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)*-) 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 approximately 0.04 nmol/min/mg protein, a value increased approximately 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 mitochondria by approximately 55%, thus suggesting that a large portion of O(2)*- exited mitochondria via these channels. These findings are discussed in terms of competitive decay pathways for O(2)*- in the intermembrane space and cytosol as well as the implications of these processes for modulating cell signaling pathways in these compartments.
Han
Derick
D
University of Southern California Research Center for Liver Disease, Keck School of Medicine, University of Southern California, Los Angeles, California 90089-9121, USA.
Antunes
Fernando
F
Canali
Raffaella
R
Rettori
Daniel
D
Cadenas
Enrique
E
D000818:Animals; D000968:Antimycin A / Q000494:pharmacology; D002458:Cell Fractionation; D003497:Cyclic N-Oxides / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D006861:Hydrogen Peroxide / Q000378:metabolism; D006878:Hydroxides / Q000378:metabolism; D007473:Ion Channels / Q000502:physiology*; D007700:Kinetics; D008297:Male; D008689:Methacrylates; D008929:Mitochondria, Heart / Q000187:drug effects / Q000378:metabolism*; D018272:Porins / Q000502:physiology*; D051381:Rats; D017208:Rats, Wistar; D013482:Superoxide Dismutase / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism*; D013844:Thiazoles / Q000494:pharmacology; D050994:Voltage-Dependent Anion Channels
D003497:Cyclic N-Oxides; D006878:Hydroxides; D007473:Ion Channels; D008689:Methacrylates; D018272:Porins; D013844:Thiazoles; D050994:Voltage-Dependent Anion Channels; D013481:Superoxides; C069977:2,2-dimethyl-5-hydroxy-1-pyrrolidinyloxy; D000968:Antimycin A; C030517:myxothiazol; C031356:hydroxide ion; D006861:Hydrogen Peroxide; D013482:Superoxide Dismutase