We investigated the effect of low micromolar Ca2+ concentrations on respiration, membrane permeability, and antioxidative defence in liver mitochondria exposed to hypoxia⁄ reoxygenation.
Trang 1prevents functional injury of mitochondria exposed to
Lorenz Schild1, Frank Plumeyer1and Georg Reiser2
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 Ca2+concentration [2]
Oxidative stress and increase in the cytosolic Ca2+
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 endoexo-genous 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 Ca2+, reactive oxygen species (ROS), and NO, which are known to change in concentration during ischaemia⁄ reperfusion, affect mitochondria Elevation of Ca2+ 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 Ca2+ in mitochondrial damage is still unclear We investigated the effect of low micromolar Ca2+ 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 Ca2+ Below and above 2 lm
Ca2+, mitochondrial function was deteriorated, as indicated by the decrease in respiratory control ratio Above 2 lm Ca2+, the mitochondrial membrane was permeabilized, as demonstrated by the sensitivity of state 3 respiration to NADH Below 2 lm Ca2+, 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 Ca2+ We conclude that
Ca2+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.
Trang 2into 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 Ca2+
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 Ca2+ 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 Ca2+ 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 Ca2+ concentration was
investigated We found that within a narrow
concentra-tion range at around 2 lm extramitochondrial Ca2+,
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 Ca2+concentration, displaying
maximal values at a concentration of about 2 lm At
this Ca2+concentration, the inhibition of NO synthesis
with l-NAME did not affect state 3 respiration From
these data we conclude that extramitochondrial Ca2+at
a narrow concentration window exerts a protective effect upon hypoxia⁄ reoxygenation by increasing the activity of antioxidative enzymes in liver mito-chondria
Results
Ca2+affects respiration and membrane permeability upon hypoxia⁄ reoxygenation
In liver ischaemia⁄ reperfusion the cytosolic Ca2+ con-centration in hepatic cells is elevated into the low micro-molar range In order to investigate the influence of extramitochondrial Ca2+ 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
Ca2+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 Ca2+ caused decrease in state 3 respiration to 45% of the normoxic control value (incubation in air saturated medium) The influence of Ca2+ on state 3 respiration obtained with hypoxia⁄ reoxygenation was characterized by a bell-shaped concentration depend-ence (Fig 1A, upper part) Increasing the Ca2+ concen-tration improved state 3 respiration Almost complete protection was seen at 2 lm extramitochondrial Ca2+ (91% of normoxic mitochondria) This was not observed when Ca2+ uptake was inhibited by 10 lm ruthenium red (data not shown) Further increase in extramitochondrial Ca2+ concentration resulted in decreased rates of state 3 respiration measured after hypoxia⁄ reoxygenation At the maximally used concen-tration of 4.4 lm Ca2+, no stimulation of oxygen con-sumption by ADP could be reached The respiration determined in the absence of ADP (state 4) had no clear
Ca2+ dependence (lower part in Fig 1A) In order to test whether the effect of hypoxia⁄ reoxygenation and elevated Ca2+on state 3 respiration was due to opening
of the MPTP, isolated rat liver mitochondria were exposed to hypoxia⁄ reoxygenation and Ca2+ in the additional presence of 2 lm of the MPTP inhibitor CSA At this concentration, CSA completely prevented
Ca2+-induced swelling of liver mitochondria (data not shown) CSA partially protected liver mitochondria against decrease in state 3 respiration at Ca2+ concen-trations below and above 2 lm (Fig 1A, upper part) Within the narrow concentration range at around 2 lm extramitochondrial Ca2+, no effect of CSA was observed The rates of state 4 respiration were slightly higher in CSA-containing incubations in comparison to
Trang 3CSA-free incubations At 2 lm extramitochondrial
Ca2+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 Ca2+ indica-ting a protective Ca2+ 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 Ca2+concentration range investigated The RCR of freshly isolated mitochondria was 6.4 ± 0.7 (n¼ 12).
To investigate the possibility of a CSA-insensitive permeabilization of the mitochondrial membrane upon hypoxia⁄ reoxygenation and Ca2+, 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 Ca2+ concentrations, is depicted in Fig 1C Up to 2 lm extramitochondrial Ca2+, 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 Ca2+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 Ca2+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 m M malate either without (state 4) or with 200 l M ADP (state 3) Subsequently 5 m M
NADH and 10 l M 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 respirrespir-ation 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)1Æmin)1 Data are presented
as mean ± SEM of five mitochondrial preparations.
Trang 4Ca2+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 Ca2+
concentration [10] To investigate how Ca2+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 Ca2+(Fig 2) At
0.2 lm Ca2+, lower activity of Mn-SOD was found (73
at 0.2 lm Ca2+vs 118 unitsÆmg)1at 2 lm Ca2+)
Sim-ilar results were obtained with Mn-SOD from bovine
erythrocytes In the presence of 2 lm Ca2+, the enzyme
activity was increased from 0.134 ± 0.021 unitsÆmg)1
(at 0.2 lm Ca2+) 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 Ca2+, but was significantly
higher than in a normoxic incubation (157 unitsÆmg)1 after hypoxia⁄ reoxygenation vs 118 unitsÆmg)1without hypoxia⁄ reoxygenation) Increase in the extramito-chondrial Ca2+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 Ca2+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 Ca2+ The activity of this enzyme didnotdependontheextramitochondrial Ca2+ concentra-tion in the low micromolar range under normoxic con-ditions (Fig 3, lower part) In Ca2+-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 Ca2+) 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 m M 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 Ca2+concentrations at 30 C After the reoxygenation period
5 m M glutamate and 5 m M 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 Ca2+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 m M glutamate plus
5 m M malate in the incubation medium or were subjected to 5 min
hypoxia and 10 min reoxygenation in the presence of various Ca2+
concentrations at 30 C After the reoxygenation period 5 m M
glutamate and 5 m M 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 Ca2+ (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.
Trang 5sensitive to Ca2+with the tendency to reach the highest
levels between 1 and 2 lm extramitochondrial Ca2+
Alternatively to the formation of H2O2 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 Ca2+, 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 Ca2+(low Ca2+
concentra-tion) partially protected rat liver mitochondria against
decrease in state 3 respiration (72 ± 4.4 vs
39 ± 3.1 nmol O2Æmin)1Æmg)1) At this Ca2+
concen-tration, the continuous presence of 50 lm haemoglobin
was also protective (78 ± 3.9 vs 39 ± 3.1 nmol O2Æ
min)1Æmg)1) When mitochondria were subjected to
hypoxia⁄ reoxygenation in the presence of 2 lm
extra-mitochondrial Ca2+ (protective Ca2+ concentration),
l-NAME did not affect respiration Likewise, at
4.4 lm Ca2+, inhibition of enzymatic NO synthesis
during hypoxia⁄ reoxygenation did not lead to a change
in state 3 respiration (high Ca2+concentration)
Discussion
Extramitochondrial Ca2+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 Ca2+ 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 Ca2+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 Ca2+ This is surprising as increases in extramitochondrial Ca2+ 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 Ca2+ 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
Ca2+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 M L -NAME in the continuous
presence of either 0.2 l M , 2 l M or 4.4 l M extramitochondrial Ca 2+
at 30 C Afterwards, 5 m M glutamate, 5 m M malate and 200 l M
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.
Trang 6Ca2+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
Ca2+ concentration [28] However, the protection
of mitochondria from hypoxia⁄ reoxygenation-induced
damage and the low amount of protein carbonyls [10]
at 2 lm Ca2+suggest relatively low superoxide radical
concentration This is consistent with our finding of
considerably increased activity of the Mn-SOD at this
Ca2+ concentration At 2 lm Ca2+, 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 Ca2+ has
to enter the mitochondrial matrix in order to cause
increase in Mn-SOD activity Both Ca2+ 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 Ca2+
in combination with hypoxia⁄ reoxygenation mediates
this antioxidant effect still remains unclear It might be
speculated that interaction of Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+-induced impairment of liver mito-chondria [31,33] Thus, the protective effect of the inhi-bition of NO synthesis at 0.2 lm Ca2+ demonstrated here (Fig 4) could be mainly attributed to the dimin-ished peroxynitrite formation In contrast, at 2 lm
Ca2+, superoxide anion radical concentration, but not
NO production, is reduced, as the increase in Ca2+ 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 Ca2+ 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 Ca2+ cause functional damage of isolated rat liver mitochon-dria Essential steps involved in the cascade of mito-chondrial injury are CSA-insensitive permeabilization
Trang 7of the mitochondrial membrane, production of ROS,
generation of NO and peroxynitrite by mitochondria
We have found a distinct extramitochondrial Ca2+
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 Ca2+concentration
Thus, we conclude that superoxide anion radicals and
peroxynitrite play a pivotal role in damaging
mitochon-dria upon hypoxia⁄ reoxygenation at Ca2+
concentra-tions up to about 2 lm At higher Ca2+concentrations,
the mechanism underlying the mitochondrial injury is
the permeabilization of the membrane As elevation of
extramitochondrial Ca2+ concentration into the low
micromolar range appears to have protective effects,
further investigations on the role of Ca2+ 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
1 mm free Mg2+at pH 7.4 Extramitochondrial Ca2+
con-centrations were adjusted by using Ca2+⁄ EGTA buffers
After preparation of the buffers, the free Ca2+
concentra-tion was checked by means of a Ca2+-selective electrode
The actual concentration of Ca2+ in the incubations was
calculated considering the complex formation with other constituents of the medium such as Mg2+ 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 N2
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
30C [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
Trang 8assay 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)
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