scholarly journals Enhanced susceptibility to suicidal death of erythrocytes from transgenic mice overexpressing erythropoietin

2007 ◽  
Vol 293 (3) ◽  
pp. R1127-R1134 ◽  
Author(s):  
Michael Föller ◽  
Ravi S. Kasinathan ◽  
Saisudha Koka ◽  
Stephan M. Huber ◽  
Beat Schuler ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Transgenic Mice ◽  
Cell Volume ◽  
Cation Channels ◽  
Forward Scatter ◽  
Membrane Asymmetry ◽  
Phosphatidylserine Exposure ◽  
Suicidal Death ◽  
Osmotic Lysis ◽  
The Difference

Eryptosis, a suicidal death of mature erythrocytes, is characterized by decrease of cell volume, cell membrane blebbing, and breakdown of cell membrane asymmetry with phosphatidylserine exposure at the cell surface. Triggers of eryptosis include increased cytosolic Ca2+ activity, which could result from activation of Ca2+-permeable cation channels. Ca2+ triggers phosphatidylserine exposure and activates Ca2+-sensitive K+ channels, leading to cellular K+ loss and cell shrinkage. The cation channels and thus eryptosis are stimulated by Cl− removal and inhibited by erythropoietin. The present experiments explored eryptosis in transgenic mice overexpressing erythropoietin (tg6). Erythrocytes were drawn from tg6 mice and their wild-type littermates (WT). Phosphatidylserine exposure was estimated from annexin binding and cell volume from forward scatter in fluorescence-activated cell sorting (FACS) analysis. The percentage of annexin binding was significantly larger and forward scatter significantly smaller in tg6 than in WT erythrocytes. Transgenic erythrocytes were significantly more resistant to osmotic lysis than WT erythrocytes. Cl− removal and exposure to the Ca2+ ionophore ionomycin (1 μM) increased annexin binding and decreased forward scatter, effects larger in tg6 than in WT erythrocytes. The K+ ionophore valinomycin (10 nM) triggered eryptosis in both tg6 and WT erythrocytes and abrogated differences between genotypes. An increase of extracellular K+ concentration to 125 mM blunted the difference between tg6 and WT erythrocytes. Fluo-3 fluorescence reflecting cytosolic Ca2+ activity was larger in tg6 than in WT erythrocytes. In conclusion, circulating erythrocytes from tg6 mice are sensitized to triggers of eryptosis but more resistant to osmotic lysis, properties at least partially due to enhanced Ca2+ entry and increased K+ channel activity.

scholarly journals Ca2+ Entry, Oxidative Stress, Ceramide and Suicidal Erythrocyte Death Following Diosgenin Treatment

2016 ◽  
Vol 39 (4) ◽  
pp. 1626-1637 ◽  
Author(s):  
Morena Mischitelli ◽  
Mohamed Jemaà ◽  
Mustafa Almasry ◽  
Caterina Faggio ◽  
Florian Lang
Keyword(s):  
Oxidative Stress ◽  
Cell Membrane ◽  
Haemoglobin Concentration ◽  
Annexin V ◽  
Cellular Mechanisms ◽  
Forward Scatter ◽  
Erythrocyte Surface ◽  
Suicidal Death ◽  
Phosphatidylserine Translocation ◽  
Surface Signaling

Background/Aims: The bioactive steroid sapogenin diosgenin is considered for a wide variety of applications including treatment of malignancy. The substance counteracts tumor growth in part by stimulating apoptosis of tumor cells. Similar to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling involved in the stimulation of eryptosis includes increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress and ceramide. The present study explored, whether diosgenin induces eryptosis and, if so, to decipher cellular mechanisms involved. Methods: Flow cytometry was employed to estimate phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ROS formation from DCF dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was quantified by determination of haemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to diosgenin significantly increased the percentage of annexin-V-binding cells (≥ 5 µM), significantly decreased forward scatter (15 µM), significantly increased Fluo3-fluorescence (≥ 10 µM), significantly increased DCF fluorescence (15 µM), significantly increased ceramide abundance (15 µM) and significantly increased hemolysis (15 µM). The effect of diosgenin (15 µM) on annexin-V-binding was significantly blunted but not abolished by removal of extracellular Ca2+. Conclusions: Diosgenin stimulates eryptosis with erythrocyte shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect paralleled by and at least in part due to Ca2+ entry, oxidative stress and ceramide.

scholarly journals Stimulation of Erythrocyte Cell Membrane Scrambling by Adarotene

2017 ◽  
Vol 41 (2) ◽  
pp. 519-529 ◽  
Author(s):  
Morena Mischitelli ◽  
Mohamed Jemaàa ◽  
MyriamFezai Fezai ◽  
Mustafa Almasry ◽  
Florian Lang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Membrane ◽  
Annexin V ◽  
Forward Scatter ◽  
Erythrocyte Surface ◽  
Suicidal Death ◽  
Phosphatidylserine Translocation ◽  
Surface Signaling ◽  
Ros Formation ◽  
Stimulation Of

Background/Aims: The atypical retinoid E23-(40-hydroxyl-30-adamantylbiphenyl-4-yl) acrylic acid (ST1926, adarotene) is used in the treatment of malignancy. The effect of ST1926 is at least in part due to stimulation of apoptosis. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal death of erythrocytes. Hallmarks of eryptosis include cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling involved in the stimulation of eryptosis includes increase of cytosolic Ca2+ activity [Ca2+]<Sub>i</Sub>, oxidative stress and ceramide. The present study explored, whether adarotene induces eryptosis and, if so, to test for the involvement of Ca2+ entry, oxidative stress and ceramide. Methods: Flow cytometry was employed to estimate phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]<Sub>i</Sub> from Fluo3-fluorescence, reactive oxygen species (ROS) formation from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Results: A 48 hours exposure of human erythrocytes to adarotene (9 µM) significantly increased the percentage of annexin-V-binding cells, an effect paralleled by significant decrease of forward scatter, as well as significant increase of Fluo3-fluorescence, DCFDA fluorescence, and ceramide abundance. The effect of adarotene (9 µM) on annexin-V-binding was significantly blunted but not abolished by removal of extracellular Ca2+. Conclusions: Adarotene stimulates phospholipid scrambling of the erythrocyte cell membrane, an effect paralleled by and at least in part due to Ca2+ entry, oxidative stress and ceramide.

scholarly journals Triggering of Erythrocyte Cell Membrane Scrambling by Emodin

2016 ◽  
Vol 40 (1-2) ◽  
pp. 91-103 ◽  
Author(s):  
Morena Mischitelli ◽  
Mohamed Jemaà ◽  
Mustafa Almasry ◽  
Caterina Faggio ◽  
Florian Lang
Keyword(s):  
Oxidative Stress ◽  
Cell Membrane ◽  
Annexin V ◽  
Microbial Infection ◽  
Cellular Mechanisms ◽  
Forward Scatter ◽  
Erythrocyte Surface ◽  
Suicidal Death ◽  
Phosphatidylserine Translocation ◽  
Surface Signaling

Background/Aims: The natural anthraquinone derivative emodin (1,3,8-trihydroxy-6-methylanthraquinone) is a component of several Chinese medicinal herbal preparations utilized for more than 2000 years. The substance has been used against diverse disorders including malignancy, inflammation and microbial infection. The substance is effective in part by triggering suicidal death or apoptosis. Similar to apoptosis of nucleated cells erythrocytes may enter suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling involved in the triggering of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress and ceramide. The present study aimed to test, whether emodin induces eryptosis and, if so, to elucidate underlying cellular mechanisms. Methods: Phosphatidylserine abundance at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ROS formation from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Results: Exposure of human erythrocytes for 48 hours to emodin (≥ 10 µM) significantly increased the percentage of annexin-V-binding cells, and at higher concentrations (≥ 50 µM) significantly increased forward scatter. Emodin significantly increased Fluo3-fluorescence (≥ 10 µM), DCFDA fluorescence (75 µM) and ceramide abundance (75 µM). The effect of emodin on annexin-V-binding was significantly blunted but not abolished by removal of extracellular Ca2+. Conclusions: Emodin triggers phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to stimulation of Ca2+ entry and paralleled by oxidative stress and ceramide appearance at the erythroctye surface.

scholarly journals Stimulating Effect of Terfenadine on Erythrocyte Cell Membrane Scrambling

2016 ◽  
Vol 38 (4) ◽  
pp. 1425-1434 ◽  
Author(s):  
Elena Signoretto ◽  
Michela Castagna ◽  
Abdulla Al Mamun Bhuyan ◽  
Florian Lang
Keyword(s):  
Cell Membrane ◽  
Human Erythrocyte ◽  
Haemoglobin Concentration ◽  
Annexin V ◽  
Human Erythrocytes ◽  
Forward Scatter ◽  
Erythrocyte Surface ◽  
Suicidal Death ◽  
Phosphatidylserine Translocation ◽  
Surface Signaling

Background/Aims: The antihistaminic drug Terfenadine may trigger apoptosis of tumor cells, an effect unrelated to its effect on histamine receptors. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal death of erythrocytes characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling triggering eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, and ceramide. The present study explored, whether Terfenadine is capable to trigger eryptosis. Methods: Flow cytometry was employed to estimate phosphatidylserine abundance at the erythrocyte surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, abundance of reactive oxygen species (ROS) from 2′,7′-dichlorodihydrofluorescein (DCF) diacetate dependent fluorescence, and ceramide abundance at the human erythrocyte surface utilizing specific antibodies. Hemolysis was quantified from haemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to Terfenadine (≥ 5 µM) significantly increased the percentage of annexin-V-binding cells and triggered hemolysis without significantly modifying the average forward scatter. Terfenadine (7.5 µM) significantly increased Fluo3-fluorescence, but did not significantly modify DCF fluorescence or ceramide abundance. The effect of Terfenadine on annexin-V-binding was significantly blunted but not abolished by removal of extracellular Ca2+. Exposure of human erythrocytes to Ca2+ ionophore ionomycin (1 µM, 15 min) triggered annexin-V-binding, an effect augmented by Terfenadine pretreatment (10 µM, 48 hours). Conclusions: Terfenadine triggers phospholipid scrambling of the human erythrocyte cell membrane, an effect in part due to entry of extracellular Ca2+ and in part due to sensitizing human erythrocyte cell membrane scrambling to Ca2+.

scholarly journals Triggering of Suicidal Erythrocyte Death by Ruxolitinib

2015 ◽  
Vol 37 (2) ◽  
pp. 768-778 ◽  
Author(s):  
Marilena Briglia ◽  
Antonella Fazio ◽  
Caterina Faggio ◽  
Stefan Laufer ◽  
Kousi Alzoubi ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Map Kinase ◽  
Kinase Inhibitor ◽  
Myeloproliferative Neoplasm ◽  
Annexin V ◽  
P38 Map Kinase ◽  
Cell Shrinkage ◽  
Forward Scatter ◽  
Mitogen Activated Protein ◽  
Suicidal Death

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.

scholarly journals Fucoxanthin Induced Suicidal Death of Human Erythrocytes

2015 ◽  
Vol 37 (6) ◽  
pp. 2464-2475 ◽  
Author(s):  
Marilena Briglia ◽  
Salvatrice Calabró ◽  
Elena Signoretto ◽  
Kousi Alzoubi ◽  
Stefan Laufer ◽  
...  
Keyword(s):  
Lipid Peroxidation ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Cell Membrane ◽  
Annexin V ◽  
Cell Shrinkage ◽  
P38 Kinase ◽  
Forward Scatter ◽  
Kinase C ◽  
Suicidal Death

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.

scholarly journals Inhibition of Suicidal Erythrocyte Death by Volasertib

2017 ◽  
Vol 43 (4) ◽  
pp. 1472-1486 ◽  
Author(s):  
Abdulla Al Mamun Bhuyan ◽  
A.K.M. Ashiqul Haque ◽  
Itishri Sahu ◽  
Hang Cao ◽  
Michael S.D. Kormann ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Membrane ◽  
Cell Surface ◽  
Annexin V ◽  
K562 Cells ◽  
Forward Scatter ◽  
Energy Depletion ◽  
Hyperosmotic Shock ◽  
Suicidal Death ◽  
Phosphatidylserine Translocation

Background/Aims: The Polo-like kinase 1 (Plk1) inhibitor volasertib is used in the treatment of malignancy. Volasertib is partially effective by triggering suicidal death or apoptosis of tumor cells. Similar to apoptosis of nucleated cells, erythrocytes may enter suicidal cell death or eryptosis, which is characterized by cell membrane scrambling with phosphatidylserine translocation to the cell surface and by cell shrinkage. Stimulators of eryptosis include energy depletion, hyperosmotic shock, oxidative stress and excessive increase of cytosolic Ca2+ activity ([Ca2+]i). The present study explored, whether volasertib impacts on eryptosis. Methods: Human erythrocytes have been exposed to energy depletion (glucose withdrawal for 48 hours), hyperosmotic shock (addition of 550 mM sucrose for 6 hours), oxidative stress (addition of 0.3 mM tert-butylhydroperoxide [tBOOH] for 50 min) or Ca2+ ionophore ionomycin (1 µM for 60 min) in absence and presence of volasertib (0.5-1.5 µg/ml) and flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3 fluorescence, reactive oxygen species from 2’,7’-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence and ceramide abundance utilizing antibodies. For comparison, annexin-V-binding and forward scatter were determined following a 48 hours exposure of human leukemic K562 cells in RPMI-1640 medium to volasertib. Results: Treatment with volasertib alone did not significantly modify annexin-V-binding or forward scatter in mature erythrocytes. Energy depletion, hyperosmotic shock, oxidative stress and ionomycin, all markedly and significantly increased the percentage of annexin-V-binding erythrocytes, and decreased the forward scatter. Volasertib significantly blunted the effect of energy depletion and hyperosmotic shock, but not of oxidative stress and ionomycin on annexin-V-binding. Volasertib did not significantly influence the effect of any maneuver on forward scatter. In K562 cells, volasertib enhanced annexin-V-binding and decreased the forward scatter. Conclusions: Volasertib is a novel inhibitor of erythrocyte cell membrane scrambling following energy depletion and hyperosmotic shock, effects contrasting the stimulation of K562 cell apoptosis.

scholarly journals Oxaliplatin Induced Suicidal Death of Human Erythrocytes

2015 ◽  
Vol 37 (6) ◽  
pp. 2393-2404 ◽  
Author(s):  
Antonella Fazio ◽  
Marilena Briglia ◽  
Caterina Faggio ◽  
Kousi Alzoubi ◽  
Florian Lang
Keyword(s):  
Cell Membrane ◽  
Annexin V ◽  
Human Erythrocytes ◽  
Cell Shrinkage ◽  
Forward Scatter ◽  
Erythrocyte Surface ◽  
Suicidal Death ◽  
Phosphatidylserine Translocation ◽  
Ros Formation ◽  
Binding Cell

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.

scholarly journals Anidulafungin-Induced Suicidal Erythrocyte Death

2016 ◽  
Vol 38 (6) ◽  
pp. 2272-2284 ◽  
Author(s):  
Thomas Peter ◽  
Rosi Bissinger ◽  
Guilai Liu ◽  
Florian Lang
Keyword(s):  
Cell Membrane ◽  
Cell Volume ◽  
Fungal Infections ◽  
Annexin V ◽  
Casein Kinase ◽  
Antifungal Drug ◽  
Cell Shrinkage ◽  
P38 Kinase ◽  
Forward Scatter ◽  
Erythrocyte Surface

Background/Aims: The novel antifungal drug Anidulafungin is used for the treatment of diverse fungal infections including candidiasis and aspergillosis. The traditional antifungal drug amphotericin B has previously been shown to trigger eryptosis, the suicidal death of erythrocytes characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide, activated protein kinase C (PKC), casein kinase 1α or p38 kinase and activated caspases. Inhibitors of eryptosis include nitric oxide (NO). The present study explored, whether Anidulafungin induces eryptosis. Methods: Flow cytometry was employed to estimate phosphatidylserine abundance at the erythrocyte surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, abundance of reactive oxygen species (ROS) from DCFDA dependent fluorescence, and ceramide abundance at the erythrocyte surface utilizing specific antibodies. Hemolysis was quantified by measuring haemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to Anidulafungin (1.5 - 6 µg/ml) significantly increased hemolysis and the percentage of annexin-V-binding cells, and significantly decreased forward scatter. Anidulafungin (6 µg/ml) slightly, but significantly inceased Fluo3-fluorescence and the effect of Anidulafungin on annexin-V-binding was slightly, but significantly blunted by removal of extracellular Ca2+. The effect of Anidulafungin on annexin-V-binding was further significantly blunted by the p38 kinase inhibitor SB203580 (2 µM) and NO donor nitroprusside (1 µM). An increase of extracellular K+ concentration significantly blunted the effect of Anidulafungin on cell volume but not on annexin-V-binding. Anidulafungin rather decreased DCFDA fluorescence and the effect of Anidulafungin on annexin-V-binding was not significantly blunted by the antioxidant N-acetylcysteine (1 mM). Moreover, the effect of Anidulafungin on annexin-V-binding was not paralleled by significant increase of ceramide abundance and was not significantly blunted by PKC inhibitor staurosporine (1 µM), casein kinase 1α inhibitor D4476 (10 µM) or pancaspase inhibitor zVAD (10 µM). Conclusions: Anidulafungin triggers hemolysis and eryptosis with cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to Ca2+ entry and activation of p38 kinase.

scholarly journals Stimulation of Suicidal Erythrocyte Death by Rottlerin

2016 ◽  
Vol 40 (3-4) ◽  
pp. 558-566 ◽  
Author(s):  
Morena Mischitelli ◽  
Mohamed Jemaà ◽  
Mustafa Almasry ◽  
Caterina Faggio ◽  
Florian Lang
Keyword(s):  
Cell Membrane ◽  
Haemoglobin Concentration ◽  
Annexin V ◽  
Decisive Role ◽  
Forward Scatter ◽  
Erythrocyte Surface ◽  
Suicidal Death ◽  
Phosphatidylserine Translocation ◽  
Surface Signaling ◽  
Stimulation Of

Background/Aims: The phytochemical polyphenol rottlerin is a potent activator of diverse Ca2+ -sensitive K+ channels. Those channels play a decisive role in the execution of eryptosis, the suicidal death of erythrocytes, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling involved in the stimulation of eryptosis includes increase of cytosolic Ca2+ activity ([Ca2+]i) and ceramide. The present study explored, whether rottlerin induces eryptosis and, if so, to test for the involvement of Ca2+ entry and ceramide. Methods: Flow cytometry was employed to estimate phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was quantified by determination of haemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to rottlerin (1 - 5 µM) significantly increased the percentage of annexin-V-binding cells, an effect paralleled by significant decrease of forward scatter. Up to 5 µM rottlerin failed to significantly increase average Fluo3-fluorescence. Rottlerin (5 µM) did, however, significantly increase the ceramide abundance. Rottlerin (5 µM) further significantly increased hemolysis. The effect of rottlerin (5 µM) on annexin-V-binding was virtually abolished by removal of extracellular Ca2+. Conclusions: Rottlerin stimulates eryptosis with erythrocyte shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect paralleled by and at least in part due to Ca2+ entry and ceramide.

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