RESUMEN
We have investigated whether increase in the oxidation rate of exogenous cytochrome c (cyto-c), induced by long-chain ceramides, might be due to an increased rate of cytosolic NADH/cyto-c electron transport pathway. This process was identified in isolated liver mitochondria and has been studied in our laboratory for many years. Data from highly specific test of sulfite oxidase prove that exogenous cyto-c both in the absence and presence of ceramide cannot permeate through the mitochondrial outer membrane. However, the oxidation of added NADH, mediated by exogenous cyto-c and coupled to the generation of a membrane potential supporting the ATP synthesis, can also be stimulated by ceramide. The results obtained suggest that ceramide molecules, by increasing mitochondrial permeability, with the generation of either raft-like platforms or channels, may have a dual function. They can promote the release of endogenous cyto-c and activate, with an energy conserving process, the oxidation of cytosolic NADH either inducing the formation of new respiratory contact sites or increasing the frequency of the pre-existing porin contact sites. In agreement with the data in the literature, an increase of mitochondrial ceramide molecules level may represent an efficient strategy to activate and support the correct execution of apoptotic program.
Asunto(s)
Apoptosis , Ceramidas/farmacología , Citocromos c/metabolismo , Citosol/efectos de los fármacos , Mitocondrias Hepáticas/efectos de los fármacos , NAD/metabolismo , Adenosina Trifosfato/biosíntesis , Adenilato Quinasa/metabolismo , Animales , Citosol/metabolismo , Transporte de Electrón , Metabolismo Energético , Técnicas In Vitro , Potencial de la Membrana Mitocondrial , Mitocondrias Hepáticas/metabolismo , Membranas Mitocondriales/metabolismo , Dilatación Mitocondrial , Oxidación-Reducción , Permeabilidad , Ratas , Sulfito-Oxidasa/metabolismo , Tripsina/farmacologíaRESUMEN
Nitric oxide ((.)NO) generated by the dissociation of S-nitrosoglutathione or added as gaseous solution, inhibits the oxidation of exogenous NADH supported by the activity of the cytosolic NADH/cyto-c electron transport pathway. The inhibition is immediate, very strong, higher at lower oxygen concentration, independent on the (.)NO concentration and remains constant as long as (.)NO is no more available and then is spontaneously removed. The data obtained, not in contrast with those reported with isolated cytochrome oxidase (Cox), strengthen a new concept: reduced cytochrome c (cyto-c) and (.)NO behave as two substrates of Cox, which promotes their oxidation with molecular oxygen as a co-substrate. In the presence of (.)NO, Cox exhibits the property of switching from cyto-c oxidase to (.)NO oxidase activity. With an "all or nothing" process Cox becomes an efficient (.)NO scavenger. The persistence of membrane potential, even in the presence of high inhibition of oxygen uptake, could be tentatively correlated to the protective effect of (.)NO on the ischaemic-reperfusion injury.
Asunto(s)
Citocromos c/metabolismo , Citosol/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Mitocondrias Hepáticas/metabolismo , NAD/metabolismo , Óxido Nítrico/farmacología , Animales , Relación Dosis-Respuesta a Droga , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Óxido Nítrico/metabolismo , Oxígeno/metabolismo , Transporte de Proteínas/efectos de los fármacos , Ratas , Ratas Wistar , Daño por Reperfusión/metabolismoRESUMEN
Cytochrome c (cyto-c), added to isolated mitochondria, activates the oxidation of extramitochondrial NADH and the generation of a membrane potential, both linked to the activity of the cytosolic NADH/cyto-c electron transport pathway. The data presented in this article show that the protective effect of magnesium ions on the permeability of the mitochondrial outer membrane, supported by previously published data, correlates with the finding that, in hypotonic but not isotonic medium, magnesium promotes a differential effect on both the additional release of endogenous cyto-c and on the increased rate of NADH oxidation, depending on whether it is added before or after the mitochondria. At the same time, magnesium prevents or almost completely removes the binding of exogenously added cyto-c. We suggest that, in physiological low-amplitude swelling, magnesium ions may have the function, together with other factors, of modulating the amount of cyto-c molecules transferred from the mitochondrial intermembrane space into the cytosol, required for the correct execution of the apoptotic programme and/or the activation of the NADH/cyto-c electron transport pathway.
Asunto(s)
Citocromos c/metabolismo , Citosol/metabolismo , Transporte de Electrón/efectos de los fármacos , Cloruro de Magnesio/farmacología , Magnesio/farmacología , NAD/metabolismo , Citocromos c/efectos de los fármacos , Citosol/efectos de los fármacos , Glucosa/metabolismo , Células HeLa/efectos de los fármacos , Células HeLa/metabolismo , Humanos , Soluciones Hipotónicas , Cinética , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Membranas Mitocondriales/efectos de los fármacos , Membranas Mitocondriales/fisiología , Rotenona/farmacologíaRESUMEN
The data reported are fully consistent with the well-known observation that exogenous cytochrome c (cyto-c) molecules do not permeate through the outer membrane of mitochondria (MOM) incubated in isotonic medium (250 mM sucrose). Cyto-c is unable to accept electrons from the sulfite/cyto-c oxido-reductase (Sox) present in the intermembrane space, unless mitochondria are solubilized. Mitochondria incubated in a very high hypotonic medium (25 mM sucrose), in contrast to any expectation, continue to be not permeable to added cyto-c even if Sox and adenylate kinase are released into the medium. The succinate/exogenous cyto-c reductase activity, very low in isotonic medium, is greatly increased decreasing the osmolarity of the medium but in both cases remains insensitive to proteolysis by added trypsin. In hypotonic medium, magnesium and potassium ions have a protective effect on the release of enzymes and on the reactivity of cyto-c as electron acceptor from both sulfite and succinate; results which are consistent with the view that MOM preserves its identity and remains not permeable to exogenous cyto-c. This report strengthens the proposal, supported by previously published data that in isotonic medium the exogenous NADH/cyto-c electron transport system is catalyzed by intact mitochondria, not permeable to added cyto-c.
Asunto(s)
Magnesio/fisiología , Membranas Mitocondriales/metabolismo , Potasio/fisiología , Animales , Transporte Biológico/fisiología , Cationes Bivalentes , Cationes Monovalentes , Citocromos c/metabolismo , Caballos , Mitocondrias Hepáticas/enzimología , Mitocondrias Hepáticas/metabolismo , Membranas Mitocondriales/enzimología , Permeabilidad , RatasRESUMEN
Cytochrome c (cyto-c) added to isolated mitochondria promotes the oxidation of extra-mitochondrial NADH and the reduction of molecular oxygen associated to the generation of an electrochemical membrane potential available for ATP synthesis. The electron transport pathway activated by exogenous cyto-c molecules is completely distinct from the one catalyzed by the respiratory chain. Dextran sulfate (500 kDa), known to interact with porin (the voltage-dependent anion channel), other than to inhibit the release of ATP synthesized inside the mitochondria, greatly decreases the activity of exogenous NADH/cyto-c system of intact mitochondria but has no effect on the reconstituted system made of mitoplasts and external membrane preparations. The results obtained are consistent with the existence of specific contact sites containing cytochrome oxidase and porin, as components of the inner and the outer membrane respectively, involved in the oxidation of cytosolic NADH. The proposal is put forward that the bi-trans-membrane electron transport chain activated by cytosolic cyto-c becomes, in physio-pathological conditions: (i) functional in removing the excess of cytosolic NADH; (ii) essential for cell survival in the presence of an impairment of the first three respiratory complexes; and (iii) an additional source of energy at the beginning of apoptosis.