VDAC-dependent permeabilization of the outer mitochondrial membrane by superoxide induces rapid and massive cytochrome c release - PubMed (original) (raw)

VDAC-dependent permeabilization of the outer mitochondrial membrane by superoxide induces rapid and massive cytochrome c release

M Madesh et al. J Cell Biol. 2001.

Abstract

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.

PubMed Disclaimer

Figures

Figure 1.

Figure 1.

Effect of ROS on Ca2+-induced PTP opening and CCR in permeabilized HepG2 cells. (A) O2 ·−-generating system (xanthine [0.1 mM] plus xanthine oxidase [20 mU/ml]) and (B) H2O2 (90 mM) augmented Ca2+-induced depolarization (three pulses, 30 μM CaCl2 each) and decreased mitochondrial Ca2+ uptake. These effects were inhibited by an O2 ·− scavenger, MnTBAP (20 μM; 68 ± 4.5% decrease in depolarization and 78 ± 13% decrease in [Ca2+]c rise at 900 s; n = 3), and catalase (Cat; 2500U/ml), respectively. At the end of the measurements, cells were exposed to FCCP (Unc; 1 μM), a protonophore that caused rapid and complete dissipation of ΔΨm. (C) Induction of CCR from mitochondria by O2 ·− but not H2O2. Cytosolic and membrane fractions were prepared after fluorometric measurements of ΔΨm and [Ca2+]c in permeabilized cells performed as shown in A and B, except that Unc was not added. Released cyto c in the cytosol was visualized by Western blotting with anti–cyto c antibody. (D) Permeabilized cells were incubated with H2O2 at three different concentrations (0.9, 9, and 90 mM) or with X + XO for 375 s. (E) Permeabilized cells were preincubated with an ONOO− scavenger, ebselen, at three different concentrations (100, 50, and 25 μM), before the exposure to X + XO. (F) Quantification of O2 ·−-induced CCR from mitochondria. Cells were exposed to X + XO (0.1 mM plus 20 mU/ml) for 375 s. Various amounts of cytosolic and membrane fraction containing cyto c were compared with cyto c standard.

Figure 3.

Figure 3.

PTP blockers fail to inhibit O2 ·− -induced CCR. Permeabilized cells were pretreated with CsA (1 μM) or BA (10 μM) before the addition of X + XO. (A) Mitochondrial depolarization and [Ca2+]c rise evoked by Ca2+ pulses added after the pretreatment with X + XO or CsA + X + XO. (B and C) Released cyto c in the cytosol was visualized by Western blotting. Cytosol samples were prepared after the measurement of ΔΨm but FCCP was not added. Permeabilized cells were pretreated with BA (10 μM) in the presence or absence of ATP-regenerating system before the X + XO treatment. Because ADP supports the binding and inhibitory effect of BA, to prevent extramitochondrial phosphorylation of ADP, ATP-regenerating system was omitted from the medium in some experiments.

Figure 2.

Figure 2.

Ca2+-induced mitochondrial depolarization is not essential for O2 ·− -dependent CCR. Permeabilized cells were treated with X (0.1 mM) and different concentrations of XO (0.1, 1, 2, 3, 4, 5, 10, or 20 mU/ml) before addition of Ca2+ pulses (30 μM each) or solvent. (A and B) ΔΨm measured simultaneously with [Ca2+]c. (C and D) cyto c in the cytosol and membrane fractions. Immunoblotting of actin in the cytosolic fraction was used to evaluate whether O2 ·− changed the distribution of nonmitochondrial proteins.

Figure 4.

Figure 4.

O2 ·− -induced depletion of mitochondrial cyto c yields mitochondrial depolarization in the absence of extramitochondrial ATP supply. (A) Effect of O2 ·− on ΔΨm was monitored in permeabilized cells pretreated with oligomycin (2.5 μg/ml), an inhibitor of the F0F1-ATPase before the addition of X + XO or CsA + X + XO. (B) Rescue of mitochondria from O2 ·−-induced depolarization by exogenously added cyto c. Permeabilized cells were preincubated with cyto c (2, 5, or 10 μM) and oligomycin (2.5 μg/ml) before exposure to X + XO.

Figure 5.

Figure 5.

Inhibition of O2 ·− -induced CCR by VDAC blockers and anti-VDAC antibody. Permeabilized cells were exposed to X + XO in the absence of Ca2+ pulses. At various times after X + XO exposure, permeabilized cells were centrifuged for the collection of cytosolic fractions. For inhibitory studies, permeabilized cells were pretreated with (A) DIDS, an inhibitor of VDAC, for 50 s before the addition of X + XO, or (B) with indicated concentrations of KP for 100 s before exposure to X + XO. (C) Prevention of O2 ·− -induced ΔΨm loss by anti-VDAC antibody (Ab#25). Permeabilized cells pretreated with or without Ab#25 (0.56 μg/μl) for 5 min, after which X + XO (0.1 mM plus 5 mU/ml) was added and changes of ΔΨ were monitored for 12 min by spectrofluorimeter. Data presented is representative of one experiment. (D) Inhibition of O2 ·− -induced CCR by anti-VDAC antibody. After the measurement of ΔΨm shown in C, the cells were centrifuged and cyto c was determined in the cytosolic fractions. (E) Translocation of Bax to the mitochondria is absent during exposure to O2 ·− but occurs during staurosporine-mediated CCR. The cytosol and mitochondrial fractions were subjected to 15% SDS-PAGE and transferred to nitrocellulose. Blots were probed with a polyclonal anti-Bax antibody.

Figure 6.

Figure 6.

O2 ·− induces caspase-3 activation; prevention by a VDAC blocker but not PTP inhibitors. (A) Cytosolic fractions (prepared as described above) were resolved on 15% SDS-PAGE followed by transfer to nitrocellulose and the blot was probed with polyclonal anticaspase-3 antibody. (B) Fluorometric assay of DEVD-AMC (12.5 μM) cleavage, in cytosol extracts (incubation for 30 min at 35°C). DEVD-AMC cleavage normalized to the activity obtained in the presence of X + XO (0.1 mM plus 5 mU/ml) is shown as mean ± SEM (n = 3).

Figure 7.

Figure 7.

O2 ·− -induced CCR in intact HepG2 cells; time-course, effect of PTP, and VDAC blockers. (A) Cells were grown in 25-cm2 flasks for 48 r and treated with X + XO for different time periods. After treatment, cells were harvested and permeabilized with 40 μg/ml digitonin containing ICM at pH 7.2 for 10 min. The cytosol was then separated from the membrane frac- tion (containing mitochondria) by centrifugation. The cytosolic fractions were resolved on SDS-PAGE and immunoblotted for cyto c. (B) Cells pretreated with DIDS, CsA, or BA for 10 min were exposed to X + XO. After 3 h, cells were isolated, permeabilized, centrifuged, and immunoblot was done as described above. (C) Confocal imaging of O2 ·−-induced CCR using cyto _c_–GFP. Cyto _c_-GFP–transfected HepG2 cells were treated with X + XO (0.1 mM and 20 mU/ml, respectively) in the absence or presence of inhibitors. Distribution of cyto _c_–GFP was imaged by confocal microscopy. The images were taken after different time periods of X + XO exposure with a 60× oil objective. (D) Quantification of cyto _c_–GFP in the cytosol. Since the nucleus is devoid of mitochondria and released cyto _c_–GFP entered the nuclear matrix, the amount of cyto _c_–GFP released into the cytosol was determined by measurements of fluorescence over the nucleus in each cell. f.a.u., fluorescence arbitrary units. To assess the amount of cyto _c_–GFP release after X + XO treatment, we counted ∼100–200 cells in three independent experiments for each condition. (E) Electron micrographs of control (left) and X + XO–treated (0.1 mM plus 20 mU/ml for 1 h; right) intact cells are shown to illustrate the typical patterns of mitochondrial morphology (>200 mitochondria were evaluated in each condition).

Figure 8.

Figure 8.

O2 ·− directly modulates purified VDAC to facilitate cyto c release. (A) Immunoblotting of purified VDAC and VDAC liposomes for VDAC and Bax. (B) Confocal imaging of FITC–cyto c efflux from plain and VDAC liposomes. Cyto c labeled with FITC was loaded into both plain and VDAC-incorporated liposomes. Images were taken before and after 7 min of exposure to X + XO (0.1 mM and 20 mU/ml). Furthermore, the effect of O2 ·− was evaluated on VDAC liposomes incubated in the presence of DIDS (100 μM). (C) Fluorometry of cyto _c_–FITC release from liposomes. Suspensions of liposomes (4 types; plain, plain loaded with cyto _c_–FITC, VDAC, and VDAC loaded with cyto _c_–FITC) were incubated in the absence or presence of X + XO (0.1 mM and 20 mU/ml) and DIDS (100 μM) for 7 min. Subsequently, the suspensions were centrifuged and FITC fluorescence was determined in the supernatants and liposomoic pellets. Cyto _c_–FITC release is shown by the FITC fluorescence measured in the supernatants. We observed that exposure to X + XO increased the fluorescence of FITC (unpublished data) and this effect could contribute to the increase of FITC in the supernatants of plain as well as VDAC liposomes. However, in response to X + XO, the VDAC liposomes exhibited a several-fold larger increase in the supernatant FITC and a considerably smaller residual pellet FITC than that of in plain liposomes (unpublished data). Data are from two separate experiments; each was performed in duplicates.

Figure 9.

Figure 9.

Execution of apoptosis in HepG2 cells exposed to O2 ·− . Mitochondrial depolarization, PS exposure, disruption of plasma membrane integrity, and DNA fragmentation in cells exposed to O2 ·−. Cells were treated with X + XO (0.1 mM plus 20 mU/ml) for various time periods in the absence or presence of DIDS (100 μM). (A) ΔΨm and PS exposure were evaluated with confocal imaging of annexin V Alexa 488 and TMRE. (B) Distribution of cyto _c_–GFP and annexin staining were visualized. (C) PS exposure and loss of plasma membrane integrity were evaluated with imaging of annexin V Alexa 488 and propidium iodide. (D) DNA fragmentation analysis was performed with cells incubated in the presence or absence of X + XO for 12 h.

Similar articles

Cited by

References

    1. Adams, J.M., and S. Cory. 1998. The Bcl-2 protein family: arbiters of cell survival. Science. 281:1322–1326. - PubMed
    1. Ankarcrona, M., J.M. Dypbukt, S. Orrenius, and P. Nicotera. 1996. Calcineurin and mitochondrial function in glutamate-induced neuronal cell death. FEBS Lett. 394:321–324. - PubMed
    1. Antonsson, B., S. Montessuit, S. Lauper, R. Eskes, and J.C. Martinou. 2000. Bax oligomerization is required for channel-forming activity in liposomes and to trigger cyto c release from mitochondria. Biochem. J. 345:271–278. - PMC - PubMed
    1. Babior, B.M. 1999. NADPH oxidase: an update. Blood. 93:1464–1476. - PubMed
    1. Basanez, G., A. Nechushtan, O. Drozhinin, A. Chanturiya, E. Choe, S. Tutt, K.A. Wood, Y. Hsu, J. Zimmerberg, and R.J. Youle. 1999. Bax, but not Bcl-xL, decreases the lifetime of planar phospholipid bilayer membranes at subnanomolar concentrations. Proc. Natl. Acad. Sci. USA. 96:5492–5497. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources