Department of General Surgery and Division of Surgical Research, University of Ulm, Ulm 89073, Germany
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ABSTRACT |
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Low arterial
blood pH and sustained nitric oxide (NO) production are critical
parameters in inflammatory events such as sepsis, and appropriate
treatment is still under debate. Because the stability of nitrogen and
oxygen intermediates is dependent on the surrounding pH, we
investigated whether the relationship among NO, peroxynitrite (ONOO), and reactive
oxygen species production also depends on the pH value, particularly
with respect to their effects on hepatocellular damage. Our studies
demonstrate that the extracellular pH influences NO and hydroxyl
radical (OH) production in hepatocytes. Acidification (pH 7.0) of the
medium revealed a significant increase
(P < 0.05) of OH-like radicals,
enhanced hepatocellular damage, and a sharp drop in cellular
glutathione (GSH) content compared with levels measured at
physiological or alkaline pH conditions. Furthermore, inhibition of NO
synthesis at all pH conditions resulted in decreased NO production and
cellular GSH levels but a simultaneous increase of OH-like radicals and
hepatocellular damage with a maximum seen at pH 7.0. Our results
suggest that hepatocellular damage is in part regulated by the
surrounding pH and that inhibition of NO synthesis at acidic conditions
(e.g., in sepsis) leads to increased reactive oxygen-mediated cell
injury.
hepatocytes; inducible nitric oxide synthase; oxygen radicals; sepsis
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INTRODUCTION |
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SEVERE INFECTIONS IN SEPTIC patients can lead to hepatic dysfunction, which can be life threatening to the critically ill (9). Low arterial blood pressure, due in part to increased nitric oxide (NO) formation, along with a decrease of blood pH and sustained acidosis, is frequently observed in severe inflammatory events (6). During sepsis the optimal treatment of metabolic dysfunction and hypotension is still a matter of debate, whereas low arterial blood pH and the resulting acidosis can be partially corrected by infusing NaHCO3. Under septic conditions the liver plays a pivotal role, resulting in numerous metabolic changes. NO is a molecule that is abundantly produced in liver cells during infections and has been linked with several "pathophysiological" changes within the liver, such as hepatic artery blood flow, glucogenolysis, prostaglandin synthesis, or hepatocellular damage (20, 22, 35, 41). On the other hand, recent publications suggest that the inhibition of NO synthase (NOS) can overcome pathophysiological effects, such as hypotension or certain forms of multiple organ dysfunction, caused by sustained NO overproduction (32, 40).
NO is produced by at least three separately identified NOS isoforms (19). NOS1 and NOS3 are constitutively expressed, whereas NOS2 is synthesized in cells and tissues after exposure to cytokines and/or various pathogens. In the liver the overproduction of NO can have profound cytostatic actions on target cells or the cells that produce NO. We have shown that NOS2 is regulated in rat, human, baboon, and mouse hepatocytes by inflammatory cytokines, endotoxin [lipopolysaccharide (LPS)], and growth factors (24, 26, 30). Increased NOS2 synthesis in hepatocytes can lead to the suppression of DNA synthesis (24, 29), profound reduction in protein synthesis (8) and in mitochondrial aconitase (36), the inhibition of GAPDH (29), and cytochrome P-450 activity (37), as well as apoptosis (24). On the other hand, NO has been suggested to protect against hepatic thrombosis and oxygen radical-mediated injury during endotoxemia (5, 15, 20, 22, 35).
It is well established that hepatocytes produce various nitrogen and
reactive oxygen intermediates (ROI) in response to cytokines, pathogens, or other forms of cell injury (1, 14, 21). Among these
oxygen species [e.g., superoxide anion
(), hydrogen peroxide
(H2O2),
hydroxyl radical (OH)], the formation of OH is considered to be
one of the major toxic radical forms (1, 14, 38). OH or OH-like
radicals can be formed when
reacts
with
H2O2
in the presence of iron or when NO reacts with superoxide to form
peroxynitrite
(ONOO
). The
latter can either spontaneously rearrange to form nitrate (
) or undergo cleavage to
generate OH-like radicals and nitrite
(
) (4). Interestingly, the
formation and stability of OH-like radicals either by the iron-catalyzed Fenton and Haber-Weiss chemistry or via
ONOO
is favored in the
presence of an acidic pH (2, 7). In the present study, we hypothesized
that pH changes in experimental sepsis interfere with the production of
reactive oxygen species and NO intermediates, hence directly affecting
the hepatocellular damage. In addition, we investigated
the consequences of NOS inhibition at different pH values on reactive
radical formation and hepatocellular damage.
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EXPERIMENTAL PROCEDURES |
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Materials.
William's medium E, penicillin-streptomycin,
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (HEPES), and calf serum were purchased from GIBCO BRL
(Paisley, Scotland). Percoll was obtained from Pharmacia (Uppsala,
Sweden). Western blot kit was supplied by Amersham [enhanced
chemiluminescence (ECL), Buckinghamshire, UK].
Corynebacterium parvum
(C. parvum) was purchased from
Immunochem Research (Hamilton, MT). Cytokines used in this study
included rat recombinant interferon- (IFN-
; Genzyme, Cambridge,
MA), human recombinant interleukin-1
(IL-1
; Cistron, Pine Brook, NJ), and murine tumor necrosis factor-
(TNF-
; Genzyme).
Collagenase H was obtained from Boehringer Mannheim (Mannheim,
Germany). NaHCO3, HCl, gelatin,
and LPS (Escherichia coli 0111:B4)
were purchased from Sigma (Deisenhofen, Germany). All other reagents
were also obtained from Sigma unless otherwise indicated.
Animals. Adult male Sprague-Dawley rats (Charles River, Sulzfeld, Germany) weighing 250 ± 50 g were used in accordance with the institutional animal welfare guidelines of the University of Ulm and the Government of the Land Baden-Württemberg. C. parvum (20 mg/kg body wt) diluted in physiological saline was injected intravenously 4 days before liver perfusion. Control animals were injected with 0.9% saline (5). Animals were kept at constant temperature (22-24°C) and humidity, under a 12:12-h dark-light cycle, and fed commercial rat chows (Altromin 1314, Forti, Lage, Germany).
Isolation of hepatocytes and cell culture.
Hepatocytes from C. parvum-injected
and control animals were isolated by a two-step collagenase perfusion
technique as previously described (31). Briefly, hepatocytes were
separated from nonparenchymal cells by differential centrifugation at
50 g and then passed over a 30%
Percoll gradient at a concentration of
106 hepatocytes/ml Percoll to
obtain a highly purified cell population. Hepatocyte purity, assessed
by microscopy, was always >95%, and viability consistently exceeded
90% by trypan blue exclusion. Freshly harvested hepatocytes were
plated onto gelatin-coated 35-mm dishes (Nunc, Roskilde,
Denmark) at a density of 5 × 104
cells/cm2, a density shown to be
associated with maximal NO synthesis in hepatocytes (31), and incubated
at 37°C in 95% air-5% CO2
for 24 h. Medium consisted of William's medium E supplemented with insulin (106 M), HEPES (15 mM), L-glutamine, penicillin,
streptomycin, and 10% low-endotoxin calf serum. At the time of
stimulation, cell cultures were exchanged to serum-free medium
supplemented with 0.5 mM
L-arginine.
Measurement of NO formation, OH-like radical,
, and
ONOO
formation.
Cell culture supernatants were assayed for the stable end-products of
NO oxidation (
plus
), using a modified procedure based
on the Griess reaction as recently described (27). Briefly, samples
were deproteinized and enzymatically reduced
(
to
) by
reductase, and then aliquots (150 µl) were screened for total
levels (representing
plus
) with a mixture of 75 µl
ice-cold Dapsone (4,4'-diaminodiphenylsulfone; 14 mM in 2 N HCl)
and 75 µl
N-(1-naphthyl)ethylenediamine (4 mM in
H2O). After incubation at room
temperature for 5 min, light absorption was measured at 550 nm in a
microplate reader (EAR 300; SLT, Crailsheim, Germany).
levels were calculated from a
standard curve.
Measurements of hepatocellular damage. To evaluate the hepatocellular damage after various treatments, cell culture supernatants from hepatocytes incubated for 24 h were tested for lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) release. Both parameters were determined by an automatic procedure using a Technitron RA-500 autoanalyzer (Technitron, Tarrytown, NY). In addition, the determination of cell viability (see Isolation of hepatocytes and cell culture) was measured after treatment to exclude differences due to cell death.
Measurement of intracellular GSH levels. Total cellular glutathione (GSH) was detected by a modified method previously reported by Griffith (13) with an enzymatic assay based on GSH reductase.
SDS-PAGE and Western blot analysis.
NOS2 protein expression after different treatments was studied using
Western blot analysis as recently described (26, 31). Hepatocytes were
plated onto 90-mm-diameter culture dishes (Nunc) and incubated
according to the experimental protocol for 12 h. Cells were then
scraped off the culture dishes, pelleted, washed three times with PBS,
and homogenized in buffer containing 20 mM
N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic
acid, 2 mM DL-dithiothreitol,
and 10% glycerol, as well as the protease inhibitors antipain (25 µg/ml), aprotinin (25 µg/ml), chymostatin (25 µg/ml), leupeptin
(25 µg/ml), pepstatin A (10 µg/ml), phenanthroline (50 µM), and
phenylmethylsulfonyl fluoride (100 µM). Cell
homogenates were then subjected to three rapid freeze-thaw cycles and
centrifuged at 100,000 g for 60 min at
4°C. The cytosolic fractions (crude cytosol) were stored at
80°C until further processing. Protein concentrations were
measured using a commercially available test based on the Lowry
reaction (Sigma). Equal amounts of total protein (50 µg) obtained
from rat hepatocyte cytosol were separated by 7% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and
electrophoretically transferred to nitrocellulose membranes in the
presence of 20% methanol, 25 mM tris(hydroxymethyl)aminomethane, and
192 mM glycine, at pH 8.3. Nonspecific binding to the membrane was
blocked by 5% nonfat dry milk in PBS-Tween 20 at 4°C overnight. The blots were washed twice in PBS-Tween 20 and then incubated with an
affinity-purified immunoglobulin G (IgG) polyclonal rabbit-anti-mouse antibody NOS2 (1:2,500; Affinity, Exeter, UK). Membranes were subjected
to three washing procedures and incubated with the secondary antibody
[goat-anti-rabbit IgG, conjugated with horseradish peroxidase (Amersham), dilution 1:1,500] at room temperature for 1 h. After incubation, membranes were washed three times with PBS-Tween 20 and
developed with 10 ml of a 1:1 mixture of solution
1 and solution 2 of
the ECL detection system (Amersham) for 1 min, dried immediately, and
exposed to a film for 0.5-15 min.
Statistical analysis. Values are expressed as means ± SE. Significance of differences was determined with the use of the analysis of variance test (Statview statistics program; Abacus Concepts). Statistical significance was established at P < 0.05.
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RESULTS |
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pH-dependent
-
formation.
Figure
1A
depicts
plus
levels measured in normal rat
hepatocytes after stimulation with CM. Incubation with CM at a
physiological pH (7.4 ± 0.1) led to a potent increase of NO
formation that was not significantly changed when the pH in the medium
was adjusted to pH 7.0 ± 0.1. In contrast, incubation of nonprimed
hepatocytes with CM at pH 7.8 ± 0.1 showed a significant increase
(P < 0.05) in NO levels compared
with values detected at pH 7.4 ± 0.1. Severe infection, as seen in
septic shock syndrome for example, is characterized by two phases, a
priming phase to trigger metabolic changes followed by a second insult
such as LPS (9, 41). Therefore, C. parvum-primed hepatocytes were exposed only to LPS to
mimic a second insult. Isolated hepatocytes from in vivo primed rats
showed a "constitutively" high level of NO production (96.2 ± 10 µM measured as
plus
) at a physiological pH. This
generation was unchanged when the pH in the medium was either decreased
to pH 7.0 or increased to pH 7.8 (Fig.
1B). However, the NO production was
increased to 239.3 ± 18 µM when C. parvum-primed hepatocytes were exposed to LPS at pH 7.4 ± 0.1. Even though an increased NO formation (96.2 ± 10 vs.
163.2 ± 5.7 µM) was detected in response to LPS at an acidic pH,
this increase was significantly (P < 0.01) lower than NO levels measured at a physiological or alkaline pH.
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pH-dependent hepatic OH-like radical formation.
The formation and stability of OH or OH-like radicals, either through
the metal-catalyzed Fenton and Haber-Weiss chemistry or via the
formation of ONOO, is
dependent on the surrounding pH levels (2, 7, 16) and has been shown to
damage cells (33, 43), including hepatocytes (38). Therefore, we
investigated whether the generation of OH or OH-like radicals also
relies on the pH in primed and normal hepatocytes when exposed to LPS
and CM, respectively. Figure 2 shows that
acidification of the medium (pH 7.0) results in a significant (P < 0.01) OH-like radical
production (measured as 2,3-DHB plus 2,5-DHB) compared with levels
measured at physiological or alkaline medium conditions. Incubation of
normal or C. parvum-primed hepatocytes with CM or LPS resulted in a further OH-like radical production (2,3-DHB plus 2,5-DHB) at all pH conditions. The increase was, however,
only significant (P < 0.05) at pH
7.0. It is interesting to note that the total amount of OH-like radical
formation decreased with the alkalinization of the surrounding pH (7.0 > 7.4 > 7.8) in stimulated and unstimulated hepatocyte cultures
(Fig. 2, A and
B).
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pH-dependent ONOO formation.
Recent evidence suggests that the formation of
ONOO
and its stability are
more likely under acidic rather than physiological or alkaline pH
conditions (2, 7, 16). Because septic patients frequently suffer from
acidosis with a low blood pH in addition to increased NO
levels, we performed studies to investigate whether we would observe an
increased ONOO
formation
under acidic conditions. In supernatants of normal and
C. parvum-primed hepatocytes we
detected a significant (P < 0.05)
increase of ONOO
after the
exposure to CM or LPS (Table 1). The
highest increase was observed at pH 7.0. It is interesting to note that
at pH 7.0 normal hepatocytes showed a high background level of oxidized dihydrorhodamine 123 that was not completely abolished after
L-NMMA addition. Although we
added catalase to the cultures, one possible explanation for this
background might be the presence of increased H2O2
formation that can cause the oxidation of dihydrorhodamine 123 in
the presence of cytochrome-c or
endogenous peroxidases.
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pH-dependent hepatocellular damage. Cell viability was consistently 88 ± 3.5% in all cell cultures after 24 h of treatment, demonstrating that the observed differences were not due to cell death. As shown in Fig. 3, A and B, the transaminase baseline changed in treated and nontreated cultures, depending on the pH in the medium. The highest release of LDH and AST was observed in nonstimulated normal and C. parvum-primed hepatocytes at acidic pH conditions. Furthermore, we observed in cytokines and/or LPS-stimulated normal or C. parvum-primed hepatocytes a significant (P < 0.05) increase of LDH release at acidic (pH 7.0) medium conditions. The lowest AST and LDH levels were measured at pH 7.8 in stimulated or nonstimulated normal and C. parvum-primed hepatocytes (Fig. 3, A and B).
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pH-dependent effects of hepatic NOS inhibition by L-NMMA. Several recent studies suggest that the inhibition of NO synthesis in sepsis promotes oxygen radical-mediated hepatic injury (5, 15, 20, 22, 35). Thus we studied OH-like formation and NO formation under acidic, physiological, and alkaline medium conditions in the presence or absence of inflammatory stimuli and/or L-NMMA. In addition, we measured the LDH and AST release under these circumstances to elucidate the hepatocellular damage caused by both the pH changes in the medium and the inhibition of NOS. Inhibition of NOS by L-NMMA in CM-stimulated normal hepatocytes resulted only at pH 7.0 in a significant (P < 0.01) release of OH (measured as 2,5-DHB) and LDH in the supernatants (Figs. 4A and 5A). In contrast, the inhibition of NOS in LPS-treated C. parvum-primed hepatocytes led to a significant increase of OH (measured as 2,5-DHB) and LDH at all pH conditions (Figs. 4B and 5B).
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Effects of pH-dependent changes and NO synthesis inhibition on GSH levels. It has been demonstrated that the presence of GSH is necessary for a functional NO synthesis of the purified enzyme (39) and in endothelial cells (12). In addition, it was shown that extracellular GSH is a protecting compound against oxidative liver damage (23). Therefore, we investigated whether hepatocellular GSH levels are dependent on the surrounding pH, NO synthesis induction, and inhibition. Figure 6, A and B, shows that GSH content is higher in normal nonprimed hepatocytes than in C. parvum-primed hepatocytes under all culture conditions. Acidification of the medium (pH 7.0) resulted in a significant decrease of GSH levels in normal and C. parvum-primed hepatocytes compared with levels detected at pH 7.4 and 7.8. The induction of NO synthesis by cytokines and/or LPS in normal or C. parvum-primed hepatocytes resulted in cellular GSH depletion under all pH conditions. Moreover, GSH content was further decreased when NO synthesis was inhibited by L-NMMA.
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pH-dependent superoxide formation.
Once superoxide is formed in a cell it can react in several ways. With
NO it can form ONOO and
react to
and
. To address the issue of whether
the dramatic drop of intracellular GSH is the result of a possible
increased oxidative damage when L-NMMA is added to hepatocyte
cultures, we measured
levels at all
culture conditions in the presence and absence of L-NMMA. As shown in Table 1 we
found the highest
level in control
supernatants, with the highest degree at pH 7.0. Addition of CM or LPS
resulted always in a drop of
levels
that was partially corrected in the presence of
L-NMMA. These results underline
the protective role of NO in hepatocytes, but they also show that
radicals can act in concert on the antioxidative GSH system.
pH-dependent NOS2 protein expression. To determine whether the changes of NO production seen at different pH conditions were associated with different hepatocellular NOS2 protein expression, Western blot analyses were performed on cell lysates from treated and nontreated hepatocyte cultures. Stimulation of normal hepatocytes with CM resulted under all pH conditions in a similar degree of NOS2 protein expression (data not shown). Although NOS2 was constitutively expressed in C. parvum-primed hepatocytes and further increased after LPS stimulation, no significant changes were observed among the different pH conditions (Fig. 7).
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DISCUSSION |
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During severe inflammatory events such as sepsis, several metabolic and pathophysiological changes take place, including the secretion of nitrogen and oxygen intermediates. Parallel to these changes local or systemic acidification is observed, followed by severe acidosis and organ dysfunction (e.g., liver). Although it is established that NO controls liver blood flow and hepatic metabolism, its role in cellular and molecular mechanisms of cell injury and/or protection is far from clear. Under septic conditions, NOS overexpression in the liver can lead to several consequences: the decrease of protein and DNA synthesis, enhanced antimicrobial activity, inhibition of GAPDH, inhibition of enzymes of mitochondrial respiration, inhibition of cytochrome P-450, apoptosis, and intracellular nonheme iron-nitrosyl formation (19). On the other hand, NO has been described to be cytoprotective in the liver by preventing oxygen-mediated cellular injury (5, 15, 20, 22, 35). Because the cytotoxic potential as well as the stability of oxygen and nitrogen intermediates seem strongly dependent on the surrounding pH (2, 7, 16, 33), the aim of the present study was to investigate the effects of pH changes on both free radical formation and hepatocellular damage.
Our experiments clearly revealed that the formation of OH or OH-like
radicals, ONOO, NO
production, and hepatocellular damage are strictly dependent on pH
conditions in normal and C. parvum-primed hepatocytes. Modest acidification (pH
7.0) of the medium as seen in sepsis resulted in an increased OH-like
radical and LDH baseline in normal and C. parvum-primed hepatocytes compared with levels measured
at physiological or alkaline medium conditions. The stimulation of normal and C. parvum-primed
hepatocytes with CM and LPS, respectively, led to an increased NO
production and a further OH-like radical generation. However,
significant hepatocellular damage (measured as LDH release) in normal
and C. parvum-primed hepatocytes was only found at pH 7.0. It is noteworthy that the NO production in
LPS-stimulated C. parvum-primed
hepatocytes is lower in acidic than in physiological or alkaline
conditions. Our results suggest that the generation of OH-like radicals
either by the superoxide-driven Fenton reaction or via
ONOO
in septic livers may
be faster and more stable under acidic conditions (2) compared with the
reaction of NO with superoxide at physiological or alkaline conditions.
In keeping with this evidence is our observation that the relative
amounts of superoxide decrease in parallel with an increased NO
production in stimulated hepatocytes at all culture conditions, which
demonstrates the antioxidative effects of NO by scavenging superoxide.
ONOO has been reported to
exert several cytotoxic effects in various cell types (4, 7, 33, 43).
In normal and C. parvum-primed
hepatocytes, we measured
ONOO
levels at all culture
conditions after the stimulation with CM and LPS. Although we were
unable to detect large amounts of
ONOO
at pH 7.4 and pH 7.8, our data are still in agreement with recent published work by Radi et
al. (33). These authors reported that cellular damage caused by
exogenous addition of ONOO
is enhanced under acidic rather than alkaline pH conditions. This
finding of pH-dependent cytotoxicity of
ONOO
was further
substantiated by data from Crow et al. (7), who demonstrated that a
less toxic ONOO
isoform is
synthesized at alkaline pH conditions. The same line of evidence
stresses our observations of low
ONOO
formation, which
correlates with the lowest percentage of cell damage. However, from our
experimental setup we were unable to distinguish between different
isoforms of ONOO
(7).
The inhibition of NO synthesis in LPS-treated primed hepatocytes caused increased hepatocellular damage at all medium conditions, with a maximum seen at acidic (pH 7.0) conditions. This hepatocellular damage, as observed when NOS is competitively blocked, is most likely due to increased ROI production, as demonstrated by increased superoxide and OH radical production. Under these conditions, increased OH production is generated because NO cannot react with superoxide to form less-toxic intermediates, thus promoting the iron-catalyzed Fenton and Haber-Weiss chemistry. Therefore, we hypothesize that generated radicals and their adducts in hepatocytes are part of a cascade that possesses higher cytotoxic capacity under acidic conditions and may play a minor role under physiological or alkaline pH conditions.
GSH has been shown to protect the liver from ROI toxicity (10, 20, 21,
23). We found that GSH content is decreased in hepatocytes under acidic
conditions and declines further when cells are exposed to cytokines
and/or LPS. In contrast, at physiological or alkaline medium
conditions we observed that reduced OH-like radical and
ONOO production was
paralleled by increased GSH levels. This increased GSH content at pH
7.4 and pH 7.8 correlated with an enhanced NO production and a
reduction of superoxide and hepatocellular damage in LPS-treated
C. parvum-primed hepatocytes.
Furthermore, increased GSH levels are associated with the necessity of
GSH for maximal NOS activity and enzyme stability (39).
Several investigators have shown that the inhibition of NO synthesis in the liver during sepsis promotes oxidative damage (5, 15, 20, 22, 35), resulting in increased ROI production. In addition, recent evidence suggests that NO itself may negatively regulate the antioxidative GSH system by inhibiting GSH reductase via the formation of S-nitroglutathione (3). Therefore, our results suggest that under certain circumstances NO itself can induce or enhance oxidative stress in cells by modifying GSH content, as recently suggested (25). The cytoprotective effects of NO in sepsis are described as follows: scavenging of radical oxygen intermediates, mainly superoxide (43), activation of soluble guanylate cyclase for guanosine 3',5'-cyclic monophosphate synthesis to inhibit platelet adherence (34) or aggregation (28), and neutrophil chemotaxis (17). In addition, there is increasing evidence that NO can antagonize the vasoconstrictive effects of the platelet-activating factor or prostaglandins to maintain hepatic blood flow (42).
On the basis of our data, it seems disadvantageous to reduce NO
formation at acidic pH conditions (pH 7.0 ± 0.1) (similar to the
acidosis observed in sepsis) because NO could not
function as a scavenger for ROI (43). Therefore, we suggest that in
sepsis, in which sustained acidosis and hypotension frequently occur, adjusting systemic pH toward the physiological value and then inhibiting NO would be more beneficial for patients than directly blocking sustained NO overproduction. This approach seems to be superior because it reduces both the high OH-like radical baseline and
the eventually formed
ONOO, because
either product is more stable at acidic than at physiological or
alkaline conditions (2, 7, 16, 33). In addition, our data imply that a
reduced OH-like radical baseline as seen under physiological or
alkaline pH conditions results in an increased intracellular GSH pool
that could additionally serve to detoxify reactive radicals and
increase NOS activity and NO production. Finally, our data emphasize
the complexity of radical formation and interactions that will need
further investigations before any application can be envisaged in vivo.
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ACKNOWLEDGEMENTS |
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This study was partially supported by grants from the Klinik Förderung of the University of Ulm (P-368 and P-453 to A. K. Nussler) and a grant from the Frauenhofer Gesellschaft InSan I 0793-v-1296 (U. B. Bruckner).
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FOOTNOTES |
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Address for reprint requests: A. K. Nussler, Div. of Surgical Research, Dept. of General Surgery, Univ. of Ulm, Parkstr. 11, 89073 Ulm, Germany.
Received 4 February 1997; accepted in final form 5 August 1997.
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