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Am J Physiol Gastrointest Liver Physiol 287: G695-G705, 2004. First published March 25, 2004; doi:10.1152/ajpgi.00415.2003
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LIVER AND BILIARY TRACT

Bax interacts with the voltage-dependent anion channel and mediates ethanol-induced apoptosis in rat hepatocytes

Masayuki Adachi,1 Hajime Higuchi,1 Soichiro Miura,2 Toshifumi Azuma,1 Sayaka Inokuchi,1 Hidetsugu Saito,1 Shinzo Kato,1 and Hiromasa Ishii1

1Department of Internal Medicine, Keio University School of Medicine, Shinjuku-ku, Tokyo, 160-8582; and 2Second Department of Internal Medicine, National Defense Medical College, Tokorozawa, Saitama, 359-8513, Japan

Submitted 24 September 2003 ; accepted in final form 22 March 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Acute ethanol exposure induces oxidative stress and apoptosis in primary rat hepatocytes. Previous data indicate that the mitochondrial permeability transition (MPT) is essential for ethanol-induced apoptosis. However, the mechanism by which ethanol induces the MPT remains unclear. In this study, we investigated the role of Bax, a proapoptotic Bcl-2 family protein, in acute ethanol-induced hepatocyte apoptosis. We found that Bax translocates from the cytosol to mitochondria before mitochondrial cytochrome c release. Bax translocation was oxidative stress dependent. Mitochondrial Bax formed a protein complex with the mitochondrial voltage-dependent anion channel (VDAC). Prevention of Bax-VDAC interactions by a microinjection of anti-VDAC antibody effectively prevented hepatocyte apoptosis by ethanol. In conclusion, these data suggest that Bax translocation from the cytosol to mitochondria leads to the subsequent formation of a Bax-VDAC complex that plays a crucial role in acute ethanol-induced hepatocyte apoptosis.

alcoholic liver disease; mitochondria; oxidative stress; cytochrome c

HEPATOCYTE APOPTOSIS IS RECOGNIZED in the liver of both clinical (19, 30) and experimental (2, 9) alcohol-related injury and is currently identified as a common feature of alcoholic liver disease. Although both oxidative stress and cytokines, i.e., TNF-{alpha} or transforming growth factor-{beta}, have been suggested as crucial mediators of hepatocyte apoptosis in alcoholic liver disease (15, 18, 28), relatively little is known regarding the intracellular mechanisms by which ethanol induces hepatocyte apoptosis. Our previous studies demonstrated that short-term ethanol intoxication causes oxidative stress, mitochondrial dysfunction (21), and apoptosis (12, 22) in primary cultured rat hepatocytes. In these studies, ethanol induced oxidative stress by an alcohol dehydrogenase-dependent mechanism (21) and was associated with loss of the mitochondrial membrane potential ({Delta}{psi}). This loss of {Delta}{psi} signified a change in the mitochondrial inner membrane permeability (22) and was associated with cytochrome c release into the cytosol (12). Cytochrome c may bind to apoptosis-activating factor-1 and procaspase-9, resulting in activation of caspase-9, followed by activation of effector caspase-3, -6, and -7 (34, 39). Indeed, we observed both caspase-3 and -9 activation in ethanol-treated hepatocytes, whereas activation of caspase-8 or Bid was not detected (12). Thus mitochondrial dysfunction, such as cytochrome c release, may initiate ethanol-induced hepatocyte apoptosis. However, the exact mechanisms responsible for the cytochrome c release by acute ethanol cytotoxicity are unclear.

Permeability of the inner mitochondrial membrane is regulated by the permeability transition pore (PTP) (3, 42). The exact nature of the PTP remains in dispute. One model suggests that the PTP is comprised of the outer membrane protein voltage-dependent anion channel (VDAC), the inner membrane protein adenine nucleotide translocator (ANT), and cyclophillin-D at outer and inner membrane contact sites (4). Although opening of the PTP is transient and does not cause swelling (14, 32), sustained opening of the PTP might cause mitochondrial swelling with secondary rupture of the outer membrane (31). This rupture of outer membrane leads to massive cytochrome c release that has been noted in both apoptotic and necrotic cell death (24). We previously reported (3) that ethanol-induced cytochrome c release and apoptosis were blocked by cyclosporin A (CsA), an inhibitor of the PTP component cylophillin-D, suggesting that ethanol-induced cytochrome c release is PTP dependent. However, the mechanism by which ethanol induces the PTP opening remains to be elucidated. Recently, the role of proapoptotic Bcl-2 family proteins in mediating the mitochondrial permeability pore has been suggested (10, 38). Therefore, we hypothesized that Bcl-2-related proteins may contribute to cytochrome c release during alcohol cytotoxicity.

Bax and Bak, proapoptotic members of the Bcl-2 family, are crucial for apoptosis (8). Bax translocates from the cytosol to the mitochondrial outer membrane in many models of apoptosis (7). Bax inserts into the mitochondrial outer membrane on apoptotic stimuli (13, 40). Bax homotypic complex or heterotypic complex with Bak promote cytochrome c release from the intermembrane space of mitochondria into the cytosol (17). An in vitro study has shown that treatment of liposomes with Bax permeabilizes lipid membranes, allowing translocation of cytochrome c from the liposomes into the media and suggesting homotypic oligomerized channel formation composed of at least four Bax molecules (33). On the other hand, heterotypic interactions of Bax with the PTP components VDAC (29) or ANT (25) have also been suggested. Therefore, Bax may regulate VDAC or ANT function via direct molecular interactions. Interestingly, Bax-VDAC heterotypic interactions can form a large pore that is permeable to cytochrome c (35). The conductance of Bax-VDAC channel was calculated as 4-fold and 10-fold greater than that of VDAC or Bax homotypic channels (35).

The overall objective of the present study was to examine the mechanisms by which acute ethanol intoxication induces mitochondrial cytochrome c release and apoptosis. To address this objective, we formulated the following questions: 1) Does Bax translocate from the cytosol to mitochondria on acute ethanol treatment? 2) Is Bax translocation oxidative stress dependent? 3) Does Bax form a complex by either homotypic oligomerization or heterotypic interactions with VDAC? 4) Are the Bax-VDAC interactions essential for ethanol-induced apoptosis? and 5) Is Bax-VDAC complex formation cyclosporin-A dependent? To assess these questions, rat primary hepatocytes were used in this study. Hepatocytes were treated with ethanol (50 mM), a sufficient concentration to induce apoptosis as established in previous studies (12, 22).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials and reagents. Anti-human VDAC monoclonal antibody (31HL) was purchased from Calbiochem-Novabiochem (La Jolla, CA). Rabbit anti-VDAC antibody for microinjection was kindly provided by Dr. S. Shimizu (Osaka University, Osaka, Japan). Mouse anti-Bax and rabbit anti-Bax polyclonal antibodies were from Pharmingen (Eugene, OR). CsA, N-acetyl-cysteine (NAC), and actinomycin D (ActD) were purchased from Sigma (St. Louis, MO). Recombinant mouse TNF-{alpha} was purchased from R&D Systems (Minneapolis, MN). N',N'-dimethylthiourea (DMTU) was purchased from Janssen Chimica (cat. no. B-2440; Geer, Belgium). 2',7'-Dichlorofluorescin diacetate (DCFH-DA), 5-(and-6-chloromethyl-2',7'-dichlorofluorescein diacetate (CM-H2DCFDA) Hoechst 33342, and MitoTracker Red CMXRos were purchased from Molecular Probes (Eugene, OR). Green fluorescent protein (GFP) was purchased from BD Biosciences Clontech (Palo Alto, CA).

Experimental protocol. Male Wistar rats with an average body weight of 250–300 g were used for the cell preparation. All animals received humane care in compliance with the National Research Council's criteria for humane care as outlined in "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Science and published by the National Institutes of Health. Rat hepatocytes were isolated and cultured as previously described (6). The viability of isolated cells was >95% as determined by the trypan blue dye exclusion test. Cells were seeded on culture dishes at a concentration of 5 x 106 cells/cm2 and incubated in DMEM (Sigma) containing 10% fetal calf serum (Invitrogen, Carlsbad, CA) for 24 h at 37°C in 5% CO2. Every precaution was taken to ensure that the additives, medium, and plastic materials used were free of endotoxin as determined by the Limulus Amebocyte Lysate Test Kit (Whittaker Bioproducts, Walkersville, MD), which has a sensitivity of 0.1 ng/ml. Rat hepatocytes were cultured for 24 h after isolation before ethanol (50 mM) exposure. This concentration of ethanol (50 mM), which is known to be toxicologically relevant, is sufficient to induce apoptosis in cultured rat hepatocytes (22). To compare the apoptotic machineries between ethanol and TNF-related apoptotic models, TNF-{alpha} (30 ng/ml) plus ActD (0.2 µM) were exposed to hepatocytes. ActD was added to cultured hepatocytes 1 h before being added to TNF-{alpha}. In some experiments, the PTP inhibitor CsA (10 µM); the antioxidant NAC (5 mM) or DMTU (10 mM); a small, permeable, and relatively nontoxic scavenger of hydrogen peroxide; and the hydroxyl radical were added to the culture medium before treatment with ethanol or TNF-{alpha} plus ActD.

Determination of reactive oxygen species. To investigate subcellular localization of oxidative stress in hepatocytes, DCFH-DA was used according to the methods of Cathcart et al. (5) with minor modification (23). Briefly, cultured rat hepatocytes on 35-mm glass-bottom Microwell culture dishes (MatTek, Ashland, MA) were incubated with DMEM (pH 7.4) containing 1 µM DCFH-DA for 30 min at 37°C in the dark. The cells were washed three times with phenol red-free DMEM to remove the extracellular fluorescence and were observed on an inverted fluorescence microscope (Diaphot TMD-2S; Nikon, Tokyo, Japan). Mitochondria were labeled by incubation of hepatocytes with 200 nM of MitoTracker Red CMXRos. A PlanApochromat x63 oil immersion objective and laser scanning confocal microscope system (Zeiss 410; Zeiss, Thornwood, NY) were used for visualization. Confocal images of 2'7'-dichlorofluorescein (DCF; an oxidized form of DCFH) fluorescence was collected by using a 488-nm excitation light from an argon/krypton laser, a 560-nm dichroic mirror, and a 500- to 550-nm band-pass barrier filter. Images of MitoTracker Red fluorescence were collected by using 568-nm excitation light from the argon/krypton laser, a 560-nm dichroic mirror, and a 590-nm long-pass filter. The intracellular formation of reactive oxygen species (ROS) was measured by using CM-H2DCFDA. Cells (2 x 105 cells) were harvested in 24-well culture plates (Corning, Acton, MA) and loaded with 1 µM CM-H2DCFDA for 30 min at 37°C. After free probes were washed with Hanks' balanced salt solution (Invitrogen, Carlsbad, CA), fluorescence was analyzed before and after ethanol treatment (20 min) under fluorescent plate reader (FLUOstar OPTIMA; BMG Labtechnologies, Durham, NC). ROS production was expressed as ROS generation equivalent to H2O2 (µmol/l) exposure for 10 min determined from an H2O2 standard, which was obtained from a fluorescence intensity from 2 x 105 cells exposed to 10–1000 µM of H2O2 for 10 min.

Immunocytochemistry of Bax. Hepatocytes were cultured on glass chamber slides (LAB-TEK; Nalge Nunc, Hanover Park, IL) and incubated with ethanol. MitoTracker Red was used for mitochondrial labeling as described in Determination of reactive oxygen species. After being washed with PBS three times, cells were fixed for 5 min using 4% paraformaldehyde in PBS and then permeabilized with 0.1% Triton X for 5 min. After being blocked with 10% fetal calf serum, cells were incubated with 1:100 dilution of rabbit anti-Bax polyclonal antibody (13686E) for 2 h at 37°C. After being washed three times, cells were incubated for 30 min with 1:250 dilution of an Oregon Green-conjugated goat anti-rabbit secondary antibody (Molecular Probes) for 45 min at 37°C. Fluorescence images were visualized by using confocal microscopy.

Preparation of protein extracts. Hepatocytes cultured on 90-mm culture dishes (Asahi Techno Glass, Tokyo, Japan) were collected by centrifugation and washed with ice-cold PBS. Cells were resuspended in 5 vol of extraction buffer (in mM: 250 sucrose, 20 HEPES pH 7.5, 1.5 MgCl2, 10 KCl, 1 sodium-EDTA, 1 sodium-EGTA, 1 dithiothreitol, 0.1 PMSF, with 10 µg/ml leupeptin and 10 µg/ml aprotinin), incubated for 30 min on ice, and lysed by homogenization with 10 strokes of a Teflon homogenizer. Homogenates were centrifuged at 750 g for 10 min to remove cell debris. The supernatants were transferred to a fresh tube and centrifuged at 10,000 g for 15 min to pellet the mitochondria. The pellet (mitochondria) was resuspended in RIPA buffer (50 mM Tris·HCl, 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS). The supernatants were then centrifuged at 100,000 g, and the resulting supernatants were designated as the cytosolic fraction (S-100). Cytosolic and mitochondrial fractions were used for immunoblot analysis. Protein concentration was determined by the bicinchoninic acid assay using BSA as the standard.

Immunoblotting. Immunoblotting for cytochrome c was performed by using the cytosolic S-100 fraction. Immunoblotting for Bax was performed by using mitochondrial or whole cell lysates from hepatocytes. Samples were resuspended in 20 µl of SDS-sample buffer and boiled at 90°C for 2 min, separated by 12% SDS-PAGE, and transferred to PVDF membranes (Immobilin-P; Millipore, Bedford, MA). After being blocked with 1% wt/vol skim milk and 3% wt/vol BSA in 20 mM Tris, 0.5 M NaCl, and 0.05% Tween 20, pH 7.0, for 30 min, membranes were incubated for 60 min with the primary antibodies: mouse anti-Bax (1:500 dilution) or mouse anti-cytochrome c (1:1,000 dilution). After being washed three times, membranes were further incubated for 60 min with peroxidase-conjugated goat anti-mouse IgG secondary antibodies (1:3,000 dilution) (Amersham, Arlington Heights, IL). Bound antibodies were detected by using enhanced chemiluminescent substrate (Amersham) and exposed to Kodak X-OMAT film. Results were confirmed by triplicate analysis.

In vivo protein cross-linking and immunoprecipitation. In vivo cross-linking for identifying Bax oligomerization or Bax-VDAC interactions was performed as described previously (1, 29). Briefly, we used the cross-linkers bis-(sulfosuccinimidyl)suberate (BS3) and disuccinimidyl suberate (DSS) (Pierce Chemical, Rockford, IL) for Bax oligomerization or 3,3'-dithio-bis(succinimidylproprionate) (DSP) and dimethyl 3,3'-dithio-bis(proprionate)·2HCl (DTBP) (Pierce Chemical) for Bax-VDAC interaction, respectively. Cells were treated with 2 mM of cross-linkers in PBS for 30 min at room temperature. After the reaction was quenched with 50 mM Tris·HCl for 10 min at 4°C, cells were washed in PBS. Cells were then lysed with lysis buffer (in mM: 10 Tris·HCl, pH 7.4, 142.5 KCl, 5 MgCl2, 1 EDTA, 1 PMSF, with 0.5% Nonidet P-40, and 20 µM leupeptin) for 30 min on ice and centrifuged to remove insoluble debris.

Immunoprecipitation was carried out as follows. Samples were precleared by mixing with 50 µl of 50% (vol/vol) protein G-Sepharose beads for 60 min at 4°C, and the beads were removed by centrifugation. The resultant supernatants were incubated with appropriate antibodies (2 µg/ml) at 4°C for 2 h. Immunoprecipitates were collected by incubating with protein G-Sepharose for 60 min, followed by centrifugation for 2 min at 4°C. The pellets were washed with lysis buffer three times. After the final wash, the beads were suspended in SDS-sample buffer, and the samples were analyzed by SDS-PAGE and Western blotting as described in Immunoblotting.

Microinjection. Microinjection was performed by using a micromanipulator (Narishige, Tokyo, Japan) as described previously (36). The rabbit anti-VDAC blocking antibodies were used. This antibody was reported to inhibit Bax-mediated cytochrome c release and membrane potential loss, without inhibiting mitochondrial respiration of cells (36). Normal rabbit IgG (NRI; Santa Cruz Biotechnology, Santa Cruz, CA) was used as a control. Either anti-VDAC antibodies (15 µg/µl) or NRI was mixed with GFP (3 µg/µl) as a marker of microinjected cells and then microinjected into the cytosol of cultured hepatocytes. One hour after the injection, cells were treated with ethanol for the following 6 h.

Quantitation of apoptosis. A cell membrane-permeable nuclear binding dye Hoechst 33342 was used for evaluation of apoptosis (12). Cells were incubated with 10 µM of Hoechst 33342 for 15 min before the addition of ethanol. The blue fluorescence was visualized by using a fluorescence microscope (excitation: 330–380 nm, emission: 460 nm). Apoptosis was evaluated by morphological criteria, i.e., condensed chromatin and fragmented nuclei, and the number of cells with apoptotic nuclei was determined within a field of view at a magnification of x400. A total of 10 randomly prechosen fields were counted per well, and the number of apoptotic cells was averaged to obtain an apoptotic index.

Caspase activity assay. Cytosolic extracts for the enzyme assay were prepared as previously described (16) with minor modifications. In brief, cells were homogenized in hypotonic buffer (in mM: 25 HEPES, 5 MgCl2, 1 EGTA, 0.5 PMSF, with 2 µg/ml pepstatin and 2 µg/ml leupeptin, pH 7.5), and centrifuged for 10 min at 1,000 g. Caspase activity was measured by adding 50 µl of cytosol to 450 µl of assay buffer containing 25 mM HEPES (pH 7.5), 10 mM DTT, 0.1% CHAPS, 0.5 mM PMSF, 100 U/ml aprotinin, and 20 µM of fluorogenic tetrapeptide substrates Ac-Asp-Glu-Val-Asp-{alpha}-(4-methyl-coumaryl-7-amide) (DEVD-MCA; Peptide Institute, Osaka, Japan) for caspase-3 or Ac-Ile-Glu-Thr-Asp-{alpha}-(4-methyl-coumaryl-7-amide) (IETD-MCA; Peptide Institute) for caspase-8. Fluorescence (excitation: 380 nm, emission: 450 nm) was quantitated by using a fluorometer (Hamamatsu Photonics, Hamamatsu, Japan) as described previously (16).

Statistical analysis. All data represent at least three independent experiments and are expressed as the means ± SD, unless otherwise indicated. Differences between groups were compared by using ANOVA for repeated measures and a post hoc Bonferroni test to correct for multiple comparisons.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Ethanol causes oxidative stress predominantly within mitochondria. Because our previous study demonstrated that ethanol-induced oxidative stress targeted mitochondria (21, 22), we first evaluated subcellular localization of oxidative stress by using the oxidant-sensitive fluorescence probe DCFH-DA. DCF, an oxidized form of DCFH, fluorescence was not visible in control hepatocytes; however, the fluorescence increased significantly within 10 min after ethanol (50 mM) treatment (Fig. 1A). The fluorescence further increased at 20 min with an increasingly dotted pattern. To determine the exact localization of the DCF, we performed dual labeling with the mitochondria-specific dye MitoTracker Red. DCF fluorescence colocalized with MitoTracker Red fluorescence (Fig. 1B), suggesting that ethanol-induced oxidative stress predominantly occurs within mitochondria. These results are consistent with our previous study showing rapid mitochondrial dysfunction after oxidative stress in ethanol-treated hepatocytes (12, 21) and emphasize the importance of oxidative stress in ethanol-induced mitochondrial injury.



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  		Fig. 1. Ethanol (EtOH)-induced 2'7'-dichlorofluorescein (DCF) oxidation was observed predominantly in mitochondria. Rat primary hepatocytes were incubated in the presence of 2',7'-dichlorofluorescin diacetate (DCFH-DA; 1 µM) for 30 min. After being washed, cells were incubated with or without EtOH (50 mM). A: representative imaging of DCF fluorescence by fluorescent microscopy. DCF fluorescence increased in rat hepatocytes exposed to EtOH within 10 min and further increased in 20 min. B: hepatocytes were double-stained with MitoTracker Red CMXRos and DCFH-DA and observed by confocal microscopy. DCF-associated green fluorescence, MitoTracker Red-associated red fluorescence, and overlaid image are shown. Note that DCF fluorescence is colocalized with the MitoTracker Red fluorescence.

 
Ethanol induces Bax translocation to mitochondria via an oxidative stress-dependent mechanism. Because mitochondrial dysfunction can be associated with translocation of cytosolic Bax to mitochondria (40), we then determined whether Bax transmigrates to mitochondria during ethanol-induced hepatocyte apoptosis. Bax immunofluorescence was initially diffuse, consistent with cytosolic localization. However, the Bax-associated fluorescence became dotted over time after ethanol treatment (Fig. 2A). To clarify the subcellular localization of Bax, mitochondria were counterstained with MitoTracker Red (Fig. 2B). Both Bax (green) and mitochondria (red)-associated fluorescence displayed the same pattern of fluorescence, and the overlay image showed a complete colocalization of Bax with mitochondria. Thus Bax transmigrates to mitochondria during ethanol treatment.



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  		Fig. 2. Bax is predominantly observed on mitochondria in EtOH-treated hepatocytes. Hepatocytes were incubated with or without EtOH (50 mM). Immunofluorescence staining for Bax and immunoblotting for Bax and cytochrome c were carried out. Experiments were repeated 3 times, and the representative images were depicted. A: Bax immunofluorescence was visualized under a confocal microscope. Subcellular localization of Bax was altered overtime by EtOH treatment. B: hepatocytes were subjected to a double staining of anti-Bax and MitoTracker Red. Bax-associated fluorescence and the MitoTracker Red fluorescence are colocalized in the overlay image. C: mitochondrial fraction (top) and the whole cell lysates (bottom) were subjected to immunoblot analysis. Mitochondrial Bax increased within 30 min after EtOH exposure, whereas the expression level of Bax in whole cell lysates was unchanged. D: release of cytochrome c from mitochondria to the cytosol was observed by immunoblot analysis. At selected time intervals, cytosolic extracts were collected from EtOH-treated hepatocytes and exposed to an immunoblot analysis. Note that cytochrome c is detected in cytosolic fraction at 60 min after the exposure to EtOH. E: caspase-3 activity was evaluated by measuring a fluorogenic substrate Ac-Asp-Glu-Val-Asp-{alpha}-(4-methyl-coumaryl-7-amide) (DEVD-MCA) cleavage activity. Caspase-3 activity was increased at 60 min of EtOH treatment and was further elevated at the following time points.

 
To confirm the transmigration of Bax to mitochondria during ethanol treatment, we performed subcellular fractionation and immunoblot analysis. Bax was initially observed in the mitochondrial fraction; however, the amount of mitochondrial Bax was significantly increased at 30 min after ethanol exposure (Fig. 2C), whereas total Bax expression levels within whole cell lysates were unchanged at all time points tested. Release of cytochrome c from mitochondria to cytosol was observed at 60 min after the addition of ethanol (Fig. 2D). In addition, the time point when release of cytochrome c starts also coincides with caspase-3 activation (Fig. 2E). This was consistent with our previous report (12). These results suggest that Bax translocates from cytosol to mitochondria before cytochrome c release and caspase-3 activation.

Previous studies (12) demonstrated that mitochondrial cytochrome c release is blocked by either antioxidants or the PTP inhibitor CsA. Therefore, we then tested the effects of these agents on ethanol-induced Bax transmigration. Both DMTU, a cell membrane-permeable antioxidant, and NAC, a glutathione precursor, prevented the increase in Bax association with mitochondria (Fig. 3A). These agents have been shown to prevent ethanol-induced elevation of DCF fluorescence (12). To confirm that the suppression of Bax translocation results in a decrease in hepatocellular apoptosis, we then evaluated the effect of antioxidants on cytochrome c release, caspase-3 activity, and apoptosis. Indeed, either NAC or DMTU inhibited cytochrome c release, caspase-3 activity, and apoptosis (Fig. 3, B–D), suggesting that Bax transmigration and subsequent apoptotic alteration in ethanol-treated hepatocytes is oxidative stress dependent.



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  		Fig. 3. Bax transmigrates to mitochondria in an oxidative stress-dependent manner. Hepatocytes were incubated with EtOH in the presence or absence of indicated inhibitors. At indicated time points, cells were harvested for mitochondria and whole cell lysate preparation. Effects of N-acetyl-cysteine (NAC; 5 mM), N',N'-dimethylthiourea (DMTU; 10 mM), or cyclosporin A (CsA; 10 µM) on EtOH-induced Bax translocation to mitochondria (at 60 min) (A), mitochondrial cytochrome c release (at 60 min) (B), caspase-3 activation (at 2 h) (C), and apoptosis (at 8 h) (D) were evaluated. A and B: results were representative of 3 independent experiments. C and D: data were expressed as means ± SD from 5 independent experiments. #P < 0.05 vs. control, *P < 0.05 vs. EtOH by ANOVA.

 
Bax is not oligomerized but interacts with the mitochondrial channel protein VDAC. Bax promotes mitochondrial cytochrome c release by either Bax homotypic oligomerization or interaction with the PTP components such as VDAC (13, 25, 29, 40). We then examined whether Bax is oligomerized in ethanol-treated hepatocyte (1). After pretreatment with the noncleavable cross-linkers BS3 and BSP, cells were lysed and Bax oligomerization was evaluated by Bax immunoprecipitation and immunoblot analysis (Fig. 4A). As a positive control for Bax oligomerization, we used protein extracts from TNF-{alpha}+ActD-treated hepatocytes. The 21-, 42-, and 63-kDa forms of Bax, corresponding to monomeric, dimeric, and trimeric forms of Bax, respectively, were observed in the TNF-{alpha}+ActD-treated cells. In contrast, only Bax monomers were detected in the ethanol-treated hepatocytes. In ethanol-treated hepatocytes, the density of the monomeric Bax band appears to be decreased compared with untreated controls (Fig. 4A). Therefore, we could not eliminate the possibility that Bax may form a larger molecular mass complex that could not be separated by the SDS-PAGE performed in the experiment.



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  		Fig. 4. Bax is not oligomerized but interacts with voltage-dependent anion channel (VDAC) in EtOH-treated hepatocytes. A: hepatocytes were treated with noncleavable cross-linkers bis-(sulfosuccinimidyl)suberate (BS3) and disuccinimidyl suberate (DSS) for 30 min. Protein extracts were immunoprecipitated with rabbit anti-Bax antisera and immunoblotted with mouse anti-Bax antibody. Bax oligomerization was not observed in both control and EtOH-treated hepatocytes. Effect of TNF-{alpha} (30 ng/ml) plus actinomycin D (ActD; 0.2 µg/ml) was also evaluated. B: hepatocytes were treated with cleavable cross-linkers 3,3'-dithio-bis(succinimidylproprionate) (DSP) and 3,3'-dithio-bis(proprionate) (DTBP) for 30 min. Protein extracts were immunoprecipitated (IP) with either rabbit anti-Bax antisera, rabbit anti-VDAC antisera, normal rabbit IgG (NRI; as a control of anti-Bax antisera), or normal mouse IgG (as a control of anti-VDAC antisera). Immunoprecipitates were immunoblotted by indicated antibodies. Representative images were shown from 3 independent experiments. Note Bax-VDAC interactions were observed in only EtOH-treated hepatocytes.

 
Bax is known to form a complex with the PTP component proteins, such as VDAC, and the importance of Bax and PTP interactions in mitochondrial cytochrome c release has been suggested (25, 29). Therefore, we then determined whether Bax-VDAC interactions were observed in ethanol-treated hepatocytes. Hepatocytes were treated with the cleavable cross-linkers DSP and DTBP, and the cell lysates were subjected to immunoprecipitation with either anti-Bax or anti-VDAC antisera. In ethanol-treated hepatocytes, VDAC coprecipitated with Bax was observed, whereas VDAC was not observed in the precipitates from the control hepatocytes or immunoprecipitates by rabbit IgG (Fig. 4B). Furthermore, when the cell lysates were exposed to immunoprecipitation using an anti-VDAC antibody, Bax was only coprecipitated in ethanol-treated cells. These results suggest that Bax binds to the PTP component protein VDAC on ethanol treatment. Interestingly, the Bax-VDAC interactions were not observed in TNF-{alpha}+ActD-treated hepatocytes, suggesting that the effect of Bax on mitochondria is different between ethanol- and TNF-{alpha}+ActD-induced apoptosis.

To compare the other apoptotic machineries between these two models (ethanol vs. TNF-{alpha}+ActD), we compared other experimental manipulations in addition to an observation of BAX-VDAC interactions. TNF-{alpha}+ActD induced activation of caspase-3 and caspase-8 (Fig. 5, A and B), whereas our previous observation has shown that caspase-8 is not activated in ethanol-treated hepatocytes (12). Interestingly, antioxidants did not prevent TNF-induced caspase activation or apoptosis (Fig. 5, A–C).



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  		Fig. 5. TNF-{alpha} plus ActD induces caspase-8- and caspase-3-dependent apoptosis, which are not sensitive to antioxidants. Hepatocytes were incubated with or without TNF-{alpha} (30 ng/ml) plus ActD (0.2 µg/ml) in the presence or absence of indicated antioxidants. Effects of NAC (5 mM) or DMTU (10 mM) on caspase-3 (at 4 h) (A), caspase-8 (at 4 h) (B), and apoptosis (at 12 h) (C) were evaluated. Data were expressed as means ± SD from 5 independent experiments. #P < 0.05 vs. control, *P < 0.05 vs. TNF-{alpha} plus ActD by ANOVA.

 
Microinjection of anti-VDAC antibody inhibits ethanol-induced apoptosis. To determine whether the Bax-VDAC complex is essential for ethanol-induced hepatocyte apoptosis, we then microinjected anti-VDAC-blocking antibody into the cells before ethanol exposure. This antibody was raised against amino acids 151–165 of human VDAC1 where they are probably exposed to the cytoplasm, are specific for human and rat VDAC, and are able to prevent Bax-VDAC interactions (36). Anti-VDAC antibody or NRI was microinjected into the cytosol of hepatocytes, and then ethanol was added 1 h after the microinjection. Six hours after treatment with ethanol, 32.6 ± 6.9% of NRI-injected hepatocytes underwent apoptosis (Fig. 6). Microinjection of anti-VDAC antibody effectively attenuated ethanol-induced hepatocyte apoptosis, 13.6 ± 3.8% (P < 0.05, Fig. 6). These results suggest that VDAC-Bax associations are essential for ethanol-induced hepatocyte apoptosis.



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  		Fig. 6. Microinjection of anti-VDAC antibodies to hepatocyte inhibits EtOH-induced apoptosis. Hepatocytes were microinjected with anti-VDAC antibody or NRI at 12 µg/µl concentration. GFP (3 µg/µl) was coinjected to identify the injected cells. After incubation with EtOH (50 mM) for 8 h, cells were stained with Hoechst 33342 and observed by a fluorescent microscope. A: apoptosis was evaluated by morphological criteria after the Hoechst 33342 nuclear staining (blue). GFP was monitored to identify the injected cells. Representative fluorographs of control NRI-injected hepatocytes (left) and anti-VDAC antibody-injected hepatocytes (right). The arrow indicates the apoptotic cell. B: apoptotic nuclei were quantitated under the microscopic fields. >100 injected cells were counted for each experiment. Data were expressed as means ± SD from 5 independent experiments. #P < 0.05 vs. GFP + NRI-injected group, *P < 0.05 vs. EtOH-treated GFP + NRI-injected group by ANOVA.

 
The PTP inhibitor does not inhibit oxidative stress, Bax translocation to mitochondria, and Bax-VDAC interactions; however, these inhibitors effectively prevent cytochrome c release, caspase activation, and apoptosis. Time course observations of oxidative stress, cytochrome c release, and Bax translocation suggest that oxidative stress and Bax translocation to mitochondria are upstream of the mitochondrial permeability transition (MPT) and mitochondrial cytochrome c release within ethanol-mediated apoptotic signaling cascade. Therefore, to confirm this hypothesis, we tested whether CsA-sensitive permeability transition affects these events. CsA did not prevent the ROS generation (Fig. 7A), the mitochondrial translocation of Bax (Fig. 7B), and the formation of Bax-VDAC complex (Fig. 7C), whereas CsA effectively attenuated caspase-3 activation (Fig. 7D) and apoptosis (Fig. 7E). Thus oxidative stress is the first event that leads the MPT, Bax translocation to mitochondria, and Bax-VDAC interactions.



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  		Fig. 7. Oxidative stress, Bax translocation to mitochondria, and Bax-VDAC interaction are upstream events of apoptotic mitochondrial permeability transition. Hepatocytes were incubated with or without 50 mM of EtOH in the presence or absence of CsA (10 µM). Effects of CsA on reactive oxygen species (ROS) generation determined by DCF fluorescence (A), Bax translocation to mitochondria (B), Bax-VDAC interaction (C), caspase-3 activity (D), and apoptosis (E) were evaluated. CsA failed to prevent oxidative-stress-associated DCF fluorescence, Bax translocation, and Bax-VDAC interaction. A, D, and E: data were expressed as means ± SD from 5 independent experiments. #P < 0.05 vs. control, *P < 0.05 vs. EtOH by ANOVA. B and C: results were representative of 3 independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The major findings of the present study relate to the cellular mechanisms of acute ethanol-induced hepatocyte apoptosis. The results indicated that 1) acute ethanol treatment induces oxidative stress in hepatocytes within mitochondria, 2) ethanol induces Bax translocation from the cytosol to mitochondria, 3) Bax translocates to mitochondria before mitochondrial cytochrome c release, 4) mitochondrial Bax interacts with the PTP component protein VDAC, and 5) inhibition of Bax-VDAC interactions by anti-VDAC antibody prevents ethanol-induced apoptosis. These data implicate a role for Bax-VDAC interactions in acute ethanol-induced hepatocyte apoptosis. These observations provide further insights into mechanisms responsible for alcoholic liver injury.

Ethanol induces a hypermetabolic state in the liver that is characterized by enhanced mitochondrial respiration. The decrease in the NAD+/NADH ratio induced by acute ethanol administration may favor mitochondrial superoxide generation by increasing the electron flow along the respiratory electron transport chain (11, 15, 21). The enhanced superoxide generation increases mitochondrial lipid peroxide generation (26). Our data support these concepts by directly demonstrating mitochondrial oxidative stress as visualized by an oxidant-sensitive fluorescent probe DCFH-DA during acute ethanol intoxication. Because mitochondria are the major source of ethanol-associated oxidant production, they are therefore also likely to be the first target in oxidative stress-associated injury.

In the present study, Bax was observed to translocate from the cytoplasm to mitochondria before mitochondrial cytochrome c release during exposure to ethanol. This observation suggests that Bax may play an important role in mitochondrial cytochrome c release. Bax-mediated mitochondrial cytochrome c release has been implicated in both death receptor-mediated and nondeath receptor pathway of apoptosis (27, 37). However, our data suggest that Bax association with mitochondria in ethanol-treated hepatocytes is distinct from the TNF-mediated death receptor signaling pathway. In the ethanol-treated hepatocytes, Bax forms a complex with the PTP component protein VDAC. In contrast, this heterotypic interaction of Bax and VDAC was not observed in TNF-{alpha}-treated cells. In death receptor-mediated pathway, Bax may form a homotypic oligomer channel on the death receptor-mediated tBid signaling (7, 20). The Bax homooligomerization may result in a formation of various oligomers of Bax complexes including dimers and trimers (1, 7). Consistent with the previous reports, we observed monomeric, dimeric, and trimeric forms of Bax in the TNF-{alpha}-treated hepatocytes. In ethanol-treated hepatocytes, Bax homotypic oligomerization was not observed. Thus ethanol may predominantly induce Bax-VDAC heterotypic interactions, whereas TNF-{alpha} may induce Bax homooligomerization.

The differences in Bax molecular complex formation may account for differences in apoptotic signals between two models (ethanol vs. TNF-{alpha}). The most significant difference is caspase-8 dependency. In the case of TNF-{alpha}, it is well accepted that death receptors such as TNF-receptor 1 can activate caspase-8. Activated caspase-8 cleaves and activates Bid. Bid and Bax (or Bak) cooperate to induce mitochondrial cytochrome c release on mitochondrial outer membrane. In contrast, our previous study (12) has shown that ethanol-mediated apoptosis is not mediated by caspase-8 and Bid. In the present study, we report some additional findings regarding the difference of apoptosis signaling between these two models: 1) antioxidants effectively inhibited Bax translocation and subsequent apoptotic signals in ethanol model, whereas antioxidants failed to inhibit TNF-induced apoptosis; and 2) ethanol induces Bax-VDAC interaction, whereas TNF does not induce detectable interaction of these two molecules. Interestingly, it has been reported that inhibitors of MPT reduced oxidative stress, whereas antioxidants reduced mitochondrial permeability in a certain caspase-8-mediated apoptosis such as bile acid (41). It would be possible that oxidative stress is more important to signal (or initiate) apoptosis in a caspase-8-independent apoptosis model.

Our previous study (12) demonstrated that acute ethanol induced an increase in the mitochondrial membrane permeability leading to massive cytochrome c release. The increase in the mitochondrial permeability was evaluated by mitochondrial calcein release assay (an indicator of the permeability of both inner and outer membranes) and was likely mediated by the PTP opening because it was prevented by the PTP inhibitor CsA. In the present study, we further evaluated whether the mitochondrial Bax transmigration and the Bax-VDAC interactions are sensitive to PTP inhibitor. CsA failed to attenuate ethanol-induced mitochondrial translocation of Bax or its interaction with VDAC, suggesting that Bax-VDAC interactions observed in the ethanol-treated hepatocytes is an upstream signal of PTP opening. Because VDAC is a major component of the PTP, it would be possible that Bax-VDAC interactions may alter the PTP status, which allows cytochrome c to leave mitochondria. Indeed, CsA effectively prevented ethanol-induced mitochondrial cytochrome c release, caspase-3 activation, and apoptosis (12).

In conclusion, the present study provides an additional mechanism for acute ethanol-induced hepatocyte apoptosis. Ethanol-associated oxidative stress induces Bax transmigration to the mitochondria. Bax interacts with the PTP component protein VDAC and likely causes PTP opening, cytochrome c release, caspase activation, and apoptosis. Prevention of the Bax-VDAC interactions by specific anti-VDAC antibody prevented the hepatocyte apoptosis. Therefore, Bax-VDAC interaction would be a potential target for prevention of alcohol-related liver injury.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.


   ACKNOWLEDGMENTS
 
The authors thank Dr. Shigeomi Shimizu (Osaka University, Osaka, Japan) for anti-VDAC antisera. We thank Dr. Gregory J. Gores (Mayo Clinic, Rochester, MN) for exciting discussion about experimental design and for manuscript preparation.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. Ishii, Dept. of Internal Medicine, School of Medicine, Keio Univ., 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Antonsson B, Montessuit S, Sanchez B, and Martinou JC. Bax is present as a high molecular weight oligomer/complex in the mitochondrial membrane of apoptotic cells. J Biol Chem 276: 11615–11623, 2001.[Abstract/Free Full Text]
  2. Benedetti A, Brunelli E, Risicato R, Cilluffo T, Jezequel AM, and Orlandi F. Subcellular changes and apoptosis induced by ethanol in rat liver. J Hepatol 6: 137–143, 1988.[CrossRef][Web of Science][Medline]
  3. Bernardi P. The permeability transition pore. Control points of a cyclosporin A-sensitive mitochondrial channel involved in cell death. Biochim Biophys Acta 1275: 5–9, 1996.[Medline]
  4. Beutner G, Ruck A, Riede B, and Brdiczka D. Complexes between porin, hexokinase, mitochondrial creatine kinase and adenylate translocator display properties of the permeability transition pore. Implication for regulation of permeability transition by the kinases. Biochim Biophys Acta 1368: 7–18, 1998.[Medline]
  5. Cathcart R, Schwiers E, and Ames BN. Detection of picomole levels of hydroperoxides using a fluorescent dichlorofluorescein assay. Anal Biochem 134: 111–116, 1983.[CrossRef][Web of Science][Medline]
  6. Drochmans P, Wanson JC, and Mosselmans R. Isolation and subfractionation on ficoll gradients of adult rat hepatocytes. Size, morphology, and biochemical characteristics of cell fractions. J Cell Biol 66: 1–22, 1975.[Abstract/Free Full Text]
  7. Eskes R, Desagher S, Antonsson B, and Martinou JC. Bid induces the oligomerization and insertion of Bax into the outer mitochondrial membrane. Mol Cell Biol 20: 929–935, 2000.[Abstract/Free Full Text]
  8. Finucane DM, Bossy-Wetzel E, Waterhouse NJ, Cotter TG, and Green DR. Bax-induced caspase activation and apoptosis via cytochrome c release from mitochondria is inhibitable by Bcl-xL. J Biol Chem 274: 2225–2233, 1999.[Abstract/Free Full Text]
  9. Goldin RD, Hunt NC, Clark J, and Wickramasinghe SN. Apoptotic bodies in a murine model of alcoholic liver disease: reversibility of ethanol-induced changes. J Pathol 171: 73–76, 1993.[CrossRef][Web of Science][Medline]
  10. Green DR and Reed JC. Mitochondria and apoptosis. Science 281: 1309–1312, 1998.[Abstract/Free Full Text]
  11. Handler JA and Thurman RG. Redox interactions between catalase and alcohol dehydrogenase pathways of ethanol metabolism in the perfused rat liver. J Biol Chem 265: 1510–1515, 1990.[Abstract/Free Full Text]
  12. Higuchi H, Adachi M, Miura S, Gores GJ, and Ishii H. The mitochondrial permeability transition contributes to acute ethanol-induced apoptosis in rat hepatocytes. Hepatology 34: 320–328, 2001.[CrossRef][Web of Science][Medline]
  13. Hsu YT, Wolter KG, and Youle RJ. Cytosol-to-membrane redistribution of Bax and Bcl-X(L) during apoptosis. Proc Natl Acad Sci USA 94: 3668–3672, 1997.[Abstract/Free Full Text]
  14. Huser J, Rechenmacher CE, and Blatter LA. Imaging the permeability pore transition in single mitochondria. Biophys J 74: 2129–2137, 1998.[Web of Science][Medline]
  15. Ishii H, Kurose I, and Kato S. Pathogenesis of alcoholic liver disease with particular emphasis on oxidative stress. J Gastroenterol Hepatol 12: S272–S282, 1997.[Web of Science][Medline]
  16. Jones B, Roberts PJ, Faubion WA, Kominami E, and Gores GJ. Cystatin A expression reduces bile salt-induced apoptosis in a rat hepatoma cell line. Am J Physiol Gastrointest Liver Physiol 275: G723–G730, 1998.[Abstract/Free Full Text]
  17. Jurgensmeier JM, Xie Z, Deveraux Q, Ellerby L, Bredesen D, and Reed JC. Bax directly induces release of cytochrome c from isolated mitochondria. Proc Natl Acad Sci USA 95: 4997–5002, 1998.[Abstract/Free Full Text]
  18. Kaplowitz N and Tsukamoto H. Oxidative stress and liver disease. Prog Liver Dis 14: 131–159, 1996.[Medline]
  19. Kawahara H, Matsuda Y, and Takase S. Is apoptosis involved in alcoholic hepatitis? Alcohol Alcohol 29, Suppl 1: 113–118, 1994.
  20. Korsmeyer SJ, Wei MC, Saito M, Weiler S, Oh KJ, and Schlesinger PH. Pro-apoptotic cascade activates BID, which oligomerizes BAK or BAX into pores that result in the release of cytochrome c. Cell Death Differ 7: 1166–1173, 2000.[CrossRef][Web of Science][Medline]
  21. Kurose I, Higuchi H, Kato S, Miura S, Watanabe N, Kamegaya Y, Tomita K, Takaishi M, Horie Y, Fukuda M, Mizukami K, and Ishii H. Oxidative stress on mitochondria and cell membrane of cultured rat hepatocytes and perfused liver exposed to ethanol. Gastroenterology 112: 1331–1343, 1997.[CrossRef][Web of Science][Medline]
  22. Kurose I, Higuchi H, Miura S, Saito H, Watanabe N, Hokari R, Hirokawa M, Takaishi M, Zeki S, Nakamura T, Ebinuma H, Kato S, and Ishii H. Oxidative stress-mediated apoptosis of hepatocytes exposed to acute ethanol intoxication. Hepatology 25: 368–378, 1997.[CrossRef][Web of Science][Medline]
  23. Kurose I, Miura S, Fukumura D, Yonei Y, Saito H, Tada S, Suematsu M, and Tsuchiya M. Nitric oxide mediates Kupffer cell-induced reduction of mitochondrial energization in hepatoma cells: a comparison with oxidative burst. Cancer Res 53: 2676–2682, 1993.[Abstract/Free Full Text]
  24. Lemasters JJ, Nieminen AL, Qian T, Trost LC, Elmore SP, Nishimura Y, Crowe RA, Cascio WE, Bradham CA, Brenner DA, and Herman B. The mitochondrial permeability transition in cell death: a common mechanism in necrosis, apoptosis and autophagy. Biochim Biophys Acta 1366: 177–196, 1998.[Medline]
  25. Marzo I, Brenner C, Zamzami N, Jurgensmeier JM, Susin SA, Vieira HL, Prevost MC, Xie Z, Matsuyama S, Reed JC, and Kroemer G. Bax and adenine nucleotide translocator cooperate in the mitochondrial control of apoptosis. Science 281: 2027–2031, 1998.[Abstract/Free Full Text]
  26. Masini A, Ceccarelli D, Gallesi D, Giovannini F, and Trenti T. Lipid hydroperoxide induced mitochondrial dysfunction following acute ethanol intoxication in rats. The critical role for mitochondrial reduced glutathione. Biochem Pharmacol 47: 217–224, 1994.[CrossRef][Web of Science][Medline]
  27. Murphy KM, Streips UN, and Lock RB. Bax membrane insertion during Fas(CD95)-induced apoptosis precedes cytochrome c release and is inhibited by Bcl-2. Oncogene 18: 5991–5999, 1999.[CrossRef][Web of Science][Medline]
  28. Nanji AA. Apoptosis and alcoholic liver disease. Semin Liver Dis 18: 187–190, 1998.[Web of Science][Medline]
  29. Narita M, Shimizu S, Ito T, Chittenden T, Lutz RJ, Matsuda H, and Tsujimoto Y. Bax interacts with the permeability transition pore to induce permeability transition and cytochrome c release in isolated mitochondria. Proc Natl Acad Sci USA 95: 14681–14686, 1998.[Abstract/Free Full Text]
  30. Natori S, Rust C, Stadheim LM, Srinivasan A, Burgart LJ, and Gores GJ. Hepatocyte apoptosis is a pathologic feature of human alcoholic hepatitis. J Hepatol 34: 248–253, 2001.[CrossRef][Web of Science][Medline]
  31. Petit PX, Goubern M, Diolez P, Susin SA, Zamzami N, and Kroemer G. Disruption of the outer mitochondrial membrane as a result of large amplitude swelling: the impact of irreversible permeability transition. FEBS Lett 426: 111–116, 1998.[CrossRef][Web of Science][Medline]
  32. Petronilli V, Miotto G, Canton M, Brini M, Colonna R, Bernardi P, and Di Lisa F. Transient and long-lasting openings of the mitochondrial permeability transition pore can be monitored directly in intact cells by changes in mitochondrial calcein fluorescence. Biophys J 76: 725–734, 1999.[Web of Science][Medline]
  33. Saito M, Korsmeyer SJ, and Schlesinger PH. BAX-dependent transport of cytochrome c reconstituted in pure liposomes. Nat Cell Biol 2: 553–555, 2000.[CrossRef][Web of Science][Medline]
  34. Shi Y. Mechanisms of caspase activation and inhibition during apoptosis. Mol Cell 9: 459–470, 2002.[CrossRef][Web of Science][Medline]
  35. Shimizu S, Ide T, Yanagida T, and Tsujimoto Y. Electrophysiological study of a novel large pore formed by Bax and the voltage-dependent anion channel that is permeable to cytochrome c. J Biol Chem 275: 12321–12325, 2000.[Abstract/Free Full Text]
  36. Shimizu S, Matsuoka Y, Shinohara Y, Yoneda Y, and Tsujimoto Y. Essential role of voltage-dependent anion channel in various forms of apoptosis in mammalian cells. J Cell Biol 152: 237–250, 2001.[Abstract/Free Full Text]
  37. Shimizu S, Narita M, and Tsujimoto Y. Bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel VDAC. Nature 399: 483–487, 1999.[CrossRef][Medline]
  38. Susin SA, Zamzami N, and Kroemer G. Mitochondria as regulators of apoptosis: doubt no more. Biochim Biophys Acta 1366: 151–165, 1998.[Medline]
  39. Thornberry NA and Lazebnik Y Caspases: enemies within. Science 281: 1312–1316, 1998.[Abstract/Free Full Text]
  40. Wolter KG, Hsu YT, Smith CL, Nechushtan A, Xi XG, and Youle RJ. Movement of Bax from the cytosol to mitochondria during apoptosis. J Cell Biol 139: 1281–1292, 1997.[Abstract/Free Full Text]
  41. Yerushalmi B, Dahl R, Devereaux MW, Gumpricht E, and Sokol RJ. Bile acid-induced rat hepatocyte apoptosis is inhibited by antioxidants and blockers of the mitochondrial permeability transition. Hepatology 33: 616–626, 2001.[CrossRef][Web of Science][Medline]
  42. Zoratti M and Szabo I. The mitochondrial permeability transition. Biochim Biophys Acta 1241: 139–176, 1995.[Medline]



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