THE MITOCHONDRIAL PERMEABILITY TRANSITION PORE IS MODULATED BY OXIDATIVE AGENTS THROUGH BOTH PYRIDINE-NUCLEOTIDES AND GLUTATHIONE AT 2 SEPARATE SITES

We studied the modulation of the permeability transition pore (MTP), a cyclosporin-A-sensitive channel, in deenergized mitochondria. Rat liv er mitochondria were incubated in a potassium gluconate medium and tre ated with uncoupler, valinomycin, oligomycin and A23187. Under these c onditions the membrane and Donnan potentials are collapsed, and no ion gradients are maintained, allowing the study of the dependence of MTP opening on the Ca2+ concentration under a variety of oxidative condit ions without the complexities arising from changes of the membrane pot ential and matrix pH, and from secondary-ion redistribution. Cross-lin king of mitochondrial dithiols with arsenite or phenylarsine oxide, or treatment with tertbutylhydroperoxide leading to complete oxidation o f glutathione, increased the sensitivity of MTP opening to Ca2+. This effect could be fully prevented by prior treatment of mitochondria wit h monobromobimane and restored by reduction with dithiothreitol. The e ffect of tert-butylhydroperoxide was not additive with that of AsO, an d it was completely blocked by modification of reduced glutathione wit h 1-chloro 2,4-dinitrobenzene through glutathione-S-transferase, indic ating that oxidized glutathione affects the pore through the AsO-react ive and PhAsO-reactive dithiol. Oxidation of mitochondrial pyridine nu cleotides by a variety of treatments also increased the sensitivity of MTP opening to Ca2+ under conditions where glutathione was maintained in the reduced state. This effect could be fully prevented when reduc ed pyridine nucleotides levels were reestablished with 2-hydroxybutyra te but not by treatment with monobromobimane or dithiothreitol. The ef fects of dithiol cross-linking or oxidation, and of pyridine nucleotid e oxidation on the MTP were additive. The contribution of each of thes e two oxidative events, when they were induced at the same time, could still be selectively blocked by monobromobimane and dithiothreitol, o r by 2-hydroxybutyrate, respectively. Complete oxidation of pyridine n ucleotides did not affect the reactivity of the dithiol towards monobr omobimane, indicating that it remained in the reduced state. After tra nsient opening of the MTP, sensitivity to pyridine nucleotide oxidatio n was lost while sensitivity to dithiol cross-linking was retained. Th ese data indicate that the dithiol belongs to an MTP regulatory compon ent which is larger than the MTP cutoff of about 1500 Da, or is non-di ffusible. Taken together, these findings indicate that the MTP is infl uenced by oxidation-reduction events at two separate sites that can be distinguished experimentally, and that these sites are not connected by common oxidation-reduction intermediates other than glutathione.