Inhibition Of Complex III Promotes Loss Of Ca2+ Dependence For Mitochondrial Superoxide Formation And Permeability Transition Evoked By Peroxynitrite
In intact U937 cells, peroxynitrite promotes the mitochondrial formation of superoxide via a Ca2+-dependent mechanism involving inhibition of complex III. Superoxide then readily dismutates to H2O2 causing lesions on different biomolecules, including DNA. Here we show that formation of H2O2 and DNA damage are suppressed by inhibition of complex I (by rotenone) or ubisemiquinone formation (by myxothiazol), as well as by a variety of manipulations preventing either the mobilization of Ca2+ or its mitochondrial accumulation. In addition, complex III inhibitors promoted rotenone- or myxothiazol-sensitive formation of H2O2 and DNA strand scission in cells exposed to otherwise inactive concentrations of peroxynitrite. However, under these conditions, the intra-mitochondrial concentration of Ca2+ remained unchanged and the effects of peroxynitrite therefore take place via Ca2+-independent mechanisms. H2O2 formation was paralleled by, and causally linked to, the loss of mitochondrial membrane potential associated with the mitochondrial release of cytochrome c and AIF, and with the mitochondrial accumulation of Bax. These events, although Ca2+ independent, were rapidly followed by death mediated by mitochondrial permeability transition, generally considered a typical Ca2+-dependent event. Thus, enforced inhibition of complex III promotes the loss of Ca2+ dependence of those mitochondrial mechanisms regulating superoxide formation and mitochondrial permeability transition evoked by peroxynitrite.