Peroxynitrite formed by simultaneous nitric oxide and superoxide generation causes cyclosporin-A-sensitive...

Nitric oxide reacts rapidly with superoxide to form the potent oxidant peroxynitrite. Mitochondria in vivo produce superoxide and in pathological situations the amount of superoxide produced increases; therefore, in the presence of nitric oxide, mitochondria will be a major site of peroxynitrite formation. Oxidative stress induces cyclosporin-A-sensitive mitochondrial calcium efflux and depolarisation which may contribute to tissue damage in pathological situations. To determine whether peroxynitrite could induce calcium efflux and depolarisation we exposed mitochondria to both nitric oxide and superoxide simultaneously, thus subjecting the mitochondria to a continual flux of peroxynitrite similar to that found in pathological situations. Our results show that: (a) exposure of mitochondria to nitric oxide plus superoxide induces cyclosporin-A-sensitive mitochondrial calcium efflux and depolarisation; (b) neither nitric oxide nor superoxide on their own induce calcium efflux or depolarisation under these conditions; (c) calcium efflux and depolarisation occur when peroxynitrite production (from nitric oxide and superoxide) exceeds about 0.99 +/- 0.03 nmol peroxynitrite.min-1 mg mitochondrial protein-1. This rate of production of peroxynitrite is similar in magnitude to superoxide formation by mitochondria in pathological situations, suggesting that mitochondria can produce sufficient peroxynitrite in vivo to cause mitochondrial calcium efflux and depolarisation. Our experiments suggest a plausible model for tissue damage when mitochondrial superoxide production increases, for example in ischaemia-reperfusion injury or following exposure to neurotoxins. In the presence of nitric oxide the mitochondrial superoxide production will lead to peroxynitrite formation which induces cyclosporin-A-sensitive mitochondrial calcium efflux and depolarisation. This disruption to mitochondrial function may in turn contribute to cell damage and death.