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Originally published In Press as doi:10.1074/jbc.M408882200 on September 10, 2004

J. Biol. Chem., Vol. 279, Issue 48, 50420-50428, November 26, 2004
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Cytochrome bc1 Regulates the Mitochondrial Permeability Transition by Two Distinct Pathways*

Jeffrey S. Armstrong{ddagger}, Hongyuan Yang, Wei Duan, and Matthew Whiteman

From the Department of Biochemistry, National University of Singapore, Singapore 117597, Singapore

Received for publication, August 4, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The mitochondrial permeability transition (MPT) pore is a calcium-sensitive channel in the mitochondrial inner membrane that plays a crucial role in cell death. Here we show that cytochrome bc1 regulates the MPT in isolated rat liver mitochondria and in CEM and HL60 cells by two independent pathways. Glutathione depletion activated the MPT via increased production of reactive oxygen species (ROS) generated by cytochrome bc1. The ROS producing mechanism in cytochrome bc1 involves movement of the "Rieske" iron-sulfur protein subunit of the enzyme complex, because inhibition of cytochrome bc1 by pharmacologically blocking iron-sulfur protein movement completely abolished ROS production, MPT activation, and cell death. The classical inhibitor of the MPT, cyclosporine A, had no protective effect against MPT activation. In contrast, the calcium-activated, cyclosporine A-regulated MPT in rat liver mitochondria was also blocked with inhibitors of cytochrome bc1. These results indicate that electron flux through cytochrome bc1 regulates two distinct pathways to the MPT, one unregulated and involving mitochondrial ROS and the other regulated and activated by calcium.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mitochondria play a vital role in cell fate by their regulation of energy metabolism and their involvement in cell death by apoptosis and necrosis (1-3). Mitochondrial function, including ion transport, biogenesis, and ATP formation, requires an intact mitochondrial transmembrane potential ({Delta}{Psi}m), which depends upon the generation of an electrochemical proton gradient ({Delta}µH+) across the mitochondrial inner membrane. The {Delta}µH+ is generated by three multisubunit protein complexes localized in the mitochondrial inner membrane including respiratory complex I (NADH:ubiquinone dehydrogenase), complex III (cytochrome bc1), and complex IV (cytochrome c oxidase) (4, 5). A crucial event that occurs in mitochondria when a cell dies is loss of the {Delta}µH+ and subsequent collapse of the {Delta}{Psi}m (6), which can occur because of opening of high conductance permeability transition (MPT)1 pores in the mitochondrial inner membrane that allow the nonselective diffusion of solutes (<1500 Da) across the membrane with resulting organelle swelling and membrane rupture (6-9). The MPT is known to be activated by Ca2+ and reactive oxygen species (ROS) and inhibited by the potent immunosuppressive agent cyclosporine A (CsA) (7-10). Although many studies have considered that the MPT is due to the formation of a preformed pore complex between the mitochondrial inner and outer membranes involving the adenine nucleotide translocator (ANT), the voltage-dependent anion channel, cyclophilin D (CyD), and a number of accessory proteins (11-15), an alternative view has been that the MPT is not the result of opening of a preformed pore, but the result of increased membrane permeability caused by oxidative damage to pre-existing membrane proteins including the ANT (8, 9).

The mechanism by which CsA inhibits the MPT has been attributed to its inhibitory effect on the peptidyl-prolyl isomerase activity of CyD, which is believed to be required for the formation of an ANT/CyD protein complex required for MPT activation (14-17). However, this MPT model has been questioned by a report showing that when CyD was overexpressed and targeted to mitochondria, it protected cells from oxidants, indicating that it was inhibiting rather than activating the MPT (18). Although in direct contrast to this Li et al. (19) recently found that overexpression of mitochondrial CyD rendered mitochondria more susceptible to the MPT and cells more sensitive to oxidant-mediated injury. The controversy concerning the role of the ANT and CyD in the MPT is further compounded, first, by the recent finding that the MPT was found to be inducible in mitochondria taken from the livers of mice with genetically inactivated ANT isoforms (20), and second, although CsA blocks the MPT in some cases, it is ineffective at MPT inhibition in others (21, 22). This led He and Lemasters (23, 24) to propose an alternative model of the MPT that would account for some of these inconsistencies (9, 11). Their model envisaged both a "CsA-regulated" and an "unregulated" form of MPT based on a study showing two possible conductance modes for the MPT. One mode was activated by Ca2+ and inhibited by CsA, and the other was unregulated.

The dualistic model of He and Lemasters (23, 24) thus reconciles two apparently divergent ideas on the MPT where both the ANT and CyD play an important regulatory role. However, other investigators have considered models of MPT that may involve the ANT and CyD but feature mitochondrial respiratory components other than the ANT as crucial MPT regulators; for example, the work of Fontaine and co-workers (25-27) clearly showed that the MPT is regulated by electron flux through the NADH:ubiquinone-dehydrogenase and that various classes of quinone analogs were important MPT regulatory molecules. Whereas we have previously shown that mitochondrial ROS activates the MPT in vivo after GSH depletion and proposed the redox target(s) include the ANT (28). These varied and somewhat controversial reports on the molecular composition and regulation of the MPT indicate that definitive knowledge on this phenomenon is lacking and suggest that concerted efforts should be made to advance our understanding of this crucial mitochondrial event associated with cell death.

In this study we show that cytochrome bc1 is a key regulatory component of the MPT in rat liver mitochondria (RLM) and in leukemic CEM and HL60 cells. Our results indicate a fundamental role for ISP subunit movement in ROS-mediated MPT activation in cells, whereas investigations with RLM indicate that cytochrome bc1 may possess a MPT channel-like function, suggesting that cytochrome bc1 is involved in two distinct pathways to the MPT.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—All of the chemicals were reagent grade and were obtained from Sigma. Tetramethylrhodamine methylester (TMRM) and dichlorodihydrofluorescein diacetate (DCFDA) was obtained from Molecular Probes (Eugene, OR). Bongkrekic acid (BgK) was obtained from Calbiochem (La Jolla, CA). Stigmatellin was obtained from Fluka Biochemika, and 2-methoxy antimycin A3 (2-MeAA) was obtained from BIOMOL Research Laboratories.

Cell Culture—CEM and HL60 cells (HL60 cells overexpressing Bcl-2 protein were used because of their sensitivity and reproducibility to GSH-induced redox stress (28, 29) were cultured in RPMI with 10% fetal calf serum and supplements as previously described (29). The cells were passaged daily to maintain them in log phase and kept at a concentration between 2.5-5 x 105/ml.

Mitochondrial Isolation and Swelling Test—RLM were isolated by conventional differential centrifugation from the livers of male adult Sprague-Dawley rats fasted overnight. Large amplitude swelling was measured by spectrophotometry in a Beckman DU 640 by recording absorbance change at 540 nm. Isolated rat liver mitochondria (~1 mg/ml) were suspended in mitochondrial isolation buffer consisting of mannitol (220 mM), sucrose (70 mM), Hepes (2 mM), EGTA (0.5 mM), and bovine serum albumin (0.1%) for all experiments.

Measurement of GSH—The CEM cells were treated with diethyl maleate (5 mM) for 0, 15, 30, 45, and 60 min. GSH levels were measured by the monochlorobimane method (30). The amount of acid-insoluble protein was determined by the Bradford method with {gamma}-globulin as a standard.

Measurement of ROS Production—After treatment with DEM (1 mM) for 0, 10, 20, 30, 40, and 50 min, the cells were stained with 10 µM DCFDA for 15 min, washed with phosphate-buffered saline containing 10 mM glucose, and analyzed immediately by flow cytometry using WinMidi software. DCFDA is a nonfluorescent ester of the dye fluorescein. DCFDA is cleaved by intracellular esterases and entrapped within the cell as the oxidant-sensitive dichlorofluorescein. ROS oxidize DCF to the fluorescent product fluorescein (31). The green fluorescence of fluorescein was measured using the FL-2 setting (log mode) after the cell debris was electronically gated out. In each analysis, 10,000 events were recorded.

Measurement of Mitochondrial Membrane Potential ({Delta}{Psi}m)—{Delta}{Psi}m was measured with the fluorescent lipophilic cationic dye TMRM (250 nM) that accumulates within mitochondria according to the {Delta}{Psi}m. After treatment with DEM for 0, 30, 60, 90, and 120 min, the cells were stained with TMRM for 15 min, and red fluorescence was measured by flow cytometry using the FL-3 setting. The protonophore CCCP (10 µM) was used to dissipate the chemiosmotic proton gradient ({Delta}µH+) and served as a control for loss of {Delta}{Psi}m. In each analysis, 10,000 events were recorded.

Electron Microscopic Examination of Cells—HL60 or CEM cells in the logarithmic proliferation phase were treated with DEM (5 mM) with or without the respiratory complex inhibitors stigmatellin (5 µM), antimycin A (5 µM), or 2-MeAA (5 µM) as described in the figure legends. The cells were 1) fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, at room temperature for 1 h, 2) washed with 0.1 M cacodylate buffer and post-fixed with 1% osmium tetraoxide in 0.1 M cacodylate buffer, and 3) dehydrated with graded series of ethanol and embedded in LX112. Thin sections were prepared and stained with uranyl acetate. Specimens were examined on a JEOL 1000x electron microscope operating at 80 KV.

Cytochrome bc1 Activity Measurements—Cytochrome bc1 activity in CEM cells was determined as described previously (29). Briefly, cytochrome bc1 complex activity from digitonin-permeabilized and sonicated CEM and HL60 cell fractions were assayed in 50 mM potassium phosphate, pH 7.0, 250 mM sucrose, 0.2 mM EDTA, 1 mM NaN3, 0.1% (w/v) and 0.01% Tween 20 at 23 °C, using 50 µM 2,3-dimethoxy-5-methyl-6n-decyl-1,4-benzoquinol (decylubiquinol) as substrate and 50 µM cytochrome c. Decylubiquinonol was synthesized in the laboratory from decylubiquinone by reduction with sodium borohydride (NaBH4) (31). The reduction of cytochrome c was monitored in a spectrophotometer at 550 versus 539 nm in dual wavelength mode. The data are expressed as percentages of control activity and were determined from five individual isolations that were assayed in triplicate.

Statistical Analysis—Statistical analyses were performed using Student's t test for unpaired data, and p values <0.05 were considered significant. The data are presented as means ± S.E.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
GSH Depletion Mediates ROS Increase in CEM and HL60 Cells That Is Decreased by Inhibition of Cytochrome bc1—Fig. 1A shows representative ROS production, and Fig. 1B shows mean ROS production, respectively, in CEM cells after treatment with 5 mM DEM with or without the respiratory complex inhibitors. Increased DCF fluorescence, a measure of ROS production (29, 32), was determined by a shift in DCF fluorescence to the right after DEM treatment (Fig. 1A, panel 1). The figures show that DEM treatment causes a time-dependent increase in ROS production. GSH levels were determined on aliquots of CEM cells treated with 5 mM DEM. Fig. 1B (inset) shows that a time-dependent loss of GSH occurs in CEM cells after cells were treated with 5 mM DEM. Approximately 90% of GSH is lost after DEM treatment for 30 min, at which time ROS increase occurs (Fig. 1B).



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  		FIG. 1.
A, representative flow cytometric analysis of CEM cells stained with DCFDA and analyzed using FL2-H channel as described under "Experimental Procedures." CEM cells were treated with DEM (5 mM) alone (panel 1), DEM (5 mM) and myxothiazol (5 µM) (panel 2), stigmatellin (5 µM) (panel 3), antimycin A (5 µM) (panel 4), 2-MeAA (5 µM) (panel 5), TTFA (5 µM) (panel 6), and rotenone (5 µM) (panel 7) for 0, 10, 20, 30, 40, and 50 min, washed in PBS, and suspended in PBS containing 10 mM glucose. The cells were loaded with DCFDA (10 µM) for 15 min, and the green fluorescence was measured by flow cytometry using the FL2-H setting. The figure shows a representative example of three independent experiments. In each analysis, 10,000 events were recorded. B, mean DCF fluorescence intensity (arbitrary units) of CEM cells treated with DEM (5 mM) ± stigmatellin (5 µM), myxothiazol (5 µM), TTFA (5 µM), rotenone (5 µM), antimycin A (5 µM), or 2-MeAA (5 µM) for 0, 10, 20, 30, 40, and 50 min, washed in PBS, and suspended in PBS containing 10 mM glucose. After treatment the cells were loaded with DCFDA (10 µM) for 15 min, and the green fluorescence was measured by flow cytometry as described above. In each analysis, 10,000 events were recorded. The data are expressed as the means ± S.E. (n = 3). Inset, CEM cells were treated with 5 mM DEM or RPMI (control) for 0, 15, 30, and 45 min. GSH levels were measured on aliquots of cells (~4 x 106/ml) using monochlorobimane. GSH concentration was plotted as nmol/mg protein. The data are expressed as the means ± S.E. (n = 3). C, relative activity of cytochrome bc1 in CEM cells treated with DEM (5 mM) with 5 µM myxothiazol, 5 µM stigmatellin, 5 µM antimycin A, 5 µM 2-MeAA, 5 µM rotenone, or 5 µM TTFA for 150 min. Cytochrome bc1 activity measurements were made as described under "Experimental Procedures." The results are expressed as percentages of control cytochrome bc1 activity ± S.E. and were determined from three independent isolations each assayed in triplicate.

 
The Distal Ubiquinol Oxidation Site of Cytochrome bc1 Is the Predominant Site of ROS Production after GSH Depletion—To determine the major mitochondrial site of ROS production after GSH depletion in CEM cells, we pharmacologically inhibited respiratory complex I (NADH ubiquinone-dehydrogenase), respiratory complex II (succinate dehydrogenase (SDH)), and respiratory complex III (cytochrome bc1) because these respiratory sites are well known to be involved in mitochondrial ROS production (33-36). The cells were co-incubated with DEM with either rotenone (5 µM), thenoyltrifluoroacetone (TTFA) (5 µM), stigmatellin (5 µM), myxothiazol (5 µM), antimycin A, and 2-MeAA (control for antimycin A, which does not inhibit respiration). Rotenone selectively blocks NADH ubiquinone-dehydrogenase (37), TTFA blocks SDH (38), stigmatellin and myxothiazol block the ubiquinol oxidation (Qo) site of cytochrome bc1 at the distal and proximal niches, respectively (39), and antimycin A blocks the ubiquinol reduction Qi site of cytochrome bc1 (33, 34). Myxothiazol, which binds to the proximal niche of the Qo ubiquinol oxidation site of cytochrome bc1, and antimycin A, which binds to the Qi site, inhibited ROS formation (Fig. 1A, panels 2 and 4), whereas stigmatellin, which binds to the distal niche of the Qo site, completely blocked DEM-mediated ROS increase (Fig. 1A, panel 3). The experiment with stigmatellin was extended up to 90 min, but we did not observe any increase in ROS production (data not shown). To confirm that antimycin A inhibited ROS production, we used the structural analog 2-MeAA as a control (this compound does not inhibit respiration) (40). 2-MeAA did not inhibit ROS production in DEM-treated CEM cells compared with antimycin A (Fig. 1A, panel 5). Rotenone and TTFA did not significantly alter ROS increase compared with DEM treatment alone (Fig. 1A, panels 6 and 7). These results indicate that the Qo distal site of cytochrome bc1 is a key source of ROS production because its inhibition abolished ROS production. However, because inhibition of the proximal Qo and the Qi sites of cytochrome bc1 also reduced ROS production, this further implicates the cytochrome bc1 as a key ROS-generating site in our experimental setting. An identical set of experiments was performed using HL60 cells with similar results (data not shown).

Cytochrome bc1 Activity Required for ROS Production after GSH Depletion—The role of cytochrome bc1 in ROS production in CEM cells was confirmed by determining cytochrome bc1 activity in isolated membrane fractions from CEM cells incubated with DEM, DEM + stigmatellin, DEM + myxothiazol, DEM + rotenone, DEM + TTFA, DEM + antimycin A, and DEM + 2-MeAA. Fig. 1C shows means ± S.E. percentage activity of cytochrome bc1 in CEM cells treated with DEM with or without respiratory inhibitors. The results show that cytochrome bc1 enzyme activity was not significantly different in CEM cells treated with DEM with or without rotenone, TTFA, or 2-MeAA compared with controls, whereas the compounds stigmatellin, myxothiazol, and antimycin A inhibited cytochrome bc1 enzyme activity ~95%.

ROS Production by Cytochrome bc1 Mediates Cell Death by Activation of the MPT—{Delta}{Psi}m was determined in CEM cells, after treatment with DEM (5 mM) to deplete GSH, by monitoring the fluorescence of the cationic potentiometric dye TMRM. Fig. 2A shows representative TMRM flow cytometric histograms of the CEM cell population monitored every 30 min for 150 min with or without the inhibitors used in the ROS experiments. Loss of {Delta}{Psi}m is indicated by a shift left of the cell population on the x axis of the histogram (log scale). Fig. 2B shows the percentage (mean ± S.E.) of CEM cells with intact {Delta}{Psi}m after treatment with DEM with or without inhibitors. Fig. 2A shows that the cytochrome bc1 inhibitors stigmatellin (5 µM) (panel 3) and antimycin A (5 µM) (panel 4) and the ANT inhibitor BgK (50 µM) (panel 8) blocked the loss of {Delta}{Psi}m after GSH depletion (Fig. 2, A and B), whereas rotenone (panel 7), TTFA (panel 6), and the antimycin A analog 2-MEAA (panel 5) did not prevent the loss of {Delta}{Psi}m. Surprisingly, myxothiazol (Fig. 2, A, panel 2, and B, top panel), which reduced ROS production, did not prevent DEM-mediated loss of {Delta}{Psi}m; paradoxically, this inhibitor increased the rate of loss of {Delta}{Psi}m compared with DEM treatment alone. The classical MPT inhibitor CyA did not inhibit the loss of {Delta}{Psi}m induced by GSH depletion, even at concentrations up to 10 µM (data not shown).



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  		FIG. 2.
A, representative TMRM flow cytometric histograms of CEM cells treated with DEM (5 mM) alone (panel 1), DEM (5 mM) with myxothiazol (5 µM) (panel 2), stigmatellin (5 µM) (panel 3), antimycin A (5 µM) (panel 4), 2-MeAA (5 µM) (panel 5), TTFA (5 µM) (panel 6), rotenone (5 µM) (panel 7), and BgK (50 µM) (panel 8) for 0, 30, 60, 90, 120, and 150 min, washed in PBS, and suspended in PBS containing 10 mM glucose. The cells were loaded with TMRM (250 nM) for 15 min, and the red fluorescence was immediately measured by flow cytometry using the FL-3 setting as described under "Experimental Procedures." The figure shows a representative example of three independent experiments. In each analysis, 10,000 events were recorded. B, top panel, percentage of CEM cells with an intact {Delta}{Psi}m determined by relative TMRM fluorescence intensity. The cells were treated with DEM (5 mM) with or without myxothiazol (5 µM) (panel 2), stigmatellin (5 µM) (panel 3), antimycin A (panel 4), and 2-MeAA (5 µM) for 0, 30, 60, 90, 120, and 150 min, washed in PBS, and suspended in PBS containing 10 mM glucose. The cells were loaded with TMRM (250 nM) for 15 min, and the red fluorescence was immediately measured by flow cytometry using the FL-3 setting as described under "Experimental Procedures." The data are expressed as the means ± S.E. (n = 3). In each analysis, 10,000 events were recorded. Bottom panel, percentage of CEM cells with an intact {Delta}{Psi}m determined by relative TMRM fluorescence intensity. The cells were treated with DEM (5 mM) with or without TTFA (5 µM), rotenone (5 µM), and BgK (50 µM) for 0, 30, 60, 90, 120, and 150 min, washed in PBS, and suspended in PBS containing 10 mM glucose. The cells were loaded with TMRM (250 nM) for 15 min, and the red fluorescence was immediately measured by flow cytometry using the FL-2 setting as described under "Experimental Procedures." The data are expressed as the means ± S.E. (n = 3). In each analysis, 10,000 events were recorded. C, the figure shows the effects of DEM treatment on CEM mitochondrial ultrastructure. Electron microscopy (TEM) was performed as described under "Experimental Procedures." The figure shows the effects of untreated cells (top left panel), DEM-treated cells (top right panel), DEM with stigmatellin (5 µM) (bottom left panel), and antimycin A (bottom right panel).

 
Inhibition of Cytochrome bc1 with Stigmatellin or Antimycin A Protects Mitochondrial Ultrastructure in Response to GSH Depletion—To confirm the protective effect of stigmatellin and antimycin A on mitochondrial integrity, electron microscopy (EM) was performed to visualize mitochondrial ultrastructure after GSH depletion. Fig. 2C shows representative electron micrographs of mitochondrial ultrastructure in control cells (top left panel), cells treated with DEM for 150 min (top right panel), cells treated with DEM for 150 min with 5 µM stigmatellin (bottom left panel), and cells treated with DEM for 150 min with 5 µM antimycin A (bottom right panel). The figure indicates that stigmatellin and antimycin A preserved mitochondrial ultrastructure structure compared with DEM treatment alone. Structural and functional studies were also performed using the HL60 B cell line to determine whether the results observed with CEM cells were a general or cell-specific phenomenon. HL60 cells were treated with DEM (5 mM), and EM and {Delta}{Psi}m values were determined every 30 min for a total of 150 min. The results show a time-dependent loss of {Delta}{Psi}m that corresponds with significant ultrastructural changes in mitochondria. At 150 min, mitochondrial ultrastructure in HL60 cells was similar to that observed in CEM cells, including increased electron opacity of mitochondrial inner membrane and cristae. These structural changes were prevented by coincubation of cells with DEM and stigmatellin or antimycin A but not with 2-MeAA (Fig. 3C).



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  		FIG. 3.
A, figure shows the effects of DEM treatment on HL60 mitochondrial ultrastructure. EM was performed as described under "Experimental Procedures." The cells were treated with control vehicle (RPMI media) or DEM (5 mM) and analyzed by EM at 0, 30, 60, 90, 120, and 150 min. Insets show representative {Delta}{Psi}m of cell population at times corresponding to the EM analyses. B, top panel shows the effects of DEM (5 mM) on HL60 mitochondrial ultrastructure in the presence of stigmatellin (5 µM) taken 150 min after treatment. Inset shows the representative {Delta}{Psi}m of HL60 cell population at a time corresponding to the EM analysis. In the analysis, 10,000 events were recorded. Bottom panel, shows bar graph of {Delta}{Psi}m of HL60 cells treated with 5 mM DEM for 0, 30, 60, 90, 120, and 150 ± 5 µM stigmatellin. The figure shows the percentage cells ± S.E. (n = 3) with intact {Delta}{Psi}m. In each analysis, 10,000 events were recorded. C, the figure shows the effects of antimycin A and 2-MeAA on ultrastructure of mitochondria in DEM-treated HL60 cells. The panels at right show the {Delta}{Psi}m of the HL60 cell population at a time corresponding to the EM analysis. Panel a, control; panel b, DEM with antimycin A (5 µM); panel c, DEM + 2-MeAA (5 µM); and panel d, CCCP (10 µM). The figure shows representative examples of at least three independent experiments. In each analysis, 10,000 events were recorded.

 
Cytochrome bc1 Inhibition Is Required for Protection against ROS-mediated Cell Death—Because inhibition of cytochrome bc1 prevented loss of {Delta}{Psi}m, we determined cell viability after incubation of CEM cells with 5 mM DEM with or without 5 µM of cytochrome bc1 inhibitors (stigmatellin, antimycin A and myxothiazol) or cyclosporin A (1-10 µM). Cell viability was performed every 30 min for 150 min using trypan blue exclusion. Cells treated with either DEM alone or DEM with rotenone, TTFA, or 2-MeAA lost cell viability over a similar time, whereas BgK prevented the loss of cell viability (Fig. 4A). Of the cytochrome bc1 inhibitors, stigmatellin blocked the loss of cell viability induced by DEM, and antimycin A reduced the rate at which cells died, whereas myxothiazol did not prevent the loss of cell viability (Fig. 4B). These results indicate that the inhibition of ROS formation by stigmatellin or antimycin A protects against redox-dependent cell death. BgK, also prevented the loss of cell viability; however, myothiazol, which reduced ROS production but did not preserve {Delta}{Psi}m after GSH depletion, did not prevent the loss of cell viability. CyA, even over a range of concentrations (1-20 µM), did not prevent the loss of cell viability after GSH depletion (data not shown). Cell death was not inhibited by broad spectrum caspase inhibitor benzyloxycarbonyl-VAD-fluoromethyl ketone or the caspase 3 inhibitor DEVD-CHO, suggesting that the predominant death pathway was by necrosis (data not shown).



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  		FIG. 4.
A, CEM cells were incubated with 5 mM DEM with or without either 5 µM rotenone, 5 µM TTFA, or 50 µM BgK, and cell viability was performed every 30 min up to 150 min using trypan blue exclusion technique. The data are expressed as the means ± S.E. (n = 3). B, CEM cells were incubated with 5 mM DEM with or without either 5 µM stigmatellin, 5 µM myxothiazol, 5 µM antimycin A, or 5 µM 2-MeAA, and cell viability was performed every 30 min up to 150 min using trypan blue exclusion technique. The data are expressed as the means ± S.E. (n = 3).

 
Inhibition of Cytochrome bc1 Blocks the Ca2+-activated MPT in RLM—Because inhibition of cytochrome bc1 prevented the redox-activated MPT in cells in situ, we determined whether inhibition of cytochrome bc1 would also prevent the MPT induced by Ca2+ in RLM. Fig. 5A shows a representative example of the effect of different respiratory complex inhibitors on the Ca2+ activated MPT in RLM. Stigmatellin (1 µM, trace a) and rotenone (1 µM, trace b) completely prevented mitochondrial swelling induced by 100 µM Ca2+ with similar potency as CsA (1 µM, trace c). TTFA did not prevent mitochondrial swelling (trace d). Fig. 5B shows a representative example of the effects of antimycin A (1 and 10 µM, traces a and c, respectively) and myxothiazol (1 µM, trace b) on the Ca2+ activated MPT in RLM. Fig. 5B (trace d) shows the effect of 2-MeAA on Ca2+-induced swelling of RLM; this trace also represents the profile of RLM swelling induced by Ca2+ alone. These results indicate that electron flux through cytochrome bc1 and the NADH:ubiquinine dehydrogenase regulates the Ca2+-activated MPT in RLM.



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  		FIG. 5.
A, the figure shows the effect of inhibition of NADH:ubiquinine dehydrogenase, SDH, and the Qo distal site of cytochrome bc1 on the Ca2+-activated MPT in RLM. RLM (~1 mg/ml) were incubated briefly with control vehicle Me2SO (Ca2+ alone trace), stigmatellin (1 µM) (trace a), rotenone (1 µM)(trace b), CsA (1 µM)(trace c), or TTFA (1 µM)(trace d). 100 µM Ca2+ was added to mitochondrial suspensions, and absorbance change was monitored at 540 nm. Large amplitude mitochondrial swelling is indicated by a decrease in absorbance at 540 nm. The cytochrome bc1 inhibitors stigmatellin and the NADH:ubiquinone dehydrogenase inhibitor rotenone blocked mitochondrial swelling induced by 100 µM Ca2+. The SDH inhibitor TTFA did not prevent mitochondrial swelling in response to 100 µM Ca2+. The figure shows representative traces from at least three independent experiments. B, the figure shows the effect of inhibition of the Qi and the Qo proximal sites of cytochrome bc1 on the Ca2+-activated MPT in RLM. RLM (~1 mg/ml) was incubated briefly with control vehicle Me2SO (Ca2+ alone trace), antimycin A (1 µM) (trace a), myxothiazol (1 µM) (trace b), antimycin A (10 µM) (trace c), or 2-MeAA (1-10 µM)(trace d). 100 µM Ca2+ was added to mitochondrial suspensions, and absorbance change was monitored at 540 nm. Large amplitude mitochondrial swelling is indicated by a decrease in absorbance at 540 nm. The cytochrome bc1 inhibitors myxothiazol and antimycin A inhibited mitochondrial swelling induced by 100 µM Ca2+. The antimycin A analog 2-MeAA did not prevent mitochondrial swelling in response to 100 µM Ca2+. The figure shows representative traces from at least three independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we show that mammalian cytochrome bc1 is a key regulator of the MPT in CEM and HL60 cells and RLM. In cells, mitochondrial ROS generated during ISP movement at the Qo site of cytochrome bc1 activates the MPT, which is insensitive to CsA and results in necrosis. In contrast, in RLM the MPT is activated by Ca2+ load and is blocked by inhibitors of the Qo site of cytochrome bc1 but is sensitive to CsA. These results indicate that cytochrome bc1 plays a key role in regulation of the regulated MPT in RLM and the unregulated MPT in cells.

DEM was used to deplete cellular GSH and induce redox stress in CEM and HL60 cells as previously shown (28, 29). The increase in mitochondrial ROS in CEM cells was blocked by inhibition of cytochrome bc1 but not NADH:ubiquinone dehydrogenase or SDH (Fig. 1B). Because these respiratory complexes have each been associated with ROS production (34), our results indicate that under these experimental conditions, cytochrome bc1 is the principal mitochondrial site of ROS formation as previously found (28, 29). Inhibition of cytochrome bc1 at either the Qo or Qi site of cytochrome bc1 inhibited ROS production (measured by the relative DCF fluorescence of cells); however, the distal Qo niche inhibitor stigmatellin completely blocked ROS production, whereas myxothiazol a proximal Qo niche inhibitor and antimycin A, which inhibits the Qi site of cytochrome bc1, reduced but did not block ROS formation (Fig. 1B, bottom panel). These results indicate that mitochondrial ROS are produced principally at the Qo site of cytochrome bc1. The difference in the Qo site-binding mechanism of the inhibitors myxothiazol and stigmatellin may be important in elucidating the mechanism involved in ROS formation at the Qo site, because these two inhibitors have dramatically different effects on the mobility of the extramembrane domain of the ISP (41-43). Stigmatellin immobilizes the ISP domain on the surface of cytochrome b inhibiting the Q cycle, whereas myxothiazol allows movement of the ISP but inhibits the enzyme by competitive inhibition at the ubiquinone oxidation site (44). These observations suggest that ROS formation by cytochrome bc1 involves the mobility of the ISP as a key feature. Although the generation of superoxide by cytochrome bc1 is not new, the concept that ISP movement is mechanistically linked to ROS during the Q cycle is noteworthy because, to our knowledge, this is the first report to propose the ISP as directly involved in ROS production. In support of this novel idea, superoxide production has recently been investigated in isolated cytochrome bc1 complexes from Saccharomyces cerevisiae by Muller et al. (39), who also showed that stigmatellin completely blocked superoxide production, whereas myxothiazol only partially prevented superoxide formation (39). A report by Sun and Trumpower (45) using bovine heart and S. cerevisiae cytochrome bc1 complexes also showed that stigmatellin eliminated superoxide formation compared with myxothiazol and antimycin A.

We next considered that if the mechanism of ROS formation involved ISP mobility, inhibition of ISP movement should not only prevent ROS formation but also prevent the toxicity from uncontrolled ROS production. Cell viability experiments clearly showed that ROS inhibition of ISP mobility with stigmatellin preserved cell viability compared with cells treated with DEM alone (Fig. 4A). The Qi site inhibitor antimycin A also preserved cell viability, although to a lesser extent than stigmatellin; however, surprisingly myxothiazol, which reduced ROS formation, did not inhibit cell death (Fig. 4A). To investigate this we first confirmed the efficacy of the respiratory complex inhibitors on cytochrome bc1 enzyme activity using membrane fractions of CEM cells treated with DEM with or without inhibitors. The results showed that cytochrome bc1 activity was almost completely (~95%) inhibited in cells treated with DEM with or without either stigmatellin, myxothiazol, or antimycin A (Fig. 1C). We next considered that myxothiazol could be intrinsically toxic at the concentration used in the experiment. To test this, we treated cells with a combination of DEM and stigmatellin with myxothiazol expecting that if myxothiazol were intrinsically toxic, inhibition of ROS production by stigmatellin would fail to rescue cells. We found that cells were rescued from loss of viability induced by DEM with myxothiazol by co-incubation with stigmatellin, indicating that myxothiazol was not intrinsically cytotoxic under the conditions of the experiment (data not shown). Next we considered that the specific topological sites of ROS production by mitochondria in the presence of myxothiazol or antimycin A could account for the observed ROS reduction using both inhibitors and yet significant differences in cytotoxicity. Because myxothiazol inhibits superoxide release by cytochrome bc1 into the intermembrane space and cytosol but does not prevent formation of superoxide release into the mitochondrial matrix, ROS will continue to be released into the mitochondrial matrix, resulting in increased intramitochondrial oxidation (Scheme 1). Moreover, because myxothiazol is not specific for cytochrome bc1 but also inhibits the NADH:ubiquinone dehydrogenase and increases superoxide formation in the mitochondrial matrix side, significantly increased mitochondrial protein oxidation might be expected using this inhibitor (48-50). There is some evidence for this proposition, because {Delta}{Psi}m of CEM cells was lost at a significantly earlier time after treatment with DEM with myxothiazol compared DEM alone (Fig. 3, B and C). The decreased level of ROS detection in CEM cells treated with DEM and myxothiazol compared with DEM alone is considered to be due to the fact that the DCF probe predominantly detects cytosolic ROS formation and not mitochondrial ROS (Fig. 1B, panel 2) (51). These results therefore indicate that cytochrome bc1 is crucial in the redox-activated MPT and that increased ROS production in the mitochondrial matrix is likely to be the major cell compartment where ROS are produced and are responsible for the loss of {Delta}{Psi}m and the MPT.



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  		SCHEME 1.
Topology of mitochondrial superoxide production in the presence of antimycin A and myxothiazol. This scheme was adapted from the work of Trumpower (46) and Boveris and Cadenas (47).

 
If ROS production in the matrix and subsequent oxidative damage to matrix proteins were involved in the redox-MPT, we would expect that cytochrome bc1 inhibition would preserve mitochondrial ultrastructure and {Delta}{Psi}m, which we determined in two independent cell lines including CEM and HL60 cells. The results clearly showed that GSH depletion caused loss of {Delta}{Psi}m together with significant mitochondrial ultrastructural changes in mitochondria compared with mitochondria in control cells (Figs. 2A and 3A). These GSH-dependent changes included a characteristic increased electron density of inner mitochondrial membranes and cristae in both HL60 and CEM cells (Figs. 2C and 3A). The ultrastructural changes resulting from GSH depletion were not observed in mitochondria of cells co-incubated with DEM and stigmatellin or antimycin A (Figs. 2C and 3, B and C) but were apparent in mitochondria of cells treated with the antimycin A analog 2-MeAA (Fig. 3C). The ANT has been previously implicated as a key protein target in the redox-MPT, because the BgK, which inhibits the ANT at the matrix side, also blocked loss of {Delta}{Psi}m and cell death (Fig. 3B). Our results suggest that ANT is a key protein involved in the redox-MPT and that the ATP/ADP-binding site of the ANT, which is excluded by binding the ligand BgK, is a key MPT oxidative target protein (52). The redox-MPT is characterized as unregulated because CsA, which was used over a wide range of concentrations (1-20 µM), did not inhibit ROS production or prevent the loss of {Delta}{Psi}m and cell death (data not shown) (23, 24). Our results suggest that cytochrome bc1-dependent ROS production activates the MPT described by the model proposed by Kowaltowski et al. (8) in 2001 (8) and recapitulates the unregulated MPT model proposed by He and Lemasters (23, 24).

Because cytochrome bc1 was clearly involved in the unregulated MPT by its ROS producing activity, we next determined whether this respiratory complex was a regulator of the Ca2+-dependent MPT. For these studies we isolated RLM by standard procedures and performed the classical mitochondrial swelling test as an indicator of MPT activation in response to either increased Ca2+ load. Stigmatellin prevented large amplitude swelling of RLM induced by 100 µM Ca2+ with similar (equimolar) efficacy as CsA. Myxothiazol and antimycin A also inhibited swelling of RLM but to a lesser extent (Fig. 5). 2-MeAA was used as a control for antimycin A and did not prevent Ca2+-induced large amplitude swelling in RLM compared with antimycin A (Fig. 3C). Taken together, our results indicate that two distinct pathways to the MPT exist, as previously suggested (8, 9, 11-15, 23, 24), and that cytochrome bc1 may be a key regulator factor in both pathways.

Although the proteins composing and regulating the MPT are still unknown, previous reports clearly indicate that the MPT is regulated by electron flux through the NADH:ubiquininone dehydrogenase in cells as well as isolated mitochondria (25-27). Our study, using inhibitors of 1) the Qo ubiquinol catalytic site, 2) the Qi site, and 3) ISP movement show that cytochrome bc1 is a key regulatory component of the MPT by its ability to generate ROS and its potential to activate the MPT in response to increased Ca2+. Metabolic flux control theory shows that the NADH:ubiquininone dehydrogenase and cytochrome bc1 may be associated as a single enzyme with coenzyme Q as a common substrate (53, 54). Schagger (54) has proposed that these two respiratory components form a stable core respirasome in humans, and similar respiratory complex associations have been found in plant mitochondria (55). Based on these notions, our work, and the investigations of others, we propose a novel MPT model regulated by a respiratory supercomplex formed by NADH:ubiquininone dehydrogenase and cytochrome bc1 as well as the ANT (Fig. 6). The role of quinones in this model is implicated from the work of Fontaine and co-workers (26, 27) as well as from our previous work (29).



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  		FIG. 6.
A model of cytochrome bc1 involvement in regulated and unregulated MPT. The model depicts the unregulated MPT activated by mitochondrial ROS generated by cytochrome bc1 in the mitochondrial matrix and the regulated MPT activated by electron flux through cytochrome bc1. In the model we show the idea that a respiratory supercomplex formed by NADH:ubiquininone dehydrogenase and cytochrome bc1 is involved in regulation of the MPT.

 

   FOOTNOTES
 
* This work was supported by Academic Research Fund Grant R183000103112 (to J. S. A.), National Medical Research Council Grants NMRC/0474/2000, NMRC/0481/2000, and NMRC/0635/2002 (to M. W.), and National of University Singapore Office of Life Science Grant R183000603712 (to M. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Dept. of Biochemistry, National University of Singapore, Kent Road, Singapore, 117597, Singapore. Tel.: 65-6874-5996; Fax: 65-6779-1453; E-mail: bchjsa{at}nus.edu.sg.

1 The abbreviations used are: MPT, mitochondrial permeability transition; ROS, reactive oxygen species; ANT, adenine nucleotide translocator; CyD, cyclophilin D; RLM, rat liver mitochondria; ISP, iron-sulfur protein; TMRM, tetramethylrhodamine methylester; DCFDA, dichlorodihydrofluorescein diacetate; BgK, bongkrekic acid; 2-MeAA, 2-methoxy antimycin A3; SDH, succinate dehydrogenase; CsA, cyclosporine A; TTFA, thenoyltrifluoroacetone; Qo, ubiquinol oxidation; Qi, ubiquinol reduction; EM, electron microscopy; PBS, phosphate-buffered saline. Back


   ACKNOWLEDGMENTS
 
We thank Yee Liu Chua for technical assistance with this work and the Electron Microscopy unit at the National University of Singapore.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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