Abstract
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.
Highlights
Min/mg protein, a value increased ϳ8-fold by the complex III inhibitor, antimycin A
In the absence of respiratory substrates, mitochondria supplemented with the spin trap, DMPO, did not generate any Electron Paramagnetic Resonance (EPR) signal (Fig. 1A)
The addition of complex I (Fig. 1B) or complex II substrates to heart mitochondria resulted in a low intensity EPR signal characteristic of the DMPO-OH spin adduct
Summary
Min/mg protein, a value increased ϳ8-fold by the complex III inhibitor, antimycin A. 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. H2O2 produced by mitochondria has been suggested to regulate several signal transduction pathways, including c-Jun N-terminal kinase (JNK1) activity [3, 4]. Alterations in mitochondrial H2O2 steady-state levels by genetic modulation of cata-. Lase expression in the mitochondrial matrix is associated with changes in cell proliferation [3, 5], tumor necrosis factor (TNF) response [6], and apoptosis [7]. A two-step model that accounts for mitochondrial H2O2 production has become widely accepted [8, 9]. The first step, shown below in Reaction 1, UQ. ϩ O2 3 UQ ϩ O2
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