RESUMO
Anemia in advanced age is often a multifactorial condition requiring an interdisciplinary approach. The contributions to the opening interdisciplinary symposium on anemia in older subjects focused on physiological and histopathological as well as on nephrological and neurogeriatric aspects and on the therapeutic implications of this underdiagnosed, yet highly frequent disease. The symposium was the kick-off event for the founding of the German Geriatric Society special interest group on anemia in advanced age.
Assuntos
Anemia/etiologia , Idoso , Doença de Alzheimer/epidemiologia , Doença de Alzheimer/etiologia , Doença de Alzheimer/terapia , Anemia/epidemiologia , Anemia/terapia , Causalidade , Eriptose/fisiologia , Geriatria , Alemanha , Humanos , Comunicação Interdisciplinar , Colaboração Intersetorial , Falência Renal Crônica/epidemiologia , Falência Renal Crônica/etiologia , Falência Renal Crônica/terapia , Prevalência , Sociedades MédicasRESUMO
Putative functions of the heterotrimeric G-protein subunit Gαi2-dependent signaling include ion channel regulation, cell differentiation, proliferation and apoptosis. Erythrocytes may, similar to apoptosis of nucleated cells, undergo eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine (PS) exposure. Eryptosis may be triggered by increased cytosolic Ca(2+) activity and ceramide. In the present study, we show that Gαi2 is expressed in both murine and human erythrocytes and further examined the survival of erythrocytes drawn from Gαi2-deficient mice (Gαi2(-/-)) and corresponding wild-type mice (Gαi2(+/+)). Our data show that plasma erythropoietin levels, erythrocyte maturation markers, erythrocyte counts, hematocrit and hemoglobin concentration were similar in Gαi2(-/-) and Gαi2(+/+) mice but the mean corpuscular volume was significantly larger in Gαi2(-/-) mice. Spontaneous PS exposure of circulating Gαi2(-/-) erythrocytes was significantly lower than that of circulating Gαi2(+/+) erythrocytes. PS exposure was significantly lower in Gαi2(-/-) than in Gαi2(+/+) erythrocytes following ex vivo exposure to hyperosmotic shock, bacterial sphingomyelinase or C6 ceramide. Erythrocyte Gαi2 deficiency further attenuated hyperosmotic shock-induced increase of cytosolic Ca(2+) activity and cell shrinkage. Moreover, Gαi2(-/-) erythrocytes were more resistant to osmosensitive hemolysis as compared to Gαi2(+/+) erythrocytes. In conclusion, Gαi2 deficiency in erythrocytes confers partial protection against suicidal cell death.
Assuntos
Eriptose , Eritrócitos/citologia , Eritrócitos/fisiologia , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/metabolismo , Animais , Sobrevivência Celular , Índices de Eritrócitos , Eritrócitos/química , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/deficiência , Humanos , Camundongos , Camundongos Knockout , Fosfatidilserinas/análiseRESUMO
The mitogen- and stress-activated kinase MSK1/2 plays a decisive role in apoptosis. In analogy to apoptosis of nucleated cells, suicidal erythrocyte death called eryptosis is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine (PS) externalization. Here, we explored whether MSK1/2 participates in the regulation of eryptosis. To this end, erythrocytes were isolated from mice lacking functional MSK1/2 (msk(-/-)) and corresponding wild-type mice (msk(+/+)). Blood count, hematocrit, hemoglobin concentration and mean erythrocyte volume were similar in both msk(-/-) and msk(+/+) mice, but reticulocyte count was significantly increased in msk(-/-) mice. Cell membrane PS exposure was similar in untreated msk(-/-) and msk(+/+) erythrocytes, but was enhanced by pathophysiological cell stressors ex vivo such as hyperosmotic shock or energy depletion to significantly higher levels in msk(-/-) erythrocytes than in msk(+/+) erythrocytes. Cell shrinkage following hyperosmotic shock and energy depletion, as well as hemolysis following decrease of extracellular osmolarity was more pronounced in msk(-/-) erythrocytes. The in vivo clearance of autologously-infused CFSE-labeled erythrocytes from circulating blood was faster in msk(-/-) mice. The spleens from msk(-/-) mice contained a significantly greater number of PS-exposing erythrocytes than spleens from msk(+/+) mice. The present observations point to accelerated eryptosis and subsequent clearance of erythrocytes leading to enhanced erythrocyte turnover in MSK1/2-deficient mice.
Assuntos
Apoptose/genética , Eritrócitos/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Animais , Índices de Eritrócitos , Eritrócitos/patologia , Feminino , Expressão Gênica , Hematócrito , Hemoglobinas , Hemólise , Humanos , Masculino , Camundongos , Camundongos Knockout , Fragilidade Osmótica , Pressão Osmótica , Fosfatidilserinas/metabolismo , Cultura Primária de Células , Contagem de Reticulócitos , Proteínas Quinases S6 Ribossômicas 90-kDa/deficiênciaRESUMO
BACKGROUND/AIMS: The cyclin-dependent kinase 4 (CDK4) participates in the regulation of apoptosis of nucleated cells by altering transcriptional regulation of genes governing cell proliferation and cell death. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine (PS) exposure at the cell surface. As mature erythrocytes lack nuclei, acute stimulation of eryptosis cannot result from altered gene expression. Eryptosis is triggered by isotonic cell shrinkage following Cl- removal (replacement with the impermeant organic anion gluconate) or by oxidative stress (exposure to 0.3 mM tertbutyl-hydroperoxide [tBOOH]). The present study explored whether CDK4 is expressed in erythrocytes and whether the CDK4 inhibitors II (NSC625987) and III (ryuvidine) influence eryptosis. METHODS: Western blotting was utilized for determination of the presence of CDK4 protein in human erythrocytes, and FACS analysis to determine Fluo3 fluorescence (reflecting cytosolic Ca2+), annexin-V-binding (reflecting PS-exposure) and forward scatter (reflecting cell volume). RESULTS: CDK4 protein was present in human erythrocytes. Cl- removal was followed by decrease of forward scatter and increase of both annexin-V-binding and Fluo3 fluorescence, an effect significantly curtailed by CDK4 inhibitors II and III. Furthermore, CDK4 inhibition blunted enhanced PS-exposure elicited by tBOOH treatment. CONCLUSIONS: The present observations disclose the presence of CDK4 protein in human erythrocytes and the suppression of suicidal erythrocyte death by pharmacological inhibition of CDK4.
Assuntos
Quinase 4 Dependente de Ciclina/metabolismo , Eritrócitos/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Apoptose , Sobrevivência Celular/efeitos dos fármacos , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Eritrócitos/citologia , Eritrócitos/enzimologia , Humanos , Fosfatidilserinas/metabolismo , terc-Butil Hidroperóxido/farmacologiaRESUMO
BACKGROUND/AIMS: Epidemiological evidence suggests that vitamin D deficiency is associated with anemia. The potent metabolite 1,25(OH)2 vitamin D3 [1,25(OH)2D3] activates various signaling cascades regulating a myriad of cellular functions including suicidal cell death or apoptosis. Suicidal death of erythrocytes or eryptosis is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine (PS) externalization. Stimulation of eryptosis may limit lifespan of circulating erythrocytes and thus cause anemia. In the present study, we explored the effect of a high vitamin D diet (10,000 I.U. vitamin D for 14 days) in mice on eryptosis. METHODS: Plasma concentrations of erythropoietin were estimated using an immunoassay kit, blood count using an electronic hematology particle counter, relative reticulocyte numbers using Retic-COUNT® reagent, PS exposure at the cell surface from annexin V binding, cell volume from forward scatter, and cytosolic Ca(2+) ([Ca(2+)]i) from Fluo3-fluorescence in FACS analysis. RESULTS: Vitamin D treatment decreased mean corpuscular volume, reticulocyte count, and plasma erythropoietin levels. Vitamin D treatment slightly but significantly decreased forward scatter but did not significantly modify spontaneous PS exposure and [Ca(2+)]i of freshly drawn erythrocytes. Vitamin D treatment augmented the stimulation of PS exposure and cell shrinkage following exposure to hyperosmotic shock (addition of 550 mM sucrose) or energy depletion (glucose removal) without significantly modifying [Ca(2+)]i. CONCLUSIONS: The present observations point to a subtle effect of exogenous vitamin D supplementation on erythrocyte survival.
Assuntos
Envelhecimento Eritrocítico/efeitos dos fármacos , Vitamina D/uso terapêutico , Vitaminas/uso terapêutico , Animais , Contagem de Células Sanguíneas , Cálcio/metabolismo , Tamanho Celular/efeitos dos fármacos , Dieta , Membrana Eritrocítica/efeitos dos fármacos , Eritropoetina/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Pressão Osmótica/efeitos dos fármacos , Fosfatidilserinas/sangueRESUMO
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.
Assuntos
Envelhecimento , Anemia/sangue , Anemia/etiologia , Eritrócitos/patologia , Adulto , Idoso de 80 Anos ou mais , Anemia/metabolismo , Anemia/patologia , Morte Celular , Tamanho Celular , Eritrócitos/citologia , Eritrócitos/metabolismo , Feminino , Hemólise , Humanos , Masculino , Estresse Oxidativo , Fosfatidilserinas/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
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.
Assuntos
Apoptose/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Fenantrenos/farmacologia , Anexina A5/metabolismo , Eritrócitos/metabolismo , Citometria de Fluxo , HumanosRESUMO
BACKGROUND/AIMS: Gedunin, an inhibitor of heat shock protein HSP90, triggers apoptosis of tumor cells and is thus effective against malignancy. Moreover, the drug has antimalarial potency. In analogy to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and by phosphatidylserine translocation to the erythrocyte surface. Eryptosis may be triggered by increase of cytosolic Ca(2+)-activity ([Ca(2+)]i). The present study explored whether gedunin stimulates eryptosis. METHODS: Forward scatter was determined to estimate cell volume, annexin V binding to identify phosphatidylserine-exposing erythrocytes, hemoglobin release to depict hemolysis, and Fluo3-fluorescence to quantify [Ca(2+)]i. RESULTS: A 48 h exposure of human erythrocytes to gedunin significantly increased [Ca(2+)]i (12 µM), significantly decreased forward scatter (24 µM) and significantly increased annexin-V-binding (12 µM). The effect of gedunin (24 µM) on annexin-V-binding was virtually abrogated by removal of extracellular Ca(2+). CONCLUSION: Gedunin stimulates suicidal erythrocyte death or eryptosis, an effect mainly if not exclusively due to stimulation of Ca(2+) entry.
Assuntos
Cálcio/metabolismo , Membrana Eritrocítica/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Limoninas/farmacologia , Fosfatidilserinas/metabolismo , Morte Celular/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Ceramidas/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , Membrana Eritrocítica/metabolismo , Eritrócitos/metabolismo , Citometria de Fluxo , Humanos , Limoninas/química , Estrutura Molecular , Fatores de TempoRESUMO
Shikonin, the most important component of Lithospermum erythrorhizon, has previously been shown to exert antioxidant, anti-inflammatory, antithrombotic, antiviral, antimicrobial and anticancer effects. The anticancer effect has been attributed to the stimulation of suicidal cell death or apoptosis. Similar to the apoptosis of nucleated cells, erythrocytes may experience eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and by phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include the increase of cytosolic Ca2+-activity ([Ca2+]i) and ceramide formation. The present study explored whether Shikonin stimulates eryptosis. To this end, Fluo 3 fluorescence was measured to quantify [Ca2+]i, forward scatter to estimate cell volume, annexin V binding to identify phosphatidylserine-exposing erythrocytes, hemoglobin release to determine hemolysis and antibodies to quantify ceramide abundance. As a result, a 48 h exposure of human erythrocytes to Shikonin (1 µM) significantly increased [Ca2+]i, increased ceramide abundance, decreased forward scatter and increased annexin V binding. The effect of Shikonin (1 µM) on annexin V binding was significantly blunted, but not abolished by the removal of extracellular Ca2+. In conclusion, Shikonin stimulates suicidal erythrocyte death or eryptosis, an effect at least partially due to the stimulation of Ca2+ entry and ceramide formation.
Assuntos
Eritrócitos/efeitos dos fármacos , Naftoquinonas/farmacologia , Fosfatidilserinas/sangue , Trifosfato de Adenosina/sangue , Transporte Biológico , Cálcio/sangue , Eritrócitos/metabolismo , Humanos , Técnicas In VitroRESUMO
BACKGROUND: Novobiocin, an aminocoumarin antibiotic, interferes with heat shock protein 90 and hypoxia inducible factor dependent gene expression and thus compromises cell survival. Similar to survival of nucleated cells, erythrocyte survival could be disrupted by eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and by phospholipd scrambling of the cell membrane with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca(2+)-activity ([Ca(2+)]i). The Ca(2+) sensitivity of phospholipid scrambling is enhanced by ceramide. The present study explored, whether novobiocin elicits eryptosis. METHODS: [Ca(2+)]i was estimated from Fluo3-fluorescence, ceramide abundance utilizing fluorescent antibodies, cell volume from forward scatter, phosphatidylserine-exposure from annexin V binding. RESULTS: A 48 hours exposure to novobiocin (500 µM) was followed by a significant increase of [Ca(2+)]i, decrease of forward scatter, increase of annexin-V-binding and enhanced ceramide formation. Removal of extracellular Ca(2+) virtually abrogated the increase of annexin-V-binding following novobiocin exposure. CONCLUSIONS: Novobiocin stimulates eryptosis, an effect at least in part due to entry of extracellular Ca(2+) and formation of ceramide.
Assuntos
Anexina A5/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Cálcio/metabolismo , Ceramidas/metabolismo , Eritrócitos/metabolismo , Novobiocina/farmacologia , Morte Celular/efeitos dos fármacos , Eritrócitos/citologia , HumanosRESUMO
The selective cyclooxygenase-2 (COX-2) inhibitor celecoxib triggers apoptosis of tumor cells and is thus effective against malignancy. The substance is at least partially effective through mitochondrial depolarization. Even though lacking mitochondria, erythrocytes may enter apoptosis-like suicidal death or eryptosis, which is characterized by cell shrinkage and by phosphatidylserine translocation to the erythrocyte surface. Eryptosis may be triggered by increase of cytosolic Ca2+-activity ([Ca2+]i). The present study explored whether celecoxib stimulates eryptosis. Forward scatter was determined to estimate cell volume, annexin V binding to identify phosphatidylserine-exposing erythrocytes, hemoglobin release to depict hemolysis, and Fluo3-fluorescence to quantify [Ca2+]i. A 48 h exposure of human erythrocytes to celecoxib was followed by significant increase of [Ca2+]i (15 µM), significant decrease of forward scatter (15 µM) and significant increase of annexin-V-binding (10 µM). Celecoxib (15 µM) induced annexin-V-binding was blunted but not abrogated by removal of extracellular Ca2+. In conclusion, celecoxib stimulates suicidal erythrocyte death or eryptosis, an effect partially due to stimulation of Ca2+ entry.
Assuntos
Inibidores de Ciclo-Oxigenase 2/farmacologia , Eritrócitos/efeitos dos fármacos , Pirazóis/farmacologia , Sulfonamidas/farmacologia , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Celecoxib , Tamanho Celular/efeitos dos fármacos , Células Cultivadas , Eritrócitos/metabolismo , Eritrócitos/patologia , Hemólise/efeitos dos fármacos , Humanos , Fosfatidilserinas/metabolismoRESUMO
BACKGROUND: Patulin, the most common mycotoxin in apples and apple-derived products, triggers apoptosis and has thus been considered for the treatment of cancer. Similar to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and by cell membrane scrambling leading to phosphatidylserine-exposure at the erythrocyte surface. Stimulators of eryptosis include increase of cytosolic Ca(2+)-activity ([Ca(2+)]i). The present study explored, whether exposure of human erythrocytes to patulin is followed by eryptosis. METHODS: Forward scatter was measured to estimate cell volume, annexin V binding to detect phosphatidylserine-exposure, hemoglobin release to quantify hemolysis, and Fluo3-fuorescence to determine [Ca(2+)]i. RESULTS: A 48 h exposure to patulin significantly increased [Ca(2+)]I (5 µM), significantly decreased forward scatter (5 µM) and significantly increased annexin-V-binding (2.5 µM). Patulin (10 µM) induced annexin-V-binding was virtually abrogated by removal of extracellular Ca(2+). CONCLUSION: Patulin stimulates Ca(2+) entry into erythrocytes, an effect triggering suicidal erythrocyte death or eryptosis.
Assuntos
Eritrócitos/efeitos dos fármacos , Micotoxinas/farmacologia , Patulina/farmacologia , Morte Celular/efeitos dos fármacos , Células Cultivadas , Eritrócitos/metabolismo , HumanosRESUMO
Honokiol ((3,5-di-(2-propenyl)-1,1-biphenyl-2,2-diol), a component of Magnolia officinalis, stimulates apoptosis and is thus considered for the treatment of malignancy. In analogy to apoptosis of nucleated cells, erythrocytes may enter eryptosis, a suicidal death characterized by cell shrinkage and by breakdown of cell membrane phosphatidylserine asymmetry with phosphatidylserine-exposure at the erythrocyte surface. Eryptosis may be triggered following increase of cytosolic Ca(2+)-activity ([Ca(2+)]i). The present study explored, whether honokiol elicits eryptosis. Cell volume has been estimated from forward scatter, phosphatidylserine-exposure from annexin V binding, hemolysis from hemoglobin release, [Ca(2+)]i from Fluo3-fluorescence, and ceramide from fluorescent antibodies. As a result, a 48 h exposure to honokiol was followed by a slight but significant increase of [Ca(2+)]i (15 µM), significant decrease of forward scatter (5 µM), significant increase of annexin-V-binding (5 µM) and significant increase of ceramide formation (15 µM). Honokiol further induced slight, but significant hemolysis. Honokiol (15 µM) induced annexin-V-binding was significantly blunted but not abrogated in the nominal absence of extracellular Ca(2+). In conclusion, honokiol triggers suicidal erythrocyte death or eryptosis, an effect at least in part due to stimulation of Ca(2+) entry and ceramide formation.
Assuntos
Compostos de Bifenilo/toxicidade , Eritrócitos/efeitos dos fármacos , Lignanas/toxicidade , Cálcio/metabolismo , Morte Celular/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Células Cultivadas , Eritrócitos/metabolismo , Eritrócitos/patologia , Hemólise/efeitos dos fármacos , Humanos , Fosfatidilserinas/metabolismoRESUMO
Withaferin A, a triterpenoid component from Withania somnifera, counteracts malignancy, an effect attributed to stimulation of apoptosis. Withaferin A is partially effective through induction of oxidative stress, altered gene expression and mitochondrial depolarization. Erythrocytes lack mitochondria and nuclei but may enter apoptosis-like 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 [Ca(2+)](i) following activation of oxidant-sensitive Ca(2+)-permeable cation channels, ceramide formation and/or ATP-depletion. The present study explored, whether withaferin A triggers eryptosis. To this end, [Ca(2+)](i) was estimated from Fluo3-fluorescence, cell volume from forward scatter, phosphatidylserine exposure from annexin-V-binding, hemolysis from hemoglobin release, oxidative stress from DCFDA-fluorescence and ceramide abundance utilizing antibodies. A 48 h exposure to withaferin A significantly decreased forward scatter (at ≥ 10 µM withaferin concentration) and increased [Ca(2+)](i) (≥ 5 µM), ROS-formation (≥ 10 µM) ceramide-formation ( ≥ 10 µM) as well as annexin-V-binding ( ≥ 5 µM). Withaferin A treatment was followed by slight but significant increase of hemolysis. Extracellular Ca(2+) removal, amiloride, and the antioxidant N-acetyl-l-cysteine significantly blunted withaferin A-triggered annexin-V-binding. The present observations reveal that withaferin A triggers suicidal erythrocyte death despite the absence of gene expression and key elements of apoptosis such as mitochondria.
Assuntos
Antineoplásicos/farmacologia , Cálcio/metabolismo , Ceramidas/metabolismo , Eritrócitos/efeitos dos fármacos , Vitanolídeos/farmacologia , Apoptose/efeitos dos fármacos , Eritrócitos/metabolismo , Hemólise/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacosRESUMO
Probucol, an antioxidant and anti-inflammatory agent counteracting atherosclerosis and restenosis, is partially effective by influencing suicidal cell death or apoptosis. In analogy to apoptosis of nucleated cells, suicidal death of erythrocytes or eryptosis is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the erythrocyte surface. Eryptosis is stimulated by increase in cytosolic Ca(2+) activity, for example, after energy depletion or oxidative stress. The present study explored whether probucol influences eryptosis. Phosphatidylserine exposure was estimated from annexin-V-binding, cell volume from forward scatter (FSC), and cytosolic Ca(2+) concentration from fluo-3 fluorescence in flow cytometry. As a result, energy depletion (48-hour glucose removal) increased annexin-V-binding, decreased FSC, and increased fluo-3 fluorescence. Probucol (≤30 µM) did not significantly modify annexin-V-binding, FSC, or fluo-3 fluorescence in the presence of glucose but (at ≥5 µM) blunted the effect of glucose depletion on annexin-V-binding. Probucol (≥20 µM) only slightly blunted the effects of glucose depletion on FSC and fluo-3 fluorescence. Ca(2+) ionophore ionomycin (1 µM) and oxidative stress (30-minute exposure to 0.3 mM of tert-butylhydroperoxide) increased annexin-V-binding, effects again blunted by 30 µM of probucol. In conclusion, probucol blunts cell membrane scrambling after energy depletion and oxidative stress, effects primarily because of interference with the scrambling effects of increased cytosolic Ca(2+) concentration.
Assuntos
Antioxidantes/farmacologia , Morte Celular/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Probucol/farmacologia , Anexina A5/metabolismo , Antioxidantes/administração & dosagem , Cálcio/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Tamanho Celular , Citosol/efeitos dos fármacos , Citosol/metabolismo , Relação Dose-Resposta a Droga , Eritrócitos/metabolismo , Citometria de Fluxo , Glucose/metabolismo , Humanos , Estresse Oxidativo/efeitos dos fármacos , Fosfatidilserinas/metabolismo , Probucol/administração & dosagemRESUMO
Fumagillin, a cyclohexane isolated from fungus Aspergillus fumigatus, has anti-infective and anti-cancer potency. Fumagillin is at least partially effective by inducing suicidal death or apoptosis. In analogy to apoptosis of nucleated cells, eryptosis is the suicidal death of erythrocytes characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Stimulators of eryptosis include increase of cytosolic Ca(2+)-activity ([Ca(2+)](i)) and ceramide. The present study explored whether fumagillin (5-100 µM) could stimulate eryptosis. To this end, [Ca(2+)](i) was estimated from Fluo3 fluorescence, ceramide by utilizing specific antibodies, cell volume from forward scatter, phosphatidylserine exposure from annexin V binding and haemolysis from haemoglobin release. As a result, a 48-hr exposure to fumagillin significantly increased [Ca(2+)](i) (≥10 µM), enhanced ceramide abundance (100 µM), triggered annexin V binding (≥10 µM) and decreased forward scatter (≥10 µM). Fumagillin exposure was followed by slight but significant increase of haemolysis. Removal of extracellular Ca(2+) significantly blunted but did not abolish the effect of fumagillin (100 µM) on annexin V binding. The present observations disclose a novel effect of fumagillin, that is, stimulation of eryptosis, paralleled by Ca(2+) entry, ceramide formation, phosphatidylserine exposure and decrease of cell volume.
Assuntos
Antibacterianos/toxicidade , Cicloexanos/toxicidade , Eritrócitos/efeitos dos fármacos , Ácidos Graxos Insaturados/toxicidade , Anexina A5/metabolismo , Cálcio/metabolismo , Morte Celular/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Ceramidas/metabolismo , Relação Dose-Resposta a Droga , Eritrócitos/metabolismo , Eritrócitos/patologia , Citometria de Fluxo/métodos , Hemólise/efeitos dos fármacos , Humanos , Fosfatidilserinas/metabolismo , Ligação Proteica , Sesquiterpenos/toxicidadeRESUMO
BACKGROUND: Sulindac sulfide, a non-steroidal anti-inflammatory drug (NSAID), stimulates apoptosis of tumor cells and is thus effective against malignancy. In analogy to apoptosis of nucleated cells, erythrocytes may undergo eryptosis, an apoptosis-like suicidal erythrocyte death, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine-exposure at the cell surface. Stimulators of eryptosis include increase of cytosolic Ca(2+)-activity ([Ca(2+)](i)) and ceramide formation. The present study explored, whether sulindac sulfide stimulates eryptosis. METHODS: [Ca(2+)](i) was estimated from Fluo-3 fluorescence, cell volume from forward scatter, phosphatidylserine-exposure from binding of fluorescent annexin-V, hemolysis from hemoglobin release, and ceramide abundance utilizing fluorescent antibodies. RESULTS: A 48 h exposure to sulindac sulfide (≤ 20 µM) was followed by significant increase of [Ca(2+)](i), enhanced ceramide abundance, decreased forward scatter and increased percentage of annexin-V-binding erythrocytes. Sulindac sulfide triggered slight but significant hemolysis. Removal of extracellular Ca(2+) significantly blunted, but did not abrogate the effect of sulindac sulfide (20 µM) on annexin-V-binding. CONCLUSION: Sulindac sulfide stimulates the suicidal death of erythrocytes or eryptosis, an effect paralleled by Ca(2+)-entry, ceramide formation, cell shrinkage and phosphatidylserine-exposure.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Antineoplásicos/farmacologia , Cálcio/metabolismo , Eritrócitos/efeitos dos fármacos , Sulindaco/análogos & derivados , Compostos de Anilina/análise , Anexina A5/metabolismo , Morte Celular/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Ceramidas/metabolismo , Eritrócitos/citologia , Eritrócitos/metabolismo , Corantes Fluorescentes/análise , Hemólise/efeitos dos fármacos , Humanos , Fosfatidilserinas/metabolismo , Sulindaco/farmacologia , Xantenos/análiseRESUMO
BACKGROUND/AIMS: Ipratropium bromide, an anticholinergic agent widely used in obstructive lung disease, has previously been shown to trigger suicidal death of nucleated cells or apoptosis. Despite their lack of mitochondria and nuclei, key organelles in the execution of apoptosis, erythrocytes may similarly undergo suicidal cell death, which is characterized by cell shrinkage and by cell membrane scrambling with phosphatidylserine-exposure at the cell surface. Triggers of eryptosis include increase of cytosolic Ca(2+)-activity ([Ca(2+)](i)). The present study explored whether ipratropium bromide triggers eryptosis. METHODS: [Ca Ca(2+)](i) was estimated utilizing Fluo3 fluorescence, cell volume from forward scatter, phosphatidylserine-exposure from annexin-V-binding, and hemolysis from hemoglobin release. RESULTS: A 48 h exposure to ipratropium bromide (1 nM) significantly increased [Ca(2+)](i), decreased forward scatter and increased annexin-V-binding. Ipratropium bromide treatment was followed by slight but significant increase of hemolysis. Removal of extracellular Ca(2+) or inhibition of Ca(2+) permeable cation channels with amiloride (1 mM) virtually abolished cell membrane scrambling. Ca(2+) ionophore ionomycin (1 µM, 30 min) increased the percentage of phosphatidylserine exposing erythrocytes to similarly high levels in the absence and presence of ipratropium bromide (1 nM). CONCLUSIONS: Ipratropium bromide triggers suicidal erythrocyte death or eryptosis, an effect mainly due to stimulation of Ca(2+)-entry.
Assuntos
Apoptose/efeitos dos fármacos , Antagonistas Colinérgicos/farmacologia , Eritrócitos/fisiologia , Ipratrópio/farmacologia , Células CACO-2 , Sinalização do Cálcio , Tamanho Celular/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Citometria de Fluxo , Humanos , Fosfatidilserinas/metabolismoRESUMO
Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone, 1), a natural product from plants with potential anticancer potency, induces apoptosis. Mechanisms involved in 1-induced apoptosis include mitochondrial depolarization, inactivation of NF-κB, and altered expression of anti- and proapoptotic Bcl proteins. Similar to nucleated cells, erythrocytes may undergo suicidal death or eryptosis, which, like apoptosis, results in cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Triggers of eryptosis include increase of cytosolic Ca(2+) activity ([Ca(2+)]i) and ceramide formation. The present study explored whether 1 stimulates eryptosis. Cell volume was estimated from forward scatter, phosphatidylserine exposure from annexin-V-binding, hemolysis from hemoglobin release, [Ca(2+)]i from Fluo-3 fluorescence, and ceramide abundance utilizing antibodies. A 48 h exposure to 1 (2 µM) decreased forward scatter and increased annexin-V-binding significantly, events paralleled by increased [Ca(2+)]i and ceramide formation. Exposure to 1 was followed by a slight but significant increase of hemolysis. Removal of extracellular Ca(2+) slightly, but significantly blunted the effect of 1 (2 µM) on annexin-V-binding. The present observations demonstrate that 1 may trigger suicidal death of erythrocytes, cells devoid of mitochondria and nuclei.
Assuntos
Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Ceramidas/metabolismo , Eritrócitos/efeitos dos fármacos , Naftoquinonas/farmacologia , Compostos de Anilina , Anexina A5/metabolismo , Morte Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Tamanho Celular/efeitos dos fármacos , Ceramidas/imunologia , Citosol/metabolismo , Hemólise/efeitos dos fármacos , Estrutura Molecular , NF-kappa B/metabolismo , Naftoquinonas/química , Fosfatidilserinas/metabolismo , Vitamina K 3/metabolismo , XantenosRESUMO
Side effects of gossypol, a polyphenolic component of Gossypium, with male contraceptive, anticancer, antimicrobial and antiviral activities include anemia due to accelerated demise of erythrocytes. Erythrocytes may be cleared from circulating blood following apoptosis-like suicidal death or eryptosis. Hallmarks of eryptosis are cell shrinkage and cell membrane scrambling with subsequent phosphatidylserine-exposure at the cell surface. Stimulators of eryptosis include increase of cytosolic Ca(2+)-activity ([Ca(2+)](i)). The present study explored, whether gossypol stimulates eryptosis of human erythrocytes. Utilizing flow cytometry, [Ca(2+)](i) was estimated from Fluo-3 fluorescence, cell volume from forward scatter, phosphatidylserine-exposure from annexin-V-binding, and hemolysis from hemoglobin release. A 48 h exposure to gossypol (0.75 µM) significantly increased [Ca(2+)](i), decreased forward scatter and increased annexin-V-binding. Gossypol exposure was followed by a slight but significant increase of hemolysis. Removal of extracellular Ca(2+) significantly blunted the effect of gossypol (1 µM) on annexin-V-binding. The present observations reveal a novel effect of gossypol on human erythrocytes, which contributes to or even accounts for the triggering of anemia by this substance.