Oxidative damage to mitochondria is mediated by the Ca(2+)-dependent inner-membrane permeability transition.
The ability of O2 metabolites derived from the xanthine-xanthine oxidase system to inhibit mitochondrial function was examined using freshly isolated rat liver mitochondria. Under 2,4-dinitrophenol-uncoupled conditions, mitochondria exposed to free radicals exhibited a significant decrease in O2 consumption supported by NAD(+)-linked substrates, but showed almost no change in O2 consumption in the presence of succinate and ascorbate. Oxidative stress caused the loss of intramitochondrial nicotinamide nucleotides, and addition of NAD+ fully prevented any fall in O2 consumption with NAD(+)-linked substrates. The activity of electron-transfer complex I (NADH oxidase and NADH-cytochrome c oxidoreductase) and the energy-dependent reduction of NAD+ by succinate were unaltered by oxidative stress. Exposure to free radicals also had an uncoupling effect at all three coupling sites. The degree of mitochondrial swelling was closely correlated with the inhibition of State-3 oxidation of site-I substrates and with the increase in State-4 oxidation of succinate. The immunosuppressive agent cyclosporin A completely prevented the mitochondrial damage induced by oxygen free radicals (swelling, Ca2+ release, sucrose trapping, uncoupling and selective inhibition of the mitochondrial respiration of site-I substrates). The same protective effect was found when Ca2+ cycling was prevented, either by chelating Ca2+ with EGTA or by inhibiting Ca2+ reuptake with Ruthenium Red. These findings suggest that the deleterious effect of free radicals on mitochondria in the present experimental system was triggered by the cyclosporin A-sensitive and Ca(2+)-dependent membrane transition, and not by direct impairment of the mitochondrial inner-membrane enzymes.
Takeyama
N
N
Department of Emergency and Critical Care Medicine, Kansai Medical University, Osaka, Japan.
Matsuo
N
N
Tanaka
T
T
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D016572:Cyclosporine / Q000494:pharmacology; D004579:Electron Transport; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000187:drug effects / Q000502:physiology; D008297:Male; D008930:Mitochondria, Liver / Q000187:drug effects* / Q000378:metabolism; D008933:Mitochondrial Swelling / Q000187:drug effects; D009243:NAD / Q000378:metabolism; D010101:Oxygen Consumption; D010539:Permeability; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000633:toxicity*; D012430:Ruthenium Red / Q000494:pharmacology
D017382:Reactive Oxygen Species; D009243:NAD; D012430:Ruthenium Red; D016572:Cyclosporine; D000255:Adenosine Triphosphate; D002118:Calcium