Investigations of in vivo ischemia showed that brains of transgenic mice which overexpressed copper⁄ zinc superoxide dismutase or manganese superoxide Keywords brain mitochondria; hypoxi
Trang 1inner membrane of brain mitochondria exposed to
Lorenz Schild1and Georg Reiser2
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 Ca2+ 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⁄ R in the presence of low micromolar Ca2+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 (H2O2) 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 H2O2 by brain mitochondria metabolizing glutamate plus malate by 80% under normoxic conditions TEMPOL partially protected brain mitochondria exposed to
H⁄ R and low micromolar Ca2+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 Ca2+ 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
Ca2+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.
Trang 2dismutase 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 H2O2 After diffusion
into the cytosol H2O2 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 Ca2+
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 Ca2+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 Ca2+ 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 Ca2+ generate significant amounts of H2O2 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 Ca2+ 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 Ca2+, 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 Ca2+ in an air atmosphere, state 3 respiration
of brain mitochondria was decreased to 81.4 ± 1.2% (n¼ 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; n¼ 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; n¼ 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 Ca2+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 Ca2+(lane 6), and the combination of both (lane 3) induced
Trang 3cytochrome c release, indicating the permeabilization at least of the mitochondrial outer membrane Neither
Ca2+-induced cytochrome c release nor H⁄ R and
Ca2+-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 H2O2 in the incuba-tion medium during H⁄ R performed in the presence or absence of 3.5 lm Ca2+ 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 H2O2 solutions To achieve hypoxic conditions, 1 mL of the incubation medium was bubbled with N2 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 H2O2 concentration in the medium was detected However, significant increase in H2O2 con-centration was seen after reoxygenation in incubations with 3.5 lm Ca2+ Under these conditions, the rate of
H2O2 increase amounted to about 1.22 ± 0.35 pmolÆ
s)1Æmg)1of 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 Ca2+ is associated with oxidative stress, we analysed changes in levels of GSH The expo-sure of brain mitochondria to 3.5 lm Ca2+ 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
Ca2+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 H2O2, as shown in Fig 3A Freshly isolated rat brain mitochon-dria contained 5.73 ± 0.23 nmolÆmg)1of 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
Ca2+, 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 Ca2+; 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 m M
glutamate plus 5 m M malate) and 200 l M ADP were added before
oxygen consumption (state 3 respiration) of mitochondria was
measured Control, 15 min incubation of mitochondria in
air-satur-ated medium; Ca2+, 3.5 l M Ca2+; H ⁄ R, 10 min hypoxia followed by
5 min reoxygenation; ADP, 5 m M ADP; AMP, 5 m M AMP The
res-piration of untreated rat brain mitochondria (100%) corresponds to
71 nmol O 2 min)1Æmg)1 from protein Data represent mean
val-ues ± SEM from five preparations of mitochondria *Significant at
P < 0.05.
Trang 4evaluated the degree by which TEMPOL reduces
extra-mitochondrial H2O2 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 H2O2-dependent oxidation of DCFH in the
presence of horseradish peroxidase was measured For
quantitative analysis, the fluorescence signal was
calib-rated by using H2O2 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
H2O2 production is shown in Fig 4B Considerable
amounts of H2O2 were released into the incubation medium, when 5 mm glutamate and 5 mm malate were added The rates of H2O2 accumulation presented as mean values ± SEM were: before substrate addition, 7.0 ± 1.5 pmol min)1Æmg)1 of mitochondrial protein (n¼ 5); after the addition of 5 mm glutamate and
5 mm malate, 88.2 ± 5.7 pmol min)1Æmg)1 of mitoch-ondrial protein (n ¼ 5); and after subsequent addition
of 10 mm TEMPOL, 21.8 ± 2.1 nmol min)1Æmg)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 H2O2measurements.
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 m M malate or 10 m M TEMPOL were added The rates of H2O2 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
H2O2production and GSH (A) H2O2accumulation was followed as
described When Ca2+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 Ca2+; 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.
Trang 5In 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 Ca2+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 Ca2+
-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
O2Æmin)1Æmg)1; state 3 respiration, 77 ± 7.1 nmol
O2Æmin)1Æmg)1; respiratory control ratio (RCR), 5.49]
Exposure to H⁄ R in the presence of 3.5 lm Ca2+
resul-ted in dramatically decreased state 3 respiration
and decreased RCR value (state 4 respiration:
11.8 ± 1.8 nmol O2Æmin)1Æmg)1; state 3 respiration,
20.6 ± 1.6 nmol O2Æmin)1Æmg)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 O2Æmin)1Æmg)1; state 4 respiration, 14.0 ± 2.8, nmol O2Æmin)1Æmg)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 Ca2+, a substantial stimulation of respiration was observed (from 20.5 ± 1.6 to 77.0 ± 5.9 nmol
O2Æmin)1Æmg)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 O2Æmin)1Æmg)1after 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 Ca2+ and ROS Therefore, we exposed isola-ted rat brain mitochondria to H⁄ R and ⁄ or Ca2+ and determined H2O2 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 Ca2+ increase in combination with H ⁄ R and
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 m M 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
O2Æmin)1Æmg protein)1 To estimate membrane permeability, 5 m M
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 Ca2+; 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.
Trang 6fusion 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 vivomodels 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 H2O2
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 Ca2+ This experimental design mimics the
situation during ischemia⁄ reperfusion in which mitochondria have to endure transient interruption of oxygen supply and increased cytosolic Ca2+ 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 Ca2+ under normoxic conditions (air saturated medium) or by H⁄ R and Ca2+ (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 Ca2+ 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 Ca2+generate
H2O2 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 Ca2+ points towards the induction of oxidative stress Although decrease in GSH may be caused by H2O2-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
Trang 7The increases in cytosolic Ca2+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 H2O2 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 Ca2+ From
the experiments presented here we conclude that
com-plete protection of mitochondria requires additional
prevention of increase in extramitochondrial Ca2+
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 KH2PO4, 0.5 mm EGTA,
60 mm KCl, 60 mm Tris, 110 mm mannitol, 1 mm free
Mg2+ at pH 7.4 and 30C Extramitochondrial calcium
was adjusted by using Ca2+⁄ 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 N2until 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 30C
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 4C, and the resulting superna-tants were centrifuged at 100 000 g for 15 min at 4C 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 H2O2production Extramitochondrial H2O2 peroxide produced by brain mitochondria was estimated by measuring the fluorescence (excitation at 488 nm, emission at 525 nm) caused by the
H2O2-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 H2O2
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
Trang 8grants from Bundesministerium fu¨r Bildung und
Fors-chung (01ZZ0107)
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