Enhanced formation of reactive oxygen species (ROS), superoxide (O2·−), and hydrogen peroxide (H2O2) may result in either apoptosis or other forms of cell death. Here, we studied the mechanisms underlying activation of the apoptotic machinery by ROS. Exposure of permeabilized HepG2 cells to O2·− elicited rapid and massive cytochrome c release (CCR), whereas H2O2 failed to induce any release. Both O2·− and H2O2 promoted activation of the mitochondrial permeability transition pore by Ca2+, but Ca2+-dependent pore opening was not required for O2·−-induced CCR. Furthermore, O2·− alone evoked CCR without damage of the inner mitochondrial membrane barrier, as mitochondrial membrane potential was sustained in the presence of extramitochondrial ATP. Strikingly, pretreatment of the cells with drugs or an antibody, which block the voltage-dependent anion channel (VDAC), prevented O2·−-induced CCR. Furthermore, VDAC-reconstituted liposomes permeated cytochrome c after O2·− exposure, and this release was prevented by VDAC blocker. The proapoptotic protein, Bak, was not detected in HepG2 cells and O2·−-induced CCR did not depend on Bax translocation to mitochondria. O2·−-induced CCR was followed by caspase activation and execution of apoptosis. Thus, O2·− triggers apoptosis via VDAC-dependent permeabilization of the mitochondrial outer membrane without apparent contribution of proapoptotic Bcl-2 family proteins.
VDAC-dependent permeabilization of the outer mitochondrial membrane by superoxide induces rapid and massive cytochrome c release
Abbreviations used in this paper: BA, bongkrekic acid; CCR, cytochrome c release; CsA, cyclosporin A; cyto c, cytochrome c; DIDS, 4,4′-diisothiocyanatostilbene-2,29-disulfonic acid; GFP, green fluorescent protein; IMM, inner mitochondrial membrane; KP, Konig's polyanion; NO, nitric oxide; OMM, outer mitochondrial membrane; ONOO−, peroxynitrite; O2·−, superoxide; PI, propidium iodide; PS, phosphatidylserine; PTP, permeability transition pore; ROS, reactive oxygen species; SOD, superoxide dismutase; VDAC, voltage-dependent anion channel; X + XO, xanthine plus xanthine oxidase.
Muniswamy Madesh, György Hajnóczky; VDAC-dependent permeabilization of the outer mitochondrial membrane by superoxide induces rapid and massive cytochrome c release . J Cell Biol 10 December 2001; 155 (6): 1003–1016. doi: https://doi.org/10.1083/jcb.200105057
Download citation file:
Sign in
Client Account
Sign in via your Institution
Sign in via your InstitutionEmail alerts
Advertisement
Advertisement