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

Abstract

Several reactions in biological systems contribute to maintain the steady-state concentrations of superoxide anion (O5.-) and hydrogen peroxide (H2O2). The electron transfer chain of mitochondria is a well documented source of H2O2; however, the release of O2.- from mitochondria into cytosol has not been unequivocally established. This study was aimed at validating mitochondria as sources of cytosolic O2.-, elucidating the mechanisms underlying the release of O2.- 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 O2.-. Inhibition of the signal in a concentration-dependent 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 O2.- release from mitochondria were estimated at ∼0.04 nmol/ min/mg protein, a value increased ∼8-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 O2.- production from mitochondria by ∼55%, thus suggesting that a large portion of O2.- exited mitochondria via these channels. These findings are discussed in terms of competitive decay pathways for O2.- in the intermembrane space and cytosol as well as the implications of these processes for modulating cell signaling pathways in these compartments.