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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 approx 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.

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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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