RESUMEN
Apoptosis is a common mechanism of programmed cell death in virtually all nucleated cells. In spite of the fact that platelets and erythrocytes are the only enucleated cells in mammals they contain most of the apoptosis machinery of other cells and undergo similar apoptotic processes as nucleated cells except those connected with nuclear and chromatin transformation. Here we compare the mechanisms of platelet and erythrocytes apoptosis induced by different stimuli namely, stimulation ofthrombin and collagen receptor (T/C), inhibitor of BclX family proteins (ABT-373) for platelets, tert-butylhydroperoxide (tBH) and calcium ionophore (A-23187) for erythrocytes. Induction of platelet apoptosis by both methods (T/C and ABT-373) lead to strong phosphoetydilserine (PS) externalization, loss of the mitochondrial membrane potential (DeltaPsim), proteolytic cleavage of some cytoskeletal and regulatory proteins, and microparticle (MP) formation. However, there are clear differences between mechanisms of platelet apoptosis induced by TC and ABT-373. T/C induced apoptotic reaction is very fast (reach the maximum at 5 min), whereas ABT-373 induced reaction is more prolonged (first apoptotic evidence appears only after 30 min and reach the maximum after 3 hours). MP formation is much more pronounced in T/C than in ABT-373 stimulated platelets, whereas caspase 3 activation is much more stronger in ABT-373 than in T/C stimulated platelets. The main differences between these two apoptotic pathways are connected with aIIbp3 integrin, which activation appears only after T/C stimulation. For tBH experiments on erythrocytes we established optimal conditions (0.25x1012 cells/L, and strong, 1500 RPM stirring) for elucidation of apoptotic processes and found two independent ways of erythrocytes apoptotic processes; calcium independent, connected with met hemoglobin (metHb) formation (tBH stimulation), and calcium dependent pathway (A-23187 stimulation). Erythrocytes apoptosis induced by tBH is characterized by formation ofmetHb, cell shrinkage, fast (95 % during 3 hours) PS externalization, yield of hemoglobin, probably by vesicle (MP) formation. These cells are transformed to stomatocytes, become highly rigid, and could not be lysed even in pure water. All these reactions are calcium independent. Whereas increase of intracellular calcium concentration by A-23187 connected with formation of exinocytes, less pronounced (17 % during 3 h, 35 % during 15 h) PS externalization and rigidity (lysed in 50 mOsm buffer).
Asunto(s)
Apoptosis/genética , Plaquetas/metabolismo , Proteínas del Citoesqueleto/metabolismo , Eritrocitos/metabolismo , Apoptosis/efectos de los fármacos , Plaquetas/citología , Plaquetas/efectos de los fármacos , Calcimicina/farmacología , Calcio/metabolismo , Caspasa 3/genética , Caspasa 3/metabolismo , Micropartículas Derivadas de Células/efectos de los fármacos , Micropartículas Derivadas de Células/metabolismo , Proteínas del Citoesqueleto/genética , Eritrocitos/citología , Eritrocitos/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Hemoglobina A/metabolismo , Humanos , Cinética , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Metahemoglobina/metabolismo , Especificidad de Órganos , Fosfatidilserinas/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/genética , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Receptores de Colágeno/agonistas , Receptores de Colágeno/genética , Receptores de Colágeno/metabolismo , Receptores de Trombina/agonistas , Receptores de Trombina/genética , Receptores de Trombina/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Proteína bcl-X/antagonistas & inhibidores , Proteína bcl-X/genética , Proteína bcl-X/metabolismo , terc-Butilhidroperóxido/farmacologíaAsunto(s)
Endotelio Vascular/fisiología , Histamina/farmacología , Ganglios Linfáticos , Vasos Linfáticos , Mesenterio/anatomía & histología , Contracción Muscular , Animales , Difenhidramina/farmacología , Relación Dosis-Respuesta a Droga , Endotelio , Agonistas de los Receptores Histamínicos/farmacología , Antagonistas de los Receptores Histamínicos H1/farmacología , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/fisiología , Vasos Linfáticos/anatomía & histología , Vasos Linfáticos/efectos de los fármacos , Vasos Linfáticos/fisiología , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/fisiología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/metabolismo , RatasAsunto(s)
Proteínas Tirosina Quinasas/fisiología , Equilibrio Hidroelectrolítico/fisiología , Amoeba , Animales , Arginina Vasopresina/fisiología , Colforsina/farmacología , Desamino Arginina Vasopresina/farmacología , Electrólitos , Inhibidores Enzimáticos/farmacología , Genisteína/farmacología , Hidroquinonas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Purinonas/farmacologíaRESUMEN
The influence of some cationic detergents on the catalytic activity of the horse blood plasma cholinesterase in reaction of hydrolysis of alpha-naphthylacetate at different pH were investigated. It was shown, that in the absence of detergents in acid pH of the reaction medium the Km value increases, but V remain constant. In the range of pH from 8.5 to 5.0 in the presence of detergents the Km and V values are not practically changed. That is why the activation of cholinesterase hydrolysis of alpha-naphthylacetate in the presence of detergents is considerably higher than that of the neutral pH.
Asunto(s)
Butirilcolinesterasa/química , Compuestos de Cetrimonio/química , Cetilpiridinio/química , Detergentes/química , Naftoles/química , Animales , Butirilcolinesterasa/sangre , Cationes , Caballos , Concentración de Iones de Hidrógeno , Hidrólisis , CinéticaAsunto(s)
Adenosina Trifosfato/metabolismo , Membranas Intracelulares/metabolismo , Canales Iónicos/metabolismo , Proteínas de la Membrana/metabolismo , Mitocondrias Hepáticas/metabolismo , Potasio/metabolismo , Talio/metabolismo , Animales , Ácido Ascórbico/farmacología , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Combinación de Medicamentos , Etilmaleimida/farmacología , Guanosina Difosfato/farmacología , Dilatación Mitocondrial/efectos de los fármacos , Palmitoil Coenzima A/farmacología , Potasio/química , Canales de Potasio , Ratas , Ratas Wistar , Tetrametilfenilendiamina/análogos & derivados , Tetrametilfenilendiamina/farmacología , Talio/química , Talio/farmacocinéticaAsunto(s)
Amoeba/fisiología , Arginina Vasopresina/farmacología , Transducción de Señal/efectos de los fármacos , Amoeba/efectos de los fármacos , Animales , Colforsina/farmacología , AMP Cíclico/farmacología , Desamino Arginina Vasopresina/farmacología , Agua Dulce , Vacuolas/efectos de los fármacos , Vacuolas/fisiologíaRESUMEN
We have studied Cd2+-induced effects on mitochondrial respiration and swelling in various media as a function of the [Cd2+] in the presence or absence of different bivalent metal ions or ruthenium red (RR). It was confirmed by monitoring oxygen consumption by isolated rat liver mitochondria that, beginning from 5 microM, Cd2+ decreased both ADP and uncoupler-stimulated respiration and increased their basal respiration when succinate was used as respiratory substrate. At concentrations higher than 5 microM, Cd2+ stimulated ion permeability of the inner mitochondrial membrane, which was monitored in this study by swelling of both nonenergized mitochondria in 125 mM KNO3 or NH4NO3 medium and succinate-energized mitochondria incubated in a medium containing 25 mM K-acetate and 100 mM sucrose. We have found substantial changes in the above-mentioned Cd2+ effects on mitochondria treated in sequence with 100 microM of Ca2+, Sr2+, Mn2+ or Ba2+(Me2+) and 7.5 microM RR, as well as the alterations in Cd2+ action on the uptake of 137Cs+ by succinate-energized mitochondria in the presence or absence of valinomycin in acetate medium (50 mM Tris-acetate and 140 mM sucrose) with or without Ca2+ or RR. The evidence obtained indicate that Ca2+ exhibits a synergestic action on all Cd2+ effects examined, whereas Sr2+ and Mn2+, conversely, are antagonistic. In the presence of RR, the Cd2+ effects on respiration [stimulation of State 4 respiration and inhibition of 2,4-dinitrophenol (DNP)-uncoupled respiration] still exist, but are observed at concentrations of cadmium more than one order higher; the inhibition of State 3 respiration by Cd2+ conversely, takes place under even lower cadmium concentrations than those determined without RR in the medium. In addition, RR added simultaneously with cadmium in the incubation medium prevents any swelling in the nitrate media, but induces an increment both in Cd2+-stimulated swelling and 137Cs+ (analog of K+) uptake in the acetate media. For the first time, we have shown that Cd2+-induced swelling in all media under study is susceptible to cyclosporin A (CSA), a high-potency inhibitor of the mitochondrial permeability transition (PT) pore. The observations are interpreted in terms of a dual effect of cadmium on respiratory chain activity and permeability transition.
Asunto(s)
Cadmio/farmacología , Cationes Bivalentes/farmacología , Mitocondrias Hepáticas/efectos de los fármacos , Desacopladores/farmacología , Animales , Respiración de la Célula/efectos de los fármacos , Radioisótopos de Cesio/farmacocinética , Interacciones Farmacológicas , Membranas Intracelulares/efectos de los fármacos , Masculino , Metales Pesados/farmacología , Permeabilidad/efectos de los fármacos , Ratas , Rojo de Rutenio/farmacologíaRESUMEN
Effects of Cd2+ and two complexes of bivalent cadmium with 1,3-bis(4-chlorbenzylidenamino)-guanidine and anabasine on ion permeability of the inner membrane and respiration of isolated rat liver mitochondria were studied. Starting from 5 microM, Cd2+ decreased state 3 and DNP-stimulated respiration of mitochondria and increased their state 4 respiration. At 30 microM, Cd2+ decreased state 4 respiration. The complexes, particularly complex of Cd2+ with 1,3-bis(4-chlorbenzylidenamino)-guanidine, inhibited the mitochondrial respiration at lower concentration of Cd2+. Nonenergized mitochondria incubated in media containing 125 mM of NH4NO3 or KNO3 showed more pronounced swelling in experiments with 10 microM of the complexes than with Cd2+. The complexes produced swelling of the mitochondria energized by 5 mM of succinate and incubated in medium containing 25 mM K-acetate and 100 mM sucrose. Uptake of 137-Cs by succinate-energized mitochondria in the presence of 10(-8) M of valinomycin was substantially decreased in experiments with 10 microM of the complexes than with Cd2+. Ruthenium red (7.5 microM) prevented this effect with 10 microM of complex of Cd2+ with 1,3-bis(4-chlorbenzylidenamino)-guanidine and especially complex of Cd2+ with anabasine and Cd2+. These results indicate that the cadmium organic complexes affect respiration and perturb ion permeability significantly stronger than Cd2+.
Asunto(s)
Compuestos de Cadmio/farmacología , Mitocondrias Hepáticas/efectos de los fármacos , Animales , Compuestos de Cadmio/química , Radioisótopos de Cesio/metabolismo , Medios de Cultivo , Metabolismo Energético , Masculino , Mitocondrias Hepáticas/metabolismo , Dilatación Mitocondrial/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Ratas , Ácido Succínico/metabolismoRESUMEN
Cuprous ions at micromolar concentrations induced swelling of rat liver mitochondria in isotonic solutions of potassium thiocyanate and potassium acetate. The swelling in K-acetate in the presence of the protonophore [corrected] carbonyl cyanide m-chloropenylhydrazone was partly inhibited by glibenclamide. In K(+)-containing media, Cu+ collapsed the mitochondrial membrane potential formed by operation of the respiratory chain with succinate or tetramethyl p-phenylenediamine + ascorbate as substrates or by the proton-pumping ATPase. In contrast, in K(+)-free media, isotonic sucrose or choline chloride, but not in NaC1, Cu+ induced a transient potassium gradient potential. These results indicate that cuprous ions at low concentrations, apart from promoting the electroneutral K+/H+ exchange, facilitate the uniport of K+, presumably by activating the mitochondrial potassium channel sensitive to glibenclamide.