Voltage-dependent Anion Channels Control the Release of the Superoxide Anion from Mitochondria to Cytosol

作者：Derick Han, Fernando Antunes, Raffaella Canali, Daniel Rettori, Enrique Cadenas · 发表于：Journal of Biological Chemistry · 年份：2003 · DOI：10.1074/jbc.m210269200 · 被引用次数：716 · 研究领域：Mitochondrial Function and Pathology、Ion channel regulation and function、Electrochemical Analysis and Applications

Several reactions in biological systems contribute to maintain the steady-state concentrations of superoxide anion (O 2 ⨪ ) and hydrogen peroxide (H 2 O 2 ). The electron transfer chain of mitochondria is a well documented source of H 2 O 2 ; however, the release of O 2 ⨪ from mitochondria into cytosol has not been unequivocally established. This study was aimed at validating mitochondria as sources of cytosolic O 2 ⨪ , elucidating the mechanisms underlying the release of O 2 ⨪ 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 ⨪ . 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 O 2 ⨪ 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 O 2 ⨪ production from mitochond...