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(The FASEB Journal. 2003;17:2194-2201.)
© 2003 FASEB

Nitric oxide produced in rat liver mitochondria causes oxidative stress and impairment of respiration after transient hypoxia

LORENZ SCHILD*,1, THOMAS REINHECKEL*,2, MICHAEL REISER{dagger}, THOMAS F. W. HORN{dagger}, GERALD WOLF{dagger} and WOLFGANG AUGUSTIN*

* Institute of Clinical Chemistry and Pathological Biochemistry, Department of Pathological Biochemistry,
{dagger} Institute of Medical Neurobiology, Faculty of Medicine, Otto-von-Guericke-University, Magdeburg, Germany

1Correspondence: Department of Pathological Biochemistry, Institute of Clinical Chemistry and Pathological Biochemistry, Otto-von-Guericke University Magdeburg Leipziger Str. 44, D-39120 Magdeburg, Germany. E-mail: lorenz.schild{at}medizin.uni-magdeburg.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Nitric oxide (NO) is produced in mammals by different isoforms of NO synthase (NOS), including the constitutive mitochondrial enzyme (mtNOS). Here we demonstrate that the concentration of NO resulting from a mitochondrial NOS activity increases under hypoxic conditions in isolated rat liver mitochondria. We show that mitochondrially derived NO mediates the impairment of active (state 3) respiration as measured in the presence of the substrates glutamate and malate after reoxygenation. Simultaneously, NO induces oxidative stress in mitochondria, characterized by an increase in the amount of protein carbonyls and a decrease in glutathione (GSH). Both the accumulation of oxidative stress markers during and the impaired respiration after reoxygenation were prevented by blocking NO production with the NOS inhibitor L-NAME. These observations suggest that mitochondria are exposed to high amounts of NO generated by a mitochondrial NOS upon hypoxia/reoxygenation. Such increased NO levels, in turn, inhibit mitochondrial respiration and may cause oxidative stress that leads to irreversible impairment of mitochondria.—Schild, L., Reinheckel, T., Reiser, M., Horn, T. F. W., Wolf, G., Augustin, W. Nitric oxide produced in rat liver mitochondria causes oxidative stress and impairment of respiration after transient hypoxia.


Key Words: oxidative phosphorylation • protein carbonyls • glutathione


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
NITRIC OXIDE (NO) is a highly diffusible free radical formed by conversion of arginine to citrulline via a family of NO synthase (NOS) isoenzymes. Besides having several biological functions, including regulation of blood flow, neurotransmission, and mediation of immune response, NO has been implicated in many pathophysiological processes such as inflammation, host defense, or neurodegeneration. Opposite effects of NO, harmful as well as protective, have been observed (1 2 3 4 5 6 7) . Furthermore, NO readily reacts with superoxide (O2· –) to form peroxynitrite (ONOO–), a potent oxidant and nitrating agent capable of attacking and modifying proteins, lipids, and DNA as well as depleting antioxidant defenses (8 , 9) .

Three distinct genes for the NOS family exist: the neuronal, endothelial, and inducible isoform (10) . The NOS genes have a similar genomic structure, suggesting a common ancestral gene. Several splice variants of NOS isoforms have been identified so far. A particular mitochondrial NOS species (mtNOS) has been found in a number of tissues by several groups (11 12 13 14) . This mtNOS was most recently shown to share characteristics with the {alpha}-splice isoform of neuronal NOS (15) . Thus, mitochondria are not only targets of NO that may regulate respiration (16 , 17) or cause deleterious effects following ischemia, hypoxia, and reperfusion (6) ; they are also NO producers.

There is evidence that NO binds in competition with oxygen to mitochondrial cytochrome oxidase (COX), the terminal enzyme of the respiratory chain, and subsequently inhibits the electron flow (16) . NO reacts with O2·– produced within the mitochondrial respiratory chain to form the highly reactive ONOO- (18) . Within the mitochondrial matrix, ONOO– can irreversibly inhibit complexes I and II of the respiratory chain as well as ATP synthase (19) . ONOO– contributes to an increase in hydroxyl radical production, which in turn causes oxidation of lipids, proteins, and DNA (20) . Other mitochondrial targets of NO are the mitochondrial Ca2+ uniporter and the mitochondrial ATP-sensitive K+ channel. In cardiomyocytes, NO inhibits mitochondrial Ca2+ uptake and increases the probability for opening of the mitochondrial ATP-sensitive K+ channel by modulating affinity to ATP (2) . These events are suggested to contribute to the protective effect mediated by NO during ischemia/reperfusion in cardiomyocytes (1) .

NOS in mitochondria of liver and kidney was first suggested to be located in the inner mitochondrial membrane and was shown to be activated by Ca2+. Recently it was reported that NO is also generated by mitochondria in heart, brain, muscle, lung, testis, and spleen by an enzyme similar to the neuronal NOS (NOS{alpha}) (15) . Alternatively, it was reported that endothelial NOS may be localized at juxtamitochondrial compartments of the endoplasmatic reticulum that are retained in the mitochondrial fraction during subcellular fractionation of sensory neurons (21 , 22) . The discovery of NOS activity in mitochondrial preparations supports the suggestion that locally produced NO, within or the immediate vicinity of mitochondria, may modulate the extent of mitochondrial and concomitantly cellular injury due ischemia/reperfusion.

Although results obtained with in vivo and cellular models of ischemia/reperfusion lead to the speculation that the effect of NO on mitochondria essentially determines the damage within the infarct area, the mechanism is still unclear. To study the distinct role of mitochondrially derived NO in damaging of mitochondria during transient hypoxia, we subjected isolated rat liver mitochondria to hypoxia/reoxygenation. Actual concentrations of NO were quantified under hypoxic conditions in mitochondrial incubations by an NO-sensitive electrode. Independently, the content of NO plus nitrite was measured in mitochondrial incubations at low oxygen tensions by applying a chemiluminescence detection method. To investigate functional impairment by hypoxia/reoxygenation, respiratory activity was assessed by measuring oxygen consumption. In addition, parameters of oxidative stress were determined: formation of protein carbonyls and the content of mitochondrial glutathione (GSH).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Reagents
Rabbit IgG directed against the 2,4-dinitrophenyl moiety was obtained from Sigma (St. Louis, MO, U.S.A.). Sheep anti-rabbit IgG conjugated with peroxidase was purchased from Boehringer Mannheim (Mannheim, Germany), and the protein assay reagent from Bio-Rad Laboratories GmbH (Munich, Germany). All other reagents were of the purest grade available.

Preparation of mitochondria
Mitochondria were prepared from livers of 220–240 g male Wistar rats in ice-cold medium containing 250 mM sucrose, 20 mM Tris/HCl (pH 7.4), 2 mM EGTA, and 1% (w/v) BSA using a standard procedure (23) . After the initial isolation, Percoll was used for purification of mitochondria from a fraction containing some endoplasmic reticulum, Golgi apparatus, and plasma membranes (24) . The mitochondria were well coupled, as indicated by a respiratory control index of >5 with glutamate plus malate as substrates.

Incubation of mitochondria
Mitochondria (1–2 mg protein/mL) were incubated in a medium containing 10 mM sucrose, 120 mM KCl, 20 mM Tris, 15 mM NaCl, 5 mM KH2PO4, 0.5 mM EGTA, and 1 mM free Mg2+ at pH 7.4 and 30°C. Hypoxia was produced by bubbling 2 mL of the incubation medium with purified nitrogen until oxygen was no longer detectable by means of a Clark electrode. Afterwards, addition of mitochondria to the medium further decreased the oxygen concentration due to oxygen consumption by the organelles. The final oxygen concentration was <2 nmol/mL, equivalent to the Km value of the cytochrome oxidase, since the mitochondrial membrane depolarized under this condition. A 2 mL volume of the air-saturated incubation medium was added to achieve reoxygenation.

Determination of mitochondrial respiration
Oxygen consumption of mitochondria was measured with a Clark-type electrode. The oxygen content of the air-saturated medium was 435 ng atoms/mL. at 30°C (25) .

Detection of NO concentration
Changes in NO concentration of the mitochondrial suspension were measured continuously by selective amperometric oxidation using an NO-sensitive electrode (ISO-NO-METER; World Precision Instruments, Berlin, Germany). The NO-sensitive electrode that is selective for NO levels in aqueous solutions was calibrated by generating stoichiometric standards from the reaction: 2KNO2 + 2KI + 2H2SO4 -> 2NO + I2 + 2H2O + 2K2SO4. Measurements of the NO concentration were performed simultaneously with measurements of oxygen consumption.

Detection of NO plus nitrite concentration plus
Alternatively, the accumulated NO metabolite nitrite plus NO was determined in samples drawn from the incubation chamber equipped with electrodes by a chemiluminescent assay using a Sievers NO Analyser in conjunction with the computerized data analysis program NOAWIN. The nitrite contained in the samples was reduced to NO by potassium iodide in the presence of acetic acid. This reaction converts nitrite but not nitrate into NO. The nitrite released NO was carried together with the NO of the sample from the reaction vessel of the analyzer to the analysis chamber by a steady flow of N2. Chemiluminescence that resulted from the reaction of ozone with NO was measured by a photomultiplier. The instrument was calibrated by injection of different NaNO2 concentrations with a fixed sample volume.

Determination of protein carbonyls
Protein carbonyls derivatives were produced by adding dinitrophenylhydrazine to the samples withdrawn from the incubations. Subsequently, the samples were prepared for reductive SDS/electrophoresis as described by Levine et al. (26) and applied to a 10% T, 3% C polyacrylamide gel using the separating system of Schägger and von Jagow (27) . After semi-dry blotting (28) , immunostaining was performed with a rabbit IgG directed against the 2,4-dinitrophenyl moiety at a dilution of 1:1500. The secondary antibody was a sheep anti-rabbit IgG conjugated with peroxidase used at a 1:1000 dilution. The quantity of protein carbonyls in the Western blot was estimated by densitometry as described before (29) .

Determination of GSSG and GSH
For the determination of glutathione (GSH) and glutathione disulfide (GSSG), 200 µL samples were taken from the mitochondrial incubations (1 mg protein/mL) and fixed by adding 100 µL of 5-sulfosalicylic acid (final concentration: 3.3%). Then the protein was sedimented by centrifugation (14.000 rpm, 6 min, Eppendorf Centrifuge 5415C, Eppendorf-Netheier-Hinz GmbH, Hamburg, Germany). Supernatant aliquots (90 µL) were neutralized with triethanolamine and buffered by a phosphate buffer in a final volume of 210 µL (4 µL vinylpyriden was added to determine GSSC). Samples of 50 µL were used for the Microtiter Plate Assay according to Baker et al. (30) . The changes in light absorption at 405 nm after adding of the sample were recorded within 10 min using an ELISA Reader (anthis HT II; Anthos Labtec Instruments, Salzburg, Austria).

Determination of protein
The protein content of the mitochondrial suspension was measured according to the method of Bradford (31) using BSA as the standard.

Statistics
Statistical analysis was performed by the Student’s t test. Actual P values are given in the figure legends. Data are presented as mean ± SE.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Hypoxia causes an increase in NO concentration in suspensions of isolated rat liver mitochondria
To estimate NO concentrations and their dependence on oxygen concentration, we simultaneously determined oxygen content and NO levels in mitochondrial incubations. The oxygen consumption of mitochondria was used to reduce the oxygen concentration in the incubation chamber. Until an O2 concentration of ~10 nmol O2/mL was reached, the content of NO remained nearly unchanged (Fig. 1 A). When lower oxygen tensions were reached, the NO concentration continuously increased at a rate of ~0.013 µM/min (left part in Fig. 1A ). Inhibition of NOS by 10 mM L-NAME almost completely prevented the rise in NO concentration (right part in Fig. 1A ). Thus, the increase in NO concentration within the mitochondrial suspension was mediated by NOS activity within the mitochondrial fraction.



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  		Figure 1. NO and nitrite concentration increase under hypoxic conditions. Rat liver mitochondria (2mg/mL) were incubated in medium also containing either 10 mM L-arginine (L-arg) or 10 mM L-NAME at 30°C. Concentrations of free NO were recorded by an NO-sensitive electrode in combination with oxygen measurements by a Clark-type electrode (A) or the concentration of free NO plus nitrite was determined applying a Sievers NO analyzer (B). Hypoxic conditions were reached after the mitochondria had consumed sufficient amounts of oxygen. A) Typical oxygen and NO traces of 5 mitochondrial preparations are shown. B) Values of the sum of free NO plus nitrite obtained from 5 mitochondrial preparations are summarized as means ± SE. *Significant difference between concentration of the sum of NO plus nitrite in the presence and absence of L-NAME, with P <= 0.05.

To investigate whether the increase in NO concentration at low oxygen tensions is paralleled by an increase in the concentration of nitrite that may derive from NO, we measured the sum of NO and nitrite in samples from mitochondrial incubations in air-saturated medium (control) or under hypoxia by applying a Sievers NO Analyzer. For this purpose the closed incubation chamber was used to control the oxygen concentration by means of a Clark-type electrode. Data obtained from these experiments are summarized in Fig. 1B . When mitochondria were incubated in air-saturated medium in the presence of 5 mM glutamate plus 5 mM malate and 10 mM L-arginine (control), no significant change in the content of NO plus nitrite was detected (white bars). In contrast, when mitochondria were additionally subjected to hypoxia, a considerable elevation was observed. Therefore, samples were held under hypoxic conditions for up to 8 min under the control of a Clark type electrode. After defined periods of hypoxia, samples were withdrawn for the quantification of NO plus nitrite content. Within the first 4 min of hypoxia, the concentration increased from 234 to 400 nM (black bars). This corresponds to a rate of increase of ~0.044 µM/min. In the additional presence of 10 mM L-NAME (hypoxia+L-NAME), hypoxia caused no increase in the content of NO plus nitrite (hatched bars). Again, the increase in the content of NO plus nitrite was caused by NOS activity as documented by the inhibitory effect of L-NAME.

Impairment of respiration after hypoxia/reoxygenation is mediated by NO
The increase in NO concentration caused by NO generation in suspensions of isolated rat liver mitochondria under hypoxic conditions led us to suggest that mitochondrially derived NO may be involved in the impairment of mitochondria upon ischemia/reperfusion. To test this hypothesis, we subjected isolated rat liver mitochondria to hypoxia/reoxygenation in the absence of exogenous substrates. In a previous study we showed by recording the mitochondrial membrane potential that freshly isolated rat liver mitochondria contain sufficient amounts of endogenous substrates for almost complete membrane polarization (32) . In the course of hypoxia, the membrane becomes depolarized in a time dependent manner. Reintroduction of oxygen and the addition of substrates result in increased membrane polarization. Therefore, this experimental approach covers effects of interruption of substrate supply and transient hypoxia as well as effects of intramitochondrially accumulated metabolites on mitochondria. It does not adequately reflect effects of intermediates released into the extramitochondrial space on mitochondria since they become diluted by a factor of ~200 in the hypoxic phase and ~400 during reoxygenation compared with hepatocytes, respectively.

We determined oxygen consumption to study the effect of NO on mitochondrial function upon hypoxia/reoxygenation. Rates of respiration under different incubation conditions are presented in Table 1 . The presence of 10 mM L-arginine in the incubation medium increased the resting respiration (oxygen consumption with 5 mM glutamate and 5 mM malate as substrates) from 6.9 ± 0.8 to 10.5 ± 2.1 ng atoms O min–1 mg–1 and significantly decreased active (state 3) respiration (80.5±4.6 vs. 49.9±3.8 ng atoms O min–1 mg–1). In consequence, the respiratory control ratio (RCR) representing the ratio between active and resting respiration dropped dramatically from 11.6 ± 0.9 down to 4.7 ± 1.7. The presence of 10 mM L-NAME in the incubation medium did not affect these respiratory parameters. Hypoxia/reoxygenation clearly impaired mitochondrial respiration indicated by decrease in active respiration (80.5±4.6 vs. 67.6±7.1 ng atoms O min–1 mg–1) and RCR (11.6±0.9 vs. 8.7±1.6). The continuous presence of 10 mM L-arginine in the incubation medium caused a further decrease in active respiration and RCR determined after hypoxia/reoxygenation (67.6±7.1 vs. 51.5±4.3 ng atoms O min–1 mg–1 and 8.7±1.6 vs. 6.4±1.7), respectively. When NOS activity was blocked by L-NAME, mitochondria were completely protected against impairment of respiration by hypoxia/reoxygenation. Thus, mitochondrially derived NO is definitely involved in the process of mitochondrial damage induced by hypoxia/reoxygenation.


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  		Table 1. NO affects mitochondrial respiration under normoxic conditions and upon hypoxia/reoxygenationa

NO causes oxidative stress during hypoxia/reoxygenation in rat liver mitochondria
Hypoxia/reoxygenation induces oxidative stress in isolated rat liver mitochondria as demonstrated by the accumulation of oxidation products of lipids and proteins (29) . In Fig. 2 , the amount of mitochondrial protein carbonyls accumulated under various conditions is depicted. Twenty-nine discrete protein carbonyl bands were found in Western blots of mitochondrial protein samples obtained from the mitochondrial suspension after 10 min of hypoxia and 5 min of reoxygenation (Fig. 2A ). The optical density of each single band was measured by densitometry; all values of one lane are summarized and depicted in Fig. 3 B. Protein carbonyls were formed in proteins ranging from 19 to 120 kDa. In the presence of L-arginine, an increase in the optical density of most of the bands was found (lanes 3 and 6 compared with lanes 2 and 5 in Fig. 2A ). The extent of this increase was different for individual bands (not shown). The highest increases in optical densities (up to threefold increase in optical density compared with freshly isolated mitochondria) were detected for some singular bands in the low molecular weight range. Protein oxidation is also reflected by increased lane intensity (Fig. 2B ). When NOS activity was inhibited by L-NAME, lower levels of optical density of individual bands were detected (lanes 4 and 7 vs. lanes 3 and 6). Under this condition the lane intensities were similar to control conditions (Fig. 2B ). Hypoxia/reoxygenation caused an additional protein oxidation indicated by increased optical densities (lanes 3 and 6 in Fig. 2A ). An increase in protein oxidation did not occur in the presence of L-NAME (lane 4 vs. 2 and lane 7 vs. 5, respectively).



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  		Figure 2. Protein carbonyl formation increases in dependence on NO formation during hypoxia/reoxygenation. Rat liver mitochondria (1 mg/mL) were incubated at 30°C and subjected to 10 min hypoxia and 5 min reoxygenation. Protein carbonyl formation was assayed by Western blot (A). The lanes represent samples obtained from liver mitochondria that were 1) freshly isolated, 2) incubated in air-saturated medium for 15 min (control), 3) incubated in air-saturated medium in the presence of 10 mM L-arginine for 15 min (control+L-arg), 4) incubated in air-saturated medium in the presence of 10 mM L-NAME for 15 min (control+L-NAME), 5) subjected to 10 min hypoxia and 5 min reoxygenation (H/R), 6) subjected to 10 min hypoxia and 5 min reoxygenation in the presence of 10 mM L-arginine (H/R+L-arg), or 7) subjected to 10 min hypoxia and 5 min reoxygenation in the presence of 10 mM L-NAME (H/R+L-NAME). B) Cumulated lane intensities are presented as means ± SE (n=5). *Significant difference between control and control + L-arg; **between control + L-arg and control + L-NAME; +between H/R and H/R + L-arg; ++between H/R + L-arg and H/R + L-NAME, with P <= 0.05.



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  		Figure 3. NO mediates oxidation of GSH upon hypoxia/reoxygenation. Rat liver mitochondria (1 mg/mL) were incubated at 30°C. Further incubation conditions were control, 15 min incubation in air-saturated medium (without) also performed in the additional presence of either 10 mM L-arginine (+L-arg) or 10 mM L-NAME (+L-NAME); H/R, 10 min hypoxia and 5 min reoxygenation (without) performed in the additional presence either of 10 mM L-arginine (+L-arg) or of 10 mM L-NAME (+L-NAME). Mean values ± SE of GSH (A) and GSSG (B) are presented from 5 mitochondrial preparations. *Significant difference between control (without) and control (+L-NAME), with P <= 0.01 (A); **between H/R (without) and H/R (+L-NAME), with P <= 0.01 (A) and P <= 0.05 (B).

A second series of experiments was performed to study the possibility of oxidative stress mediated by NO during hypoxia/reoxygenation, and the mitochondrial content of GSH and oxidized glutathione (GSSG) was analyzed. The data are presented in Fig. 3 . Hypoxia/reoxygenation caused a considerable decrease in mitochondrial GSH (4.8 vs. 6.4 nmol/mg, Fig. 3A ). The continuous presence of 10 mM L-arginine in the incubation medium resulted in lower GSH levels both during normoxic conditions in air-saturated medium and after hypoxia/reoxygenation (5.2 vs. 6.4 nmol/mg in controls and 4.2 vs. 4.8 nmol/mg after hypoxia/reoxygenation). When NO synthesis was blocked with L-NAME, high GSH levels of ~8.7 nmol/mg were found whether mitochondria were subjected to hypoxia/reoxygenation or not. These results demonstrate that GSH oxidation is mediated by mitochondrially derived NO. Under the experimental conditions investigated, GSSG content remained low, between 0.22 and 0.38 nmol/mg (Fig. 3B ). Inhibition of NOS by L-NAME caused a slight decrease in the GSSG content under normoxic conditions and upon hypoxia/reoxygenation (0.23 vs. 0.29 nmol/mg in controls and 0.22 vs. 0.37 nmol/mg after hypoxia/reoxygenation). However, changes in GSSG content did not counterbalance the changes in GSH content. Thus, the decrease in GSH content mainly reflects cleavage of glutathione.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
CONCLUSIONS
 REFERENCES
 
NO concentration in mitochondrial suspensions increases during hypoxia
Here we show that the concentration of NO increases during hypoxia in suspensions of isolated rat liver mitochondria. This NO accumulation was found to be L-NAME sensitive, pointing to an NOS-mediated NO generation. A most likely source is the recently described mtNOS (15) located within either the mitochondria or juxtamitochondrial compartments of the endoplasmic reticulum that are retained in the mitochondrial fraction (21 , 22) . In any case, this locally formed NO is, because of short diffusion distances, able to reach mitochondrial components that are highly sensitive to NO.

Oxygen and Ca2+ concentration are known to regulate NO production by NOS. Within the ischemic period, deficiency of oxygen (hypoxia) and an increase in the cytosolic Ca2+ concentration occur in liver cells. One would expect that under hypoxic conditions, NO production and its subsequent concentration of NO decrease due to low availability of oxygen, a substrate of NOS. However, despite the relatively high Km value of NOS for oxygen in the micromolar range (33 34 35 36) , NO can still be produced at low oxygen tensions. Here we show the opposite, that an increase in NO concentration occurs in hypoxic suspensions of isolated rat liver mitochondria. The concentration of NO displays the difference between NO production and consumption. Therefore, a reason for increase in NO concentration at low oxygen concentration might be the release of NO from reversible binding sites such as cytochrome oxidase as well as lower rates of NO consumption by the formation of nitrogen oxides such as NO2 and N2O3, thereby overcompensating the decrease in NO production.

Our experiments clearly reveal that the concentration of NO dramatically increased during hypoxia even at low extramitochondrial Ca2+ concentration. A rate of NO production of ~1,6 nmol NO min–1 mg–1 was reported for normoxic and Ca2+ free incubations of isolated rat liver mitochondria at 35°C determined by monitoring the oxidation of oxymyoglobin in the presence of succinate (37) . We determined that under hypoxic conditions at 30°C with glutamate and malate in Ca2+-free incubations of isolated rat liver mitochondria, the rate of increase in the concentration of NO plus nitrite is 0.044 nmol min–1 mg–1. Only ~30% of the total increase are contributed by free NO.

Hypoxia-induced increase in NO concentration impairs mitochondrial respiration
NO is a competitive inhibitor of cytochrome oxidase (14) . Moreover, ONOO– derived from NO and O2· – impairs the electron flow through the complexes I, II, III, and V of the respiratory chain (19) . We provide evidence that in incubations with isolated rat liver mitochondria, the source for increased NO concentrations during hypoxia/reoxygenation are the mitochondria themselves.

The involvement of NO in the impairment of respiration in brain mitochondria upon ischemia/reperfusion has been demonstrated in perinatal anoxia. The decrease in respiration was attributed to an impairment of complexes II and III of the respiratory chain (38) . Furthermore, it was reported that the NO-mediated impairment of respiration in cardiac muscle subjected to hypoxia/reoxygenation is irreversible (39) . Hypoxia/reoxygenation caused a decrease in the activities of the complexes I and III of the respiratory chain in isolated rat liver mitochondria (29 , 40) . We now demonstrate that this decrease in respiration is mediated exclusively by NO, which was synthesized by an NOS within the mitochondrial suspension since the presence of L-NAME abolished the effect of NO.

Oxidative stress is involved in the impairment of tissues by ischemia/reperfusion (41) . Since oxygen is required for the generation of reactive oxygen species, oxidative stress mainly occurs after reperfusion. Based on our data on isolated mitochondria, we suggest a new mechanism of NO-mediated damage of mitochondria upon hypoxia/reoxygenation. An increase in extramitochondrial NO concentration accumulated within the hypoxic period may cause inhibition of cytochrome oxidase (complex IV of the respiratory chain) at the moment of reoxygenation (42) . This in turn increases the probability for one electron transfer to oxygen at the level of ubiquinone (43) . Subsequently, formation of superoxide anion radicals by the respiratory chain becomes enhanced. NO and superoxide anion radicals are converted to the highly reactive ONOO– by a diffusion-limited reaction that further induces the formation of a variety of reaction products (44) . Alternatively, superoxide anion radicals are dismutated, catalyzed by superoxide dismutase to H2O2. In the presence of Fe2+, it forms the highly reactive hydroxyl radical via the Fenton reaction. Both pathways of superoxide anion radical reactions, the formation of H2O2 and the formation of ONOO–, lead to a decrease in GSH. ONOO– consumes GSH to form NO2– or GSNO. GSH is also consumed by the elimination of H2O2 via the glutathione peroxidase reaction.

Our results clearly demonstrate a NO-mediated decrease in GSH. Since this decrease was not adequately reflected by increased GSSG concentrations, we conclude that the ONOO–-induced formation of GSNO may contributed significantly to the decrease of GSH during reoxygenation in isolated rat liver mitochondria.

Superoxide anion radical reaction that forms H2O2 and generated ONOO– lead to nonenzymatic generation of hydroxyl radicals very likely responsible for oxidative modification of mitochondrial proteins. Indeed, we found an increased amount of protein carbonyls by Western blot analysis in mitochondria after hypoxia/reoxygenation compared with normoxic controls (Fig. 2) . Thus, NO causes increased oxidative stress most likely by stimulation of superoxide anion radical and ONOO– generation. We suggest that this is the underlying mechanism of the NO-mediated impairment of mitochondria upon hypoxia/reoxygenation.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
REFERENCES
 
Here we demonstrate that the concentration of NO generated within or in the near vicinity of mitochondria increases at the mitochondrial location during hypoxia/reoxygenation, resulting in diminished active respiration and ATP production.

Such energetic deficit can cause an influx of Ca2+ into the cytosol from extracellular space and intracellular stores resulting in an elevation in cytosolic Ca2+. It has been shown by Ghafourifar et al. (45) that in isolated rat liver mitochondria, Ca2+ induces a cyclosporin A-insensitive release of cytochrome c. The liberation of cytochrome c from mitochondria into the cytosol can trigger apoptosis by activation of caspase 9 (46 , 47) . Moreover, NO can induce the release of Ca2+ also from neuronal mitochondria in a permeability transition pore (PTP) -dependent mechanism (48) , contributing to further increased cytosolic Ca2+ levels and initiating a vicious cycle.

The data, presented here provide evidence for NO-mediated oxidative stress during hypoxia/reoxygenation in isolated liver mitochondria. In combination with elevated cytosolic Ca2+ concentrations, oxidative stress increases the probability for opening of the mitochondrial PTP (49 , 50) . When that unspecific pore stays open permanently, it causes a collapse of the mitochondrial membrane potential and swelling, finally resulting in the rupture of mitochondrial membrane. In this way, NO produced within mitochondria may contribute to tissue infarction in liver as has been observed in in vivo models of ischemia/reperfusion (51) . Mitochondrial NO production may comprise a prime target for pharmacological intervention to prevent from pathological consequences upon ischemia/reperfusion.


   ACKNOWLEDGMENTS
 
This work was supported by DFG WO 474/II-4 and Sachsen-Anhalt grant 2997A/0088G


   FOOTNOTES
 
2 Present address: Institute of Molecular Medicine, Albert-Ludwigs-University, Freiburg, Germany.

doi: 10.1096/fj.02-1170com

Received for publication January 31, 2003. Accepted for publication August 5, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 

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