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J. Biol. Chem., Vol. 279, Issue 17, 17197-17204, April 23, 2004

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Opening of the Mitochondrial Permeability Transition Pore Induces Reactive Oxygen Species Production at the Level of the Respiratory Chain Complex I^*

Cécile Batandier, Xavier Leverve, and Eric Fontaine

From the INSERM E-0221, Bioénergétique Fondamentale et Appliquée, Université Joseph Fourier, F-38041 Grenoble Cedex 09, France

Received for publication, September 17, 2003 , and in revised form, December 23, 2003.

	ABSTRACT

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We have investigated the consequences of permeability transition pore (PTP) opening on the rate of production of reactive oxygen species in isolated rat liver mitochondria. We found that PTP opening fully inhibited H₂O₂ production when mitochondria were energized both with complex I or II substrates. Because PTP opening led to mitochondrial pyridine nucleotide depletion, H₂O₂ production was measured again in the presence of various amounts of NADH. PTP opening-induced H₂O₂ production began when NADH concentration was higher than 50 µM and reached a maximum at over 300 µM. At such concentrations of NADH, the maximal H₂O₂ production was 4-fold higher than that observed when mitochondria were permeabilized with the channel-forming antibiotic alamethicin, indicating that the PTP opening-induced H₂O₂ production was not due to antioxidant depletion. Moreover, PTP opening decreased rotenone-sensitive NADH ubiquinone reductase activity, whereas it did not affect the NADH FeCN reductase activity. We conclude that PTP opening induces a specific conformational change of complex I that (i) dramatically increases H₂O₂ production so long as electrons are provided to complex I, and (ii) inhibits the physiological pathway of electrons inside complex I. These data allowed the identification of a novel consequence of permeability transition that may partly account for the mechanism by which PTP opening induces cell death.

	INTRODUCTION

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Besides their well established role in energy metabolism, mitochondria are now recognized to play a key role in the commitment to cell death (1–4). Several intermembrane space proteins, such as cytochrome c, AIF, Smac/DIABLO, endonuclease G, and Omi/HtrA2, that have no pro-apoptotic activity when they remain inside mitochondria promote cell death once they are released into the cytosol. There is evidence to suggest that two different pathways may make the outer mitochondrial membrane permeable to these pro-apoptotic proteins. One relies on outer membrane channel(s) involving Bcl-2 family proteins, whereas the other is due to opening of an inner membrane channel, the permeability transition pore (PTP)¹.

PTP opening in vitro leads to the collapse of the proton motive force, ATP hydrolysis, the disruption of ionic homeostasis, and mitochondrial swelling. In this situation, mitochondrial swelling is due to the presence of matrix proteins that cannot diffuse through the open pore, thus creating an oncotic pressure gradient (5). The role of PTP in the commitment to cell death is supported by (i) the fact that mitochondrial swelling leads to outer membrane rupture and release of pro-apoptotic intermembrane space proteins (6–8), (ii) the demonstration that PTP opening occurs in intact cells (9–13), and (iii) the finding that different PTP inhibitors have a protective effect in several models of cell death (9, 14–22). However, the mechanisms by which PTP opening leads to the release of pro-apoptotic proteins in vivo needs to be clarified, especially whether PTP opening induces mitochondrial swelling in intact cells (or not) when mitochondria are surrounded by cytosolic proteins. Indeed, PTP opening in the absence of cytochrome c release has been reported in intact cells (7), whereas massive cytochrome c release after PTP opening has been observed in the absence of mitochondrial swelling (23).

It has been suggested that PTP opening increases ROS production in vivo (24), which may be relevant in the commitment to cell death. However, because PTP opening leads to mitochondrial uncoupling whereas mitochondrial uncoupling has been shown to decrease ROS production (25–28), this idea did not get much attention until recently, when it was reported that PTP opening increases ROS production in isolated mitochondria (29).

To characterize the mechanism by which PTP opening increases ROS production, knowing that mitochondria produce ROS at the level of complexes I and III (30–32), we have examined in this work the role of the respiratory substrates in ROS production before and after pore opening. Surprisingly, we found that PTP opening fully inhibited the H₂O₂ production of isolated mitochondria, regardless of the respiratory substrates. Because PTP opening led to pyridine nucleotide depletion, H₂O₂ production was measured again in the presence of NADH. Under this condition, PTP opening specifically increased H₂O₂ production. Moreover, PTP opening dramatically decreased rotenone-sensitive NADH ubiquinone reductase activity. We conclude that PTP opening induces a specific conformational change of complex I that (i) dramatically increases H₂O₂ production so long as electrons are provided to complex I, and (ii) inhibits the physiological pathway of electrons inside complex I.

	MATERIALS AND METHODS

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Rat liver mitochondria were prepared according to standard differential centrifugation procedures in a medium containing 250 mM sucrose, 20 mM Tris-HCl (pH 7.4), and 1 mM EGTA-Tris.

ROS production was assessed fluorometrically either in the presence of 20 units of horseradish peroxidase (HRP) and 100 µM homovanillic acid (HVA), as described in Ref. 33 (excitationemission, 319420 nm), or in the presence of 5 µM H₂DCFDA, as described in Ref. 34 (excitationemission, 503521 nm). Pyridine nucleotide oxidation-reduction status was estimated based upon endogenous fluorescence of NAD(P)H (excitationemission, 340450 nm), as described previously (35). Mitochondrial volume changes were measured from the light scattering changes at 520 nm. Fluorometric assays and light scattering measurements were performed at 30 °C simultaneously with a double-beam PTI Quantamaster C61 fluorometer.

Because the excitation and emission spectra of NADH and HVA are close, the measured signals (namely, F₃₁₉₄₂₀ and F₃₄₀₄₅₀) in the presence of these two fluorochromes correspond to

(Eq. 1)

(Eq. 2)

where F^NADH and F^HVA correspond to the fluorescence of NADH and HVA, respectively.

Given that = F^NADH₃₁₉₄₂₀/F^NADH₃₄₀₄₅₀ and F^HVA₃₄₀₄₅₀/F^HVA₃₁₉₄₂₀, then:

(Eq. 3)

(Eq. 4)

After the addition of NADH, which led to a sudden increase in fluorescence (F) in the two channels, was determined as F^NADH₃₁₉₄₂₀/ F^NADH₃₄₀₄₅₀. The experimental value of was 0.161 ± 0.013 (mean ± S.E., n = 63). Importantly, remained constant in the range of concentrations used (see Fig. 2). Under our experimental conditions, H₂O₂ addition led to a sudden increase in fluorescence in F₃₁₉₄₂₀ but not in F₃₄₀₄₅₀ (data not shown), indicating that was almost equal to zero. Therefore, H₂O₂ production (i.e. F^HVA₃₁₉₄₂₀) was obtained after the deconvolution of the two channels using the following equation

(Eq. 5)

View larger version (12K): [in this window] [in a new window]   FIG. 2. Relationship between NADH concentration and NADH fluorescence. Experimental conditions were as in Fig. 1. Experiments were started by the addition of 1 mg mitochondria followed by the addition of 400 µM Ca2+ to open the PTP (not shown). A, F340450 () and F319420 () represent the fluorescence in the presence of the indicated concentration of NADH minus the fluorescence before NADH addition. Results represent one typical experiment. B, relationship between FNADH319420/FNADH340450 and NADH concentration. Results are mean ± S.E. (n = 9).

Succinic acid, rotenone, valinomycin, alamethicin, HRP, HVA, CCCP, DUb, FeCN, EGTA, Tris, HCl, glutamic acid, CaCl₂, and CsA were purchased from Sigma-Aldrich; phosphoric acid, sucrose, malic acid, and NADH were purchased from Merck. KCN was purchased from Prolabo, and H₂DCFDA was obtained from Molecular Probes.

Results are expressed as mean ± S.E. Statistically significant differences were assessed by a Student's t test (Stat View, Abacus Concepts, Inc., Berkeley, CA).

	RESULTS

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Effect of PTP Opening on ROS Production—In the experiment in Fig. 1, we measured the production of H₂O₂ before and after PTP opening, with mitochondria energized either with glutamate plus malate (gray traces) or succinate (in the absence of rotenone) (black traces). Because the HRP/HVA method is known to be sensitive to NADH auto-fluorescence, we simultaneously recorded NADH (340450 nm) and the HVA (319420 nm) fluorescence. In agreement with previous reports (28, 37, 38), the production of H₂O₂ before PTP opening was higher in the presence of complex II than complex I substrates (Fig. 1, lower signal), whereas the NADH signal remained stable (Fig. 1, upper signal). As expected, PTP opening led to NADH oxidation, which interfered with the HVA signal. However, once the NADH signal had reached a new steady state (i.e. when NADH did not affect the slope of the HVA signal), the production of H₂O₂ was almost undetectable. Therefore, PTP opening decreased the H₂O₂ production of isolated mitochondria regardless of the respiratory substrates.

View larger version (15K): [in this window] [in a new window]   FIG. 1. Effect of PTP opening on H2O2 production rate. The incubation medium contained 250 mM sucrose, 10 µM EGTA, 20 mM Tris-HCl, 20 units of HRP, 100 µM HVA, and 5 mM Pi-Tris. The final volume was 2 ml, pH 7.4, at 30 °C. Experiments were started by the addition of 1 mg of mitochondria (data not shown). Where indicated, 5 mM glutamate-Tris plus 2.5 mM malate-Tris (gray traces) or 5 mM succinate-Tris (black traces) and 400 µM Ca2+ were added.

319420

319420

340450

As shown in Fig. 3A, in the presence of 150 µM NADH, PTP opening led to NADH consumption (trace a) and to an increase in the HVA signal (trace b). As expected, the addition of NADH did not interfere with H₂O₂ production after deconvolution of the two channels (trace c), and the production of H₂O₂ induced by PTP opening was then easily observed. Similar experiments were performed with various concentration of NADH. As shown in Fig. 3B, PT-induced H₂O₂ production () started when NADH concentration was higher than 50 µM and was maximal from 300 µM.

View larger version (15K): [in this window] [in a new window]   FIG. 3. Effect of NADH on H2O2 production rate after PTP opening. Experimental conditions were as in Fig. 1. The final volume was 2 ml, pH 7.4, 30 °C. A, experiments were started by the addition of 1 mg of mitochondria (data not shown). Where indicated, 150 µM NADH and 400 µM Ca2+ were added. Deconvolution was performed as indicated under "Materials and Methods." B, H2O2 production in the presence of the indicated concentrations of NADH either after PTP opening () or after addition of 1.5 µM alamethicin (). Results are mean ± S.E. (n = 6 for PTP opening, n = 3 for addition of alamethicin).

Using the H₂O₂-sensitive probe H₂DCFDA, it has been reported previously that PTP opening induced ROS production in the absence of NADH addition (29). Because this observation was in complete disagreement with the results in Fig. 1, we next measured the effect of PTP opening on the rate of H₂DCFDA oxidation.

Because mitochondrial volume changes optimally affect light scattering at 520 nm, which is close to the excitation-emission wavelengths recommended for H₂DCFDA, we first checked whether mitochondrial swelling could affect the signal assessed with this probe. In the experiment depicted in Fig. 4, we simultaneously recorded H₂DCFDA oxidation (upper signal, 503521 nm) and light scattering (lower signal, 520520 nm) when mitochondrial volume was modified by K⁺ movements across the inner membrane, a condition where mitochondrial swelling is not due to PTP opening. As expected, the addition of K⁺ ionophore valinomycin led to mitochondrial swelling as a consequence of the -driven accumulation of K⁺ inside mitochondria, whereas the addition of uncoupler CCCP led to mitochondrial shrinkage as a result of K⁺ release after had been abolished. As shown in Fig. 4, such changes of mitochondrial volume interfered with the signal coming from the H₂DCFDA probe (503521 nm). However, because the slope of the signal remained similar to the control when mitochondrial volume had stabilized, H₂DCFDA oxidation seemed to be a convenient assay of ROS production, except during volume transition.

View larger version (12K): [in this window] [in a new window]   FIG. 4. Effect of mitochondrial volume on H2DCFDA oxidation. The incubation medium contained 250 mM sucrose, 100 µM EGTA, 20 mM Tris-HCl, 5 mM glutamate-Tris, 2.5 mM malate-Tris, and 5 µM H2DCFDA. The final volume was 2 ml, pH 7.4, 30 °C. Experiments were started by the addition of 2 mg of mitochondria (data not shown). Where indicated, 5 mM potassium Pi, 1.25 µg/ml valinomycin, and 250 nM CCCP were added. The dashed line denotes H2DCFDA oxidation in the absence of valinomycin and CCCP.

View larger version (11K): [in this window] [in a new window]   FIG. 5. Effect of PTP opening on H2DCFDA oxidation. The incubation medium contained 250 mM sucrose, 10 µM EGTA, 20 mM Tris-HCl, 5 µM H2DCFDA, and either 5 mM glutamate-Tris plus 2.5 mM malate-Tris (gray traces) or 5 mM succinate-Tris (black traces). The final volume was 2 ml, pH 7.4, 30 °C. Experiments were started by the addition of 2 mg of mitochondria (data not shown). Where indicated, 5 mM Pi-Tris and 150 µM Ca2+ were added.

View larger version (21K): [in this window] [in a new window]   FIG. 6. Effect of PTP opening on oxygen consumption rate. The incubation medium contained 250 mM sucrose, 10 µM EGTA, 20 mM Tris-HCl, and either 5 mM glutamate-Tris plus 2.5 mM malate-Tris (A) or 5 mM succinate-Tris (B). The final volume was 2 ml, pH 7.4, 30 °C. Experiments were started by the addition of 2 mg of mitochondria (data not shown). Where indicated, 1 µM CsA (traces a and c), 1 mM NADH (traces a and b), 400 µM Ca2+ (traces b and d), or 1.5 µM alamethicin (traces a, b, and c) were added. The histograms represent cumulative data of at least eight different experiments ± S.E. of oxygen consumption rate. ***, p < 0.001, Student's t test. State 4 (St 4) denotes oxygen consumption rate before PTP opening. Inset, represents one typical experiment showing the relationship between NADH concentration and the oxygen consumption rate of mitochondria after PTP opening.

View larger version (21K): [in this window] [in a new window]   FIG. 7. Effect of PTP opening on complex I activity. NADH FeCN reductase activity (A) and NADH DUb reductase activity (B and C) were measured after the inner membrane had been either broken by hypoosmotic shock (Control) or permeabilized by PTP opening (After PTP opening). For PTP opening, 50 µg (A) or 250 µg (B and C) of mitochondria were pre-incubated in the medium of Fig. 6 supplemented with 5 mM glutamate-Tris plus 2.5 mM malate-Tris and then challenged for 10 min with 100 µM Ca2+. PTP opening was checked by mitochondrial swelling. For hypoosmotic shock-induced inner membrane rupture, 50 µg (A) or 250 µg (B and C) of mitochondria were pre-incubated for 10 min in a hypoosmotic medium containing only 5 mM glutamate-Tris plus 2.5 mM malate-Tris, pH 7.4. The medium was supplemented with stock solutions to be identical to the medium in which the PTP opening was performed. The final volume was 2 ml, pH 7.4, 30 °C. The incubation medium was then supplemented with 1.5 mM KCN (all panels) and 2 µM rotenone (C). FeCN reduction (A) and NADH oxidation (B and C) were measured after the addition of 500 µM FeCN plus 200 µM NADH (A) or 200 µM NADH plus 150 µM DUb. Results are means ± S.E. of at least seven separate determinations. **, p < 0.01, Student's t test.

View larger version (13K): [in this window] [in a new window]   FIG. 8. Effect of PTP opening and rotenone on H2O2 production rate. The incubation medium contained 250 mM sucrose, 10 µM EGTA, 20 mM Tris-HCl, 20 units HRP, 100 µM HVA, 5 mM Pi-Tris, and 5 mM glutamate-Tris plus 2.5 mM malate-Tris. The final volume was 2 ml, pH 7.4, 30 °C. Experiments were started by the addition of 1 mg of mitochondria (data not shown). Where indicated, 1 µM rotenone (gray trace) or 500 µM NADH plus 400 µM Ca2+ (black trace) were added. PT-induced H2O2 production was obtained after deconvolution as indicated under "Materials and Methods." No deconvolution was required for rotenone-induced H2O2 production because NADH remained constant (data not shown).

	DISCUSSION

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Respiratory Inhibition after PTP Opening—It has been reported that PT leads to a decrease in oxygen consumption rate when mitochondria are energized with NAD-linked substrates, a finding previously imputed to pyridine nucleotide depletion through the open pore (39). In this work, we show that complex I activity remains partly inhibited after PTP opening, even in the presence of saturating amounts of NADH (Fig. 6, Inset). It must be noted that such inhibition is not due to a kinetic control during NADH diffusion across the inner membrane, because under this condition (i.e. after PTP opening and in the presence of NADH), the NADH FeCN reductase activity was 10 times higher than the respiratory chain activity (compare Figs. 6 and 7). Importantly, once the PTP opened, we checked that whatever the concentration of mitochondria used, chelation of Ca²⁺ and addition of succinate led to mitochondrial repolarization (data not shown), demonstrating that PTP opening did not lead to inner membrane rupture.

Complex I catalyzes the transfer of electrons from NADH to the ubiquinone pool with an electron flux that is coupled to proton-pumping and inhibited by rotenone (40, 41). However, electrons can be transferred to electron acceptors that bind complex I at non-physiologic sites. Such electron flux is not coupled to proton pumping and is rotenone insensitive; thus, it does not represent the physiologic electron pathway inside complex I (40, 41). Among artificial electron acceptors, DUb binds simultaneously physiologic and non-physiologic sites, whereas FeCN binds non-physiologic site(s) only (36). The fact that PTP opening decreased the rotenone-sensitive NADH DUb reductase activity indicates that PTP opening per se inhibits the physiologic pathway of electrons inside complex I. Although the diameter of the open PTP is comparable with that of the pores induced by alamethicin (5, 42), the fact that alamethicin addition did not lead to complex I inhibition in the presence of NADH indicates that the PT-induced complex I inhibition is not due to the release of a putative complex I activating factor. Therefore, the PT-induced complex I inhibition is most probably due to a conformational change of complex I. It should be noted that PT did not affect either the NADH FeCN or the rotenone-insensitive NADH DUb reductase activities, which suggests that such conformational change does not affect the affinity of complex I for NADH.

Mechanism of ROS Production after PT—The two methods used in this work to assess the mitochondrial production of H₂O₂ (namely, HRP/HVA and H₂DCFDA) gave noticeably different results. H₂DCFDA is widely used for measuring H₂O₂ in intact cells, and indeed, PT-induced ROS production was described using this probe (24, 29). It must be kept in mind, however, that H₂DCFDA is prone to auto-oxidation. On the contrary, the HRP/HVA technique cannot be used in intact cells but is a reference method for H₂O₂ production in isolated mitochondria.

With this latter technique, PTP opening inhibited H₂O₂ production (Fig. 1) unless NADH was added (Fig. 3), regardless of the respiratory substrate used before PT. These findings are in total agreement with numerous works showing that (i) ROS production decreases with the mitochondrial membrane potential when electrons are provided to complex II (25–28), or (ii) electrons are not provided anymore to complex I because of NADH leak through the open pore (39, 43). On the contrary, PTP opening transiently increased or did not affect H₂DCFDA oxidation in the presence of complex I and complex II substrates, respectively (Fig. 5). The fact that PTP opening totally inhibited H₂O₂ production in the absence of NADH, whereas it did not decrease H₂DCFDA oxidation in the same condition, is most probably because of a large auto-oxidation of that probe. The reason why PTP opening transiently increased H₂DCFDA oxidation in the presence of glutamate plus malate remains unclear. However, because H₂DCFDA oxidation was not affected by catalase addition (data not shown), we propose that PTP opening may increase H₂DCFDA auto-oxidation under this particular condition of incubation, independently of any production of ROS by the respiratory chain.

Because the previously published works reporting that PTP opening induces ROS production have been performed with H₂DCFDA (24), the question arises as to whether PTP opening really induces H₂O₂ production in intact cells. It must, however, be kept in mind that the mean concentration of NADH in cells is within the millimolar range, a concentration at which PT-induced H₂O₂ production occurs (Fig. 3).

Mitochondria produce ROS at the level of complexes I and III (30–32). The fact that PT did not induce ROS production when electrons were provided directly to complex II (i.e. downstream complex I) strongly suggests that the PT-induced H₂O₂ production observed in the presence of NADH occurred in complex I but not in complex III.

Because complex I inhibitors have been shown to increase ROS production in the presence of NAD-linked substrates (26, 28, 44, 45), it was tempting to speculate that the PT-induced ROS production observed in the presence of NADH was due to the PT-induced inhibition of complex I. However, as shown in Fig. 8, rotenone slightly favored H₂O₂ production, whereas PTP opening dramatically increased H₂O₂ production. Because complex I inhibition could not per se account for the PT-induced ROS production, we propose that PTP opening induces a specific conformational change of complex I that dramatically increases H₂O₂ production so long as electrons are provided to complex I, and inhibits the physiologic pathway of electrons inside complex I. Moreover, depending on the incubation conditions, it may also catalyze H₂DCFDA oxidation in the absence of NADH.

Implications for PTP Opening-induced Cell Death—It has long been known that oxidative stress can induce cell death (46). Although ROS can trigger PTP opening both in vitro (5) and in intact cells (9), ROS have been shown to induce cytochrome c release and subsequent cell death, whereas PTP remained closed (47). Evidence suggests that in the absence of outer membrane rupture, the release of pro-apoptotic factors occurs by means of large pores that are the result of the insertion of Bcl-2 family protein BAX into the outer membrane (1–3). Voltage-dependent anion channel (VDAC) has also been implicated, alone or in interaction with Bcl-2 family proteins, in outer membrane permeabilization (48–50). In support of the idea that ROS affect outer membrane permeability, it has been reported that (i) H₂O₂ induced BAX translocation in cardiomyocytes (51), (ii) NO· induced BAX translocation in SH-SY5Y neuroblastoma (52), and (iii) induced VDAC-dependent cytochrome c release in HepG2 cells (48). Moreover, it has been shown that ROS production induced by the addition of transforming growth factor decreased the expression and level of anti-apoptotic protein Bcl-x_L in fetal hepatocytes (53).

Although cell death does not necessarily require PTP opening, the involvement of the PTP in the commitment to cell death is supported by a large body of evidence based on the protective effect of different PTP inhibitors in several models of cell death (9–13). During in vitro assays, PT is followed by mitochondrial swelling, outer membrane rupture, and the release of apoptotic proteins (6–8). In intact cells, however, PTP opening does not systematically lead to cytochrome c release (7). This may be due to transient PTP opening (7), although it must be noted that the cytosolic oncotic pressure may prevent PT-induced mitochondrial swelling. Indeed, PT-induced cytochrome c release has been reported to occur in the absence of measurable mitochondrial swelling (23), suggesting that PTP opening could trigger a cell signaling process that, in turn, would lead to outer membrane permeabilization. Because PTP opening induces ROS production, whereas ROS induces PTP opening, it is tempting to speculate that PTP opening will propagate to the whole cell, leading to a massive oxidative stress that directly induces BAX- or VDAC-dependent outer membrane permeabilization.

Several outer membrane proteins such BAX and Bcl-2 regulate PTP opening in vitro (54, 55). Our proposal implies that, in addition, PTP opening could affect proteins involved in outer membrane permeabilization. In this scenario, PTP opening would not represent an alternative to BAX- or VDAC-dependent outer membrane permeabilization but would be a highly regulated pathway located upstream to this event.

	FOOTNOTES

 
^* This work was supported by Grants from INSERM, the Ministère de l'Enseignement de la Recherche et de la Technologie, the Association pour la Recherche sur le Cancer, the Ligue Nationale Contre le Cancer (Comité de la Drôme), and the Région Rhône-Alpes (Programmes Émergence). 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.

To whom correspondence should be addressed: INSERM E-0221, Bioénergétique Fondamentale et Appliquée, Université Joseph Fourier, BP 53X, F-38041 Grenoble Cedex 09, France. Tel.: 33-47663-5601; Fax: 33-47651-4218; E-mail: eric.fontaine{at}ujf-grenoble.fr.

¹ The abbreviations used are: PTP, permeability transition pore; ROS, reactive oxygen species; HVA, homovanillic acid; HRP, horseradish peroxidase; CCCP, carbonyl cyanide m-chlorophenylhydrazone; DUb, decylubiquinone; H₂DCFDA, 2',7'-dichlorofluorescin diacetate; CsA, cyclosporin A; , transmembrane electric potential difference; VDAC, voltage-dependent anion channel.

	ACKNOWLEDGMENTS

 
We thank Prof. Paolo Bernardi for helpful discussions and Baby Lenhard and Dr. Dan Veale for their help in correcting the manuscript.

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