Báo cáo khoa học: Oxidative stress is involved in the permeabilization of the inner membrane of brain mitochondria exposed to hypoxia/reoxygenation and low micromolar Ca2+ Lorenz Schild1 and Georg Reiser2 doc

Oxidative stress is involved in the permeabilization of the
inner membrane of brain mitochondria exposed to
hypoxia/reoxygenation and low micromolar Ca
2+
Lorenz Schild
1
and Georg Reiser
2
1 Bereich Pathologische Biochemie des Instituts fu
¨
r Klinische Chemie und Pathologische Biochemie, Otto-von-Guericke-Universita
¨
t
Magdeburg, Germany
2 Institut fu
¨
r Neurobiochemie, Medizinische Fakulta
¨
t, Otto-von-Guericke-Universita
¨
t Magdeburg, Germany
Oxidative stress seems to be involved in the patho-
genesis of neurodegenerative processes such as tissue
infarction resulting from transient ischemia in stroke
[1,2]. Investigations of in vivo ischemia showed that
brains of transgenic mice which overexpressed copper ⁄
zinc superoxide dismutase or manganese superoxide
Keywords
brain mitochondria; hypoxia, calcium;
membrane permeabilization; oxidative stress
Correspondence

L. Schild, Bereich Pathologische Biochemie,
Institut fu
¨
r Klinische Chemie und
Pathologische Biochemie, Medizinische
Fakulta
¨
t, Otto-von-Guericke-Uneversita
¨
t
Magdeburg, Leipziger Strasse 44,
39120 Magdeburg, Germany
Fax: +49 391 67 190176
Tel: +49 391 67 13644
E-mail: Lorenz.Schild@Medizin.
Uni-Magdeburg.de
(Received 23 March 2005, revised 11 May
2005, accepted 19 May 2005)
doi:10.1111/j.1742-4658.2005.04781.x
From in vivo models of stroke it is known that ischemia ⁄ reperfusion indu-
ces oxidative stress that is accompanied by deterioration of brain mito-
chondria. Previously, we reported that the increase in Ca
2+
induces
functional breakdown and morphological disintegration in brain mitochon-
dria subjected to hypoxia⁄ reoxygenation (H ⁄ R). Protection by ADP indica-
ted the involvement of the mitochondrial permeability transition pore in
the mechanism of membrane permeabilization. Until now it has been
unclear how reactive oxygen species (ROS) contribute to this process. We
now report that brain mitochondria which had been subjected to H ⁄ Rin

the presence of low micromolar Ca
2+
display low state 3 respiration (20%
of control), loss of cytochrome c, and reduced glutathione levels (75%
of control). During reoxygenation, significant mitochondrial generation of
hydrogen peroxide (H
2
O
2
) was detected. The addition of the membrane
permeant superoxide anion scavenger TEMPOL (4-hydroxy-2,2,6,6-tetra-
methylpiperidine-N-oxyl) suppressed the production of H
2
O
2
by brain
mitochondria metabolizing glutamate plus malate by 80% under normoxic
conditions. TEMPOL partially protected brain mitochondria exposed to
H ⁄ R and low micromolar Ca
2+
from decrease in state 3 respiration (from
25% of control to 60% of control with TEMPOL) and permeabilization of
the inner membrane. Membrane permeabilization was obvious, because
state 3 respiration could be stimulated by extramitochondrial NADH. Our
data suggest that ROS and Ca
2+
synergistically induce permeabilization of
the inner membrane of brain mitochondria exposed to H ⁄ R. However, per-
meabilization can only partially be prevented by suppressing mitochondrial
generation of ROS. We conclude that transient deprivation of oxygen and

glucose during temporary ischemia coupled with elevation in cytosolic
Ca
2+
concentration triggers ROS generation and mitochondrial permeabili-
zation, resulting in neural cell death.
Abbreviations
CSA, cyclosporin A; DCFH, dichlorofluorescin; GSH, reduced glutathione; GSSH, oxidised glutathione; H ⁄ R, hypoxia ⁄ reoxygenation; mPTP,
mitochondrial permeability transition pore; RCR, respiratory control ratio; ROS, reactive oxygen species; TEMPOL, 4-hydroxy-2,2,6,6-
tetramethylpiperidine-N-oxyl.
FEBS Journal 272 (2005) 3593–3601 ª 2005 FEBS 3593
dismutase were protected against deleterious conse-
quences of stroke [3,4]. Moreover, the increase in lipid
peroxidation after ischemia also points towards the
involvement of oxidative stress [5,6].
Further investigations using animal models of stroke
made it clear that mitochondria are injured during cer-
ebral ischemia and postischemic reperfusion. Decreased
respiratory capacity and changes in ultrastructure of
mitochondria have been reported [7,8]. Cortical neur-
onal mitochondria after transient ischemia showed
condensation, increased matrix density, and deposits
of electron-dense material, and finally disintegration.
It was further shown that mitochondria swell due to
transient focal ischemia, whereas permanent ischemia
causes loss of matrix density [9]. Another mitochond-
rial response to cerebral ischemia is membrane permea-
bilization resulting in the release of mitochondrial
proteins, such as cytochrome c, caspase 9, and SMAC-
Diablo [10,11].
It is conceivable that reactive oxygen species (ROS)

generated outside the mitochondria can cause damage
of these organelles finally resulting in their rupture. In
fact, this pathway has been demonstrated in isolated
mitochondria, using iron ⁄ ascorbate as an external
ROS generating system [12]. Mitochondria themselves
are generators of ROS which may also cause damage.
Up to 2% of the oxygen consumed by mitochondria
can be converted to superoxide anion radicals by the
mitochondrial respiratory chain under reduced condi-
tions, which have been shown to occur when complex
III is blocked with antimycin A. Subsequently, these
superoxide anion radicals are converted by the man-
ganese-superoxide dismutase to H
2
O
2
. After diffusion
into the cytosol H
2
O
2
mediates the damage of cellular
components such as proteins, nucleic acids and lipids.
The generation of ROS by the electron transport chain
has been mainly attributed to complex III [13]. An
additional source of superoxide anion radicals is com-
plex I [14]. The degree of production of ROS by the
respiratory chain depends on the type of tissue, the
kind of substrates driving oxidative phosphorylation
(either complex I-dependent or complex II-dependent

substrates) and the polarization of the mitochondrial
membrane [15,16]. Until now it is unclear how mito-
chondrially generated ROS contribute to mitochond-
rial damage by causing membrane permeabilization
upon brain ischemia ⁄ reperfusion. A second factor
determining mitochondrial damage upon ischemia ⁄
reperfusion is the increase in cytosolic Ca
2+
concentra-
tion, which causes swelling of mitochondria, induction
of ROS generation and functional failure [17,18].
Investigations on isolated mitochondria subjected to
hypoxia ⁄ reoxygenation (H ⁄ R) allow the study of the
effect of single factors such as elevated Ca
2+
and hyp-
oxia on mitochondria [19,20]. In previous work we
showed that in isolated brain mitochondria H ⁄ R in the
presence of low micromolar Ca
2+
concentrations pro-
vokes the permeabilization of the inner membrane [21].
In this study we report that isolated brain mitochon-
dria exposed to H ⁄ R in the presence of low micro-
molar Ca
2+
generate significant amounts of H
2
O
2

during reoxygenation. After this treatment reduced
state 3 respiration with glutamate plus malate as sub-
strate, increased cytochrome c release, and reduced
glutathione (GSH) levels in comparison to normoxic
controls were found. Detoxification of mitochondrially
generated supoxide anion radicals by the membrane
permeant superoxide anion scavenger TEMPOL (4-
hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) resulted
in increased state 3 respiration and reduced membrane
permeabilization. Membrane permeabilization was
measured as stimulation of state 3 respiration by extra-
mitochondrial NADH.
Results
We first investigated the effect of H ⁄ R and extramito-
chondrial Ca
2+
on mitochondrial respiration. There-
fore, isolated rat brain mitochondria were exposed
either to 10 min hypoxia followed by 5 min reoxygena-
tion or to 3.5 lm Ca
2+
, or to the combination of both.
After each treatment, 5 mm glutamate, 5 mm malate,
and 200 lm ADP were added to the incubation to
induce state 3 respiration and oxygen consumption
was analysed. The corresponding values are shown in
Fig. 1. After 15 min of incubation in the presence of
3.5 lm Ca
2+
in an air atmosphere, state 3 respiration

of brain mitochondria was decreased to 81.4 ± 1.2%
(n ¼ 5) of normoxic control. Exposure to 10 min
hypoxia and 5 min reoxygenation also resulted in a
suppression of state 3 respiration (61.8 ± 2.8% of
normoxic control; n ¼ 5). Most impressively, the com-
bination of the two treatments caused tremendous
effects on mitochondrial function measured as state 3
respiration (21.1 ± 3.3% of normoxic control; n ¼ 5).
A high degree of protection was reached in the pres-
ence of 5 mm ADP (84.9 ± 2.7% with ADP; n ¼ 5)
but not of 5 mm AMP during the treatment.
The damage to respiration of brain mitochondria
exposed to H ⁄ R and ⁄ or Ca
2+
was always accompanied
by the release of cytochrome c into the medium (Fig. 2).
Under normoxic control conditions no cytochrome c
was found in the incubation medium (lane 5). In this
case cytochrome c is localized in the intermembrane
space of mitochondria. H ⁄ R (lane 2), 3.5 lm Ca
2+
(lane
6), and the combination of both (lane 3) induced
Mitochondrial permeabilization by transient hypoxia L. Schild and G. Reiser
3594 FEBS Journal 272 (2005) 3593–3601 ª 2005 FEBS
cytochrome c release, indicating the permeabilization at
least of the mitochondrial outer membrane. Neither
Ca
2+
-induced cytochrome c release nor H ⁄ R and

Ca
2+
-induced cytochrome c release was prevented by
cyclosporin A (lane 7 and lane 4, respectively).
To test the possibility that mitochondrially generated
ROS could be involved in the mechanism of membrane
permeabilization, we determined H
2
O
2
in the incuba-
tion medium during H ⁄ R performed in the presence or
absence of 3.5 lm Ca
2+
. The mitochondrial incuba-
tions were carried out in the cuvette of the lumines-
cence spectrophotometer at 30 °C in the presence of
dichlorofluorescin (DCFH) and horseradish peroxi-
dase. Before the experiment, the fluorescence signal
was calibrated using standard H
2
O
2
solutions. To
achieve hypoxic conditions, 1 mL of the incubation
medium was bubbled with N
2
for 5 min. The cuvette
was closed after mitochondria had been added. Reoxy-
genation was performed by opening the cuvette and

adding 1 mL of air saturated medium. In Fig. 3A, a
typical protocol observed with five mitochondrial pre-
parations is shown. In the hypoxic phase no relevant
increase in H
2
O
2
concentration in the medium was
detected. However, significant increase in H
2
O
2
con-
centration was seen after reoxygenation in incubations
with 3.5 lm Ca
2+
. Under these conditions, the rate of
H
2
O
2
increase amounted to about 1.22 ± 0.35 pmolÆ
s
)1
Æmg
)1
of mitochondrial protein. The decrease in the
fluorescence signal recorded at the moment of reoxy-
genation is caused by dilution due to the addition of
1 mL medium.

To investigate whether the generation of ROS by
brain mitochondria during H ⁄ R at low micromolar
extramitochondrial Ca
2+
is associated with oxidative
stress, we analysed changes in levels of GSH. The expo-
sure of brain mitochondria to 3.5 lm Ca
2+
caused a
decrease in GSH of 0.81 ± 0.02 nmolÆmg
)1
of mito-
chondrial protein (Fig. 3B). Hypoxia ⁄ reoxygenation
had a smaller effect on GSH concentration. This treat-
ment resulted in a decrease of 0.29 ± 0.19 nmolÆmg
)1
of mitochondrial protein. The combination of 3.5 lm
Ca
2+
and H ⁄ R led to the substantial decrease in GSH
of 1.27 ± 0.15 nmolÆmg
)1
of mitochondrial protein.
This correlates well with increased amounts of H
2
O
2
,as
shown in Fig. 3A. Freshly isolated rat brain mitochon-
dria contained 5.73 ± 0.23 nmolÆmg

)1
of mitochondrial
protein GSH. The reduction in the GSH concentration
was partially reflected by increased amounts of oxidized
glutathione (GSSG) (data not shown).
To directly test whether mitochondrially derived ROS
are involved in damaging the organelles under H ⁄ R and
Ca
2+
, we used TEMPOL which permeates biological
membranes and scavenges superoxide anions. First, we
Fig. 2. Induction of cytochrome c release from rat brain mitochon-
dria by H ⁄ R and Ca
2+
. Brain mitochondria ( 0.5 mg proteinÆmL
)1
)
were incubated at 30 °C under the conditions indicated: –Ca
2+
,
15 min in incubation medium; Ca
2+
, 15 min in incubation medium
plus 3.5 l
M Ca
2+
;H⁄ R, 10 min hypoxia followed by 5 min reoxy-
genation; CSA, 2 l
M cyclosporin A. A volume of 2 mL of the incu-
bation mixture was centrifuged at 12 000 g for 10 min and the

resulting supernatant at 100 000 g for 15 min at 4 °C. Cyto-
chrome c was detected in the supernatant by western blot analy-
sis. As positive controls 6 ng and 15 ng cytochrome c were applied
to the gel (lane 1 and lane 8, respectively). The western blot pre-
sented is typical for four preparations of mitochondria.
Fig. 1. Effect of H ⁄ R and low micromolar Ca
2+
on state 3 respir-
ation of brain mitochondria. Rat brain mitochondria (0.5 mg pro-
teinÆmL
)1
) were incubated at 30 °C. The substrates (5 mM
glutamate plus 5 mM malate) and 200 lM ADP were added before
oxygen consumption (state 3 respiration) of mitochondria was
measured. Control, 15 min incubation of mitochondria in air-satur-
ated medium; Ca
2+
, 3.5 lM Ca
2+
;H⁄ R, 10 min hypoxia followed by
5 min reoxygenation; ADP, 5 m
M ADP; AMP, 5 mM AMP. The res-
piration of untreated rat brain mitochondria (100%) corresponds to
71 nmol O
2
min
)1
Æmg
)1
from protein. Data represent mean val-

ues ± SEM from five preparations of mitochondria. *Significant at
P < 0.05.
L. Schild and G. Reiser Mitochondrial permeabilization by transient hypoxia
FEBS Journal 272 (2005) 3593–3601 ª 2005 FEBS 3595
evaluated the degree by which TEMPOL reduces extra-
mitochondrial H
2
O
2
accumulation caused by brain
mitochondria metabolizing glutamate plus malate under
normoxic conditions (incubation in air saturated med-
ium). Therefore, the fluorescence of dichlorofluorescein
formed by H
2
O
2
-dependent oxidation of DCFH in the
presence of horseradish peroxidase was measured. For
quantitative analysis, the fluorescence signal was calib-
rated by using H
2
O
2
standard solutions which were
added to the incubation medium. The corresponding
calibration is shown in Fig. 4A. A typical result simi-
larly obtained in five mitochondrial preparations dem-
onstrating the effect of the addition of substrates on
H

2
O
2
production is shown in Fig. 4B. Considerable
amounts of H
2
O
2
were released into the incubation
medium, when 5 mm glutamate and 5 mm malate were
added. The rates of H
2
O
2
accumulation presented as
mean values ± SEM were: before substrate addition,
7.0 ± 1.5 pmol min
)1
Æmg
)1
of mitochondrial protein
(n ¼ 5); after the addition of 5 mm glutamate and
5mm malate, 88.2 ± 5.7 pmol min
)1
Æmg
)1
of mitoch-
ondrial protein (n ¼ 5); and after subsequent addition
of 10 mm TEMPOL, 21.8 ± 2.1 nmol min
)1

Æmg
)1
of
mitochondrial protein (n ¼ 5). The reduction of the
increase in fluorescence intensity by TEMPOL by about
75% demonstrates the power of TEMPOL to effectively
scavenge superoxide anion radicals within the mito-
chondria.
Fig. 4. Effect of substrate supply and TEMPOL on H
2
O
2
generation
by brain mitochondria. (A) Calibration curve of H
2
O
2
measurements.
At the points indicated 88 pmol H
2
O
2
standard solution were added
to 2 mL incubation medium at 30 °C and the fluorescence intensity
(excitation at 488 nm, emission at 525 nm) was monitored. (B) Rat
brain mitochondria (0.5 mgÆmL
)1
) were incubated at 30 °C in incu-
bation medium. At the times indicated 5 m
M glutamate plus 5 mM

malate or 10 mM TEMPOL were added. The rates of H
2
O
2
produc-
tion are given in the text. The data are presented as mean ± SEM
from five mitochondrial preparations.
A
B
Fig. 3. Effect of H ⁄ R and low micromolar Ca
2+
on mitochondrial
H
2
O
2
production and GSH. (A) H
2
O
2
accumulation was followed as
described. When Ca
2+
was present, as indicated, the concentration
was 3.5 l
M. The traces shown are typical for four preparations of
brain mitochondria. (B) GSH concentrations were determined as
described. Incubations were: Ca
2+
, 15 min in incubation medium

plus 3.5 l
M Ca
2+
;H⁄ R+Ca
2+
, 10 min hypoxia followed by 5 min
reoxygenation in the presence of 3.5 l
M Ca
2+
. The data are presen-
ted as mean values ± SEM. *Significant at P < 0.05.
Mitochondrial permeabilization by transient hypoxia L. Schild and G. Reiser
3596 FEBS Journal 272 (2005) 3593–3601 ª 2005 FEBS
In the next series of experiments, we applied 10 mm
TEMPOL to brain mitochondria exposed to 10 min
hypoxia followed by 5 min reoxygenation (H ⁄ R) in the
presence of 3.5 lm Ca
2+
and analysed the respiration.
The data in Fig. 5 are presented as mean values ± SEM
from five mitochondrial preparations. Under control
conditions, that is, incubation of mitochondria in Ca
2+
-
free and air saturated medium in the presence of 5 mm
glutamate and 5 mm malate, oxidative phosphorylation
was well coupled [state 4 respiration, 14.0 ± 1.7 nmol
O
2
Æmin

)1
Æmg
)1
; state 3 respiration, 77 ± 7.1 nmol
O
2
Æmin
)1
Æmg
)1
; respiratory control ratio (RCR), 5.49].
Exposure to H ⁄ R in the presence of 3.5 lm Ca
2+
resul-
ted in dramatically decreased state 3 respiration
and decreased RCR value (state 4 respiration:
11.8 ± 1.8 nmol O
2
Æmin
)1
Æmg
)1
; state 3 respiration,
20.6 ± 1.6 nmol O
2
Æmin
)1
Æmg
)1
; RCR, 1.74). The pres-

ence of 10 mm TEMPOL during H ⁄ R partially preven-
ted the decrease in state 3 respiration (state 3
respiration, 40.4 ± 7.4 nmol O
2
Æmin
)1
Æmg
)1
; state 4
respiration, 14.0 ± 2.8, nmol O
2
Æmin
)1
Æmg
)1
; RCR,
2.88).
The most important experiment which helped us to
understand the mechanism of damage was to study
the sensitivity of state 3 respiration to extramitoch-
ondrial NADH. Thus, we were able to detect permea-
bilization of the inner mitochondrial membrane.
Under control conditions and in the presence of
10 mm TEMPOL, extramitochondrial NADH had no
influence on state 3 respiration (Fig. 5) which is due
to the tight membrane. In contrast, when NADH was
added to mitochondria which had been subjected to
10 min hypoxia followed by 5 min reoxygenation and
3.5 lm Ca
2+

, a substantial stimulation of respiration
was observed (from 20.5 ± 1.6 to 77.0 ± 5.9 nmol
O
2
Æmin
)1
Æmg
)1
), indicating permeabilization of the
membrane system. Even in the additional presence
of 10 mm TEMPOL, significant permeabilization of
the mitochondrial membrane occurred, which was
shown by the NADH sensitivity of state 3 respiration
(40.0 ± 7.4 vs. 84.5 nmol O
2
Æmin
)1
Æmg
)1
after NADH
addition).
Discussion
In animal models of stroke, mitochondria are injured
upon ischemia ⁄ reperfusion. This injury is characterized
by swelling [22] and membrane perturbation which
results in release of cytochrome c [23]. Attempts to
prevent mitochondrial membrane permeabilization in
in vivo models of stroke were only partially successful.
Applying the immunosuppressive compound cyclospo-
rin A which is known to prevent opening of the mito-

chondrial permeability transition pore (mPTP) resulted
in an incomplete protection from neurodegeneration.
Cyclosporin A diminished the size of the infarct, but
was not able to prevent general necrotic cell death [24].
These findings demonstrate, however, the involvement
of mitochondrial membrane permeabilization in the
damage of mitochondria upon ischemia ⁄ reperfusion.
Only in vitro studies on mitochondria allow investiga-
tion of the impact of single factors and their interplay,
such as Ca
2+
and ROS. Therefore, we exposed isola-
ted rat brain mitochondria to H ⁄ R and ⁄ or Ca
2+
and
determined H
2
O
2
concentration, membrane permeabil-
ity, and, as a parameter of mitochondrial function,
oxygen consumption.
Indirect evidence for the suggestion that oxidative
stress may also contribute to permeabilization of the
mitochondrial membrane and subsequently to the
impairment of mitochondria upon ischemia⁄ reper-
Fig. 5. Influence of Ca
2+
increase in combination with H ⁄ Rand
extramitochondrial NADH on respiration of brain mitochondria. Rat

brain mitochondria (0.5 mgÆmL
)1
) were incubated at 30 °C in incu-
bation medium. The substrates (5 m
M glutamate plus 5 mM malate)
were added before oxygen consumption of mitochondria was
measured. Control measurements and H ⁄ R were performed as
described in the Experimental procedures. State 4 respiration was
measured after the addition of substrates. State 3 respiration was
induced by the addition of 200 l
M ADP. The state 3 respiration of
untreated rat brain mitochondria (100%) corresponds to 71 nmol
O
2
Æmin
)1
Æmg protein
)1
. To estimate membrane permeability, 5 mM
NADH was added to the incubations after the state 3 respiration
had been analysed. Designations: H ⁄ R+Ca
2+
± 10 min hypoxia
and 5 min reoxygenation in the presence of 3.5 l
M Ca
2+
; TEMPOL,
10 m
M TEMPOL. Data represent mean values ± SEM from five
preparations of mitochondria. §, Difference between state 4 and

state 3 respiration is significant at P < 0.05; *Difference between
state 3 respiration with and without NADH significant at P < 0.05.
L. Schild and G. Reiser Mitochondrial permeabilization by transient hypoxia
FEBS Journal 272 (2005) 3593–3601 ª 2005 FEBS 3597
fusion comes from in vivo investigations using gene
knock-out animals, which were deficient in the mito-
chondrial antioxidant enzyme manganese superoxide
dismutase. In this model, increased cytochrome c
release was found after ischemia ⁄ reperfusion in com-
parison to wild type animals [25,26]. Remarkable
protection from brain injury was achieved by
administration of metalloporphyrin catalytic antioxi-
dants [27]. However, investigations of the therapeutic
efficacy of antioxidant compounds both in animal
models and humans [28–31] generated contradictory
results. Consequently, the initial enthusiasm for the
use of antioxidants to treat acute brain injury subsi-
ded. As a reason for the failure, the bioavailability of
antioxidants was discussed. However, the protective
effect varied depending on the type of brain ischemia
(focal or global) and the animal species (rat or mouse).
The in vivo application of the Mito Tracker Red
CMH(2)Xros, a rosamine derivative used for the detec-
tion of mitochondrial free radicals, identified mito-
chondria as generators of free radicals primarily in
vulnerable neurons following focal cerebral ischemia
[32]. Another piece of evidence for mitochondrial ROS
production during ischemia ⁄ reperfusion comes from
in vivo models of stroke using hydroethidine oxidation
by superoxide anion radicals [33]. Thus, it may be con-

cluded that mitochondria contribute to the induction
of oxidative stress during ischemia ⁄ reperfusion in
brain.
There is a growing body of information concerning
the mechanism of ROS generation by the respiratory
chain in mitochondria. Complex I and complex III
were identified as generators of superoxide anion radi-
cals in brain mitochondria [34–38]. Depending on the
animal species and incubation conditions, different
fractions for the generation of ROS were attributed to
the two respiratory chain complexes. When succinate
was used as substrate, almost all superoxide anion
radicals are produced in complex I of the respirarory
chain by reversed electron transfer [38]. Although it
seems that the substrate pair glutamate and malate
induces the production of relatively small amounts of
ROS, in our experiments we determined H
2
O
2
genera-
tion by brain mitochondria in the presence of these
electron donors. This is of relevance in brain, because
in this tissue glucose is metabolized providing the
NADH-yielding substrate pyruvate. Under these con-
ditions, some succinate is formed by the citric acid
cycle, oxidizing malate, even in the case of the
NADH-depending substrate supplementation.
In our experiments, we subjected isolated brain
mitochondria to H ⁄ R in the presence of low micro-

molar Ca
2+
. This experimental design mimics the
situation during ischemia ⁄ reperfusion in which
mitochondria have to endure transient interruption of
oxygen supply and increased cytosolic Ca
2+
concentra-
tions. Permeabilization of at least the outer mitoch-
ondrial membrane, indicated by cytochrome c release,
and dramatic functional injury seen as decrease of
ADP-stimulated respiration, are consequences of this
treatment. The high degree of protection by ADP sug-
gests that the mitochondrial injury is caused by open-
ing of the mPTP. There is evidence that ADP inhibits
opening of the mPTP by occupying binding sites
located in the inner and outer mitochondrial mem-
brane [39,40]. The binding of ADP stabilizes the
matrix conformation of the adenine nucleotide translo-
cator which is known to prevent pore opening [36].
The depolarization of the mitochondrial membrane
which occurs within the hypoxic phase and probably
also during reoxygenation supports opening of the
mPTP. It is a particular property of brain mitochon-
dria that opening of the mPTP can happen even in the
presence of CSA [41]. Two different mechanisms seem
to be responsible for the release of cytochrome c
induced either by Ca
2+
under normoxic conditions

(air saturated medium) or by H ⁄ R and Ca
2+
(Fig. 2).
In the first case, only the outer membrane was pemea-
bilized, as reported earlier [42]. This process was not
sensitive to CSA. In contrast, H ⁄ R in combination
with low micromolar Ca
2+
caused CSA insensitive
permeabilization of the inner mitochondrial mem-
brane, indicated by the stimulatory effect of NADH
on state 3 respiration (Fig. 5). In this situation it is
likely that mitochondria swell, which then results in
the rupture of the outer membrane accompanied by
the release of cytochrome c. It had already been shown
that especially in brain, CSA-insensitive permeabiliza-
tion of the mitochondrial membrane may occur
[21,41,43].
We report here that brain mitochondria subjected to
H ⁄ R in the presence of low micromolar Ca
2+
generate
H
2
O
2
during reoxygenation that is released into the
extramitochondrial space. This is in line with in vivo
studies of ischemia ⁄ reperfusion showing increased
ROS production by mitochondria [32,33,44]. The

decreased levels of GSH found after exposure to H ⁄ R
in the presence of low micromolar Ca
2+
points
towards the induction of oxidative stress. Although
decrease in GSH may be caused by H
2
O
2
-dependent
oxidation and ⁄ or by decrease in the reduction of
GSSG due to permeabilization of the mitochondrial
inner membrane, the reduction of GSH concentration
indicates decrease in antioxidative capacity. Conse-
quently, the concentration of free ROS may enhance
oxidative stress.
Mitochondrial permeabilization by transient hypoxia L. Schild and G. Reiser
3598 FEBS Journal 272 (2005) 3593–3601 ª 2005 FEBS
The increases in cytosolic Ca
2+
and ROS concentra-
tion are two essential factors that favour opening of
the mPTP [45]. Thereby oxidation of SH-groups of the
adenine nucleotide translocator stimulates pore open-
ing. We hypothesize that elimination of ROS may
reduce the permeabilization of the mitochondrial
membrane and subsequently exert beneficial effects on
mitochondria. In fact, we were able to demonstrate
that the reduction of H
2

O
2
concentration in the pres-
ence of TEMPOL significantly protected mitochondria
from permeabilization of the inner membrane upon
H ⁄ R in the presence of low micromolar Ca
2+
. From
the experiments presented here we conclude that com-
plete protection of mitochondria requires additional
prevention of increase in extramitochondrial Ca
2+
concentration into the low micromolar range during
H ⁄ R.
Experimental procedures
All chemicals were of analytical grade. 2¢7¢-dichlorofluore-
scin-diacetate and TEMPOL were from Sigma (St. Louis,
MO, USA). Horseradish peroxidase was from Boehringer
(Mannheim, Germany). Mn-SOD, catalase and glutathione
reductase were from Sigma (Deisenhofen, Germany).
Preparation and incubation of brain mitochondria
This work was conducted in accordance with the regula-
tions of the National Act, the use of Experimental Animals
(Germany). Mitochondria were prepared from the brains of
220–240 g male Wistar rats in ice-cold medium containing
250 mm mannitol, 20 mm Tris, 1 mm EGTA, 1 mm EDTA,
and 0.3% (w ⁄ v) BSA at pH 7.4 (isolation medium) by
using a modified standard procedure [21,46]. The mitochon-
dria were well coupled, as indicated by a respiratory control
ratio > 4 with glutamate plus malate as substrates. Protein

content was measured according to the Bradford method
[47] using BSA as the standard. In separate experiments we
determined protein values with the Bradford and Lowry
methods. From the comparison, a correction factor of 1.4
was estimated. This was used to correct the protein values
of the Bradford method.
Mitochondria (0.5–1.0 mg proteinÆmL
)1
) were incubated
in a medium containing 10 mm KH
2
PO
4
, 0.5 mm EGTA,
60 mm KCl, 60 mm Tris, 110 mm mannitol, 1 mm free
Mg
2+
at pH 7.4 and 30 °C. Extramitochondrial calcium
was adjusted by using Ca
2+
⁄ EGTA buffers. For calculating
the concentration of free calcium, we used the complexing
constants according to Fabiato et al. [48].
Hypoxia was produced by bubbling 2 mL of the incuba-
tion medium with N
2
until oxygen was not detectable any
more by means of a Clark electrode, as described previ-
ously [21]. Afterwards, the mitochondria added to the med-
ium further decreased the oxygen concentration via the

respiratory chain until depolarization of the mitochondrial
membrane was reached (not shown). A 2 mL volume of
air-saturated incubation medium was added to achieve
reoxygenation.
Measurement of respiration
Oxygen uptake of the mitochondria was measured at 30 °C
in a thermostat-controlled chamber equipped with a Clark-
type electrode. For the calibration of the oxygen electrode,
the oxygen content of the air-saturated incubation medium
was taken to be 217 nmolÆmL
)1
[49].
Immunoblotting of cytochrome c
A volume of 2 mL of the incubation mixture was centrifuged
at 12 000 g for 10 min at 4 °C, and the resulting superna-
tants were centrifuged at 100 000 g for 15 min at 4 °C. The
supernatants were used for western blot analysis [50].
The mouse anti-(cytochrome c) Ig (PharMingen, Heidelberg,
Germany) was applied in a dilution of 1 : 6000 and the sec-
ondary sheep antimouse horseradish conjugated antibody
(Chemicon, Chandlers Ford, UK) in a dilution of 1 : 3000.
Detection of H
2
O
2
production
Extramitochondrial H
2
O
2

peroxide produced by brain
mitochondria was estimated by measuring the fluorescence
(excitation at 488 nm, emission at 525 nm) caused by the
H
2
O
2
-dependent oxidation of DCFH to the fluorescent
compound dichlorofluorescein in the presence of horserad-
ish peroxidase [51]. Prior to the experiments, DCFH was
obtained from dichlorofluorescin-diacetate by treatment
with alkali. Fluorescence signals were calibrated by measur-
ing the fluorescence changes upon addition of known
amounts of H
2
O
2
.
Determination of glutathione
The measurement of GSH and GSSG was based on the
reaction with 5,5¢-dithio-bis-2-nitrobenzoic acid using a
microplate assay according to Baker et al. [52].
Statistical analysis
Data are presented as mean values ± SEM. The signifi-
cance of differences was checked by using Student’s t-test
of paired values.
Acknowledgements
The expert technical assistance of Mrs R. Widmayer is
greatfully acknowledged. The work was supported by
L. Schild and G. Reiser Mitochondrial permeabilization by transient hypoxia

FEBS Journal 272 (2005) 3593–3601 ª 2005 FEBS 3599
grants from Bundesministerium fu
¨
r Bildung und Fors-
chung (01ZZ0107).
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FEBS Journal 272 (2005) 3593–3601 ª 2005 FEBS 3601