RESUMO
Oxidative damage of mammalian mitochondria induced by Ca2+ and prooxidants is mediated by the attack of mitochondria-generated reactive oxygen species on membrane protein thiols promoting oxidation and cross-linkage that leads to the opening of the mitochondrial permeability transition pore (Castilho et al., 1995). In this study, we present evidence that deenergized potato tuber (Solanum tuberosum) mitochondria, which do not possess a Ca2+ uniport, undergo inner membrane permeabilization when treated with Ca2+ (>0.2 mM), as indicated by mitochondrial swelling. Similar to rat liver mitochondria, this permeabilization is enhanced by diamide, a thiol oxidant that creates a condition of oxidative stress by oxidizing pyridine nucleotides. This is inhibited by the antioxidants catalase and dithiothreitol. Potato mitochondrial membrane permeabilization is not inhibited by ADP, cyclosporin A, and ruthenium red, and is partially inhibited by Mg2+ and acidic pH, well known inhibitors of the mammalian mitochondrial permeability transition. The lack of inhibition of potato mitochondrial permeabilization by cyclosporin A is in contrast to the inhibition of the peptidylprolyl cis-trans isomerase activity, that is related to the cyclosporin A-binding protein cyclophilin. Interestingly, the monofunctional thiol reagent mersalyl induces an extensive cyclosporin A-insensitive potato mitochondrial swelling, even in the presence of lower Ca2+ concentrations (>0.01 mM). In conclusion, we have identified a cyclosporin A-insensitive permeability transition pore in isolated potato mitochondria that is induced by reactive oxygen species.
Assuntos
Cálcio/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Cálcio/metabolismo , Ciclosporina/farmacologia , Técnicas In Vitro , Potenciais da Membrana , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Dilatação Mitocondrial/efeitos dos fármacos , Permeabilidade , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Ratos , Solanum tuberosum/metabolismo , Compostos de Sulfidrila/químicaRESUMO
The ability of low concentrations (5-15 microM) of long-chain fatty acids to open the permeability transition pore (PTP) in Ca(2+)-loaded mitochondria has been ascribed to their protonophoric effect mediated by mitochondrial anion carriers, as well as to a direct interaction with the pore assembly [M.R. Wieckowski and L. Wojtczak, FEBS Lett. 423 (1998) 339-342]. Here, we have compared the PTP opening ability of arachidonic acid (AA) with that of carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) at concentrations that cause similar quantitative dissipation of the membrane potential (DeltaPsi) in Ca(2+)-loaded rat liver mitochondria respiring on succinate. The initial protonophoric effects of AA and FCCP were only slightly modified by carboxyatractyloside and were followed by PTP opening, as indicated by a second phase of DeltaPsi disruption sensitive to EGTA, ADP, dithiothreitol and cyclosporin A. This second phase of DeltaPsi dissipation could also be prevented by rotenone or NAD(P)H-linked substrates which decrease the pyridine nucleotide (PN) oxidation that follows the stimulation of oxygen consumption induced by AA or FCCP. These results suggest that, under the experimental conditions used here, the PTP opening induced by AA or FCCP was a consequence of PN oxidation. Exogenous catalase also inhibited both AA- and FCCP-induced PTP opening. These results indicate that a condition of oxidative stress associated with the oxidized state of PN underlies membrane protein thiol oxidation and PTP opening.
Assuntos
Mitocôndrias Hepáticas/enzimologia , NADP/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Apoptose , Ácido Araquidônico/farmacologia , Cálcio/metabolismo , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Eletrofisiologia , Ácidos Graxos/farmacologia , Feminino , Potenciais da Membrana/efeitos dos fármacos , Necrose , Oxirredução , Consumo de Oxigênio/efeitos dos fármacos , Ratos , Ratos Wistar , Rotenona/farmacologia , Desacopladores/farmacologiaRESUMO
In this communication, we show that the plant uncoupling mitochondrial protein (PUMP) present in potato tuber mitochondria is induced by aging at 28 degrees C and that this induction is strongly stimulated when the potato tubers are stored at low temperature (4 degrees C). PUMP activity was detected by the degree of linoleic acid (LA)-induced ATP-sensitive mitochondrial uncoupling measured as a function of the decrease in membrane potential (delta psi). The PUMP content was evaluated by immunoblot analysis using polyclonal antibodies raised against potato PUMP that specifically detected a 32 kDa band. In agreement with the effect of LA on delta psi, the content of the 32 kDa band increased during storage and was stimulated by low temperature. These results support the proposed role of PUMP in plant thermogenesis and possibly in fruit ripening and senescence.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas de Plantas/metabolismo , Solanum tuberosum/metabolismo , Temperatura , Immunoblotting , Canais Iônicos , Potenciais da Membrana , Proteínas Mitocondriais , Fatores de Tempo , Proteína Desacopladora 1RESUMO
The mitochondrial membrane permeability transition induced by Ca2+ is inhibited by quinine in a dose-dependent fashion. Competition experiments strongly suggest that quinine displaces Ca2+ bound to the inner membrane. This is supported by experiments showing that quinine inhibits Ca2+-dependent but not Ca2+-independent mitochondrial swelling induced by phenylarsine oxide. As with Ca2+ chelators, quinine induces permeability transition pore closure preventing the contraction induced by poly(ethylene glycol) 2000 in mitochondria preswollen by incubation in KSCN medium containing Ca2+ and inorganic phosphate. These results suggest that quinine dislodges Ca2+ bound to the protein site, which triggers pore opening.