Cytochrome c release occurs via Ca2+-dependent and Ca2+-independent mechanisms that are regulated by Bax

Release of cytochrome c from mitochondria is a key initiative step in the apoptotic process, although the mechanisms regulating this event remain elusive. In the present study, using isolated liver mitochondria, we demonstrate that cytochrome c release occurs via distinct mechanisms that are either Ca2+-dependent or Ca2+-independent, An increase in mitochondrial matrix Ca2+ promotes the opening of the permeability transition (PT) pore and the release of cytochrome c, an effect that is significantly enhanced when these organelles are incubated in a reaction buffer that is based on a physiologically relevant concentration of K+ (150 mM KCI) versus a buffer composed of mannitol/sucrose/Hepes. Moreover, low concentrations of Ca2+ are sufficient to induce mitochondrial cytochrome c release without measurable manifestations of PT, though inhibitors of PT effectively prevent this release, indicating that the critical threshold for PT varies among mitochondria within a single population of these organelles. In contrast, Ca2+-independent cytochrome c release is induced by oligomeric Bax protein and occurs without mitochondrial swelling or the release of matrix proteins, although our data also indicate that Pax enhances permeability transition-induced cytochrome c release. Taken together, our results suggest that the intramitochondrial Ca2+ concentration, as well as the reaction buffer composition, are key factors in determining the mode and amount of cytochrome c release. Finally, oligomeric Bax appears to be capable of stimulating cytochrome c release via both Ca2+-dependent and Ca2+-independent mechanisms.