Inducible nitric oxide synthase in the lung and exhaled nitric
oxide after hyperoxia
Giovanni
Cucchiaro1,
Arthur H.
Tatum2,
Michael C.
Brown3,
Enrico M.
Camporesi1,
John W.
Daucher2, and
Tawfic S.
Hakim1
Departments of
1 Anesthesiology,
2 Pathology, and
3 Anatomy and Cellular
Biology, State University of New York-Health Science Center,
Syracuse, New York 13210
 |
ABSTRACT |
The effect of hyperoxia on nitric oxide (NO)
production in intact animals is unknown. We described the effects of
hyperoxia on inducible nitric oxide synthase (iNOS) expression and NO
production in the lungs of rats exposed to high concentrations of
oxygen. Animals were placed in sealed Plexiglas chambers and were
exposed to either 85% oxygen (hyperoxic group) or 21% oxygen
(negative control group). Animals were anesthetized after 24 and 72 h
of exposure and were ventilated via a tracheotomy. We measured NO production in exhaled air (ENO) by chemiluminescence. The
lungs were then harvested and processed for detection of iNOS by
immunohistochemistry and Western blotting analysis. The same
experiments were repeated in animals exposed to hyperoxia for 72 h
after they were infused with L-arginine. We used rats that
were injected intraperitoneally with Escherichia
coli lipopolysaccharide to induce septic shock as a
positive control group. Hyperoxia and septic shock induced expression
of iNOS in the lung. However, ENO was elevated only in
septic shock rats but was normal in the hyperoxic group. Exogenous infusion of L-arginine after hyperoxia did not increase
ENO. To exclude the possibility that in the hyperoxic group
NO was scavenged by oxygen radicals to form peroxynitrite, lungs were
studied by immunohistochemistry for the detection of nitrotyrosine.
Nitrotyrosine was found in septic shock animals but not in the
hyperoxic group, further suggesting that NO is not synthesized in rats
exposed to hyperoxia. We conclude that hyperoxia induces iNOS
expression in the lung without an increase in NO concentration in the
exhaled air.
hyperoxia; septic shock
 |
INTRODUCTION |
OXYGEN TOXICITY IN ANIMALS and humans is a well-known
phenomenon, with acute lung injury occurring within a few days of
exposure to high concentrations of oxygen. Although the pathophysiology of oxygen toxicity has been widely described, the mechanism of lung
injury is still poorly understood. It has been suggested that oxygen
free radicals play an important role in oxygen toxicity (10, 21, 48,
51). Superoxide anion and its product
H2O2 can directly produce cellular damage (13). They can also co-react to
form hydroxyl radicals (15) or can react with other free radicals, like
nitric oxide (NO), to yield secondary more cytotoxic species such as
peroxynitrite anion (ONOO
)
(3, 25). ONOO
is a strong
oxidizing agent that can diffuse across cell membranes (27) and
initiate lipid peroxidation (37).
Several inflammatory processes involving the lung, such as asthma,
chronic bronchitis, and sepsis, are associated with an elevation in NO
production (2, 4, 29). The lung damage induced by hyperoxia also has a
strong inflammatory component. Therefore, it has been suggested that NO
might be involved in the lung injury induced by hyperoxia (33, 34).
However, the effect of hyperoxia on endogenous NO production is
controversial and depends on the experimental conditions. It has been
shown that oxygen can affect the expression of constitutive NO synthase and NO production in cultured cells exposed to hyperoxia (26). Furthermore, macrophages obtained from bronchoalveolar lavage of
animals exposed to oxygen have been reported to produce NO (49). Nozik
et al. (33, 34) have shown that the infusion of L-arginine,
a substrate for the synthesis of NO, enhances injury in the isolated
rabbit lung during hyperoxia. High concentrations of inhaled NO have
also been shown to enhance hyperoxic lung injury (11). However, there
is no direct evidence that hyperoxia induces NO production in intact animals.
In this study, we describe the effect of hyperoxia on NO production and
inducible nitric oxide synthase (iNOS) expression in the lungs of rats
exposed to toxic concentrations of oxygen.
 |
METHODS |
Male Sprague-Dawley rats, weighing between 300 and 400 g, were exposed
to either oxygen (study group, n = 12)
or room air (negative control group, n = 6). Animals were placed in a sealed Plexiglas chamber (20 × 24 × 30 in.), and 85-90% oxygen or room air (21% oxygen) was
supplied to the chamber at a flow rate sufficient to exchange the
entire chamber volume 10 times per hour. Oxygen concentration in the
chamber was monitored using an oxygen analyzer (OM-; Sensormedics,
Anaheim, CA). CO2 in the chamber
was maintained at <1% by using bara lime.
CO2 levels were monitored using a
Nellcor gas analyzer with gases sampled from the chamber. Animals had free access to food and water during the experiments.
The animals were exposed for 24 or 72 h. Rats were then anesthetized
with an intraperitoneal injection of pentobarbital sodium (50 mg/kg). A
tracheotomy was performed, and the animals were ventilated with a
rodent ventilator (model 683; Harvard Apparatus, South Natick, MA). The
ventilatory parameters used were the same for the two groups of animals
studied. Exhaled gases were collected from the expiratory port of the
ventilator for 3 min. The concentration of NO (parts per billion, ppb)
was measured by chemiluminescence using a highly sensitive NO analyzer
(model 270B; Sievers, Boulder, CO). The right carotid artery was then
cannulated to monitor the blood pressure and to sample arterial blood
for blood gases. The animals were heparinized, a sternotomy was
performed, and the pulmonary artery was cannulated. After
exsanguination, the lungs were washed with heparinized normal saline
and then infused with paraformaldehyde (4% wt/vol) in 0.1 M PBS
(pH 7.3) at a pressure not exceeding 20 mmHg. The left lung was
removed, cut in 1.5-mm-thin pieces, and left in paraformaldehyde (4%
wt/vol) for 40 min. The specimens were then fixed overnight in
Formalin. Five-micrometer-thin sections were prepared for
immunohistochemistry. The right lung was snap-frozen in liquid nitrogen
and was kept at
70°C for Western blot analysis.
The positive control group consisted of rats that were injected with
Escherichia coli lipopolysaccaride
(LPS; 20 mg/kg ip). LPS has been shown to increase NO production in
many organs including the lung (44). Four hours after LPS injection,
the animals were anesthetized and ventilated via a tracheotomy, and
exhaled air was analyzed for the presence of NO as previously
described. Arterial blood pressure and blood gases were
determined. The lungs were fixed and processed for immunostaining and
Western blot analysis as in the other groups.
To exclude the possibility that the availability of
L-arginine could affect the production and detection of
exhaled NO (ENO) in animals exposed to hyperoxia, a group
of four rats received exogenous L-arginine. The animals,
after 72 h of exposure to hyperoxia, were anesthetized and mechanically
ventilated via a tracheotomy. A sample of exhaled air was collected for
NO determination. The animals then received a bolus of 300 mg/kg
L-arginine, followed by an infusion at 50 mg · kg
1 · min
1
for 60 min. Samples of exhaled air were obtained every 15 min for
determination of ENO. The lungs were then harvested for
immunohistochemistry analysis.
Immunohistochemistry Labeling
iNOS. After antigen retrieval with 10 mM citrate acid solution (pH 6), specimens were preincubated with goat
serum for 5 min at 42°C and then were incubated overnight at
4°C with polyclonal anti-iNOS (Oxford Biomedical, Rochester Hills,
MI), specific for the 130-kDa enzyme in rats, or PBS (control).
Anti-iNOS binding was detected using biotinylated secondary antibody
(goat anti-mouse IgG; Vector Laboratories, Burlingame, CA) for 10 min
at 42°C. The specimens were then incubated with
streptavidin-peroxidase complex (Vector Laboratories) for 5 min at
42°C followed by incubation with 3,3-diaminobenzidine
tetrahydrochloride (DAB; Biogenex, San Ramon, CA) for 3 min at
42°C. Slides were counterstained and mounted.
Nitrotyrosine detection. The major
product from the reaction of
ONOO
with proteins is
nitrotyrosine (9). Antibodies that specifically recognize nitrotyrosine
can be used as a marker of
ONOO
production. After
antigen retrieval with 10 mM citrate acid solution (pH 6), sections
were blocked with PBS-1% BSA for 1 h at room temperature and then were
incubated with 2% goat serum for 5 min at 42°C. Specimens were
then incubated overnight at 4°C with an anti-nitrotyrosine
monoclonal antibody (Upstate Biotechnology, Lake Placid, NY) or PBS
(control). The sections were then incubated with biotinylated secondary
antibody for 10 min at 42°C followed by streptavidin-peroxidase
complex and DAB staining. Slides were counterstained and mounted.
Western Blotting for Detection of iNOS
Rat lung lysates obtained from three animals for each experimental
group (control, septic shock, and hyperoxia) were prepared by fine
mincing and dounce homogenization of snap-frozen rat lung tissues in 10 volumes (wt/vol) of lysis buffer containing 10 mM Tris · HCl, pH 7.6, 150 mM NaCl, 1 mM EDTA, 0.1%
deoxycholine, 1% Triton X-100, and a mixture of protease
inhibitors (Complete; Boehringer Mannheim, Indianapolis, IN). The
lysate was clarified at 14,500 g for
15 min at 4°C. Aliquots of tissue lysate (500 µg) were incubated
with 10 µl of anti-iNOS rabbit polyclonal antibody (Oxford Biomedical
Research, Oxford, MI) for 4 h at 4°C followed by the addition of
protein A/G agarose (Santa Cruz Biotechnology, Santa Cruz, CA). After
incubation for 90 min at 4°C, the immunoprecipitates were washed
extensively in lysis buffer, then boiled in 2× SDS-PAGE sample
buffer. The samples, including 10 µl of human iNOS Western blot
standard from A172 cells (R&D, Richmond, CA), were
processed by SDS-PAGE on 10% polyacrylamide gels, transferred in
Towbin's transfer buffer to Immobilon-NC (Millipore, Bedford, MA), and then blocked overnight with 10 mM Tris · HCl, pH 7.5, 150 mM NaCl, 0.2% Tween 20, and 3% BSA. The polyclonal antiserum
(anti-iNOS; Oxford Biomedical) was diluted 1:50 in blocking buffer and
incubated for 4 h at room temperature. After extensive washing in 10 mM Tris · HCl, pH 7.5, 150 mM NaCl, and 0.2% Tween 20, a secondary goat anti-rabbit IgG-horseradish peroxidase (Sigma, St.
Louis, MO) was diluted in washing buffer at 1:15,000 and was incubated at room temperature for 1 h. The blot was washed with washing buffer
and then incubated with enhanced chemiluminescence (Amersham) reagents
for 2 min followed by exposure of the blot to Kodak X-OMAT film for up
to 10 min.
Statistical Analysis
Differences in arterial blood pressure, blood gases, and
ENO between the three study groups were tested using ANOVA.
P < 0.05 was considered to be significant.
 |
RESULTS |
The oxygen concentration in the chamber of animals exposed to hyperoxia
ranged from 85 to 90% and was 21% in the control group for the
duration of the studies. The hyperoxia experiments were terminated
after 72 h because the animals appeared to experience discomfort and
dyspnea after 3 days of exposure. Blood samples for blood gases were
always obtained after anesthesia and while the animals were
mechanically ventilated with room air. Blood gases were normal for the
first 48 h of exposure to hyperoxia (data not shown). However, after 72 h of exposure, the animals were severely hypoxic, (Table
1) with normal pH and arterial PCO2
(PaCO2). In the control and septic shock
groups, blood gases were always within normal ranges (Table 1). The
arterial blood pressure and heart rate were normal in the hyperoxia and control groups, whereas septic shock animals were severely hypotensive (Table 1). ENO was significantly elevated in the
septic shock group (>100 ppb). In contrast, ENO was
normal in both control and hyperoxia-exposed animals (1.4 ± 1.4 and 4 ± 5 ppb, respectively). ENO levels were
also normal after 72 h of exposure to hyperoxia (Table 1).
The infusion of L-arginine in animals exposed to
hyperoxia for 72 h did not affect the levels of ENO, which
remained within normal ranges during the 60-min infusion (3 ± 2 ppb). Immunostaining of the lungs of control animals with antibody
specific for iNOS showed occasional signal in macrophages and
inflammatory cells (Figs. 1 and
2) in specimens that were otherwise
normal. In septic shock animals, the staining was more prominent:
macrophages and alveolar lining cells were diffusely stained (Fig.
3). In animals exposed to hyperoxia, there
was microscopic evidence of interstitial edema, with intra-alveolar
exudates and infiltration of macrophages and neutrophils after 24 h of
exposure. In these animals, there was a marked positive
immunoreactivity for iNOS in macrophages and alveolar cells (Fig.
4). Endothelial cells of peribronchial vessels were also positive for iNOS (Fig.
5). Similar findings were seen in lung
sections obtained from animals exposed for 72 h to hyperoxia (Fig.
6). These findings were surprising given the fact that ENO levels in animals exposed to hyperoxia
were normal. Western blot analysis confirmed the presence of iNOS in samples from septic shock and hyperoxic animals (after both 24 and 72 h
of exposure), although no signal for iNOS was found in control animals
(Fig. 7). We also examined the levels of
paxillin expression in the same samples. Paxillin was chosen as an
irrelevant, arbitrary protein for internal control purposes to
determine whether the different experimental treatments resulted in a
general, nonspecific stimulation or inhibition of protein synthesis.
The observation that paxillin levels were similar in control, septic
shock, and hyperoxic animals, although we observed a clear induction of
iNOS after septic shock and hyperoxia, further suggests that induction of iNOS is specific to the experimental conditions.
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Table 1.
Blood pressure and blood gas analysis in animals exposed to 21%
oxygen (control group), 100% oxygen (hyperoxic group) and in
animals injected with Escherichia coli lipopolysaccharide (septic shock
group)
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Fig. 1.
Immunoreactivity for inducible nitric oxide synthase (iNOS) in rats
exposed to normoxia for 72 h (control group). Staining was observed in
a few macrophages. Figure represents 1 of the 6 animals studied, and
the findings were consistent in all 6 specimens.
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Fig. 2.
Immunoreactivity for iNOS in rats exposed to normoxia for 72 h (control
group). Staining was found occasionally in macrophages and inflammatory
cells beneath the alveolar epithelium.
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Fig. 3.
Immunoreactivity for iNOS in rats injected with lipopolysaccharide
(LPS; septic shock group). Staining was observed in macrophages and
alveolar cells. Figure represents 1 of the 6 animals studied, and the
findings were consistent in all 6 specimens.
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Fig. 4.
Immunoreactivity for iNOS in rats exposed to hyperoxia for 24 h. Marked
staining was observed in macrophages and alveolar cells. Figure
represents 1 of the 6 animals studied, and the findings were consistent
in all 6 specimens.
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Fig. 5.
Immunoreactivity for iNOS in rats exposed to hyperoxia for 24 h.
Staining was observed in macrophages and endothelial cells of
peribronchial vessels.
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Fig. 6.
Immunoreactivity for iNOS in rats exposed to hyperoxia for 72 h. Figure
shows diffuse staining in macrophages and alveolar cells and represents
1 of the 6 animals studied. Findings were consistent in all 6 specimens.
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Fig. 7.
SDS-PAGE and Western blot analysis of lung homogenates after
immunoprecipitation. Aliquots have been incubated with anti-iNOS
polyclonal antibody. Immunoreactivity at 130 kDa was found only in
sepsis (lane 2) and in animals
exposed to hyperoxia for 24 and 72 h (lane
3 and 4,
respectively). No immunoreactivity was detected in animals exposed to
normoxia (lane 1). Figure is
representative of 3 separate experiments.
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We observed a marked immunoreactivity for nitrotyrosine in the
connective tissue around pulmonary vessels and alveoli and in
capillaries and peribronchial vessels of animals in septic shock (Fig.
8). Staining for nitrotyrosine was negative
in both control and hyperoxic animals after 24 (Fig.
9) and 72 h.

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Fig. 8.
Immunoreactivity for nitrotyrosine in rats injected with LPS (septic
shock immunoreactivity for nitrotyrosine in rat group). Intense
staining in endothelial cells, subendothelial inflammatory cells, and
connective tissue surrounding bronchial vessels is seen.
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Fig. 9.
Immunoreactivity for nitrotyrosine in rats exposed to hyperoxia. No
staining was detected after 24 h of exposure.
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 |
DISCUSSION |
In this study, we have shown that hyperoxia upregulates iNOS expression
in the lung without increasing emission of NO in exhaled air. Lung
tissue damage after hyperoxia was verified by histological examination.
Upregulation of iNOS expression with subsequent increased production of
NO characterizes several experimental and clinical conditions. For
instance, the hemodynamic and metabolic changes seen in septic shock
are associated with an overproduction of NO (23, 32, 46). These
findings were confirmed by our study: we observed an increased
expression of iNOS in epithelial cells and macrophages with increased
production of NO in rats injected with LPS. We also observed an
increased expression of iNOS in the lungs of rats exposed to hyperoxia.
The increase in iNOS expression during hyperoxia seems to contradict
results reported in a recent study by Arkovitz at al. (1) in which
hyperoxia did not induce the expression of iNOS in the lungs of mice.
One possible explanation that can account for such differences may
include the use of different species. We used a rat model, whereas
Arkovitz et al. used mice, and these observations can be species
specific. Furthermore, in this study, we looked for the protein (iNOS)
using a Western blot analysis, whereas Arkovitz et al. looked for
the messenger of the same protein (iNOS mRNA) using a Northern blot.
The Northern blot technique is not as sensitive as the RT-PCR; very low
amounts of messenger that are sufficient for the synthesis of a protein can be undetected by Northern blot analysis. There is also the possibility that induction of iNOS is due primarily to
posttranscriptional mechanisms.
The unexpected finding in this study was that the expression of iNOS in
animals exposed to hyperoxia was not associated with an increased
amount of NO in exhaled air. In contrast, septic shock animals
exhibited an increase in ENO. There are four possible explanations why ENO of animals exposed to hyperoxia
remained at normal levels despite an increased expression of
iNOS: 1) NO could not reach the
alveolar space because of severe lung damage; 2) NO production was not
possible because of low levels of substrate; 3) NO was formed and was immediately
scavenged by proteins and free radicals; and
4) iNOS was expressed but was not
functioning in the presence of hyperoxia.
The first possible explanation to our findings is that NO could not be
detected in the exhaled air because the severe lung damage caused by
hyperoxia affected the diffusion of NO into the alveolar space. We
indeed observed severe pulmonary edema and diffuse areas of atelectasis
after 72 h of exposure to hyperoxia. The pulmonary diffusing capacity
of NO is lower than that of CO2, approximately one-sixth (16), but three to five times greater than
CO2-diffusing capacity (30). The
blood gases obtained from animals exposed for 72 h to oxygen
showed a slightly increased PaCO2
associated with hypoxemia. This indicates that the diffusing capacity
of gases was partially affected. However, it seems unlikely that these
changes in the lung structure could completely prevent NO from
diffusing into the alveolar space while still allowing a partial
exchange of CO2 and oxygen.
With respect to the second hypothesis that relatively low levels of
L-arginine during hyperoxia limited NO production, recent studies have shown that arginase can compete with iNOS for a common substrate, L-arginine. The amount of end product (urea and
NO, respectively) is inversely related to the activation of either enzyme (6). Plasma levels of L-arginine (8) can also affect the production of NO by iNOS, in particular when the extracellular supply of L-arginine is limited. The effects of hyperoxia
on arginase activity and L-arginine availability are
unknown. However, we could not detect increased ENO when
exogenous L-arginine was infused in animals exposed to
hyperoxia. This suggests that normal levels of ENO
concomitant with increased expression of iNOS during hyperoxia are not
due to a low supply of L-arginine.
We examined the possibility that NO was actually produced but
immediately reacted with O
2 to form
ONOO
(19).
ONOO
is a very strong
oxidizing agent, and it has been considered by several authors as a
possible mediator of oxygen-induced toxicity (3, 37). We could not
detect any nitrotyrosine, which we chose as an indicator of
ONOO
production, in the
lungs of the rats exposed to oxygen. However, nitrotyrosine was
identified in the lungs of septic shock rats. This may indicate that NO
is not produced in rats exposed to oxygen or, alternatively,
ONOO
preferentially reacts
with other molecules in the presence of hyperoxia, producing
by-products other than nitrotyrosine. In contrast to our findings,
Haddad et al. (14) detected the presence of nitrotyrosine in the lungs
of rats exposed to hyperoxia, using an experimental model similar to
ours. The type of primary antibody that we used can explain this
discrepancy: we used a monoclonal antibody for detection of
nitrotyrosine, whereas Haddad et al. used a polyclonal antibody. This
could explain the relatively high signal observed in the control
animals of Haddad et al.'s study.
The absence of nitrotyrosine by immunoperoxidase staining alone can not
be used as an indication that
ONOO
is not formed in the
presence of hyperoxia. Several authors have demonstrated that
unsaturated fatty acids can react with reactive nitrogen species, in
particular ONOO
, yielding
to the formation of nitrogen-containing lipid products (35, 39). It is
unknown if and to what extent hyperoxia redirects the metabolism of
nitrogen reactive compounds.
It should also be emphasized that there are several possible pathways
for NO elimination besides the formation of
ONOO
that were not tested
in this study. It was originally thought that endogenous and inhaled NO
are promptly removed from the intravascular compartment by hemoglobin.
NO is readily converted to nitrate with parallel formation of
methemoglobin in oxygenated blood (47, 52). Although inefficient (18),
the direct conversion of NO to nitrite is also possible in biological
fluids. There is also evidence that in vivo nitrosation of
thiol-containing molecules, such as glutathione, cysteine, and serum
albumin (11, 42), is an important mechanism of transport of NO in the
presence of hyperoxia. These molecules can serve as an extracellular
and intracellular reservoir of NO (40, 41, 43). NO can also bind to
cytochrome P-450,
PGI2 synthase, and thromboxane
A2 synthase and inactivate them
(31, 50). In the present study, we did not assess if hyperoxia promotes
the binding of NO to other scavenging molecules such as hemoglobin and
thiol-containing molecules. Exposure to high concentrations of inhaled
oxygen induced severe inflammatory changes in the lung with loss of the
alveolar-endothelial barrier. This may have promoted the scavenging of
NO from the lung by plasma proteins. Further studies are needed to
evaluate this possibility.
The fourth possible explanation for our findings is that iNOS activity
was inhibited in the presence of hyperoxia. iNOS generates NO from
L-arginine in an NADPH-dependent stepwise reaction (24, 45). The enzymes are composed of the following three main regions: a
COOH-terminal half, which contains the flavin
mononucleotide, FAD, and NADPH binding sites; a
central calmodulin-binding site; and an
NH2-terminal region, which
contains the heme moiety, the arginine binding site, and the
tetrahydrobiopterin site (5, 40).
Experimental evidence shows that it is possible to inhibit NO synthase:
haloperidol (20) and chlorzoxazone (12), a cytochrome P-4502E1 substrate, can inhibit the
activity of iNOS and neuronal NOS. Structural analogs of CuZn
superoxide dismutase (28) and H2O2
(36) can inhibit the activity of neuronal NO synthase by interfering
with the electron transport processes that are involved in the
synthesis of NO or by a competitive inhibitor mechanism.
Our data do not allow us to conclude whether hyperoxia affects iNOS
activity or "reroutes" the oxidative metabolism of NO. Therefore,
further studies are needed to determine the relationship between
hyperoxia and iNOS activity. If the inhibition of iNOS by hyperoxia is
confirmed, some of the toxic effects induced by hyperoxia may be
explained by the absence of endogenous production of NO. There is
evidence that endogenous NO may have a protective role in the presence
of hyperoxia. Capellier et al. (7) showed that the administration of
nitro-L-arginine methyl ester, an NO synthase inhibitor, in
intact animals during hyperoxia resulted in earlier death. Wink et al.
(54) have shown that NO protects against cellular damage from oxygen
radicals. The work done by Rubbo et al. (38) suggests that NO may have
a protective role in acute inflammatory reactions depending on the
production rate and concentration of NO versus that of other reactive
oxygen species. The inhibition of endogenous NO production during
hyperoxia may undermine a possible physiological mechanism of defense
against oxygen toxicity. The possibility of iNOS activity inhibition by hyperoxia has far-reaching implications because iNOS is a member of the
heme enzyme family, which includes the cytochrome
P-450 hemoproteins (53). Therefore,
others in this group of enzymes could be equally affected by hyperoxia.
In conclusion, this study shows that there is induction of iNOS but no
increase in ENO in animals exposed to hyperoxia. This suggests that hyperoxia may inhibit iNOS activity and subsequent endogenous production of NO or may reroute NO metabolism. Further studies are needed to determine which of these two mechanisms is
responsible for our observation.
 |
FOOTNOTES |
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: T. S Hakim,
Dept. of Anesthesiology, SUNY-Health Science Center-Syracuse, 750 E. Adams St., Syracuse, NY 13210 (E-mail:
HAKIMT{at}mailbox.hscsyr.edu).
Received 29 May 1998; accepted in final form 7 April 1999.
 |
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