Publications (6)20.16 Total impact
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Article: The impact of erythrocyte age on eryptosis.
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ABSTRACT: Mature, circulating erythrocytes undergo senescence, which limits their life span to approximately 120 d. Upon injury, erythrocytes may undergo suicidal erythrocyte death or eryptosis, which may accelerate senescence and shorten their survival. Eryptosis is defined as cell shrinkage and exposure of phosphatidylserine at the cell surface. Triggers of eryptosis include oxidative stress. The present study addresses the impact of erythrocyte age on the relative susceptibility to eryptosis. Erythrocytes were separated into five fractions, based on age-associated differences in density and volume. Cell membrane scrambling was estimated from binding of annexin V to phosphatidylserine at the erythrocyte surface, the cell volume from forward scatter, and the Ca(2+) level from Fluo-3-dependent fluorescence. In addition, glutathione (GSH) concentrations were measured by an enzymatic/colourimetric method. After 48 h incubation in Ringer solution, Annexin V binding increased significantly with erythrocyte age. The differences were not accompanied by altered GSH concentrations, but were reversed by addition of the antioxidant N-acetyl-L-cysteine in vitro. Also, N-acetyl-L-cysteine significantly prolonged the half-life of circulating mouse erythrocytes in vivo. Thus, the susceptibility to eryptosis increases with the age of the erythrocytes, and this effect is at least partially due to enhanced sensitivity to oxidative stress.British Journal of Haematology 03/2012; 157(5):606-14. · 4.94 Impact Factor -
Article: Potential roles of the NFκB and glutathione pathways in mature human erythrocytes.
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ABSTRACT: Anucleated erythrocytes were long considered as oxygen-transporting cells with limited regulatory functions. Components of different nuclear signaling pathways have not been investigated in those cells, yet. Surprisingly, we repeatedly found significant amounts of transcription factors in purified erythrocyte preparations, i.e. nuclear factor κB (NFκB), and major components of the canonical NFκB signaling pathway. To investigate the functional role of NFκB signaling, the effects of the preclinical compounds Bay 11-7082 and parthenolide on the survival of highly purified erythrocytes were investigated. Interestingly, both inhibitors of the NFκB pathway triggered erythrocyte programmed cell death as demonstrated by enhanced phospholipid scrambling (phosphatidylserine exposure) and cell shrinkage. Anucleated erythrocytes are an ideal cellular model allowing the study of nongenomic mechanisms contributing to suicidal cell death. As NFκB inhibitors might also interfere with the anti-oxidative defense systems of the cell, we measured the levels of reduced glutathione (GSH) after challenge with the inhibitors. Indeed, incubation of erythrocytes with Bay 11-7082 clearly decreased erythrocyte GSH levels. In conclusion, the pharmacological inhibitors of the NFκB pathway Bay 11-7082 and parthenolide interfere with the survival of erythrocytes involving mechanisms other than disruption of NFκB-dependent gene expression. Besides affecting erythrocyte survival, NFκB inhibition and induction of erythrocyte phosphatidylserine exposure may influence blood clotting. Future studies will be aimed at discriminating between NFκB-dependent and NFκB-independent GSH-mediated effects of Bay 11-7082 and parthenolide on erythrocyte death.Cellular & Molecular Biology Letters 11/2011; 17(1):11-20. · 1.50 Impact Factor -
Article: The NFĸB pathway inhibitors Bay 11-7082 and parthenolide induce programmed cell death in anucleated Erythrocytes.
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ABSTRACT: The preclinical compounds Bay 11-7082 and parthenolide trigger apoptosis, an effect contributing to their antiinflammatory action. The substances interfere with the activation and nuclear translocation of nuclear factor NFκB, by inhibiting NFκB directly (parthenolide) or by interfering with the inactivation of the NFκB inhibitory protein IκB-α (Bay 11-7082). Beyond that, the substances may be effective in part by nongenomic effects. Similar to apoptosis of nucleated cells, erythrocytes may undergo apoptosis-like cell death (eryptosis) characterized by cell membrane scrambling with phosphatidylserine exposure, and cell shrinkage. Thus, erythrocytes allow the study of nongenomic mechanisms contributing to suicidal cell death, e.g. Ca(2+) leakage or glutathione depletion. The present study utilized Western blotting to search for NFκB and IκB-α expression in erythrocytes, FACS analysis to determine cytosolic Ca(2+) (Fluo3 fluorescence), phosphatidylserine exposure (annexin V binding), and cell volume (forward scatter), as well as an enzymatic method to determine glutathione levels. As a result, both NFκB and IκB-α are expressed in erythrocytes. Targeting the NFκB pathway by Bay 11-7082 (IC(50) ≈ 10 μM) and parthenolide (IC(50) ≈ 30 μM) triggered suicidal erythrocyte death as shown by annexin V binding and decrease of forward scatter. Bay 11-7082 treatment further increased intracellular Ca(2+) and led to depletion of reduced glutathione. The effects of Bay 11-7082 and parthenolide on annexin V binding could be fully reversed by the antioxidant N-acetylcysteine. In conclusion, the pharmacological inhibitors of NFκB, Bay 11-7082 and parthenolide, interfere with the survival of erythrocytes involving mechanisms other than disruption of NFκB-dependent gene expression.Cellular Physiology and Biochemistry 01/2011; 27(1):45-54. · 2.86 Impact Factor -
Article: Targeting glutathione by dimethylfumarate protects against experimental malaria by enhancing erythrocyte cell membrane scrambling.
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ABSTRACT: The balance between GSH-levels and oxidative stress is critical for cell survival. The GSH-levels of erythrocytes are dramatically decreased during infection with Plasmodium spp. We therefore investigated the consequences of targeting GSH for erythrocyte and Plasmodium survival in vitro and in vivo using dimethylfumarate (DMF) at therapeutically established dosage. We first show that noninfected red blood cells (RBC) exposed to DMF undergo changes typical of apoptosis or eryptosis, such as cell shrinkage and cell membrane scrambling with subsequent phosphatidylserine (PS) exposure. DMF did not induce appreciable hemolysis. DMF-triggered PS exposure was mediated by intracellular GSH depletion and reversed by the antioxidative N-acetyl-l-cysteine. DMF treatment controlled intraerythrocyte DNA amplification and in vitro parasitemia of Plasmodium falciparum-infected RBC. In vivo, DMF treatment had no effect on RBC count or GSH levels in noninfected mice. Consistent with its effects on infected RBC, DMF treatment abrogated parasitemia and enhanced the survival of mice infected with Plasmodium berghei from 0% to 60%. In conclusion, DMF sensitizes the erythrocytes to the effect of Plasmodium infection on PS exposure, thus accelerating the clearance of infected erythrocytes. Accordingly, DMF treatment favorably influences the clinical course of malaria. As DMF targets mechanisms within the host cell, it is not likely to generate resistance of the pathogen.AJP Cell Physiology 10/2010; 299(4):C791-804. · 3.54 Impact Factor -
Article: Inhibition of suicidal erythrocyte death by nitric oxide.
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ABSTRACT: Nitric oxide (NO) is known to counteract apoptosis by S-nitrosylation of protein thiol groups. NO is generated and stored in erythrocytes, which may undergo eryptosis, a suicidal cell death similar to apoptosis of nucleated cells. Eryptosis is triggered by increased cytosolic Ca2+ activity and/or ceramide and characterized by cell shrinkage and phosphatidylserine exposure at the cell surface. The present study explored whether nitric oxide could interfere with the machinery underlying eryptosis. To this end, erythrocyte phosphatidylserine exposure (annexin V-binding) and cell volume (forward scatter) were determined by flow cytometry. The Ca2+ ionophore ionomycin (0.1 microM) increased cytosolic Ca2+ activity, triggered annexin binding, and decreased forward scatter. The annexin binding and decrease of forward scatter but not the increase of cytosolic Ca2+ activity were reversed by the NO-donor nitroprusside (1 microM) and papanonoate (100 microM). Higher concentrations of nitroprusside (0.1 and 1 mM) stimulated eryptosis. Glucose depletion, exposure to C6-ceramide (3 microM), hypertonic (addition of 550 mM sucrose), and isotonic (replacement of Cl- with gluconate) cell shrinkage all triggered annexin V binding, effects all reversed by nitroprusside (1 microM). Dibutyryl-cGMP (1 mM) blunted the ionomycin- but not the ceramide-induced annexin V binding. Ionomycin decreased protein nitrosylation and thioredoxin activity, effects reversed by the NO-donor papanonoate. Clearance of erythrocytes from circulating blood was significantly faster in eNOS knockout mice than in their wild-type littermates. In conclusion, nitric oxide participates in the regulation of erythrocyte survival, an effect partially mimicked by cGMP and paralleled by alterations of protein nitrosylation and thioredoxin activity.Pflügers Archiv - European Journal of Physiology 06/2008; 456(2):293-305. · 4.46 Impact Factor -
Article: Stimulation of erythrocyte cell membrane scrambling by amiodarone.
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ABSTRACT: Side effects of amiodarone, an effective antiarrhythmic drug, include anemia, which may be caused by decreased formation or accelerated death of erythrocytes. Suicidal erythrocyte death (eryptosis) is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine exposure at the cell surface. Stimulators of erythrocyte membrane scrambling include increase of cytosolic Ca2+ concentration ([Ca2+]i) following activation of Ca2+-permeable cation channels. Moreover, eryptosis is triggered by ceramide. The present study has been performed to test for an effect of amiodarone on eryptosis. Erythrocytes from healthy volunteers were exposed to amiodarone and phosphatidylserine exposure (annexin V binding), cell volume (forward scatter), [Ca2+]i (Fluo3-dependent fluorescence), and ceramide formation (anti-ceramide-FITC antibody and radioactive labelling) determined by flow cytometry. Exposure of erythrocytes to amiodarone (1 microM) increased [Ca2+]i and triggered annexin V binding, but did not significantly decrease forward scatter and did not significantly influence ceramide formation. Amiodarone augmented the increase of annexin binding following hypertonic shock (addition of 550 mM sucrose) but did not significantly alter the enhanced annexin binding following Cl- removal (replacement with gluconate). Amiodarone did not significantly modify the decrease of forward scatter following hypertonic shock or Cl- removal. The present observations disclose a novel action of amiodarone which may contribute to the side effects of the drug.Cellular Physiology and Biochemistry 02/2007; 20(6):1043-50. · 2.86 Impact Factor
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2010–2012
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Eberhard-Karls-Universität Tübingen
- Institute for Physiology
Tübingen, Baden-Wuerttemberg, Germany
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