Voltage-dependent Anion Channels Control the Release of the Superoxide Anion from Mitochondria to Cytosol*

Derick HanDagger , Fernando Antunes§, Raffaella Canali, Daniel Rettori, and Enrique Cadenas||

From the  Department of Molecular Pharmacology and Toxicology, School of Pharmacy and the Dagger  University of Southern California Research Center for Liver Disease, Keck School of Medicine, University of Southern California, Los Angeles, California 90089-9121 and the § Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa, P-1749-06 Lisboa, Portugal

Received for publication, October 8, 2002, and in revised form, December 2, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Several reactions in biological systems contribute to maintain the steady-state concentrations of superoxide anion (O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>) and hydrogen peroxide (H2O2). The electron transfer chain of mitochondria is a well documented source of H2O2; however, the release of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> from mitochondria into cytosol has not been unequivocally established. This study was aimed at validating mitochondria as sources of cytosolic O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>, elucidating the mechanisms underlying the release of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> from mitochondria into cytosol, and assessing the role of outer membrane voltage-dependent anion channels (VDACs) in this process. Isolated rat heart mitochondria supplemented with complex I or II substrates generate an EPR signal ascribed to O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>. Inhibition of the signal in a concentration-dependent manner by both manganese-superoxide dismutase and cytochrome c proteins that cannot cross the mitochondrial membrane supports the extramitochondrial location of the spin adduct. Basal rates of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> release from mitochondria were estimated at ~0.04 nmol/min/mg protein, a value increased ~8-fold by the complex III inhibitor, antimycin A. These estimates, obtained by quantitative spin-trapping EPR, were confirmed by fluorescence techniques, mainly hydroethidine oxidation and horseradish peroxidase-based p-hydroxyphylacetate dimerization. Inhibitors of VDAC, 4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS), and dextran sulfate (in a voltage-dependent manner) inhibited O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> production from mitochondria by ~55%, thus suggesting that a large portion of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> exited mitochondria via these channels. These findings are discussed in terms of competitive decay pathways for O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> in the intermembrane space and cytosol as well as the implications of these processes for modulating cell signaling pathways in these compartments.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Mitochondria from various aerobic organisms have been recognized as effective sources of hydrogen peroxide (H2O2) (1, 2). H2O2 produced by mitochondria has been suggested to regulate several signal transduction pathways, including c-Jun N-terminal kinase (JNK1) activity (3, 4). Alterations in mitochondrial H2O2 steady-state levels by genetic modulation of catalase expression in the mitochondrial matrix is associated with changes in cell proliferation (3, 5), tumor necrosis factor (TNF) response (6), and apoptosis (7).

A two-step model that accounts for mitochondrial H2O2 production has become widely accepted (8, 9). The first step, shown below in Reaction 1, 
<UP>UQ&cjs1138;</UP>+<UP>O<SUB>2</SUB></UP>→<UP>UQ</UP>+<UP>O</UP><SUB><UP>2</UP></SUB><SUP><UP>&cjs1138;</UP></SUP>

<UP><SC>Reaction 1</SC></UP>
entails the autoxidation of ubisemiquinone in the respiratory chain to generate O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>, which is released into the mitochondrial matrix (10, 11). The second step entails the conversion of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> to H2O2 catalyzed by manganese superoxide dismutase (Mn-SOD), which resides in the mitochondrial matrix. H2O2 diffuses rapidly through membranes (12), and the release of H2O2 from mitochondria to cytosol reflects the balance between H2O2 production and consumption reactions, with the latter mainly involving reduction of the hydroperoxide to H2O via matrix glutathione peroxidase. Mitochondria contribute ~20-30% to the cytosolic steady-state concentration of H2O2 (~10-8 M) (13).

Two recent findings have modified and extended this two-step mechanism for mitochondrial production of H2O2. First, part of the O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> generated during mitochondrial electron transfer is vectorially released into the intermembrane space (14). Evidence for this, obtained with mitoplasts (mitochondria devoid of the outer membrane), consisted of abrogation of the EPR spin adduct signal by superoxide dismutase, competitive inhibition by cytochrome c, and broadening of the signal by membrane-impermeable spin-broadening agents (14). The mechanism underlying the release of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> into the intermembrane space considers the formation of ubisemiquinone (Reaction 1) at two sites in the ubiquinone pool, the QI site that lies near the matrix and the QO site in the vicinity of the intermembrane space (15, 16). Autoxidation of ubisemiquinone at the QO site (UQ<UP><SUB>O</SUB><SUP>&cjs1138;</SUP></UP>) results in release of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> into the cytosolic side of the mitochondrial inner membrane. Of note, O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> cannot cross membranes except in the protonated form (a small fraction of the O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> pool at physiological pH; pKa = 4.8) (17). Taken together, this finding suggests that H2O2 could be formed both at the intermembrane space and matrix from O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> generated toward the respective compartments.

Second, the release of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> toward the intermembrane space would be in functional relationship to the localization of a superoxide dismutase activity in this compartment. Indeed, the occurrence of a Cu,Zn-SOD in the intermembrane space of mitochondria has recently been confirmed both in rat liver (18, 19) and yeast (20). These findings ended a 30-year debate following the initial report on the presence of Cu,Zn-SOD in the intermembrane space (21), which was challenged on the basis of a contamination by lysosomes (22).

The vectorial release of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> into the intermembrane space raises the question of whether O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> generated in this manner can diffuse into the cytosol across the outer membrane and both contribute to the cytosolic steady-state levels of this species and participate in the regulation of cell signaling cascades. The intermembrane space contains several O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>-scavenging pathways (cytochrome c and the intermembrane space Cu,Zn-SOD) as well as pores for O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> diffusion across the outer membrane, in particular, the voltage-dependent anion channel (VDAC).1 Physiologically, VDAC functions as the major channel allowing passage of low molecular weight solutes and proteins between the intermembrane space and cytoplasm (23, 24) and, more recently, its role both in the release of the pro-apoptotic cytochrome c and in the mitochondrial permeability transition pore has been described (25, 26). The channel formed by VDAC has a diameter of 2-4 nm and is more selective for the passage of anions than cations (24). Erythrocyte membranes have been shown to contain an anion channel through which O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> can diffuse (24).

VDAC may act similarly as a channel that allows the movement of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> from the intermembrane space to the cytoplasmic surface of the outer membrane of mitochondria. This study was aimed at validating mitochondria as sources of cytosolic O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>, elucidating the mechanisms underlying the release of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> from mitochondria into cytosol, and assessing the role of outer membrane VDACs in this process.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Chemicals and Biochemicals-- 5,5'-Dimethyl-1-pyrroline-N-oxide (DMPO), Cu,Zn-SOD, Mg-SOD, myxothiazol, bovine heart cytochrome c, dextran sulfate (Mr 8,000), bovine serum albumin, antimycin A, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and rotenone were from Sigma. 4-Hydroxy-2,2,6,6-tetramethyl-piperidine-N-oxyl (TEMPOL) was from Aldrich. 4,4'-Diisothioc-yano-2,2'-disulfonic acid stilbene (DIDS), hydroethidine, and tetramethylrhodamine methyl ester (TMRM) were from Molecular Probes (Eugene, OR).

Isolation of Heart Mitochondria-- Heart mitochondria were isolated from adult male Wistar rats by differential centrifugation using Nagarse (27). Rat hearts were excised, washed, chopped into fine pieces, and suspended in an isolation buffer consisting of 230 mM mannitol, 70 mM sucrose, 1 mM EDTA, and 5 mM Trizma/HCl buffer, pH 7.4. The chopped heart was treated with Nagarse (1 mg/heart) for 5 min and homogenized by 10 strong strokes of a loose fitting Potter-Elvehjem Teflon pestle. The homogenate was centrifuged at 800 × g for 8 min, the pellet was removed, and the centrifugation process was repeated. The supernatant was centrifuged at 8,000 × g for 10 min, washed with isolation buffer, and the centrifugation was repeated. Isolated mitochondria were resuspended in isolation buffer and placed in ice for the duration of the experiments.

Determination of Mitochondria Integrity-- Isolated heart mitochondria integrity was assessed in terms of respiratory control ratios, defined as the state 3 to state 4 ratio. O2 uptake was measured polarographically with a Clark-type electrode (Hansatech) in respiration buffer containing 230 mM mannitol, 70 mM sucrose, 30 mM Tris-HCl, 5 mM KH2PO4, 1 mM EDTA, and 0.1% bovine serum albumin, pH 7.4. Mitochondria, with a respiratory control ratio of >4 using glutamate/malate as substrates and 2.5 using succinate, were used for all experiments. State 3 respiration was not changed upon the addition of cytochrome c (84 ± 10 and 85 ± 10 ng of atoms of O/min/mg of protein in the presence of 2 mM of cytochrome c), thus confirming the integrity of the mitochondrial outer membrane.

Electron Paramagnetic Resonance (EPR) Spectroscopy-- Heart mitochondria were suspended in 230 mM mannitol, 70 mM sucrose, 20 mM Tris, pH 7.4 (adjusted with MOPS) buffer in the absence or presence of respiratory substrates or inhibitors. DMPO was present at a concentration of 160 mM. EPR spectra were recorded with a Bruker ECS106 spectrometer (operating at X-band) equipped with a cylindrical room temperature cavity operating in TM110 mode. Samples were promptly transferred to bottom sealed Pasteur pipettes (volume, 200 µl) and measured at room temperature with the following instrument settings: receiver gain, 5 × 105; microwave power, 20 mW; microwave frequency, 9.77 GHz; modulation amplitude, 1 G; time constant, 164 ms; scan time, 87 s; and scan width, 80 G. The DMPO-OH signal generated from heart mitochondria was quantified by comparison with a TEMPOL standard after double integration of both signals. All spectra shown are an accumulation of seven scans.

Fluorescence Spectroscopy-- Fluorescence measurements were performed on a PerkinElmer LS-5 spectrofluorometer equipped with a thermal controlled, magnetic stirring sample compartment. For all measurements, the mitochondria were incubated in 230 mM mannitol, 70 mM sucrose, 20 mM Tris, pH 7.4 (adjusted with MOPS) at 25 °C. Membrane potential was measured using TMRM (28) (lambda ex = 550 nm; lambda em = 590 nm). H2O2 production by heart mitochondria was measured by monitoring the fluorescence of p-hydroxyphenylacetate oxidation in the presence of horseradish peroxidase with or without superoxide dismutase (29, 30) (lambda ex = 320 nm; lambda em = 400 nm). Hydroethinine (HE) oxidation to fluorescence ethidine (E+) (lambda ex = 470 nm; lambda em = 590 nm) was used as a measure of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> (31, 32) in mitochondria suspensions supplemented with 3 µM hydroethinine at 37 °C.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Release of Superoxide Anion by Mitochondria-- The release of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> by isolated heart mitochondria was investigated by spin-trapping EPR. In the absence of respiratory substrates, mitochondria supplemented with the spin trap, DMPO, did not generate any EPR signal (Fig. 1A). The addition of complex I (Fig. 1B) or complex II (not shown) substrates to heart mitochondria resulted in a low intensity EPR signal characteristic of the DMPO-OH spin adduct (quartet signal with intensity ratios of 1:2:2:1; aN = aH = 14.9). Antimycin, which increases the steady-state levels of the UQ<UP><SUB>O</SUB><SUP>&cjs1138;</SUP></UP> by inhibiting electron transfer at the bc1 segment (complex III), caused a 8-fold increase of the EPR signal intensity (Fig. 1, C and D). The signal was abolished by Cu,Zn-SOD (Fig. 1E), thus establishing O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> as the source of the DMPO-OH signal, which is formed by spontaneous decay of the superoxide spin adduct (DMPO-OOH) (33). The addition of myxothiazol to antimycin-treated mitochondria resulted in a complete inhibition of the DMPO-OH signal in agreement with an expected inhibition of ubisemiquinone formation caused by myxothiazol (Fig. 1F).


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Fig. 1.   Formation of superoxide by heart mitochondria. Under the assay conditions, the reaction mixture consisted of mitochondria (0.3 mg protein ml-1) in buffer (230 mM mannitol/70 mM sucrose/20 mM Tris, pH 7.4), supplemented with 160 mM DMPO, catalase (200 units), and respiratory substrate and/or inhibitor. A, no treatment. B, glutamate/malate. C, glutamate/malate plus antimycin. D, succinate plus rotenone plus antimycin. E, glutamate/malate plus antimycin plus Cu,Zn-SOD. F, succinate plus antimycin plus myxothiazol. When present, glutamate/malate = 7.5 mM; succinate = 7.5 mM; rotenone = 2 µM; antimycin = 1 µg/mg protein; myxothiazol = 1 µM; and superoxide dismutase = 400 units/ml.

Localization of Superoxide Anion Production-- The outer mitochondrial membrane is considered porous to molecules of 5 kDa, and small molecules like DMPO can diffuse from the cytoplasmic side of the outer membrane to the intermembrane space and vice versa. Thus, the DMPO-OH signal observed from isolated heart mitochondria (Fig. 1) may originate from the reaction of DMPO with O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> in the intermembrane space. The compartmentalization of this reaction requires the release of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> generated at the UQO pool into the intermembrane space. The effect of agents that cannot cross the mitochondrial inner membrane on the DMPO-OH signal is illustrated in Fig. 2; cytochrome c and Mn-SOD elicited a concentration-dependent loss of the DMPO-OH signal generated by heart mitochondria respiring on complex I substrates and in the presence of antimycin A. The outer mitochondrial membrane is impermeable to both cytochrome c (12 kDa) (34, 35) and superoxide dismutase (80 kDa) (36); hence, their effects on the EPR signals (Figs. 1E and 2) are expected to take place on the cytosolic side of the outer mitochondrial membrane. This also suggests that the generation of the spin adduct occurred with O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> diffusing from the intermembrane space and across the outer mitochondrial membrane. The data shown in Fig. 2 may be viewed as a competitive inhibition entailing the different reactivities for O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> of the spin trap, cytochrome c, and Mn-SOD as seen below in Reactions 2, 3, and 4, respectively,


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Fig. 2.   Inhibitory effect of cytochrome c and superoxide dismutase on DMPO-OH spin adduct generated by superoxide released from mitochondria. Mitochondria treated with glutamate/malate (5 mM) plus antimycin (1 µg/mg) and 160 mM DMPO were incubated with various amounts of cytochrome c () and Mn-SOD (open circle ).


<UP>O</UP><SUB><UP>2</UP></SUB><SUP><UP>&cjs1138;</UP></SUP>+<UP>H<SUP>+</SUP></UP>+<UP>DMPO</UP>→<UP>DMPO-OOH</UP>

<UP><SC>Reaction 2</SC></UP>

<UP>O</UP><SUB><UP>2</UP></SUB><SUP><UP>&cjs1138;</UP></SUP>+<UP>cyt</UP> c<SUP><UP>3+</UP></SUP>→<UP>O<SUB>2</SUB></UP>+<UP>cyt</UP> c<SUP><UP>2+</UP></SUP>

<UP><SC>Reaction 3</SC></UP>

<UP>O</UP><SUB><UP>2</UP></SUB><SUP><UP>&cjs1138;</UP></SUP>+<UP>O</UP><SUB><UP>2</UP></SUB><SUP><UP>&cjs1138;</UP></SUP>+<UP>2H<SUP>+</SUP></UP>→<UP>O<SUB>2</SUB></UP>+<UP>H<SUB>2</SUB>O<SUB>2</SUB></UP>

<UP><SC>Reaction 4</SC></UP>
where k2 = 10 M-1s-1 (for the spin trap; Ref. 33), k3 = 2.5 × 105 M-1s-1 (for cytochrome c, Ref. 37), and k4 = 1.2 × 109 M-1s-1 (for Mn-SOD; Ref. 38).

Quantitative Determination of Superoxide Anion Production by EPR-- Isolated liver mitoplasts were shown to reduce DMPO-OH adducts to EPR-silent hydroxylamines, thus rendering quantification of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> difficult (14). The effect of mitochondria on the stability of the DMPO-OH adduct was assessed in a system consisting of DMPO-OH (generated by mixing DMPO with H2O2 and Fe2+) supplemented with isolated heart mitochondria, and its decomposition (kdecay) followed. The apparent rate of DMPO-OH decomposition (kobs) depended on the amount of mitochondria (Fig. 3A); kdecay (slope value in Fig. 3B) was estimated by fitting data to a first-order kinetics, and a value of 0.0138 s-1 mg-1 protein was obtained.


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Fig. 3.   Effect of mitochondria on the stability of the DMPO-OH spin adduct. The DMPO-OH adduct was generated upon incubation of 100 µM H2O2, 20 µM ferrous ammonium sulfate, and 100 mM DMPO. Mixtures containing various concentrations of mitochondria (200 µl final assay mixture volume), glutamate/malate (7.5 mM), antimycin (1 µg/mg protein), catalase (200 units), and superoxide dismutase (400 units/ml) were added and decay of the DMPO-OH signal monitored by EPR. A, decomposition of DMPO-OH signal by mitochondria. open circle , absence of mitochondria; , mitochondria 0.3 mg of protein ml-1; black-square, mitochondria 0.6 mg protein ml-1. The slope of the plot gives the apparent first-order rate constant of DMPO-OH decomposition. B, DMPO-OH decomposition rate constants versus mitochondria protein. kdecay was calculated from the slope shown here.

Fig. 4A shows the formation of the DMPO-OH adduct versus time from antimycin-treated mitochondria in the presence of glutamate/malate. For each sample, the rate of DMPO-OH formation (upsilon DMPO-OH) could be calculated from the slope of Fig. 4B. The rate of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> formation was calculated under the assumption that it is equal to the rate of the spin adduct (DMPO-OH) formation as shown in Equation 1,
<UP>+</UP>d[<UP>O</UP><SUB><UP>2</UP></SUB><SUP><UP>&cjs1138;</UP></SUP>]<UP>/</UP>d<UP>t</UP>=<UP>&ugr;<SUB>DMPO-OH</SUB></UP> (Eq. 1)
and upsilon DMPO-OH can be obtained from Equation 2,
d[<UP>DMPO-OH</UP>]<SUB><UP>obs</UP></SUB><UP>/</UP>d<UP>t</UP>=<UP>&ugr;<SUB>DMPO-OH</SUB>−</UP>k<SUB><UP>decay</UP></SUB>[<UP>DMPO-OH</UP>]<SUB><UP>obs</UP></SUB> (Eq. 2)
where [DMPO-OH]obs is the DMPO-OH concentration measured by EPR. Resolution of the differential equation leads to Equation 3,
[<UP>DMPO-OH</UP>]<SUB><UP>obs</UP></SUB>=<UP>&ugr;<SUB>DMPO-OH</SUB>/</UP>k<SUB><UP>decay</UP></SUB>(<UP>1</UP>−<UP>1/e</UP><SUP>k</SUP><SUB><UP>decay</UP></SUB><SUP><UP>t</UP></SUP>) (Eq. 3)
shown above. Using the outlined calculations, O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> generation into cytoplasm by heart mitochondria in the presence or absence of substrates and antimycin was estimated at 25 °C (Fig. 5; Table I). In the presence of glutamate/malate, heart mitochondria are estimated to release 0.041 nmol/min/mg O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> into the cytoplasm, a value that increased to 0.33 nmol/min/mg O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> in the presence of antimycin.


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Fig. 4.   Rate of DMPO-OH generation by mitochondria. A, formation of DMPO-OH adduct versus time by antimycin-treated mitochondria (0.16 mg protein/ml) supplemented with glutamate/malate (7.5 mM). DMPO-OH was measured by EPR at the time points indicated. Experiments were carried out in buffer (230 mM mannitol/70 mM sucrose/20 mM Tris, pH 7.4) in the presence of 200 mM DMPO and catalase (200 units). B, [DMPO-OH]obs versus 1-1/ekdecayt; slope = upsilon DMPO-OH/kdecay. The kdecay used was calculated from Fig. 3.

                              
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Table I
O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> and H2O2 production by heart mitochondria
Heart mitochondria (0.2 mg/ml) were incubated in 230 mM mannitol, 70 mM sucrose, 20 mM Tris/ HCl buffer, pH 7.4 (adjusted with MOPS) at 25°C with horseradish peroxidase (10 units) and p-hydroxyphenylacetate (1 mM). Estimated values were calculated according to Equation in the text (+d[O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>]/dt = 2 × [(d [H2O2]/dt)+SOD - (d[H2O2]/dt)-SOD]). EPR measurements were carried out as described under "Materials and Methods." When present, concentration of reactants was as follows: 7.5 mM glutamate/malate; 7.5 mM succi-nate; 2 µM rotenone; 1 µg antimycin A/mg of protein; 10 µM myxothiazol; 1 µM CCCP; and 400 units of superoxide dismutase per milliliter. Values are expressed as mean ± S.D from three experiments.


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Fig. 5.   Effect of substrate concentrations on superoxide production by mitochondria. Antimycin-treated mitochondria (0.3 mg of protein ml-1) were supplemented with various amounts of substrates. Experiments were carried out in buffer (230 mM mannitol/70 mM sucrose/20 mM Tris, pH 7.4) in the presence of 200 mM DMPO and 200 units of catalase. , glutamate/malate plus antimycin; black-triangle, glutamate/malate; open circle , succinate plus rotenone plus antimycin; triangle , succinate plus rotenone.

Quantitative Determination of Superoxide Anion Production Using Redox-sensitive Fluorescent Dyes-- The information on mitochondrial O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> production gathered by EPR approaches (Figs. 1-3) was further evaluated with fluorescent redox dyes. Two types of redox dyes were used: (i) p-hydroxyphenylacetate and horseradish peroxidase to detect H2O2 (O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> is the stoichiometric precursor of H2O2; Refs. 10 and 39); and (ii) hydroethidine, a dye that reacts specifically with O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>.

Isolated heart mitochondria generated low amounts of H2O2 in the presence of substrates (Table I); Mn-SOD elicited a 3-fold increase of H2O2 production, thus strengthening the notion that part of the O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> generated by heart mitochondria was released across the outer membrane. The rate of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> production, seen below in Equation 4,
+d[<UP>O</UP><SUB><UP>2</UP></SUB><SUP><UP>&cjs1138;</UP></SUP>]<UP>/</UP>dt=<UP>2×</UP>[(d[<UP>H<SUB>2</SUB>O<SUB>2</SUB></UP>]<UP>/</UP>dt)<SUP><UP>+SOD</UP></SUP><UP>−</UP>(d[<UP>H<SUB>2</SUB>O<SUB>2</SUB></UP>]<UP>/</UP>dt)<SUP><UP>−SOD</UP></SUP>] (Eq. 4)
was calculated by the difference between the production of H2O2 in the presence of Mn-SOD and the production of H2O2 in the absence of the enzyme (Table I). Using this estimate, the rate of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> production, calculated by EPR, and the rate of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> production, calculated from H2O2, were in good agreement (Table I). Myxothiazol, which inhibits UQ<UP><SUB>O</SUB><SUP>&cjs1138;</SUP></UP> formation, inhibited O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> production detected by both EPR (Fig. 1F) and fluorescence (Table I) spectroscopy approaches. In agreement with previous findings (27), mitochondrial H2O2 generation (and, by extension, O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> generation) was substantially enhanced by the mitochondrial uncoupler CCCP.

O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>, but not H2O2, HOCl, or ONOO-, oxidizes hydroethidine to ethidium (E+); E+ fluorescence is considered a specific measure of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>, albeit a qualitative one (31, 32). Similar to what was observed with EPR approaches (Fig. 1), E+ fluorescence increased in heart mitochondria upon supplementation with substrates and antimycin A, whereas the signal was decreased by myxothiazol (Table II). Exogenous Mn-SOD completely abolished E+ fluorescence, thus indicating the specificity of hydroethidine toward O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>.

                              
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Table II
Estimation of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> production in heart mitochondria using hydroethidine
Heart mitochondria (0.2 mg/ml) were supplemented with 5 mM hydroethinine and incubated in 230 mM mannitol, 70 mM sucrose, 20 mM Tris/HCl buffer, pH 7.4 (adjusted with MOPS) at 37 °C. E+ fluorescence was monitored as described under "Materials and Methods." When present, concentration of reactants was as follows: 7.5 mM glutamate/malate; 7.5 mM succinate; 2 µM rotenone; 1 µg antimycin A/mg of protein; 1 µM myxothiazol; 400 units of superoxide dismutase per milliliter. Values are expressed as mean ± S.D from three experiments. n.d., not detected.

Control of Superoxide Anion Diffusion through VDAC-- The role of VDAC in mediating the diffusion of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> from the intermembrane space to the cytoplasmic surface of mitochondria was assessed by means of the inhibitors of this channel, DIDS and dextran sulfate (40). Because respiratory substrates such as succinate and other metabolites enter mitochondria through VDAC (41), the treatment of isolated mitochondria with either VDAC inhibitor resulted in a ~82% decrease of state 4 respiration supported by either glutamate/malate or succinate (data not shown). However, as expected, VDAC inhibitors did not affect mitochondria respiration where respiratory substrates were not limiting, i.e. respiration supported by endogenous substrates or in the presence of antimycin A (data not shown). Therefore, antimycin-treated mitochondria in the presence of glutamate/malate, where O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> production is maximal and the substrate not limiting, were treated with VDAC inhibitors to assess their effect on mitochondrial O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> release. The treatment of isolated heart mitochondria with DIDS resulted in a dose-dependent inhibition of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> production detected by spin-trapping EPR; a maximum inhibition of 55% was observed (Fig. 6).


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Fig. 6.   Effect of DIDS on superoxide production by isolated heart mitochondria. Heart mitochondria (0.3 mg protein ml-1) were supplemented with various concentrations of DIDS, and superoxide production was measured by EPR. All experiments were carried in the presence of DMPO (160 mM), antimycin (1 µg/mg protein), glutamate/malate (5 mM), and catalase (200 units) in buffer (230 mM mannitol, 70 mM sucrose, 30 mM Tris-HCl, 5 mM KH2PO4, 1 mM ETDA, pH 7.4).

Polyvalent anions such as dextran sulfate modulate VDAC closure, particularly in the presence of a membrane potential across the phospholipid bilayer (or in a voltage-dependent manner) (42). Although the mitochondrial outer membrane is permeable to small solutes, large proteins, impermeable through VDAC, could effectively generate a Nernst potential across the outer membrane. In fact, it has been speculated that large cytoplasmic proteins modulate membrane potential across the outer membrane and possibly regulate VDAC opening/closing (43).

Thus, an experimental model consisting of mitochondria treated with both albumin and dextran sulfate was devised to assess whether O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> production by mitochondria was also inhibited by dextran sulfate in a voltage-dependent manner. Albumin, because of its large size (67 kDa; impermeable across the outer membrane) and its net charge (-11), was ideal to generate a Nernst potential across the outer membrane. Membrane potential measurements using TMRM demonstrated that albumin generates negative potential on the outer membrane in the presence of antimycin A (to collapse the inner membrane potential; see Fig. 7, inset). (Higher levels of BSA led to a quenching of TMRM fluorescence because of binding between BSA and TMRM (data not shown)). Treatment with dextran sulfate resulted in a concentration-dependent inhibition of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> production by isolated heart mitochondria (Fig. 7); the extent of inhibition was amplified when an outer membrane potential was generated by albumin (Fig. 7). Approximately 70% of the O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> signal was inhibited with dextran sulfate when a membrane potential was formed across the outer membrane by albumin.


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Fig. 7.   Effect of dextran sulfate and BSA on superoxide production by isolated heart mitochondria. Antimycin-treated mitochondria (0.3 mg of protein ml-1) were supplemented with various concentrations of dextran sulfate in buffer in the presence and absence of BSA (1%). open circle , no BSA; , BSA. Superoxide was measured by EPR in the presence of DMPO (160 mM), glutamate/malate (5 mM), and catalase (200 units) in buffer (230 mM mannitol, 70 mM sucrose, 30 mM Tris-HCl, 5 mM KH2PO4, 1 mM ETDA, pH 7.4). Inset, membrane potential was measured using the fluorescent dye TMRM (0.5 mM). Mitochondria were pretreated with antimycin to destroy the potential of the inner membrane.

The effects on either DIDS or dextran sulfate on superoxide generation by antimycin-treated mitochondria in the presence of glutamate/malate was not due to an inhibitory effect on the electron transport chain, for neither compound affected submitochondrial particle respiration supported by complex I (NADH) substrates (data not shown).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The mitochondrial respiratory chain has long been recognized as an effective source of H2O2 originating from the disproportionation of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>. Diffusion of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> from mitochondria is the consequence of two major events: (i) UQ<UP><SUB>O</SUB><SUP>&cjs1138;</SUP></UP> autoxidation to generate O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> into the intermembrane compartment; and (ii) diffusion of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> from the intermembrane space to the cytoplasm through VDAC.

The notion of UQ<UP><SUB>O</SUB><SUP>&cjs1138;</SUP></UP> oxidation as the source of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> in the intermembrane space is supported by the mechanism underlying the effect of the complex III inhibitors antimycin and myxothiazol. The former blocks electron transfer from cytochrome bH to the inner ubiquinone pool (UQI) as seen in,
<UP>UQ<SUB>I</SUB></UP>+<UP>cyt </UP>b<SUB><UP>H</UP></SUB><SUP><UP>2+</UP></SUP>→<UP>UQ</UP><SUB><UP>I</UP></SUB><SUP><UP>&cjs1138;</UP></SUP>+<UP>cyt </UP>b<SUB><UP>H</UP></SUB><SUP><UP>3+</UP></SUP>

<UP><SC>Reaction 5</SC></UP>
thus resulting, through Reaction 6, 
<UP>cyt </UP>b<SUB><UP>H</UP></SUB><SUP><UP>3+</UP></SUP>+<UP>cyt </UP>b<SUB><UP>L</UP></SUB><SUP><UP>2+</UP></SUP>→<UP> cyt </UP>b<SUB><UP>H</UP></SUB><SUP><UP>2+</UP></SUP><UP> + cyt </UP>b<SUB><UP>L</UP></SUB><SUP><UP>3+</UP></SUP>

<UP><SC>Reaction 6</SC></UP>
in decreased cytochrome bL3+ levels and, ultimately, an increase of UQO&cjs1138; level by limiting the rate, as seen in Reaction 7, 
<UP>cyt </UP>b<SUB><UP>L</UP></SUB><SUP><UP>3+</UP></SUP>+<UP>UQ</UP><SUB><UP>O</UP></SUB><SUP><UP>&cjs1138;</UP></SUP> → <UP>cyt </UP>b<SUB><UP>L</UP></SUB><SUP><UP>2+</UP></SUP>+<UP>UQ<SUB>O</SUB></UP>

<UP><SC>Reaction 7</SC></UP>
of the electron transfer (15, 44). The latter, myxothiazol, binds to the QO site of the ubiquinone pool, thereby blocking electron transfer from ubiquinol (UQH2 or its monoanionic form, UQH-) to iron-sulfur clusters (45), as seen in Reaction 8, 
<UP>UQ<SUB>O</SUB>H<SUP>−</SUP></UP>+[<UP>Fe<SUB>2</SUB>S<SUB>2</SUB></UP>]<SUP><UP>ox</UP></SUP>→<UP>UQ</UP><SUB><UP>0</UP></SUB><SUP><UP>&cjs1138;</UP></SUP>+[<UP>Fe<SUB>2</SUB>S<SUB>2</SUB></UP>]<SUP><UP>red</UP></SUP>

<UP><SC>Reaction 8</SC></UP>
resulting in the inhibition of UQ<UP><SUB>O</SUB><SUP>&cjs1138;</SUP></UP> formation (15, 44, 46). The effects of antimycin A and myxothiazol reported here (Fig. 1 and Table I) are consistent with this electron transfer sequence (Reactions 5-8) and strengthen the QO site of the ubiquinone pool in O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> generation by mitochondria.

A role for VDAC in O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> diffusion from the intermembrane space to the cytoplasm is supported by the effect of inhibitors. VDAC is the major channel in the outer membrane responsible for the passage of low molecular weight solutes and proteins between the intermembrane space and the cytoplasm. The concept that VDAC may act as a O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> channel is not entirely surprising, because in erythrocyte membranes an anion channel that allows the diffusion of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> has been previously characterized (47). In this study, two VDAC inhibitors, DIDS and dextran disulfate, decreased O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> production (in a voltage-dependent manner) in isolated heart mitochondria. Although VDAC inhibition by DIDS is nonspecific, inhibition by dextran sulfate in a voltage dependent manner is a feature that has been only observed for VDAC (42). Based on the work with VDAC inhibitors, our data suggest that the opening and closing of VDAC modulates the flux of mitochondrially generated O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> into cytosol. However, the physiological regulators of VDAC are not known.

For topological and structural reasons, the diffusion of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> through VDAC may be an important pathway for removal of this species from the intermembrane space even in the presence of an intermembrane space Cu,Zn-SOD. First, the selectivity of VDAC for anions over cations has been attributed to positively charged amino acids in the core of the channel attracting negatively charged compounds (43). Thus, analogous to the positive charges of copper in Cu,Zn-SOD attracting O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>, positive charges in VDAC may similarly attract O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>. Topologically, the inner membrane where O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> is produced remains close to the VDAC. In areas where the inner membrane and outer membrane are parallel, the mean width of intermembrane space is estimated to be 22 nm (48). At points near the contact junctions, where the inner and outer membrane meet, the inner membrane lies extremely close to the cytoplasm. Mathematical modeling suggests that the mean displacement of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>, generated from the respiratory chain, is 400 nm in the matrix, taking into account the presence of Mn-SOD (49). If the estimated O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> mean displacement is applied to the intermembrane space, O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> generated toward the intermembrane space could diffuse across the outer membrane, especially near the contact junctions.

Interestingly, neither DIDS nor dextran disulfate inhibited O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> production by mitochondria completely. This suggests that either the VDAC inhibitors failed to completely close VDAC, or that O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> diffused through channels in the outer membrane other than VDAC. The latter view is sustained by the occurrence of two other channels in addition to VDAC in the outer membrane, i.e. translocase of the outer membrane (TOM) and the peptide-sensitive channel (PSC), both of which are believed to participate in protein translocation (23). It has been suggested that NADH diffusion through the outer membrane is mediated by TOM when VDAC is inactivated (50). Similarly, VDAC-deficient yeast still remains viable and can grow on nonfermentable carbon sources below 37 °C (50). It has been suggested that TOM might serve as a backup metabolic channel (50, 51) when VDAC loses function.

Diffusion of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> from mitochondria into cytosol is difficult to assess because conventional methods to measure O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>, such as epinephrine oxidation or cytochrome c reduction, lack sensitivity and suffer from interference with mitochondrial enzymic activities. In this study, evidence obtained with spin-trapping EPR, which allows signal accumulation, supports the notion that O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> can be vectorially released from mitochondria into cytosol at basal rates of ~0.041 nmol min-1 mg-1 protein; expectedly, this rate was substantially enhanced (~8-fold) by antimycin A. The basal rate of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> release from mitochondria (0.041 nmol min-1 mg-1 protein) is probably an underestimation and probably represents only a portion of the total O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> that is generated toward the intermembrane space. The levels of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> in this compartment is probably determined by several competing pathways: (i) diffusion to cytosol through VDAC and other outer membrane channels; (ii) reaction with cytochrome c3+ (reaction 3 above; k3 = 2.5 × 105 M-1s-1); and (iii) dismutation to H2O2 that is either spontaneous (k = 4.5 × 105 M-1s-1) or catalyzed by intermembrane space Cu,Zn-SOD (k = 0.62 × 109 M-1s-1). Based on its fast catalytic activity, the latter may represent the prevalent scavenging pathway for O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>. However, although the existence of intermembrane space Cu,Zn-SOD has been demonstrated in liver and yeast (18, 20), its concentration and even its existence in heart mitochondria is, at present, unknown. Because of a modest rate constant, the reaction between cytochrome c and O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> would represent another O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> scavenging pathway in the intermembrane space. However much of cytochrome c is believed to be ionically bound to the inner membrane, limiting its mobility (52) and ability to scavenge O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>.

The physiological and pathophysiological implications for O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> released toward the intermembrane space and cytosol remain to be explored. O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> released into the cytoplasm from mitochondria may play an important role in cell signaling, because O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> has been implicated in several signaling events. In addition, cytoplasmic aconitase and other cytoplasmic enzymes susceptible to O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> may be targets of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> released from mitochondria (53). Another important decay pathway of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> released from mitochondria may involve its reaction (at a diffusion-controlled rate) with nitric oxide (·NO) to yield the peroxynitrite. This O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> decay pathway may be of significance in the intermembrane compartment, because nitrosation of cytochrome c and pro-apoptotic caspases that reside in the intermembrane space have been demonstrated to occur prior to apoptosis by some unidentified mechanism (54). O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> generated toward the intermembrane space may play an important role in nitration of these pro-apoptotic enzymes through ONOO- formation when ·NO is present. In liver, 92% of total superoxide dismutase activity is found in the cytoplasm (55), whereas only 8% is in mitochondria. Cytoplasmic Cu,Zn-SOD may therefore function to protect cytoplasmic proteins from mitochondrial O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> as well as from cytoplasmic O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> sources.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants RO1 AG 16718 and RO1 ES 11342 and a grant from the L. K. Whittier Foundation.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.

|| To whom correspondence should be addressed: School of Pharmacy, University of Southern California, 1985 Zonal Ave., Los Angeles, CA 90089-9121. Tel.: 323-442-1918; Fax: 323-224-7473; E-mail: cadenas@usc.edu.

Published, JBC Papers in Press, December 12, 2002, DOI 10.1074/jbc.M210269200

    ABBREVIATIONS

The abbreviations used are: VDAC, voltage-dependent anion channels; SOD, superoxide dismutase; DMPO, 5,5'-dimethyl-1-pyrroline-N-oxide; CCCP, carbonyl cyanide m-chlorophenylhydrazone; TEMPOL, 4-hydroxy-2,2,6,6-tetramethyl-piperidine-N-oxyl; DIDS, 4,4'-diisothiocyano-2,2' disulfonic acid stilbene; TMRM, tetramethylrhodamine methyl ester; EPR, electron paramagnetic resonance; MOPS, 4-morpholinepropanesulfonic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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