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CELLULAR METABOLISM
Institut für Physiologische Chemie, Universitätsklinikum Essen, Essen, Germany
Submitted 5 July 2006 ; accepted in final form 20 December 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2 h of incubation. NO exposure inhibited H2O2 degradation, assessed after addition of 50 µM, 200 µM, or 4 mM authentic H2O2, significantly in both cell types. However, again, early and delayed inhibition was observed. The late inhibition of H2O2 degradation in endothelial cells was paralleled by a decrease in glutathione peroxidase activity. Glutathione peroxidase inactivation was prevented by hypoxia or by ascorbate, suggesting inactivation by reactive nitrogen oxide species (NOx). Early inhibition of H2O2 degradation by NO, in contrast, could be mimicked by the catalase inhibitor azide. Together, these results suggest that the cooperative effect of NO and H2O2 is due to inhibition of H2O2 degradation by NO, namely to inhibition of catalase by NO itself (predominant in hepatocytes) and/or to inhibition of glutathione peroxidase by NOx (prevailing in endothelial cells). nitrogen monoxide; catalase; glutathione peroxidase
–, yielding peroxynitrite, has been studied extensively (23, 24), we and others have reported a cooperative action of NO and hydrogen peroxide (H2O2) in their cytotoxicity against Fu5 rat hepatoma cells, rat liver endothelial cells, HepG2 cells, murine lymphoma cells, rabbit gastric mucosal cells, embryonic chick cardiomyocytes, and a human epithelial ovarian cancer cell line (17, 19, 21, 26, 28, 49, 65). An explanation for this cooperative action of NO and H2O2 has not yet been provided. A chemical reaction as originally proposed by Noronha-Dutra et al. (47) turned out to be very unlikely (10). Since, on the other hand, high-affinity inhibition of isolated catalase by NO is known (10, 17) and effects on glutathione peroxidase have also been reported (3), we speculated that inhibition of H2O2 degradation by NO might be responsible for the cooperative effect [as also suggested by Stadler et al. (62)]. The present study with isolated rat liver endothelial cells and hepatocytes was performed to decipher the mechanistic basis for the cooperative effect of NO and H2O2 and showed that both, inhibition of catalase by NO and inhibition of glutathione peroxidase by reactive nitrogen oxide species (NOx), are likely to contribute and that it is dependent on cell type and H2O2 levels which of these effects prevails. |
MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Male Wistar rats (180–280 g) were obtained from the Zentrales Tierlaboratorium (Universitätsklinikum Essen). All animals received humane care in compliance with the German Law for the Protection of Animals and the institutional guidelines, and permission for the use of the animals for liver cell isolations was obtained from the local authorities.
Materials
Catalase (from bovine liver), horseradish peroxidase (grade I), and glucose oxidase (GOD, from A. niger) were purchased from Roche Molecular Biochemicals (Mannheim, Germany). Leibovitz L-15 medium, diethylenetriaminepentaacetic acid (DTPA), buthionine sulfoximine (BSO), scopoletin (7-hydroxy-6-methoxy-2H-1-benzopyran-2-one), and H2O2 were obtained from Sigma (Taufkirchen, Germany). SpermineNONOate (SpNO) was from Situs (Düsseldorf, Germany) and RPMI 1640 medium from GIBCO (Karlsruhe, Germany). All other chemicals used were purchased from Merck (Darmstadt, Germany).
Cell Isolation and Culture
A rat liver endothelial cell line, derived from the liver of a male Wistar rat and characterized as described previously (50), and hepatocytes isolated from male Wistar rats as described previously (14) were used for experiments. The endothelial cells were cultured in RPMI 1640 medium supplemented with fetal calf serum (20%), L-glutamine (2 mM), penicillin/streptomycin (50 U/ml and 50 µg/ml, respectively), and dexamethasone (1 µM). Subcultures were obtained by trypsinization. For the experiments, the cells were split 1:3 and seeded onto collagen-coated 12.5- or 25-cm2 culture flasks and were used for experiments on day 6 or 7 after subcultivation. Hepatocytes were seeded onto collagen-coated 12.5- or 25-cm2 culture flasks (Falcon, Heidelberg, Germany), cultured in L-15 medium supplemented with 5% fetal calf serum, L-glutamine (2 mM), glucose (8.3 mM), bovine serum albumin (0.1%), NaHCO3 (14.3 mM), gentamicin (50 µg/ml), and dexamethasone (1 µM), and were used for experiments 20–24 h after cell isolation.
Experimental Procedures
At the beginning of the experiments, cells were washed three times with Hanks' balanced salt solution (HBSS) and then covered with 2.5 ml of modified Krebs-Henseleit buffer (KH; 115 mM NaCl, 25 mM NaHCO3, 5.9 mM KCl, 1.2 mM MgCl2, 1.2 mM NaH2PO4, 1.2 mM Na2SO4, 2.5 mM CaCl2, 20 mM HEPES, pH 7.4, supplemented with 10 or 30 mM glucose). All experiments were performed at 37°C. The NO donor SpNO and/or the catalase inhibitor azide were added to the incubation solution 15 min before H2O2 or GOD. Some cultures were pretreated with BSO (0.5 mM in cell culture medium) for 16 h before the experiment.
For hypoxic incubations, cells were covered with KH buffer that had been saturated with 95% N2-5% CO2, and each culture flask was then gently flushed with 95% N2-5% CO2 through cannulas piercing the rubber stoppers of the flasks as described in Ref. 14.
Experiments in the absence of cells were performed under the same conditions as given for the experiments with cells. Bovine liver catalase (7.8 ng/ml) and 100 µM H2O2 were used to study the effect of NO on the isolated enzyme.
Assays
Determination of SpNO decomposition.
SpNO decomposition was assessed spectrophotometrically. One millimolar SpNO was added to KH buffer supplemented with 100 µM DTPA in the absence and the presence of 4 mM H2O2, and the decrease in absorbance at 
= 252 nm was monitored. Decomposition was calculated using 
= 8.5 mM/cm (43).
Determination of H2O2 steady-state levels. H2O2 concentrations in the incubation medium were determined fluorimetrically by monitoring the horseradish peroxidase-dependent oxidation of scopoletin, using the assay conditions described previously [for low H2O2 concentrations (53) and for higher H2O2 concentrations (52)].
Determination of glutathione peroxidase activity. Glutathione peroxidase activity was assessed in cell lysates by the enzymatic assay described by Flohé and Gunzler (20). Briefly, in this assay the glutathione peroxidase-catalyzed, H2O2-dependent glutathione oxidation (with exogenous substrates) is coupled to the glutathione reductase-mediated rereduction of glutathione (in the presence of an excess of added NADPH and of glutathione reductase), and the NADPH consumption during the latter reaction is followed spectrophotometrically at 340 nm.
Determination of cellular glutathione content. Total glutathione (GSH and GSSG) and GSSG were determined with the method of recycling GSH with glutathione reductase and NADPH according to Griffith (22) with minor modifications.
Assessment of cellular lactate dehydrogenase release. Lactate dehydrogenase (LDH) activity was measured using a standard assay; released LDH is given as percentage of total LDH activity (54).
Statistics
All experiments were performed in duplicate and repeated three to five times. Data are expressed as means ± SD. Data obtained from two groups were compared by means of Student's t-test and comparisons among multiple groups were performed using an ANOVA with Student-Newman-Keuls or Dunnett post hoc comparisons, as appropriate. A P value of <0.05 was considered significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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When confluent cultures of liver endothelial cells were incubated in KH buffer with the H2O2-generating system glucose (10 mM)/GOD, GOD activities in excess of 50 U/l were required to cause significant injury within 7 h (data not shown). GOD activities of 20 U/l or lower did not cause any loss of viability (Fig. 1A).
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Enhancement of H2O2 Toxicity by NO/NOx
When liver endothelial cells were exposed simultaneously to SpNO (1 mM) and glucose/GOD (10 mM; 20 U/l), more than 80% of cells lost viability within 5 h (Fig. 1A). NO alone (in the absence of GOD) caused significantly less cell injury (33 ± 14% after 5 h). The enhancing effect of the NO donor on H2O2 toxicity could be attributed to the NO released without doubts as neither the decomposition product spermine (1 mM; Fig. 1A) nor nitrite (the major product of the reaction of NO with reactive oxygen species or molecular oxygen in the hemoglobin-free cell cultures, 2 mM; data not shown) enhanced H2O2 toxicity. The enhancement was slight with 0.1 mM SpNO, moderate with 0.5 mM SpNO, and most marked with 1 mM SpNO (data not shown).
In cultured hepatocytes, the addition of 1 mM SpNO did not lead to any loss of viability, neither in the absence nor in the presence of GOD (100 U/l; data not shown). However, when glutathione-depleted hepatocytes were used [a model that is of interest, for example, with regard to the role of NO/NOx in acetaminophen toxicity (30); hepatocytes were pretreated for 16 h with BSO, which decreased hepatocellular glutathione content from 36.8 ± 9.3 to 9.2 ± 8.8 nmol/106 cells], the combined exposure to NO (1 mM SpNO) and H2O2 (30 mM glucose + 100 U/l GOD) caused marked cell injury (Fig. 1B). Also, in these cells, SpNO (1 mM) alone did not cause any cell injury over the incubation period used (3 h). SpNO decomposition, as assessed spectrophotometrically, did not differ significantly in the absence and the presence of H2O2 (4 mM; data not shown).
Effect of NO/NOx on H2O2 Steady-State Levels
In cell-free KH buffer, the addition of SpNO did not affect H2O2 generation by the glucose/GOD system (20 U/l yielded H2O2 levels, at 5 h, of 137.6 ± 33.0 µM in the presence of 1 mM SpNO and 122.2 ± 47.7 µM in the absence of SpNO). H2O2 steady-state levels in cell-free KH buffer in the absence of GOD were below 0.4 µM (0.21 ± 0.15 µM in the absence and 0.09 ± 0.04 µM in the presence of SpNO), i.e., the mechanism of H2O2 generation by reaction of peroxynitrite with HEPES (36) did not considerably contribute to H2O2 generation in the system described here.
In liver endothelial cell cultures incubated in KH buffer in the absence of GOD, H2O2 steady-state levels were 0.06 ± 0.01 µM, and the addition of SpNO (1 mM) increased the H2O2 levels slightly to 0.14 ± 0.05 µM. The addition of 20 U/l GOD, in the absence of SpNO, led to an increase in H2O2 steady-state levels that reached a maximum at 1 to 2 h of incubation (Fig. 2A), levels at 1 h being 5.1 ± 0.7 µM. When SpNO (1 mM) was added in addition to 20 U/l GOD, the initial rise in H2O2 levels, up to 1 h, was similar to the one in incubations with GOD alone. However, in the presence of SpNO H2O2 steady-state levels continued to rise beyond 1 h of incubation (Fig. 2A). In contrast to 1 mM SpNO, and in line with its only marginal effect on cell viability, 0.1 mM SpNO did not alter H2O2 steady-state levels (data not shown).
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Effect of NO/NOx on H2O2 Degradation
As the addition of SpNO increased H2O2 steady-state levels in the cell cultures, but did not affect H2O2 generation by the glucose/GOD system and only slightly increased cellular H2O2 generation, inhibition of H2O2 degradation by NO/NOx appeared to be the most likely explanation for the effects of SpNO. Therefore, we assessed the capacity of the cells to degrade H2O2, added as a bolus, in the absence and in the presence of SpNO. In line with the time course of H2O2 steady-state levels (cf. Fig. 2A), preincubation of endothelial cells with 1 mM SpNO for 15 min before the addition of a 50 µM H2O2 bolus did not inhibit H2O2 degradation (Fig. 3A). After 45 min of preincubation with SpNO, a slight but still nonsignificant inhibition of H2O2 degradation was observed (data not shown), and after 75 min of preincubation with SpNO, marked inhibition of H2O2 degradation was present (Fig. 3A; please note, that this delay with which the inhibitory effect developed in these cells fits well with the effect of SpNO on H2O2 steady-state levels as presented in Fig. 2A). In contrast to this delayed effect of SpNO on the degradation of a 50 µM H2O2 bolus, the degradation of 200 µM H2O2 was already inhibited after a 15-min preincubation with the NO donor (Fig. 3C). Similarly, in hepatocytes the addition of SpNO (1 mM) strongly inhibited H2O2 degradation (Fig. 3, B and D). In contrast to liver endothelial cells, however, in hepatocytes this inhibition was already present after 15-min preincubation with the NO donor, irrespective of the concentration of H2O2 applied.
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As the addition of SpNO uniformely caused inhibition of H2O2 degradation in both cell types, although with different time courses, we assessed the effects of SpNO on the different cellular systems involved in H2O2 degradation, namely the glutathione/glutathione peroxidase system and catalase. When liver endothelial cells were incubated in KH buffer supplemented with 10 mM glucose, addition of 20 U/l GOD was not sufficient to decrease cellular GSH or increase cellular GSSG content (Fig. 4A). The addition of SpNO, however, strongly decreased cellular GSH levels in the absence as well as in the presence of GOD. The decrease in GSH levels was not accompanied by an increase in cellular GSSG content (Fig. 4A) nor was it accompanied by an increased glutathione (total glutathione, GSH + GSSG) extrusion (data not shown).
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Effect of NOx on Cellular Activity of Glutathione Peroxidase
In liver endothelial cells incubated in the absence or presence of SpNO, cellular activity of glutathione peroxidase did not change significantly over 1 h of incubation (Fig. 5A). Thereafter, however, endothelial cell glutathione peroxidase activity decreased in the presence of SpNO, being significantly lower than in the absence of SpNO after 2 h of incubation. This inhibition was concentration dependent, requiring an SpNO concentration 0.25 mM to decrease significantly (Fig. 5B). The delay in the inhibition of glutathione peroxidase (Fig. 5A), which fits well with the delayed effects of SpNO on H2O2 degradation in the lower range of H2O2 concentrations in these cells (Fig. 3A) and with the delayed effect of SpNO on H2O2 steady-state levels in these cells (Fig. 2A), might suggest an involvement of NO-derived NOx rather than NO itself in glutathione peroxidase inhibition. We therefore tested the effects of the NOx scavenger ascorbate (4, 16, 34, 35, 66) and of hypoxic conditions (under which NOx formation is inhibited) on the SpNO-induced inactivation of glutathione peroxidase: both, ascorbate (100 µM) and hypoxia inhibited the inactivation of endothelial glutathione peroxidase (Fig. 5C), thereby strongly suggesting that endothelial glutathione peroxidase is inactivated by NOx.
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Effect of NO on Catalase Activity
While in endothelial cells the effect of NOx on glutathione peroxidase activity can account for the (delayed) inhibition of H2O2 degradation in the range of low H2O2 concentrations (Fig. 3A and also Fig. 2A), the early inhibition of the degradation of high H2O2 concentrations in endothelial cells and of H2O2 degradation in all concentration ranges in hepatocytes can neither be explained by inhibition of glutathione peroxidase nor by the decrease in glutathione levels (that is similar for endothelial cells and for hepatocytes; Fig. 4). However, in contrast to liver endothelial cells, hepatocytes are known to possess high catalase activities, and catalase, a heme-containing enzyme, is known to be inhibited by NO in a cell-free system (10, 17). Therefore, we tested whether inhibition of catalase could explain the early inhibition of H2O2 degradation. Isolated catalase could be inhibited by NO (released by SpNO) in the concentrations used in the cellular experiments (Fig. 6), and the inhibitory effect of 1 mM SpNO on hepatocellular H2O2 degradation was similar to the effect of 50 µM azide, a well-known inhibitor of catalase (Fig. 3D). Similarly, H2O2 steady-state levels in hepatocyte cultures exposed to the glucose/GOD system were not only increased by 1 mM SpNO but also, with similar kinetics, by 50 µM azide (data not shown). As 1 mM SpNO, azide did not increase cell injury in normal hepatocytes exposed to 100 U/l GOD or in GSH-depleted hepatocytes in the absence of GOD (data not shown), but it did increase injury in GSH-depleted hepatocytes exposed to 100 U/l GOD (Fig. 1B). With regard to these results, inhibition of catalase by NO is likely to account for the inhibition of H2O2 degradation in hepatocytes treated with SpNO.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Evidence that NO can bind to catalase was produced by Keilin and Hartree (32) 70 years ago. This observation has subsequently been confirmed by various other groups. Two mechanisms appear to be responsible for the inhibition of catalase by NO (for a review, see Ref. 67). Just as it is known from other heme-containing enzymes, NO can bind to the heme moiety of native (basal state) catalase, resulting in heme nitrosylation (formation of a complex between the heme iron and NO). This heme nitrosylation prevents the interaction of H2O2 with the iron center and thus H2O2 degradation. Alternatively, reaction of NO with the compound I form of catalase has been proposed, based on the observation that catalase can catalyze the consumption of NO in the presence of H2O2 (10). Reaction of compound I (formed by the reaction of ferric catalase with one H2O2 molecule) with NO, leading to the formation of nitrite and compound II, which then rapidly reacts with an additional NO (resulting in the formation of an additional nitrite anion), prevents reaction of compound I with a second H2O2 molecule (67). These reactions, consuming 2 NO and 1 H2O2 and producing 2 nitrite anions per cycle, thus retard the degradation of H2O2 at the expense of NO metabolism. Regardless of which of these two mechanisms is the predominant one, in fact, one would expect this to vary with the concentrations of both NO and H2O2, NO inhibits catalase reversibly and with high affinity [Ki of 0.18 µM in the study by Brown (10), 80% inhibition of H2O2 degradation by 
15 µM NO in the study by Farias-Eisner et al. (17)]. The NO levels which inhibited catalase in the current study were in the range of 1–10 µM [half-maximal inhibition of isolated catalase with 0.1 mM SpNO which, in the system used, gives steady-state levels of NO of 1.5 µM (unpublished results), and, in the cellular system, pronounced inhibition with 1 mM SpNO, which gives a steady-state level of >5 µM NO (15, 48)]. As the inhibition is accomplished by NO itself, the effect could already be observed at an early time point in the cellular system.
Glutathione peroxidase is the second H2O2-degrading enzyme in endothelial cells and hepatocytes (25). In contrast to catalase, data on inhibition of glutathione peroxidase by NO are controversial. While Asahi et al. (3) reported inhibition of glutathione peroxidase by NO, Farias-Eisner et al. (17) and Keese et al. (31) did not find this inhibition. This discrepancy may have arisen because the inhibition described by Asahi et al. results from direct transnitrosation by the NO donor SNAP, or because it was mediated by NOx such as peroxynitrite, as inhibition appears to be dependent on oxidative modification of a thiol group of the enzyme. In line with the results of Farias-Eisner et al. and Keese et al., our experiments do not provide any evidence for inhibition of glutathione peroxidase by NO itself: H2O2 degradation by endothelial cells (except for the high concentration range) was only inhibited at late time points, already suggesting the involvement of NOx. Direct assessment of glutathione peroxidase activity confirmed a delayed inhibition (Fig. 5A), and this inhibition was prevented by ascorbate, a potent scavenger of NOx (4, 16, 34, 35, 66), and under hypoxic conditions that should strongly decrease the formation of NOx (Fig. 5C). Thus inhibition of glutathione peroxidase appears to be mediated by NOx. At ambient oxygen levels, the NO levels required for the inhibition of glutathione peroxidase were in the same range as those required for the inhibition of catalase [half-maximal inhibition with 0.5 mM SpNO (Fig. 5B), i.e., a donor concentration which resulted in NO steady-state levels of 3–4 µM in the same cellular model (15)].
SpNO, the NO donor used here, has a half-life of 37 min at 37°C (18). Thus NO release is highest within the first half hour but substantially lowered thereafter. However, NO steady-state levels behave somewhat differently: they slowly build up, reach a maximum at 20–60 min and slowly decline thereafter, so that in the liver endothelial cell and hepatocyte systems incubated at 21% O2, as used here, steady-state levels of NO at 120 min were still at 50% of peak NO levels (15, 48, and unpublished results). Thus, also the reversible inhibition of catalase by NO can be expected to be present over a major part of the incubation periods of the experiments presented here, although it is likely to be less pronounced at the later stages of the experiments. Possibly, this might contribute to the time course of the injury observed in hepatocytes (Fig. 1B) with a major part of the injury occurring during the early stages of the experiment.
Ascorbate, used here to scavenge NOx, has been shown to be about three times more effective than GSH in inhibiting N2O3-dependent nitrosation reactions in cell-free systems (66) and to be highly effective in inhibiting N2O3-mediated nitrosation reactions in cellular systems (4, 16). Furthermore, ascorbate is an effective scavenger of NO2 (34, 35). The reaction of ascorbate with peroxynitrous acid, in contrast, only has a low rate constant (5, 60). However, in the presence of CO2, i.e., under conditions that are relevant for biological systems and that were used here, the toxicity of peroxynitrite is mediated by NO2 and CO3 radicals, formed in the reaction of CO2 with peroxynitrite, and not by peroxynitrous acid (12, 39, 61). Ascorbate reacts rapidly with both, NO2 and CO3 radicals, much faster than GSH or other thiols do (34, 55). Thus ascorbate can be regarded as the central cellular antioxidant against NOx (34, 35). In the absence of ascorbate, NOx might lead to the oxidation or to the S-nitrosation of GSH as well as to the oxidation or S-nitrosation of a crucial thiol group of the selenocysteine enzyme glutathione peroxidase, which is the likely reason for glutathione peroxidase inactivation.
Besides inhibition of catalase and glutathione peroxidase, NO and/or its derivatives had a pronounced effect on cellular glutathione homeostasis: the addition of the NO donor led to a relatively rapid decrease in the cellular content of reduced glutathione that could neither be accounted for by oxidation of glutathione to GSSG (as might have been possible with regard to the effect of SpNO on H2O2 steady-state levels) nor by cellular glutathione extrusion. A likely explanation for this decrease in reduced glutathione is that part of the glutathione reacts with NOx to form nitrosoglutathione. However, this decrease in cellular reduced glutathione did not appear to be of a major functional significance as it was also present under conditions where no inhibition of H2O2 degradation was observed (i.e., in endothelial cells at early time points; Figs. 2A and 3A vs. 4A).
For most cells, including endothelial cells (33, 56, 63) and cardiomyoblasts (59), cellular glutathione peroxidase activity is considered to be predominantly responsible for H2O2 degradation. This is especially true at low H2O2 levels. The data on H2O2 degradation in liver endothelial cells using the GOD system and the smaller bolus of H2O2 are in line with this concept. Hepatocytes, on the other hand, have high glutathione peroxidase activities but also very high catalase activities (24). In these cells, catalase apparently played a major role in H2O2 degradation; this is shown by the effects of azide on H2O2 degradation in this cell type, and the consistently early effects of SpNO on hepatocellular H2O2 degradation under all conditions studied here also fit with a predominant role of catalase. At high H2O2 levels, glutathione peroxidase gets saturated and glutathione is depleted, while catalase is not saturable. Therefore, at higher H2O2 levels, catalase becomes increasingly more important, as has also been shown for endothelial cells (56) and in isolated heart mitochondria (2); this can explain the different behavior (inhibition by azide, early inhibition by SpNO) of liver endothelial cells exposed to a higher concentration H2O2 bolus (Fig. 3C). Thus inhibition of catalase by NO, an early effect, apparently caused the inhibition of H2O2 degradation in hepatocytes and contributed to the inhibition of H2O2 degradation in endothelial cells exposed to high H2O2 levels. Inhibition of glutathione peroxidase by NOx required time, appeared to be less pronounced in hepatocytes, in which a major part of the NOx apparently was scavenged by the glutathione present in high levels in these cells and, most likely, by endogenous ascorbate, but appeared to be the most important effect in endothelial cells in the lower and pathophysiologically more interesting regions of H2O2 concentrations.
H2O2 is highly membrane permeable. Nevertheless, H2O2 gradients form if the H2O2 production site and the H2O2 degradation site are separated by membranes (1, 8, 58). For Jurkat T-cells exposed to an extracellular H2O2 source, the cytosolic H2O2 concentration has been estimated to be by a factor of 7 below extracellular levels (1). Similar H2O2 gradients have been reported for bacteria (58) and for yeast cells (8). However, due to the high membrane permeability of H2O2, the equilibrium between extra- and intracellular levels of H2O2 has been reported to be reached extremely rapidly, i.e., within 1 s in Jurkat T cells (1). Thus alterations in cellular H2O2-degrading capacity between different experimental groups can safely be judged by monitoring extracellular H2O2 levels, as done here. However, one should keep in mind that intracellular H2O2 levels are bound to be substantially lower than the extracellular levels measured.
Although the inhibition of H2O2 degradation shown here is a sufficient explanation for the increase in H2O2 toxicity mediated by NO/NOx, we cannot rule out the possibility that other effects of NO on cellular metabolism may also contribute to this phenomenon. In addition to catalase and glutathione peroxidase, inhibition of a variety of other enzymes by NO/NOx has been reported (13, 38, 67). One of the best studied cases in which inhibition can be produced directly by NO and in which inhibition is likely to have severe consequences for the cell is inhibition of cytochrome oxidase. However, binding of NO to the heme moiety of this enzyme, which has been reported to occur with high affinity (6, 11, 13, 41, 57), is also known to occur in competition to oxygen (11). In our experimental systems, there was no evidence for a contribution of inhibition of cytochrome oxidase by NO to cell injury. Although hepatocytes crucially depend on mitochondrial energy production, even 1 mM SpNO hardly affected viability in the absence of H2O2, a finding that is due to the relatively high oxygen partial pressure (atmospheric oxygen content) used; 1 mM SpNO at lower oxygen levels or 2 mM SpNO at 21% oxygen does cause an energy deficiency-related injury in cultured hepatocytes (48). Liver endothelial cells are especially in the presence of glucose far less susceptible to adverse effects of inhibition of the mitochondrial respiratory chain than hepatocytes (15, 51). Nevertheless, these cells were also injured by the NO donor in the absence of H2O2 (far less than in the presence of H2O2 but still considerably; Fig. 1A). This toxicity induced by SpNO alone was due to NOx as shown in previous studies (15, 27).
NO concentrations assumed to occur in vivo are below 1 µM under physiological conditions (11, 13, 23); however, under pathophysiological conditions, NO concentrations in the low micromolar range are considered to be reached locally (9, 13, 42). As under diverse pathophysiological conditions where NO formation is increased, e.g., during endotoxemia or during inflammatory reactions (e.g., Ref. 40), formation of H2O2 can also be expected to be increased (e.g., Ref. 29), the inhibition of catalase and/or glutathione peroxidase by NO or NOx as described here is likely to be of pathophysiological relevance.
In conclusion, we showed here that inhibition of H2O2 degradation via either inhibition of catalase by NO and/or inhibition of glutathione peroxidase by NOx can explain the enhancement of H2O2 toxicity by NO.
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ACKNOWLEDGMENTS |
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Present address of T. Li: Max-Planck-Institut für Molekulare Physiologie, Otto-Hahn-Str. 11, 45227 Dortmund, Germany.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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