Báo cáo khoa học: Ca2+ rise within a narrow window of concentration prevents functional injury of mitochondria exposed to hypoxia ⁄reoxygenation by increasing antioxidative defence pdf

Ca
2+
rise within a narrow window of concentration
prevents functional injury of mitochondria exposed to
hypoxia ⁄ reoxygenation by increasing antioxidative defence
Lorenz Schild
1
, Frank Plumeyer
1
and Georg Reiser
2
1 Bereich Pathologische Biochemie der Medizinischen Fakulta
¨
t der Otto-von-Guericke-Universita
¨
t Magdeburg, Germany
2 Institut fu
¨
r Neurobiochemie der Medizinischen Fakulta
¨
t der Otto-von-Guericke-Universita
¨
t Magdeburg, Germany
It has been shown in animal models that transient isch-
aemia in liver results in mitochondrial damage. The
involvement of oxidative stress in the impairment of the
organelle was demonstrated by the finding that glutathi-
one (GSH) exerts a protective role [1]. A hallmark of
ischaemia ⁄ reperfusion in liver is a significant increase
in cytosolic and mitochondrial Ca
2+

concentration [2].
Oxidative stress and increase in the cytosolic Ca
2+
concentration favour opening of the mitochondrial
permeability transition pore (MPTP) mediating mito-
chondrial damage. In fact, cyclosporin A (CSA), a speci-
fic inhibitor of MPTP, has been demonstrated to
prevent mitochondrial and liver dysfunction in the re-
perfusion phase [3,4]. Long-lasting ischaemia in liver
was shown to induce cytochrome c release and necrosis,
whereas short ischaemia with reperfusion results in the
release of cytochrome c and apoptosis [5].
A further factor determining the outcome after liver
ischaemia ⁄ reperfusion is nitric oxide (NO). However,
reports about the effect of NO on mitochondrial and
tissue damage are still controversial. Using either exo-
genous NO donors, or endogenous NO precursors or
inhibitors of NO synthesis, protective [6,7] as well as
harmful effects [8,9] have been found with in vivo
models of liver ischaemia.
Investigations on isolated liver mitochondria have
clearly shown that extramitochondrial Ca
2+
, reactive
oxygen species (ROS), and NO, which are known to
change in concentration during ischaemia ⁄ reperfusion,
affect mitochondria. Elevation of Ca
2+
concentrations
Keywords

glutathione peroxidase; mitochondrial
permeability transition pore; manganese
super oxide dismutase; nitric oxide;
oxidative stress
Correspondence
L. Schild, Bereich Pathologische Biochemie
der Medizinischen Fakulta
¨
t der Otto-von-
Guericke-Universita
¨
t Magdeburg, Leipziger
Str. 44, 39120 Magdeburg, Germany
Tel: +49 0931 6713644
Fax: +49 0931 6719176
E-mail: lorenz.schild@medizin.
uni-magdeburg.de
(Received 18 July 2005, revised 15 September
2005, accepted 19 September 2005)
doi:10.1111/j.1742-4658.2005.04978.x
Injury of liver by ischaemia crucially involves mitochondrial damage. The
role of Ca
2+
in mitochondrial damage is still unclear. We investigated the
effect of low micromolar Ca
2+
concentrations on respiration, membrane
permeability, and antioxidative defence in liver mitochondria exposed to
hypoxia ⁄ reoxygenation. Hypoxia ⁄ reoxygenation caused decrease in state 3
respiration and in the respiratory control ratio. Liver mitochondria were

almost completely protected at about 2 lm Ca
2+
. Below and above 2 lm
Ca
2+
, mitochondrial function was deteriorated, as indicated by the
decrease in respiratory control ratio. Above 2 lm Ca
2+
, the mitochondrial
membrane was permeabilized, as demonstrated by the sensitivity of state 3
respiration to NADH. Below 2 lm Ca
2+
, the nitric oxide synthase inhib-
itor nitro-l-arginine methylester had a protective effect. The activities of
the manganese superoxide dismutase and glutathione peroxidase after
hypoxia showed maximal values at about 2 lm Ca
2+
. We conclude that
Ca
2+
exerts a protective effect on mitochondria within a narrow concentra-
tion window, by increasing the antioxidative defence.
Abbreviations
CSA, cyclosporin A; GPx, glutathione peroxidase; GSH, reduced glutathione; Mn-SOD, manganese superoxide dismutase; MPTP,
mitochondrial permeability transition pore;
L-NAME, nitro-L-arginine methylester; NO, nitric oxide; RCR, respiratory control ratio; ROS,
reactive oxygen species; TPP
+
, tetraphenyl phosphonium cation.
5844 FEBS Journal 272 (2005) 5844–5852 ª 2005 The Authors Journal Compilation ª 2005 FEBS

into the low micromolar range causes reduction of state
3 respiration [10] and permeabilization of the mito-
chondrial membrane [11–16]. ROS can impair function
and integrity of mitochondria [17]. NO controls the
electron flow through the respiratory chain by compet-
itive inhibition of cytochrome oxidase [18].
There is a body of evidence showing that mitochon-
dria are equipped with a constitutive and Ca
2+
sensi-
tive NO synthase [19,20]. Thus, NO generated by
mitochondria may influence oxidative phosphorylation.
Moreover, mitochondria are major producers of super-
oxide anion radicals within the respiratory chain in
hepatocytes which in the presence of NO allow the
formation of the highly reactive peroxynitrite. Peroxy-
nitrite is known to contribute to functional damage of
components of the mitochondrial electron transport
chain [21]. Hypoxia ⁄ reoxygenation, an important com-
ponent of ischaemia ⁄ reperfusion, leads to the impair-
ment of isolated rat liver mitochondria with the
involvement of oxidative stress [22]. In our previous
work, we demonstrated that mitochondrially derived
NO is significantly involved in the deterioration of iso-
lated liver mitochondria upon hypoxia ⁄ reoxygenation
[23]. In this way, mitochondria may exacerbate their
own injury upon ischaemia ⁄ reperfusion. Although the
effect of single factors on mitochondria, such as ele-
vated extramitochondrial Ca
2+

concentration, hypo-
xia ⁄ reoxygenation, and NO, have been elucidated,
their interplay during ischaemia ⁄ reperfusion is still
poorly understood.
Here, we subjected isolated rat liver mitochondria to
hypoxia and reoxygenation in combination with extra-
mitochondrial Ca
2+
concentrations up to 5 lm. After-
wards, we determined state 3 and state 4 respiration
with glutamate and malate as substrates. We also meas-
ured membrane permeability, and the activities of the
antioxidative enzymes glutathione peroxidase (GPx)
and manganese superoxide dismutase (Mn-SOD). Addi-
tionally, the effect of permanent inhibition of mito-
chondrial NO synthesis by nitro-l-arginine methylester
(l-NAME) on respiration upon hypoxia ⁄ reoxygenation
in combination with elevated Ca
2+
concentration was
investigated. We found that within a narrow concentra-
tion range at around 2 lm extramitochondrial Ca
2+
,
mitochondria were almost completely protected against
decrease in active respiration and increase in membrane
permeability. The activities of the antioxidative enzymes
GPx and Mn-SOD were stimulated by hypoxia ⁄ reoxy-
genation and increase in Ca
2+

concentration, displaying
maximal values at a concentration of about 2 lm.At
this Ca
2+
concentration, the inhibition of NO synthesis
with l-NAME did not affect state 3 respiration. From
these data we conclude that extramitochondrial Ca
2+
at
a narrow concentration window exerts a protective
effect upon hypoxia ⁄ reoxygenation by increasing
the activity of antioxidative enzymes in liver mito-
chondria.
Results
Ca
2+
affects respiration and membrane
permeability upon hypoxia ⁄ reoxygenation
In liver ischaemia⁄ reperfusion the cytosolic Ca
2+
con-
centration in hepatic cells is elevated into the low micro-
molar range. In order to investigate the influence of
extramitochondrial Ca
2+
on the impairment of mito-
chondria by ischaemia ⁄ reperfusion, isolated rat liver
mitochondria were subjected to 5 min hypoxia followed
by 10 min reoxygenation in the continuous presence of
Ca

2+
at concentrations varying from 0.2 up to 4.4 lm.
Rates of respiration were determined after hypoxia ⁄
reoxygenation with 5 mm glutamate and 5 mm malate
as substrates. Transient hypoxia in the presence of
0.2 lm Ca
2+
caused decrease in state 3 respiration to
45% of the normoxic control value (incubation in air
saturated medium). The influence of Ca
2+
on state 3
respiration obtained with hypoxia ⁄ reoxygenation was
characterized by a bell-shaped concentration depend-
ence (Fig. 1A, upper part). Increasing the Ca
2+
concen-
tration improved state 3 respiration. Almost complete
protection was seen at 2 lm extramitochondrial Ca
2+
(91% of normoxic mitochondria). This was not
observed when Ca
2+
uptake was inhibited by 10 lm
ruthenium red (data not shown). Further increase in
extramitochondrial Ca
2+
concentration resulted in
decreased rates of state 3 respiration measured after
hypoxia ⁄ reoxygenation. At the maximally used concen-

tration of 4.4 lm Ca
2+
, no stimulation of oxygen con-
sumption by ADP could be reached. The respiration
determined in the absence of ADP (state 4) had no clear
Ca
2+
dependence (lower part in Fig. 1A). In order to
test whether the effect of hypoxia ⁄ reoxygenation and
elevated Ca
2+
on state 3 respiration was due to opening
of the MPTP, isolated rat liver mitochondria were
exposed to hypoxia ⁄ reoxygenation and Ca
2+
in the
additional presence of 2 lm of the MPTP inhibitor
CSA. At this concentration, CSA completely prevented
Ca
2+
-induced swelling of liver mitochondria (data not
shown). CSA partially protected liver mitochondria
against decrease in state 3 respiration at Ca
2+
concen-
trations below and above 2 lm (Fig. 1A, upper part).
Within the narrow concentration range at around 2 lm
extramitochondrial Ca
2+
, no effect of CSA was

observed. The rates of state 4 respiration were slightly
higher in CSA-containing incubations in comparison to
L. Schild et al. Ca
2+
protects mitochondria during hypoxia
FEBS Journal 272 (2005) 5844–5852 ª 2005 The Authors Journal Compilation ª 2005 FEBS 5845
CSA-free incubations. At 2 lm extramitochondrial
Ca
2+
both values were equal (Fig. 1A, lower part).
In order to evaluate precisely the functional injury
of mitochondria, the coupling of oxidative phosphory-
lation was quantified by calculating respiratory con-
trol ratios (RCR), which are given by the ratios of
state 3 and state 4 respiration. The resulting data are
presented in Fig. 1B. Highest RCR values were found
between 1 and 2 lm extramitochondrial Ca
2+
indica-
ting a protective Ca
2+
concentration range. Below
and above this concentration range, loss of mito-
chondrial coupling was observed. Inhibition of pore
opening by CSA had no significant effect on RCR
over the whole Ca
2+
concentration range investigated.
The RCR of freshly isolated mitochondria was
6.4 ± 0.7 (n ¼ 12).

To investigate the possibility of a CSA-insensitive
permeabilization of the mitochondrial membrane upon
hypoxia ⁄ reoxygenation and Ca
2+
, we used a different
approach. The membrane impermeable pyridine
nucleotide NADH (5 mm) and cytochrome c (10 lm)
were added to mitochondria respiring under state 3
conditions. Both compounds have no effect on state 3
respiration in intact mitochondria. However, permeabi-
lization of the membrane allows access of NADH and
cytochrome c to the respiratory chain resulting in sti-
mulation of state 3 respiration. The relative changes of
state 3 respiration without and with NADH addition
plus cytochrome c, measured after exposure of mito-
chondria to hypoxia ⁄ reoxygenation and various Ca
2+
concentrations, is depicted in Fig. 1C. Up to 2 lm
extramitochondrial Ca
2+
, no stimulation of state 3 res-
piration by NADH plus cytochrome c was observed,
documenting the tightness of the mitochondrial mem-
brane. Even in the presence of 2 lm CSA, elevation of
the Ca
2+
concentration from 2 lm to 4.4 lm was par-
alleled by an increase in the ratio of state 3 respiration
without and with NADH addition plus cytochrome c
clearly. This indicates permeabilization of the mito-

chondrial membrane. The permeabilization (Fig. 1C)
was associated with loss of mitochondrial function
(Fig. 1B).
Fig. 1. Influence of cyclosporin A on the Ca
2+
sensitivity of res-
piration upon hypoxia ⁄ reoxygenation. Rat liver mitochondria
(1 mgÆmL
)1
) were subjected to 5 min hypoxia and 10 min reoxy-
genation with and without 2 l
M CSA in the presence of various
Ca
2+
concentrations at 30 °C. Afterwards, respiration was deter-
mined in the presence of 5 m
M glutamate, 5 mM malate either
without (state 4) or with 200 l
M ADP (state 3). Subsequently 5 mM
NADH and 10 lM cytochrome c were added to demonstrate per-
meabilization of the mitochondrial membrane. The rates of respir-
ation (A), RCR (B) and the ratio of the rates of state 3 respiration
before and after the addition of NADH and cytochrome c (C) are
presented. The rate of state 3 respiration of freshly isolated mito-
chondria was 82.3 ± 6.8 nmol O
2
Æmg
)1
Æmin
)1

. Data are presented
as mean ± SEM of five mitochondrial preparations.
Ca
2+
protects mitochondria during hypoxia L. Schild et al.
5846 FEBS Journal 272 (2005) 5844–5852 ª 2005 The Authors Journal Compilation ª 2005 FEBS
Ca
2+
and hypoxia ⁄ reoxygenation regulate
antioxidative activity in liver mitochondria
In our previous work we have shown that hypoxia ⁄
reoxygenation induces oxidative stress indicated by the
formation of protein carbonyls (marker of oxidative
protein modification) which depends on the Ca
2+
concentration [10]. To investigate how Ca
2+
modulates
oxidative stress during hypoxia ⁄ reoxygenation, we
measured the activity of the Mn-SOD in normoxic incu-
bation and after hypoxia ⁄ reoxygenation. The enzyme
activity in the normoxic incubation was maximal in the
presence of 2 lm extramitochondrial Ca
2+
(Fig. 2). At
0.2 lm Ca
2+
, lower activity of Mn-SOD was found (73
at 0.2 lm Ca
2+

vs. 118 unitsÆmg
)1
at 2 lm Ca
2+
). Sim-
ilar results were obtained with Mn-SOD from bovine
erythrocytes. In the presence of 2 lm Ca
2+
, the enzyme
activity was increased from 0.134 ± 0.021 unitsÆ mg
)1
(at 0.2 lm Ca
2+
) to 1.851 ± 0.056 unitsÆmg
)1
. This sti-
mulation could be reversed by the addition of 2 mm
EGTA. The activity of the enzyme determined after
hypoxia ⁄ reoxygenation also reached a maximum value
in the presence of 2 lm Ca
2+
, but was significantly
higher than in a normoxic incubation (157 unitsÆmg
)1
after hypoxia⁄ reoxygenation vs. 118 unitsÆmg
)1
without
hypoxia ⁄ reoxygenation). Increase in the extramito-
chondrial Ca
2+

concentration from 0.2 to 2 lm resulted
in a 2.6-fold increase in the activity of Mn-SOD,
whereas in normoxic incubations the elevation was only
1.6-fold. Thus, the combination of hypoxia⁄ reoxygena-
tion and 2 lm extramitochondrial Ca
2+
caused a con-
siderable increase in the activity of this antioxidative
defence enzyme.
In a further series of experiments we investigated
whether the activity of a second antioxidative enzyme,
that is GPx, is sensitive to hypoxia ⁄ reoxygenation and
extramitochondrial Ca
2+
. The activity of this enzyme
didnotdependontheextramitochondrialCa
2+
concentra-
tion in the low micromolar range under normoxic con-
ditions (Fig. 3, lower part). In Ca
2+
-free incubations,
the activity of GPx was double after 5 min hypoxia fol-
lowed by 10 min reoxygenation (819 vs. 405 unitsÆmg
)1
at 0.2 lm extramitochondrial Ca
2+
). The activity
determined after hypoxia ⁄ reoxygenation was slightly
Fig. 3. Influence of hypoxia ⁄ reoxygenation and Ca

2+
on the activity
of glutathione peroxidase (GPx). Rat liver mitochondria (1 mgÆmL
)1
)
were either incubated at various Ca
2+
concentrations and 5 mM glu-
tamate plus 5 m
M malate in the incubation medium or were subjec-
ted to 5 min hypoxia and 10 min reoxygenation in the presence of
various Ca
2+
concentrations at 30 °C. After the reoxygenation period
5m
M glutamate and 5 mM malate were added. For the determin-
ation of GPx activity, 500 lL samples were withdrawn from the
incubations. The data are presented as mean ± SEM from five
preparations of mitochondria. The differences between GPx activities
of incubations with and without hypoxia ⁄ reoxygenation in the pres-
ence of similar Ca
2+
concentrations were significant with P < 0.01.
Fig. 2. Change of Ca
2+
-sensitivity of Mn-SOD activity by hypoxia ⁄
reoxygenation. Rat liver mitochondria (1 mgÆmL
)1
) were either incu-
bated at various Ca

2+
concentrations and 5 mM glutamate plus
5m
M malate in the incubation medium or were subjected to 5 min
hypoxia and 10 min reoxygenation in the presence of various Ca
2+
concentrations at 30 °C. After the reoxygenation period 5 mM
glutamate and 5 mM malate were added. For the determination of
Mn-SOD activity, 500 lL samples were withdrawn from the incuba-
tions. The data are presented as mean ± SEM from five prepara-
tions of mitochondria. Additional student’s t-test analysis gave a
significant difference in Mn-SOD activities between the values at
0.1 and 2.0 l
M Ca
2+
(P < 0.01), both without and with hypoxia ⁄
reoxygenation. *Differences in Mn-SOD activities of incubations
with and without hypoxia ⁄ reoxygenation were significant with
P < 0.05.
L. Schild et al. Ca
2+
protects mitochondria during hypoxia
FEBS Journal 272 (2005) 5844–5852 ª 2005 The Authors Journal Compilation ª 2005 FEBS 5847
sensitive to Ca
2+
with the tendency to reach the highest
levels between 1 and 2 lm extramitochondrial Ca
2+
.
Alternatively to the formation of H

2
O
2
by the
Mn-SOD reaction, superoxide anion radicals produced
within the respiratory chain can react with mitochond-
rially generated NO to form the highly reactive per-
oxynitrite. In order to estimate the involvement of NO
and ⁄ or peroxynitrite in the impairment of mitochond-
rial function by hypoxia ⁄ reoxygenation and extra-
mitochondrial Ca
2+
, we studied to what degree the
inhibition of NO synthesis by l-NAME affects respir-
ation measured after 5 min hypoxia followed by
10 min reoxygenation. We could not find any signifi-
cant difference in state 4 respiration by comparing
incubations without and with l-NAME. Therefore,
only the rates of state 3 respiration are depicted in
Fig. 4. The continuous presence of l-NAME during
hypoxia ⁄ reoxygenation performed in the presence of
0.2 lm extramitochondrial Ca
2+
(low Ca
2+
concentra-
tion) partially protected rat liver mitochondria against
decrease in state 3 respiration (72 ± 4.4 vs.
39 ± 3.1 nmol O
2

Æmin
)1
Æmg
)1
). At this Ca
2+
concen-
tration, the continuous presence of 50 lm haemoglobin
was also protective (78 ± 3.9 vs. 39 ± 3.1 nmol O
2
Æ
min
)1
Æmg
)1
). When mitochondria were subjected to
hypoxia ⁄ reoxygenation in the presence of 2 lm extra-
mitochondrial Ca
2+
(protective Ca
2+
concentration),
l-NAME did not affect respiration. Likewise, at
4.4 lm Ca
2+
, inhibition of enzymatic NO synthesis
during hypoxia ⁄ reoxygenation did not lead to a change
in state 3 respiration (high Ca
2+
concentration).

Discussion
Extramitochondrial Ca
2+
can amplify or attenuate
the impairment of liver mitochondria by
hypoxia ⁄ reoxygenation
Isolated mitochondria have been successfully used to
study the effect of distinct factors impairing mitochon-
dria which are relevant in pathophysiological situations
such as ischaemia ⁄ reperfusion [24–26]. Both the in vivo
studies of ischaemia and the cell culture investigation
on hypoxia ⁄ reoxygenation require a relatively long
period of hypoxia to achieve significant injury. How-
ever, in isolated mitochondria a few minutes of
hypoxia are sufficient to cause dramatic damage.
Differences in local oxygen concentration may be the
reason for this different time required to reach injury
either in vivo or in isolated mitochondria. We have
found that at elevated extramitochondrial Ca
2+
con-
centrations, ADP at physiological concentration
protects mitochondria from hypoxia ⁄ reoxygenation-
induced damage [27,28]. Only when all the ADP is
converted into AMP, mitochondrial damage occurs.
This finding may contribute to the fact that longer
periods of ischaemia are required to achieve damage in
tissue, in comparison with results obtained with iso-
lated mitochondria, which have to be exposed only for
a short period of time to hypoxia in order to induce

damage.
In previous papers we reported that hypoxia ⁄ reoxy-
genation reduces state 3 respiration in isolated rat liver
mitochondria [22] and that extramitochondrial Ca
2+
in
the low micromolar range modulates mitochondrial
damage and oxidative stress [10]. Now, by testing the
action of CSA and measuring RCR we show that open-
ing of the MPTP is not significantly involved in func-
tional impairment of liver mitochondria exposed to
hypoxia ⁄ reoxygenation and Ca
2+
. This is surprising as
increases in extramitochondrial Ca
2+
concentration
and oxidative stress are known to be major factors for
increasing the probability for pore opening [29–33].
Reasons for the CSA-independent injury of mitochond-
rial function might be the increase in oxidative stress
below and above 2 lm Ca
2+
as demonstrated earlier
[10], possibly causing damage to respiratory chain com-
plexes, and ⁄ or CSA-insensitive permeabilization of the
mitochondrial membrane. In fact, CSA-insensitive per-
meabilization of the mitochondrial membrane was
found after hypoxia ⁄ reoxygenation in the presence of
Ca

2+
concentrations higher than 2 lm (Fig. 1C).
Fig. 4. Modulation of the effect of NO on state 3 respiration after
hypoxia ⁄ reoxygenation by extramitochondrial Ca
2+
. Rat liver mito-
chondria (1 mgÆmL
)1
) were subjected to 5 min hypoxia and 10 min
reoxygenation with and without 10 m
ML-NAME in the continuous
presence of either 0.2 l
M,2lM or 4.4 lM extramitochondrial Ca
2+
at 30 °C. Afterwards, 5 mM glutamate, 5 mM malate and 200 lM
ADP were added to stimulate state 3 respiration. Data are presen-
ted as mean ± SEM of five preparations of mitochondria. *State 3
respiration with and without
L-NAME is different with P < 0.05
according to Student’s t-test.
Ca
2+
protects mitochondria during hypoxia L. Schild et al.
5848 FEBS Journal 272 (2005) 5844–5852 ª 2005 The Authors Journal Compilation ª 2005 FEBS
Ca
2+
affects the balance between oxidative and
antioxidative processes during hypoxia⁄
reoxygenation
As we have demonstrated earlier, hydrogen peroxide

which is formed from superoxide anion radicals accu-
mulates during reoxygenation in the presence of high
Ca
2+
concentration [28]. However, the protection
of mitochondria from hypoxia ⁄ reoxygenation-induced
damage and the low amount of protein carbonyls [10]
at 2 lm Ca
2+
suggest relatively low superoxide radical
concentration. This is consistent with our finding of
considerably increased activity of the Mn-SOD at this
Ca
2+
concentration. At 2 lm Ca
2+
, no protection of
state 3 respiration was seen during hypoxia⁄ reoxygena-
tion in the presence of ruthenium red (data not
shown). Therefore, it can be concluded that Ca
2+
has
to enter the mitochondrial matrix in order to cause
increase in Mn-SOD activity. Both Ca
2+
and hypox-
ia ⁄ reoxygenation synergistically contribute to this
effect. Under these conditions, protein levels of the
Mn-SOD remained unchanged as determined by west-
ern blot analysis (data not shown). This is not surpri-

sing, as protein synthesis of this enzyme takes place
within the cytosolic compartment [34]. Thus chemical
modification is responsible for the change in the activ-
ity of the enzyme. It has been shown that inactivation
of the enzyme may result from tyrosine nitration by
peroxynitrite [35]. In the in vitro model of isch-
aemia ⁄ reperfusion applied here we did not observe
decrease in the activity of Mn-SOD. Instead, increase
in activity was found. The mechanism by which Ca
2+
in combination with hypoxia ⁄ reoxygenation mediates
this antioxidant effect still remains unclear. It might be
speculated that interaction of Ca
2+
with components
of the active site as well as oxidation of certain amino
acids in the catalytic domain may modulate enzymatic
activity. It has been shown that replacement of His30
with Asn30 resulted in dramatic decrease of enzyme
activity, because it did not participate in the hydrogen
bond network of the active site [36]. Therefore, it is
reasonable to assume that metal ions and ROS may
modify the active site of the enzyme leading to changes
in enzyme activity.
Similarly, the activity of glutathione peroxidase was
affected by hypoxia ⁄ reoxygenation. However, only a
slight Ca
2+
dependence was observed. As the enzyme
is synthesized within the cytosolic compartment, modi-

fication of the enzyme should be responsible for
increase in the activity caused by hypoxia ⁄ reoxygena-
tion. The mechanism by which hypoxia ⁄ reoxygenation
causes increase in the activity of mitochondrial gluta-
thione peroxidase still has to be elucidated.
In our previous study [10] we have demonstrated
that increasing the extramitochondrial Ca
2+
concen-
tration above 2 lm caused high levels of protein car-
bonyls indicating a high degree of oxidative stress. On
the other hand, we here report high activities of
Mn-SOD and GPx in this range of Ca
2+
concentra-
tion. The CSA-insensitive permeabilization of the mito-
chondrial membrane occurring under this condition
(Fig. 1C) may explain the apparent discrepancy. Per-
meabilization of the membrane is known to be associ-
ated with energetic failure and efflux of mitochondrial
constituents such as glutathione. Consequently, the
antioxidative defence decreases.
At Ca
2+
concentrations lower than 2 lm, impair-
ment of oxidative phosphorylation by hypoxia ⁄ reoxy-
genation was caused by oxidative stress as high
amounts of protein carbonyls were determined [10].
This fits well with our observation that in this range
of Ca

2+
concentration relatively low activities of
Mn-SOD and GPx were determined (Figs 2 and 3).
Thereby, the mitochondrial respiratory chain is the
source of reactive superoxide anion radicals generated
within the complexes I and III.
In experiments with isolated rat liver mitochondria,
we have earlier demonstrated that NO accumulates
during hypoxia ⁄ reoxygenation [23]. Subsequently, the
highly reactive peroxynitrite can be formed which is
known to injure components of the respiratory chain.
It has been demonstrated that peroxynitrite is
involved in Ca
2+
-induced impairment of liver mito-
chondria [31,33]. Thus, the protective effect of the inhi-
bition of NO synthesis at 0.2 lm Ca
2+
demonstrated
here (Fig. 4) could be mainly attributed to the dimin-
ished peroxynitrite formation. In contrast, at 2 lm
Ca
2+
, superoxide anion radical concentration, but not
NO production, is reduced, as the increase in Ca
2+
concentration stimulates NO synthesis. Here, no effect
of l-NAME on state 3 respiration was seen.
Conclusions
We used an in vitro model of ischaemia ⁄ reperfusion of

liver to study the effect of hypoxia ⁄ reoxygenation in
combination with elevated extramitochondrial Ca
2+
concentration into the nonphysiological concentration
range up to 4.4 lm. In this model we were able to clarify
how mitochondrially generated ROS, NO and permea-
bilization of the mitochondrial membrane are involved
in mitochondrial damage. Our data demonstrate that
hypoxia ⁄ reoxygenation and extramitochondrial Ca
2+
cause functional damage of isolated rat liver mitochon-
dria. Essential steps involved in the cascade of mito-
chondrial injury are CSA-insensitive permeabilization
L. Schild et al. Ca
2+
protects mitochondria during hypoxia
FEBS Journal 272 (2005) 5844–5852 ª 2005 The Authors Journal Compilation ª 2005 FEBS 5849
of the mitochondrial membrane, production of ROS,
generation of NO and peroxynitrite by mitochondria.
We have found a distinct extramitochondrial Ca
2+
con-
centration range around 2 lm, in which isolated rat liver
mitochondria are almost completely protected against
decrease in oxidative phosphorylation. Similar effects
were also found in brain and heart mitochondria (data
not shown). There was a clear correlation of increase in
the activities of Mn-SOD, GPx, and insensitivity of inhi-
bition of NO-synthesis at the 2 lm Ca
2+

concentration.
Thus, we conclude that superoxide anion radicals and
peroxynitrite play a pivotal role in damaging mitochon-
dria upon hypoxia ⁄ reoxygenation at Ca
2+
concentra-
tions up to about 2 lm. At higher Ca
2+
concentrations,
the mechanism underlying the mitochondrial injury is
the permeabilization of the membrane. As elevation of
extramitochondrial Ca
2+
concentration into the low
micromolar range appears to have protective effects,
further investigations on the role of Ca
2+
in hypoxia⁄
reoxygenation at the cellular level and in in vivo studies
of ischaemia should be performed.
Experimental procedures
Reagents
Cyclosporin A, l-NAME and xanthin oxidase were pur-
chased from Sigma (Deisenhofen, Germany). All other
chemicals were of analytical grade.
Preparation of mitochondria
Liver mitochondria were prepared from 220 to 240 g male
Wistar rats in ice-cold medium containing 25 mm sucrose,
20 mm Tris (pH 7.4), 2 mm EGTA, and 1% (w ⁄ v) bovine
serum albumin using a standard procedure [37]. After the

initial isolation, Percoll was used for purification of mito-
chondria from a fraction containing some endoplasmatic
reticulum, Golgi apparatus and plasma membranes [38].
This work was conducted in accordance with the regula-
tions of the National Act for the use of Experimental
Animals (Germany).
Incubation of mitochondria
Mitochondria (1–2 mg proteinÆmL
)1
) were incubated in a
medium containing 10 mm sucrose, 120 mm KCl, 20 mm
Tris, 15 mm potassium phosphate, 0.5 mm EGTA and
1mm free Mg
2+
at pH 7.4. Extramitochondrial Ca
2+
con-
centrations were adjusted by using Ca
2+
⁄ EGTA buffers.
After preparation of the buffers, the free Ca
2+
concentra-
tion was checked by means of a Ca
2+
-selective electrode.
The actual concentration of Ca
2+
in the incubations was
calculated considering the complex formation with other

constituents of the medium such as Mg
2+
and adenine
nucleotides. For the calculation, the complexing constants
were used according to Fabioto et al. [39]. Hypoxia was
produced by bubbling 2 mL of incubation medium with N
2
until an oxygen content of less than 1% of air saturation
was reached. Afterwards, mitochondria were added to this
oxygen-free medium and the incubation chamber was
closed. The mitochondria themselves consumed most of the
remaining oxygen resulting in very low oxygen concentra-
tions reflected by collapse of the mitochondrial membrane
potential (not shown). Reoxygenation was achieved by add-
ing another 2 mL of incubation medium, which was air sat-
urated, to the incubation tube [22].
Determination of mitochondrial respiration
Oxygen consumption of mitochondria was measured in an
incubation chamber equipped with a Clark-type electrode.
The experimental approach was calibrated using the oxygen
content of air saturated medium of 435 ng atomsÆmL
)1
at
30 °C [40].
Determination of the mitochondrial membrane
potential
The mitochondrial membrane potential was calculated from
the distribution of the lipophilic cation tetraphenyl phos-
phonium (TPP
+

) according to [41]. The extramitochondrial
TPP
+
concentration was determined by means of a TPP
+
-
sensitive electrode in the presence of 1 lm extramitochond-
rial TPP
+
. For the calculation of the membrane potential
a matrix volume of 1 lLÆmg
)1
mitochondrial protein was
assumed. The TPP
+
-sensitive electrode was calibrated by
applying standard TPP
+
solutions.
Determination of Mn-SOD activity
The determination of Mn-SOD activity was based on the
consumption of superoxide anion radicals generated by
the xathine ⁄ xanthine oxidase system [42]. The reduction
of cytochrome c was spectrophotometrically followed at
550 nm. 500 lL samples were withdrawn from mitochond-
rial incubations and stored in liquid nitrogen. Before use
samples were subjected to a threefold cycle of freezing and
thawing.
Determination of glutathione peroxidase activity
Five-hundred microlitre samples were withdrawn from the

mitochondrial incubations and stored in fluid nitrogen.
After threefold freezing and thawing, samples were used for
the determination of glutathione peroxidase activity. The
Ca
2+
protects mitochondria during hypoxia L. Schild et al.
5850 FEBS Journal 272 (2005) 5844–5852 ª 2005 The Authors Journal Compilation ª 2005 FEBS
assay is based on the oxidation of glutathione and the sub-
sequent oxidation of NADPH [43] which was followed
photometrically at 340 nm.
Determination of protein
The protein content of the mitochondrial suspension was
measured according to the Bradford method [44] using
bovine serum albumin as the standard.
Statistics
Statistical analysis was performed by the Student’s t-test.
Actual P -values are given in the legends of the figures. Data
are presented as mean ± SEM.
Acknowledgements
This work was supported by Deutsche Forschungs-
gemeinschaft (Project Re 847 ⁄ 3–1).
References
1 Grattagliano I, Vendemiale G & Lauterburg BH
(1999) Reperfusion injury of the liver: role of mito-
chondria and protection by glutathione ester. J Surg
Res 86, 2–8.
2 Isozaki H, Fujii K, Nomura E & Hara H (2000) Cal-
cium concentration in hepatocytes during liver ischae-
mia-reperfusion injury and the effects of diltiazem and
citrate on perfused rat liver. Eur J Gastroenterol Hepatol

12, 291–297.
3 Leducq N, Delmas-Beauvieux MC, Bourdel-Marchasson
I, Dufour S, Gallis JL, Canioni P & Diolez P (2000)
Mitochondrial and energetic dysfunctions of the liver
during normothermic reperfusion: protective effect of
cyclosporine and role of the mitochondrial permeability
transition pore. Transplant Proc 32, 479–480.
4 Leducq N, Delmas-Beauvieux MC, Bourdel-Marchasson
I, Dufour S, Gallis JL, Canioni P & Diolez P (1998)
Mitochondrial permeability transition during hypother-
mic to normothermic reperfusion in rat liver demon-
strated by the protective effect of cyclosporin A.
Biochem J 336, 501–506.
5 Soeda J, Miyagawa S, Sano K, Masumoto J, Taniguchi
S & Kawasaki S (2001) Cytochrome c release into cyto-
sol with subsequent caspase activation during warm
ischemia in rat liver. Am J Physiol Gastrointest Liver
Physiol 281, G1115–G1123.
6 Rivera-Chavez FA, Toledo-Pereyra LH, Dean RE,
Crouch L & Ward PA (2001) Exogenous and endogen-
ous nitric oxide but not iNOS inhibition improves func-
tion and survival of ischemically injured livers. J Invest
Surg 14, 267–273.
7 Morisue A, Wakabayashi G, Shimazu M, Tanabe M,
Mukai M, Matsumoto K, Kawachi S, Yoshida M,
Yamamoto S & Kitajima M (2003) The role of nitric
oxide after a short period of liver ischemia-reperfusion.
J Surg Res 109, 101–109.
8 Meguro M, Katsuramaki T, Nagayama M, Kimura H,
Isobe M, Kimura Y, Matsuno T, Nui A & Hirata K

(2002) A novel inhibitor of inducible nitric oxide synthase
(ONO-1714) prevents critical warm ischemia-reperfusion
injury in the pig liver. Transplantation 73, 1439–1446.
9 Ozakyol AH, Tuncel N, Saricam T, Uzuner K, Ak D &
Gurer F (2000) Effect of nitric oxide inhibition on rat
liver ischemia reperfusion injury. Pathophysiology 7,
183–188.
10 Schild L, Plumeyer F, Reinheckel T & Augustin W
(1997) Micromolar calcium prevents isolated rat liver
mitochondria from anoxia-reoxygenation injury.
Biochem Mol Biol Int 43, 35–45.
11 Andreyev A & Fiskum G (1999) Calcium induced
release of mitochondrial cytochrome c by different
mechanisms selective for brain versus liver. Cell Death
Differ 6, 825–832.
12 Gogvadze V, Robertson JD, Zhivotovsky B & Orrenius
S (2001) Cytochrome c release occurs via Ca
2+
-depend-
ent and Ca
2+
-independent mechanisms that are regula-
ted by Bax. J Biol Chem 276, 19066–19071.
13 Petronilli V, Cola C & Bernardi P (1993) Modulation of
the mitochondrial cyclosporin A-sensitive permeability
transition pore. II. The minimal requirements for pore
induction underscore a key role for transmembrane elec-
trical potential, matrix pH, and matrix Ca
2+
. J Biol

Chem 268, 1011–1016.
14 Davies AM, Hershman S, Stabley GJ, Hoek JB, Peter-
son J & Cahill A (2003) A Ca
2+
-induced mitochondrial
permeability transition causes complete release of rat
liver endonuclease G activity from its exclusive location
within the mitochondrial intermembrane space. Identifi-
cation of a novel endo-exonuclease activity residing
within the mitochondrial matrix. Nucleic Acids Res 31,
1364–1373.
15 Xue L, Borutaite V & Tolkovsky AM (2002) Inhibition
of mitochondrial permeability transition and release of
cytochrome c by anti-apoptotic nucleoside analogues.
Biochem Pharmacol 64, 441–449.
16 Kantrow SP & Piantadosi CA (1997) Release of cyto-
chrome c from liver mitochondria during permeability
transition. Biochem Biophys Res Commun 232, 669–671.
17 Wiswedel I, Tru
¨
mper L, Schild L & Augustin W (1988)
Injury of mitochondrial respiration and membrane
potential during iron ⁄ ascorbate-induced peroxidation.
Biochim Biophys Acta 934, 80–86.
18 Shiva S, Brookes PS, Patel RP, Anderson PG &
Darley-Usmar VM (2001) Nitric oxide partitioning into
mitochondrial membranes and the control of respiration
L. Schild et al. Ca
2+
protects mitochondria during hypoxia

FEBS Journal 272 (2005) 5844–5852 ª 2005 The Authors Journal Compilation ª 2005 FEBS 5851
at cytochrome c oxidase. Proc Natl Acad Sci USA 98,
7212–7217.
19 Giulivi C (2003) Characterization and function of mito-
chondrial nitric-oxide synthase. Free Radic Biol Med 34,
397–408.
20 Ghafourifar P & Richter C (1997) Nitric oxide synthase
activity in mitochondria. FEBS Lett 418, 291–296.
21 Cassina A & Radi R (1996) Differential inhibitory action
of nitric oxide and peroxynitrite on mitochondrial elec-
tron transport. Arch Biochem Biophys 328, 309–316.
22 Schild L, Reinheckel T, Wiswedel I & Augustin W
(1997) Short-term impairment of energy production in
isolated rat liver mitochondria by hypoxia ⁄ reoxygena-
tion: involvement of oxidative protein modification.
Biochem J 328, 205–210.
23 Schild L, Reinheckel T, Reiser M, Horn TF, Wolf G &
Augustin W (2003) Nitric oxide produced in rat liver
mitochondria causes oxidative stress and impairment of
respiration after transient hypoxia. FASEB J 17, 2194–
2201.
24 Willet K, Detry O & Sluse FE (2000) Resistance of
isolated pulmonary mitochondria during in vitro
anoxia ⁄ reoxygenation. Biochim Biophys Acta 1460,
346–352.
25 Chang YJ & Chang KJ (2002) Calcium concentration
in rat liver mitochondria during anoxic incubation.
J Formos Med Assoc 101, 136–143.
26 Manzo-Avalos S, Perez-Vazquez V, Ramirez J, Aguil-
era-Aguirre L, Gonzalez-Hernandez JC, Clemente-

Guerrero M, Villalobos-Molina R & Saavedra-Molina
A (2002) Regulation of the rate of synthesis of nitric
oxide by Mg
2+
and hypoxia: studies in rat heart mito-
chondria. Amino Acids 22, 381–389.
27 Schild L, Huppelsberg J, Kahlert S, Keilhoff G &
Reiser G (2003) Brain mitochondria are primed by
moderate Ca
2+
rise upon hypoxia ⁄ reoxygenation for
functional breakdown and morphological desintegra-
tion. J Biol Chem 278, 25454–25460.
28 Schild L & Reiser G (2005) Oxidative stress is involved
in the permeabilization of the inner membrane of brain
mitochondria exposed to hypoxia ⁄ reoxygenation and
low micromolar Ca
2+
. FEBS J 272, 3593–3601.
29 Ichas F & Mazat JP (1998) From calcium signaling to
cell death: two conformations for the mitochondrial per-
meability transition pore: switching from low- to high-
conductance state. Biochim Biophys Acta 1366, 33–50.
30 Brustovetsky N & Klingenberg M (1996) Mitochondrial
ADP ⁄ ATP carrier can be reversibly converted into a
large channel by Ca
2+
. Biochemistry 35, 8483–8488.
31 Ghafourifar P, Schenk U, Klein SD & Richter C (1999)
Mitochondrial nitric-oxide synthase stimulation causes

cytochrome c release from isolated mitochondria:
evidence for intramitochondrial peroxynitrite formation.
J Biol Chem 274, 31185–31188.
32 Kowaltowski AJ, Castilho RF & Vercesi AE (2001)
Mitochondrial permeability transition and oxidative
stress. FEBS Lett 495, 12–15.
33 Petronilli V, Costantini P, Scorrano L, Colonna R,
Passamonti S & Bernardi P (1994) The voltage sensor
of the mitochondrial permeability transition pore is
tuned by the oxidation-reduction state of vicinal thiols:
increase of the gating potential by oxidants and its
reversal by reducing agents. J Biol Chem 269, 16638–
16642.
34 Sato M, Sasaki M & Hojo H (1995) Antioxidative roles
of metallothionein and manganese superoxide dismutase
induced by tumor necrosis factor-alpha and interleukin-
6. Arch Biochem Biophys 316, 738–744.
35 Macmillan-Crow LA & Cruthirds DL (2001) Invited
review: manganese superoxide dismutase in disease. Free
Radic Res 34, 325–336.
36 Ramilo CA, Leveque V, Guan Y, Lepock JR, Tainer
JA, Nick HS & Silverman DN (1999) Interrupting the
hydrogen bond network at the active site of human
manganese superoxide dismutase. J Biol Chem 274,
27711–27716.
37 Johnson D & Lardy HA (1967) Isolation of liver or
kidney mitochondria. Methods Enzymol 10, 94–96.
38 McCormack JG & Denton RMJ (1998) Influence of cal-
cium ions on mammalian intramitochondrial dehydro-
genases. Methods Enzymol 174, 95–118.

39 Fabiato A & Fabiato F (1979) Calculator programs for
computing the composition of the solutions containing
multiple metals and ligands used for experiments in
skinned muscle cells. J Physiol (Paris) 75, 463–505.
40 Reynafarje B, Costa LE & Lehninger AL (1985) O
2
solubility in aqueous media determined by a kinetic
method. Anal Biochem 145, 406–418.
41 Kamo N, Muratsugu M, Hongoh R & Kobatake Y
(1979) Membrane potential of mitochondria measured
with an electrode sensitive to tetraphenyl phosphonium
and relationship between proton electrochemical poten-
tial and phosphorylation potential in steady state.
J Membr Biol 49, 105–121.
42 Flohe
´
L (1984) Superoxide dismutase assays. Methods
Enzymol 105, 93–104.
43 Paglia DE & Valentine WN (1967) Studies on the quan-
titative and qualitative characterization of erythrocyte
glutathione peroxidase. J Laboratory Clin Med 70,
158–169.
44 Bradford MM (1976) A rapid and sensitive method for
the quantitation of microgram quantities of protein util-
izing the principle of protein-dye binding. Anal Biochem
72, 248–254.
Ca
2+
protects mitochondria during hypoxia L. Schild et al.
5852 FEBS Journal 272 (2005) 5844–5852 ª 2005 The Authors Journal Compilation ª 2005 FEBS