Voltage-dependent Anion Channels Control the Release
of the Superoxide Anion from Mitochondria to Cytosol*
Derick
Han
,
Fernando
Antunes§,
Raffaella
Canali¶,
Daniel
Rettori¶, and
Enrique
Cadenas¶
From the ¶ Department of Molecular Pharmacology and
Toxicology, School of Pharmacy and the
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 |
Several reactions in biological systems
contribute to maintain the steady-state concentrations of superoxide
anion (O
) and hydrogen peroxide
(H2O2). The electron transfer chain of
mitochondria is a well documented source of
H2O2; however, the release of O
from
mitochondria into cytosol has not been unequivocally established. This
study was aimed at validating mitochondria as sources of cytosolic
O
, elucidating the mechanisms underlying the release of
O
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
. 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
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
production from mitochondria by ~55%, thus suggesting
that a large portion of O
exited mitochondria via these channels. These findings are discussed in terms of competitive decay
pathways for O
in the intermembrane space and cytosol as well
as the implications of these processes for modulating cell signaling
pathways in these compartments.
 |
INTRODUCTION |
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,
entails the autoxidation of ubisemiquinone in the respiratory
chain to generate O
, which is released into the mitochondrial
matrix (10, 11). The second step entails the conversion of O
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
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
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
) results in
release of O
into the cytosolic side of the mitochondrial
inner membrane. Of note, O
cannot cross membranes except in
the protonated form (a small fraction of the O
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
generated
toward the respective compartments.
Second, the release of O
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
into the intermembrane space
raises the question of whether O
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
-scavenging pathways (cytochrome
c and the intermembrane space Cu,Zn-SOD) as well as pores
for O
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
can diffuse
(24).
VDAC may act similarly as a channel that allows the movement of
O
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
, elucidating the
mechanisms underlying the release of O
from mitochondria into
cytosol, and assessing the role of outer membrane VDACs in this process.
 |
MATERIALS AND METHODS |
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) (
ex = 550 nm;
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)
(
ex = 320 nm;
em = 400 nm). Hydroethinine
(HE) oxidation to fluorescence ethidine (E+)
(
ex = 470 nm;
em = 590 nm) was used as a
measure of O
(31, 32) in mitochondria suspensions
supplemented with 3 µM hydroethinine at 37 °C.
 |
RESULTS |
Release of Superoxide Anion by Mitochondria--
The release of
O
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
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
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.
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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
in the
intermembrane space. The compartmentalization of this reaction requires
the release of O
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
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
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 ( ).
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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
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. , absence of mitochondria; , mitochondria 0.3 mg of
protein ml 1; , 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.
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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 (
DMPO-OH) could be calculated from
the slope of Fig. 4B. The rate of O
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,
|
(Eq. 1)
|
and
DMPO-OH can be obtained from Equation 2,
|
(Eq. 2)
|
where [DMPO-OH]obs is the DMPO-OH concentration
measured by EPR. Resolution of the differential equation leads to
Equation 3,
|
(Eq. 3)
|
shown above. Using the outlined calculations, O
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
into the cytoplasm, a value that increased to
0.33 nmol/min/mg O
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 = DMPO-OH/kdecay. The
kdecay used was calculated from Fig. 3.
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Table I
O 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 ]/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; ,
glutamate/malate; , succinate plus rotenone plus antimycin; ,
succinate plus rotenone.
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Quantitative Determination of Superoxide Anion Production Using
Redox-sensitive Fluorescent Dyes--
The information on mitochondrial
O
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
is the
stoichiometric precursor of H2O2; Refs. 10 and
39); and (ii) hydroethidine, a dye that reacts specifically with
O
.
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
generated by heart mitochondria was released across the outer membrane.
The rate of O
production, seen below in Equation 4,
|
(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
production,
calculated by EPR, and the rate of O
production, calculated from H2O2, were in good agreement (Table I).
Myxothiazol, which inhibits UQ
formation, inhibited O
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
generation) was
substantially enhanced by the mitochondrial uncoupler CCCP.
O
, but not H2O2, HOCl, or
ONOO
, oxidizes hydroethidine to ethidium
(E+); E+ fluorescence is considered a specific
measure of O
, 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
.
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Table II
Estimation of O 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.
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Control of Superoxide Anion Diffusion through VDAC--
The role
of VDAC in mediating the diffusion of O
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
production is maximal
and the substrate not limiting, were treated with VDAC inhibitors to
assess their effect on mitochondrial O
release. The treatment
of isolated heart mitochondria with DIDS resulted in a
dose-dependent inhibition of O
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).
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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
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
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
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%). , 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.
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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 |
The mitochondrial respiratory chain has long been recognized as an
effective source of H2O2 originating from the
disproportionation of O
. Diffusion of O
from
mitochondria is the consequence of two major events: (i) UQ
autoxidation to generate O
into the intermembrane
compartment; and (ii) diffusion of O
from the intermembrane
space to the cytoplasm through VDAC.
The notion of UQ
oxidation as the source of O
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,
thus resulting, through Reaction 6,
in decreased cytochrome bL3+
levels and, ultimately, an increase of UQO
level by
limiting the rate, as seen in Reaction 7,
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,
resulting in the inhibition of UQ
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
generation by mitochondria.
A role for VDAC in O
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
channel is not entirely surprising, because in erythrocyte membranes an
anion channel that allows the diffusion of O
has been
previously characterized (47). In this study, two VDAC inhibitors, DIDS
and dextran disulfate, decreased O
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
into cytosol. However, the
physiological regulators of VDAC are not known.
For topological and structural reasons, the diffusion of O
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
, positive charges in VDAC may similarly attract
O
. Topologically, the inner membrane where O
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
, generated from the respiratory
chain, is 400 nm in the matrix, taking into account the presence of
Mn-SOD (49). If the estimated O
mean displacement is applied
to the intermembrane space, O
generated toward the
intermembrane space could diffuse across the outer membrane, especially
near the contact junctions.
Interestingly, neither DIDS nor dextran disulfate inhibited O
production by mitochondria completely. This suggests that either the
VDAC inhibitors failed to completely close VDAC, or that O
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
from mitochondria into cytosol is difficult to
assess because conventional methods to measure O
, 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
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
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
that is generated toward the intermembrane space. The
levels of O
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
. 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
would represent another O
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
.
The physiological and pathophysiological implications for O
released toward the intermembrane space and cytosol remain to be
explored. O
released into the cytoplasm from mitochondria may
play an important role in cell signaling, because O
has been
implicated in several signaling events. In addition, cytoplasmic
aconitase and other cytoplasmic enzymes susceptible to O
may
be targets of O
released from mitochondria (53). Another
important decay pathway of O
released from mitochondria may
involve its reaction (at a diffusion-controlled rate) with nitric oxide
(·NO) to yield the peroxynitrite. This O
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
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
as well as from
cytoplasmic O
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.
 |
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