Voltage-dependent anion channels control the release of the superoxide anion from mitochondria to cytosol

Several reactions in biological systems contribute to maintain the steady-state concentrations of superoxide anion (O-2(radical anion)) and hydrogen peroxide (H2O2). The electron transfer chain of mitochondria is a well documented source of H2O2; however, the release of O-2(radical) (anion) from mito chondria into cytosol has not been unequivocally established. This study was aimed at validating mitochondria as sources of cytosolic O-2(radical anion) elucidating the mechanisms underlying the release of W from mitochondria into cytosol, and assessing the role of outer membrane voltage-dependent anion channels (VDACs) in this process. Isolated rat heart mitochondria supplemented with complex I or II substrates generate an EPR signal ascribed to O-2(radical anion) Inhibition of the signal in a concentration-depend ent manner by both manganese-superoxide dismutase and cytochrome c proteins that cannot cross the mitochondrial membrane supports the extramitochondrial location of the spin adduct. Basal rates of O-2(radical anion) release from mitochondria were estimated at similar to0.04 nmol/ min/mg protein, a value increased similar to8-fold by the complex III inhibitor, antimycin A. These estimates, obtained by quantitative spin-trapping EPR, were confirmed by fluorescence techniques, mainly hydroethidine oxidation and horseradish peroxidase-based p-hydroxyphylacetate dimerization. Inhibitors of VDAC, 4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS), and dextran sulfate (in a voltage-dependent manner) inhibited O-2(radical anion) production from mitochondria by similar to55%, thus suggesting that a large portion of O-2(radical anion) exited mito chondria via these channels. These findings are discussed in terms of competitive decay pathways for O-2(radical anion) in the intermembrane space and cytosol as well as the implications of these processes for modulating cell signaling pathways in these compartments.