca(2+) and by the addition of amiloride (1mM). The present observations reveal a completely novel effect of oridonin, i.e. triggering of Ca(2+) entry and ceramide formation as well as suicidal death of erythrocytes.
    Archives of Biochemistry and Biophysics 07/2011; 511(1-2):14-20. · 3.37 Impact Factor
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    ABSTRACT: Phlorhizin interferes with glucose transport. Glucose depletion triggers suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and cell membrane scrambling. Eryptosis is further triggered by oxidative stress. The present study explored whether phlorhizin influences eryptosis following glucose depletion or oxidative stress. Cell membrane scrambling was estimated from annexin binding, cell volume from forward scatter (FSC), and cytosolic Ca(2+) concentration from Fluo-3 fluorescence. Phlorhizin (10-100 μM) added alone did not modify scrambling, FSC, or Fluo-3 fluorescence. Glucose depletion (48 h) significantly increased Fluo-3 fluorescence, decreased FSC, and increased annexin binding, effects in part significantly blunted by phlorhizin (annexin binding ≥ 10 μM, FSC ≥ 50 μM). Oxidative stress (30 min 0.3 mM tert-butylhydroperoxide) again significantly increased Fluo-3 fluorescence and triggered annexin binding, effects again in part significantly blunted by phlorhizin (Fluo-3 fluorescence ≥ 50 μM, annexin-binding ≥ 10 μM). Phlorhizin did not blunt the cell shrinkage induced by oxidative stress. The present observations disclose a novel effect of phlorhizin, that is, an influence on suicidal erythrocyte death following energy depletion and oxidative stress.
    Journal of Agricultural and Food Chemistry 06/2011; 59(15):8524-30. · 3.11 Impact Factor
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    ABSTRACT: Klotho-hypomorphic (Klotho(hm)) mice suffer from renal salt wasting and hypovolemia despite hyperaldosteronism. The present study explored the effect of Klotho on renal Na(+)/K(+) ATPase activity. According to immunohistochemistry and confocal microscopy Na(+)/K(+) ATPase protein abundance in isolated collecting ducts was lower in Klotho(hm) mice than in their wild type littermates (Klotho(+/+)). Analysis with dual electrode voltage clamp recording showed that expression of Klotho in Xenopus oocytes increased the Na(+)/K(+) ATPase pump current. Treatment of Xenopus oocytes with Klotho protein similarly increased the pump current. In conclusion, Klotho increases the membrane abundance and activity of the Na(+)/K(+) ATPase. Decreased Na(+)/K(+) ATPase activity could thus contribute to the volume-depletion of klotho(hm) mice.
    FEBS letters 06/2011; 585(12):1759-64. · 3.54 Impact Factor
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    ABSTRACT: Blebbistatin, a myosin II inhibitor, interferes with myosin-actin interaction and microtubule assembly. By influencing cytoskeletal dynamics blebbistatin counteracts apoptosis of several types of nucleated cells. Even though lacking nuclei and mitochondria, erythrocytes may undergo suicidal cell death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Triggers of eryptosis include energy depletion and osmotic shock, which enhance cytosolic Ca(2+) activity with subsequent Ca(2+)-sensitive cell shrinkage and cell membrane scrambling. The present study explored the effect of blebbistatin on eryptosis. Cell membrane scrambling was estimated from binding of annexin V to phosphatidylserine at the erythrocyte surface, cell volume from forward scatter in fluorescence-activated cell sorting analysis and cytosolic Ca(2+) concentration from Fluo3 fluorescence. Exposure to blebbistatin on its own (1-50 μM) did not significantly modify cytosolic Ca(2+) concentration, forward scatter, or annexin V binding. Glucose depletion (48 h) was followed by a significant increase of Fluo3 fluorescence and annexin V binding, effects significantly blunted by blebbistatin (Fluo3 fluorescence ≥ 25 μM, annexin V binding ≥ 10 μM). Osmotic shock (addition of 550 mM sucrose) again significantly increased Fluo3 fluorescence and annexin binding, effects again significantly blunted by blebbistatin (Fluo3 fluorescence ≥ 25 μM, annexin V binding ≥ 25 μM). The present observations disclose a novel effect of blebbistatin, i.e., an influence on Ca(2+) entry and suicidal erythrocyte death following energy depletion and osmotic shock.
    AJP Cell Physiology 05/2011; 301(2):C490-8. · 3.71 Impact Factor
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    ABSTRACT: Activation of AMP-activated protein kinase (AMPK) upon energy depletion stimulates energy production and limits energy utilization. Erythrocytes lacking AMPK are susceptible to suicidal cell death (eryptosis). A hallmark of eryptosis is cell membrane scrambling with phosphatidylserine exposure at the erythrocyte surface, which can be identified from annexin V-binding. AMPKα1-deficient mice (ampk(-/-)) suffer from anemia due to accelerated clearance of erythrocytes from circulating blood. To determine the link between AMPK and the eryptotic phenotype, we performed a global proteome analysis of erythrocytes from ampk(-/-) mice and wild-type mice using high-accuracy mass spectrometry and label-free quantitation and measured changes of expression levels of 812 proteins. Notably, the p21-activated kinase 2 (PAK2), previously implicated in apoptosis, was detected as downregulated in erythrocytes of ampk(-/-) mice, pointing to its potential role in eryptosis. To validate this, we showed that specific inactivation of PAK2 with the inhibitor IPA3 in human and murine ampk(+/+) erythrocytes increases the binding of annexin V and augments the stimulating effect of glucose deprivation on annexin V-binding. Inhibition of PAK2 failed to significantly modify annexin V-binding in ampk(-/-) erythrocytes, showing that AMPK and PAK2 exert similar phenotypes upon inactivation in erythrocytes. This study presents the first large-scale analysis of protein expression in erythrocytes from AMPKα1-deficient mice and reveals a role of PAK2 kinase in eryptosis.
    Journal of Proteome Research 02/2011; 10(4):1690-7. · 5.06 Impact Factor
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    ABSTRACT: The heterotetrameric K(+)-channel KCNQ1/KCNE1 is expressed in heart, skeletal muscle, liver and several epithelia including the renal proximal tubule. In the heart, it contributes to the repolarization of cardiomyocytes. The repolarization is impaired in ischemia. Ischemia stimulates the AMP-activated protein kinase (AMPK), a serine/threonine kinase, sensing energy depletion and stimulating several cellular mechanisms to enhance energy production and to limit energy utilization. AMPK has previously been shown to downregulate the epithelial Na(+) channel ENaC, an effect mediated by the ubiquitin ligase Nedd4-2. The present study explored whether AMPK regulates KCNQ1/KCNE1. To this end, cRNA encoding KCNQ1/KCNE1 was injected into Xenopus oocytes with and without additional injection of wild type AMPK (AMPKα1 + AMPKβ1 + AMPKγ1), of the constitutively active (γR70Q)AMPK (α1β1γ1(R70Q)), of the kinase dead mutant (αK45R)AMPK (α1(K45R)β1γ1), or of the ubiquitin ligase Nedd4-2. KCNQ1/KCNE1 activity was determined in two electrode voltage clamp experiments. Moreover, KCNQ1 abundance in the cell membrane was determined by immunostaining and subsequent confocal imaging. As a result, wild type and constitutively active AMPK significantly reduced KCNQ1/KCNE1-mediated currents and reduced KCNQ1 abundance in the cell membrane. Similarly, Nedd4-2 decreased KCNQ1/KCNE1-mediated currents and KCNQ1 protein abundance in the cell membrane. Activation of AMPK in isolated perfused proximal renal tubules by AICAR (10 mM) was followed by significant depolarization. In conclusion, AMPK is a potent regulator of KCNQ1/KCNE1.
    Molecular Membrane Biology 02/2011; 28(2):79-89. · 3.13 Impact Factor