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Published ahead of print on September 15, 2005, doi:10.1165/rcmb.2005-0182OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 33, pp. 582-588, 2005
© 2005 American Thoracic Society
DOI: 10.1165/rcmb.2005-0182OC

Pulmonary and Systemic Nitric Oxide Metabolites in a Baboon Model of Neonatal Chronic Lung Disease

David A. Munson, Peter H. Grubb, Jay D. Kerecman, Donald C. McCurnin, Bradley A. Yoder, Stanley L. Hazen, Philip W. Shaul and Harry Ischiropoulos

The Joseph Stokes Jr. Research Institute, Children's Hospital of Philadelphia; Department of Biochemistry and Biophysics, The University of Pennsylvania, Philadelphia, Pennsylvania; San Antonio Military Pediatric Center, Department of Pediatrics, and Department of Pathology, University of Texas Health Science Center and the Southwest Foundation for Biomedical Research, San Antonio, Texas; Departments of Cell Biology and Cardiovascular Medicine, Center for Cardiovascular Diagnostics and Prevention, Cleveland Clinic Foundation, Cleveland, Ohio

Correspondence and requests for reprints should be addressed to Harry Ischiropoulos, Joseph Stokes Jr. Research Institute, Children's Hospital of Philadelphia, 3516 Civic Center Blvd., 416D Abramson Research Center, Philadelphia, PA 19104. E-mail: ischirop{at}mail.med.upenn.edu


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
We report on developmental changes of pulmonary and systemic nitric oxide (NO) metabolites in a baboon model of chronic lung disease with or without exposure to inhaled NO. The plasma levels of nitrite and nitrate, staining for S-nitrosothiols and 3-nitrotyrosine in the large airways, increased between 125 d and 140 d of gestation (term 185 d) in animals developing in utero. The developmental increase in NO-mediated protein modifications was not interrupted by delivery at 125 d of gestation and mechanical ventilation for 14 d, whereas plasma nitrite and nitrate levels increased in this model. Exposure to inhaled NO resulted in a further increase in plasma nitrite and nitrate and an increase in plasma S-nitrosothiol without altering lung NO synthase expression. These data demonstrate a developmental progression in levels of pulmonary NO metabolites that parallel known maturational increases in total NO synthase activity in the lung. Despite known suppression of total pulmonary NO synthase activity in the chronic lung disease model, pulmonary and systemic NO metabolite levels are higher than in the developmental control animals. Thus, a deficiency in NO production and biological function in the premature baboon was not apparent by the detection and quantification of these surrogate markers of NO production.

Key Words: 3-nitrotyrosine • S-nitrosocysteine • S-nitrosothiols • nitric oxide • chronic lung disease • prematurity • baboon


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Endogenously produced nitric oxide (NO) decreases fetal lung fluid (1), decreases pulmonary vascular and airway resistance, and improves compliance in the newborn lung (2). In experimental models such as the fetal sheep model, stimulation of NO synthesis by acetylcholine or bradykinin leads to decreased production of fetal lung fluid before birth (3), and this activity can be replicated by supplying exogenous NO (1, 4). Inhibition of NO synthase (NOS) in newborn piglets causes increased resistance and decreased compliance (5). Because of these important physiologic functions, there is interest in the use of inhaled NO (iNO) to improve pulmonary function in premature infants and consequently decrease the incidence and severity of chronic lung disease (CLD) of prematurity. Schreiber and colleagues (6) recently demonstrated such an effect, and two ongoing multicenter trials are evaluating the utility of inhaled NO in premature infants.

Critical to the understanding of the role of NO in developing lungs is the exploration of NOS expression and evaluation of NO metabolites during development. Although studies have reported developmental changes in NOS expression and activity, NO metabolites have not been evaluated during development. Developmental changes in the expression of particular NOS activities have been reported in sheep (7, 8) and baboons during the third trimester. In baboons, the increase in total NOS activity between 125 d and 140 d of gestation was associated with increased exhaled NO and with improved resistance and compliance as the baboons grew older (9). When baboons are delivered prematurely at 125 d gestation and placed on a mechanical ventilator for 14 d, there is a 90% decrease in lung neuronal and endothelial NOS activity (10) compared with animals that remain in utero over the 14-d time course, implying that inhaled NO may be beneficial in the premature mammal receiving mechanical ventilation. However, in the same model, after a few days of suppressed levels, exhaled NO levels increase to become comparable to in utero control animals. This implies that although the production of NO was initially suppressed, it may increase over this time course in spite of the decrease in total NOS activity. Alternatively, there may be changes in NO metabolism that allow for an increase in exhaled NO. Furthermore, McCurnin and colleagues (11) recently described an increase in lung volume and weight with administration of inhaled NO in the baboon model of CLD. Whether this growth effect is a consequence of NO replacement in the setting of a deficiency or a pharmacologic effect is not known.

This study evaluated developmental changes in the pulmonary and systemic levels of NO-mediated protein modifications (S-nitrosocysteine and 3-nitrotyrosine) and NO metabolites in baboons over the third trimester. We hypothesized that with the marked increase in NOS activity seen between 125 d and 140 d gestation (8), there would be increases in the levels of S-nitrosocysteine, nitrate, and nitrite. In addition, we evaluated the effect of mechanical ventilation in extremely premature baboons (the CLD model) on the levels of S-nitrosothiols (SNO), 3-nitrotyrosine, and nitrate and nitrite. Finally, the same metabolites and the expression of NOS enzymes in the lung tissue were evaluated in premature baboons on mechanical ventilation with exposure to inhaled NO.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Animal Model
Animal studies were performed at the Southwest Foundation for Biomedical Research Primate Center in San Antonio, TX as previously described (12). Pregnancies in baboons (Papio papio) were timed with cycle dates and fetal growth parameters on ultrasound at 115 d gestation. Lung specimens, bronchoalveolar lavage fluid, and plasma were collected from animals delivered and killed at 125 d (68% term), 140 d (75% term), 160 d (85% term), and 175 d gestation (95% term).

For the CLD model, animals were delivered by C-section at 125 ± 2 d gestation. They were weighed, sedated, and intubated. Surfactant (4 ml/kg) was administered (Survanta; Ross Laboratories, Columbus, OH), and animals were ventilated with a pressure-limited ventilator (InfantStar; Infrasonics, San Diego, CA) or a high-frequency oscillatory ventilator (Sensormedics, Yorba Linda, CA). Animals in the experimental group were given 5 ppm inhaled NO via the INOvent (iNO Therapeutics, Clinton, NJ) starting at 1 h after delivery. Ventilation and supplemental oxygen adjustments were made according to a strict blood gas monitoring and radiography protocol. Details of animal care have been published elsewhere (12). The group that received inhaled NO is referred to as the CLD plus iNO group, and the ventilated control animals are referred to as the CLD group. Tracheal aspirate and plasma samples were collected at several time points over the 14 d of ventilation.

Lung tissue preparation for histopathologic analysis followed previously published procedures. The right lower lobe was removed, weighed, and intrabronchially fixed for 24 h at room temperature with phosphate-buffered 4% paraformaldehyde at 20 cm H2O constant pressure. The lobe was cut transversely into three equal tissue blocks, and sections from each block were prepared for light microscopy. The specimens were dehydrated in ethanol, embedded in paraffin, and cut at a thickness of 4 µm.

Measurement of Nitrate and Nitrite
Nitrite and nitrate levels were evaluated by reduction to NO and detection of NO by chemiluminescence on the Sievers 280 NO analyzer. Plasma, bronchoalveolar lavage, or tracheal aspirate samples were injected into a purge container containing 5 ml of 0.05 M Vanadium chloride in 1 N HCl heated to 95°C. Plasma was first treated with ethanol to precipitate protein. The reading in millivolts was compared with a standard curve, obtaining a total concentration of nitrate plus nitrite. This reductive system reports on the total levels of nitrate, nitrite, SNO, N-nitrosamines, and metal nitrosyl as low molecular mass or protein adducts. However, the predominant species in plasma are nitrate and nitrite, and therefore the concentrations measured using this reductive system are reported as nitrate and nitrite.

Measurement of SNO
Plasma was collected, and the metal chelator diethylenetriaminepentaacetic acid and the alkylating agent N-ethylmaleimide were added to a final concentration of 100 µM and 5 mM, respectively. Protein SNO and low-molecular-weight SNO were measured by chemiluminescence-based assay using potassium iodide in glacial acetic acid as the reducing agent. Potassium iodine (5 ml of 50 mM) and glacial acetic acid were placed in the purge container of the Sievers 280 NO analyzer. Sulfanilamide was added to each sample at a final concentration of 0.1% to eliminate nitrite. Plasma was injected into the purge container, and the reading in millivolts was compared with a standard curve generated with S-nitrosoglutathione, obtaining a concentration of SNO.

Measurement of 3-Nitrotyrosine
Snap-frozen lung tissue was pulverized and homogenized in 10 mM potassium phosphate buffer (pH 7.4) with 0.1 mM diethylenetriaminepentaacetic acid. The samples were centrifuged at 14,000 x g, and the supernatant was removed. The levels of 3-nitrotyrosine were obtained by HPLC with on-line electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS) using stable isotope dilution methodology and an ion trap mass spectrometer (LCQ Deca; ThermoFinigann, San Jose, CA) as described previously (13, 14). Nitrotyrosine contents of proteins are expressed as a product/precursor ratio (i.e., nitrotyrosine/tyrosine; µmol/mol). BAL and plasma were analyzed by the same method.

Immunohistochemical Analysis of Lung Tissue
Lung sections were deparaffinized and stained with rabbit polyclonal anti–3-nitrotyrosine antibody (polyclonal 432) raised against a synthetic peptide as described previously (15) or rabbit polyclonal anti-S-nitrosocysteine antibodies from A.G. Scientific (San Diego, CA). Antibodies were diluted 1:200 in 5% goat serum and applied overnight after antigen retrieval in boiling 0.1 M citrate (pH 6) and after blocking with 50% goat serum for 1 h. Immunostaining was performed using a Vector Elite ABC staining kit for 3,3'-diaminobenzidine (DAB). Dithionite or para-hydroxybenzomercuric acid was used to generate the appropriate negative controls for nitrotyrosine and nitrosothiol antibody staining, respectively. The sections were counterstained with hematoxylin and mounted for light microscopy.

NOS Enzymatic Activity
Measurements of NOS enzymatic activity in the presence of excess substrate and cofactors provide a reliable quantitative assessment of enzyme abundance and the capacity to assess the relative abundance of different NOS isoforms. NOS enzymatic activity was determined using previously described methods in the proximal one-third of lung parenchyma obtained at necropsy at 14 d of postnatal life in the baboons delivered at 125 d gestation (control) and in identically aged animals who had received inhaled NO gas since the first hour of life. The calcium-dependent and calcium-independent subfractions of NOS enzymatic activity were distinguished by the addition of 7.5 mM EGTA to the incubation mixture (10, 16). To partition the calcium-dependent NOS activity into that derived from neuronal NOS (nNOS) versus endothelial NOS (eNOS), additional enzymatic activity assays were performed in the absence and presence of the nNOS-selective antagonist S-methyl-L-thiocitrulline (10–8 M) (17). Four to six animals were used in each analysis.

NOS Protein Levels
The abundance of nNOS and eNOS protein was determined in the same proximal lung parenchyma samples by immunoblot analysis. The methods were similar to those previously reported (1). Although inducible NOS (iNOS) enzymatic activity was detectable as calcium-independent NOS activity, iNOS protein was not detectable in any specimens.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Fetal Development
Pulmonary metabolites. Microscopic evaluation of the lung sections showed maturation of the lung early in the third trimester as evidenced by the thinning of alveolar walls as previously described (Figure 1) (12). Immunohistochemical evaluation with antibodies against S-nitrosocysteine revealed minimal staining for SNO in the large airways in 125-d gestational control animals. The staining increased in the large airways in 140-d gestational control animals and remained stable thereafter (Figure 2). Staining with antibodies against tyrosine-nitrated proteins was minimal in the large airways of 125-d gestational control animals. Increased immunoreactivity for nitrated proteins was observed in large airways by 140 d of gestation and remained stable through the remainder of the third trimester (Figure 3). There was no apparent staining for SNO or 3-nitrotyrosine in the alveoli at any gestational age. Quantification of pulmonary 3-nitrotyrosine in proximal lung tissue by LC/ESI/MS/MS also showed an increase between 125 and 140 d gestation, although this change was not statistically significant (P = 0.126 by Student's t test). Lavage levels of 3-nitrotyrosine did not change between 125 and 140 d gestation (Figure 3). Lavage levels of nitrate and nitrite decreased over the third trimester without achieving statistical significance (P = 0.352, one-way ANOVA) (Figure 4A).



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  		Figure 1. Representative pictures of alveoli after staining for S-nitrosocysteine in lung tissue using polyclonal rabbit anti-S-nitrosocysteine antibody. 125 Gest, 125-d gestational control; 140 Gest, 140-d gestational control; 160 Gest,160-d gestational control; 175 Gest, 175-d gestation control; CLD, 125-d gestation mechanically ventilated for 14 d (CLD group); CLD + iNO, 125-d gestation mechanically ventilated and exposed to inhaled NO at 5 ppm for 14 d (CLD plus iNO group).

 


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  		Figure 2. Representative pictures of large airways after staining for -nitrosocysteine in lung tissue using polyclonal rabbit anti–S-nitrosocysteine antibody. Staining indicated by arrows. 125 Gest, 125-d gestational control; 140 Gest, 140-d gestational control; 160 Gest, 160-d gestational control; 175 Gest, 175-d gestation control; CLD, 125-d gestation mechanically ventilated for 14 d; CLD + iNO, 125 d gestation mechanically ventilated and exposed to inhaled NO at 5 ppm for 14 d.

 


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  		Figure 3. Representative pictures of the immunohistochemical staining for 3-nitrotyrosine in the large airways from the same model using polyclonal rabbit anti–3-nitrotyrosine. Staining indicated by arrows. 125 Gest, 125-d gestational control; 140 Gest, 140-d gestational control; CLD, 125-d gestation mechanically ventilated for 14 d; CLD + iNO, 125-d gestation mechanically ventilated and exposed to inhaled NO at 5 ppm for 14 d. (A) Quantification of 3-nitrotyrosine by liquid chromatography/tandem mass spectroscopy in lung homogenate from 125-d gestational, 140-d gestational, CLD, and CLD plus iNO animals. (B) Quantification of 3-nitrotyrosine by liquid chromatography/tandem mass spectroscopy (LC-MS/MS) in lung fluid from 125-d gestational, 140-d gestational, CLD, and CLD plus iNO animals.

 


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  		Figure 4. Lavage and plasma nitrite and nitrate levels were measured by chemiluminescence. (A) Nitrite and nitrate in bronchoalveolar lavage in gestational control animals delivered at four different time points during the third trimester. The trend toward a decrease in levels over the third trimester was not statistically significant (P = 0.352, one-way ANOVA). (B) Quantification of nitrite and nitrate in plasma from gestational control animals delivered at four different time points during the third trimester. The increase over the third trimester is statistically significant (P = 0.003, one-way ANOVA).

 
Systemic metabolites. Total plasma SNO levels (protein and low molecular weight) did not change over the third trimester in the gestational control animals (means ranged from 1.07–1.3 µM at each time point). Plasma levels of 3-nitrotyrosine did not change significantly early in the third trimester (means ranged from 6.6 to 18 µmole 3-nitrotyrosine/mole tyrosine, P = 0.245, t test between 125- and 140-d gestational control animals). Plasma nitrate and nitrite levels increased over the third trimester (P = 0.003, one-way ANOVA) (Figure 4B).

Course of CLD
Pulmonary metabolites. Immunohistochemical evidence for the presence of SNO and 3-nitrotyrosine in the large airways at the end of the course of CLD is depicted in Figures 2 and 3. The location and staining intensities seemed to be comparable to that seen in the 140-d gestational control animals of similar postconceptual age, and 3-nitrotyrosine quantified in pulmonary homogenate was no different from the levels measured in 140-d gestational control animals (Figure 3). There was no staining for SNO or 3-nitrotyrosine in the alveoli of the animals in the CLD group.

Tracheal aspirate levels of nitrate and nitrite in the CLD model did not change significantly over the 2-wk time course of mechanical ventilation (Figure 5A). At the end of the study of CLD animals, the levels were higher than in 140-d gestational control animals at similar postconceptual age (P < 0.001, t test comparing 14-d postnatal age group to 140-d gestational control animals).



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  		Figure 5. Plasma and tracheal aspirate nitrite and nitrate levels were measured by chemiluminescence. (A) Nitrite and nitrate in tracheal aspirates collected at different time points from animals delivered at 125 d gestation and mechanically ventilated with or without inhaled NO for 14 d. There was no difference between the CLD plus NO (filled squares) and CLD (open squares) groups (P = 0.841, two-way ANOVA controlling for time). However, tracheal aspirate levels in the CLD model were higher than gestational control lavage levels (P < 0.001, t test comparing 14-d postnatal age group to 140-d gestational control animals [circles]). (B) Nitrite and nitrate in plasma collected at different time points from CLD and CLD plus iNO animals. Levels are significantly higher in animals receiving inhaled NO (P < 0.001, two-way ANOVA controlling for time).

 
Systemic metabolites. In the CLD model, by Day 3 of ventilation after delivery at 125 d of gestation, the mean plasma concentration of nitrate plus nitrite was 52 µM (Figure 5B), which represents a tripling of the 125-d gestation level of 17 µM. Plasma nitrate and nitrite rose over time during the 2 wk of ventilation, reaching a level of 88 µM on Day 14 (P = 0.059 by one-way ANOVA) (Figure 5B). At the end of the course of study, the baboons in the CLD model had elevated plasma levels of total nitrate and nitrite compared with gestational control animals (t test comparing 2-wk time point to 140-d gestational control animals, P = 0.004). Plasma 3-nitrotyrosine was unchanged compared with gestational control animals (means 11.5 and 18 µmol 3-nitrotyrosine/mole tyrosine in the140-d gestation and CLD groups, respectively; P = 0.515, t test between the two groups). Plasma SNO did not change over time in the CLD group, and at the end of study they were unchanged compared with gestational control animals (Figure 6).



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  		Figure 6. SNO (protein plus low-molecular-weight) levels were quantified by chemiluminescence. SNO levels in plasma collected at different time points from animals from the CLD (open squares) and CLD plus iNO (filled squares) groups. SNO levels were higher in animals administered inhaled NO compared with the CLD group (see Figure 3; P < 0.011, two-way ANOVA controlling for time).

 
Inhaled NO Treatment
Pulmonary metabolites. Staining for SNO and 3-nitrotyrosine showed the same pattern as that seen in the CLD model. Inhaled NO did not result in an increase in staining for these protein modifications in the alveoli (Figure 2) or in the large airways (Figure 3).

Lavage levels of 3-nitrotyrosine were not changed with exposure to inhaled NO compared with the CLD group or developmental control animals (Figure 3). There was also no difference in the tracheal aspirate levels of nitrate and nitrite between the CLD plus iNO and CLD groups (P = 0.841, two-way ANOVA controlling for time) (Figure 5A).

Systemic metabolites. Plasma levels of 3-nitrotyrosine remained unchanged in the CLD plus iNO group (means ranged from 6.6–18 µmol 3-nitrotyrosine/mol tyrosine across all groups, P = 0.633, one-way ANOVA). SNO levels were higher in animals administered inhaled NO compared with CLD control animals (P < 0.011, two-way ANOVA controlling for time) (Figure 6). Premature baboons exposed to inhaled NO (CLD plus iNO group) had significantly higher plasma levels of nitrate and nitrite compared with ventilated control animals (CLD group) (P < 0.001, two-way ANOVA controlling for time) (Figure 5B). The levels in the CLD plus iNO were comparable to full-term baboons on postnatal day of life 2–3 that had a mean plasma concentration of 134 µM. Plasma nitrate and nitrite rose over the 2-wk time course. The pattern of rise mirrored that seen in the CLD model but had a much larger rise in plasma levels early in the time course.

NOS
There was no difference in total NOS activity between the CLD and CLD plus inhaled NO groups. The addition of a calcium chelator showed that calcium-dependent and independent isoforms were unchanged (Figure 7A). When pulmonary eNOS and nNOS were evaluated, protein levels and activities were similar between the CLD and CLD plus iNO groups (Figure 7B and 7C).





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  		Figure 7. (A) Total NOS activity was measured in baboons delivered at 125 d of gestation and ventilated for 14 d with (CLD plus iNO) or without the administration of inhaled NO (CLD). Total NOS activity (stippled bars) was differentiated into calcium-dependent (solid bars) and calcium-independent (striped bars) fractions. There was no difference noted between the two groups. (B) eNOS protein abundance was evaluated by immunoblot, and eNOS specific enzymatic activity was quantified. (C) nNOS protein abundance was evaluated by immunoblot, and nNOS specific enzymatic activity was quantified. There was no statistically significant difference between the CLD and CLD plus iNO groups for either enzyme.

 

   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
In the baboon, pulmonary levels of eNOS and nNOS rise sharply between 125 and 140 d gestation, and this enzymatic activity is focused in the large airways (9). If a baboon is delivered at 125 d gestation and the ensuing 2 wk of development occur on a mechanical ventilator, levels of eNOS and nNOS do not rise and are 90% lower than those seen in the 140-d gestational control animals (10). In this study, we evaluated NO metabolites in pulmonary fluid, pulmonary parenchyma, and plasma over the course of baboon fetal development and in a baboon model of prematurity. The data presented herein provide evidence for the first time that changes in NO metabolites mirror closely the developmental changes in pulmonary eNOS and nNOS expression. Mechanical ventilation had little effect on NO-mediated protein modifications within the lung parenchyma and circulation but significantly increased the levels of nitrite and nitrate in lung fluid and plasma. Exposure to inhaled NO resulted in an increase in the systemic levels of nitrate, nitrite, and SNO.

We hypothesized that the dramatic rise in eNOS and nNOS activity early in the third trimester of the fetal baboon (9) would be reflected in a rise in NO metabolites within the lung tissue, lung fluid, and plasma. We found that although systemic levels of the low-molecular-weight metabolites nitrate and nitrite rose during the third trimester of fetal development, bronchoalveolar lavage levels did not. Although the rise in circulating metabolites may reflect an increase in systemic production of NO, it may also reflect changes in pulmonary production consistent with the known "sink" effect of hemoglobin-catalyzed conversion of NO to nitrate because lung tissue is highly vascularized (18). Alternatively, the nitrite and nitrate produced within the lung rapidly equilibrate with the plasma. Previous studies that used intratracheal instillation of 13N-nitrite or 13N-nitrate to mice and rabbits showed a rapid movement of the anions into circulation. Moreover, within 10 min after intratracheal instillation of 13N-nitrite, 70% of the labeled nitrogen was found as nitrate in the plasma, indicating a rapid oxidation of nitrite to nitrate in the lung or in circulation (19). Consistent with these data, changes in the levels of nitrite and nitrate are apparent in circulation, possibly reflecting the rapid diffusion of NO or of the anions into the blood stream.

In the parenchyma, immunoreactivity for S-nitrosocysteine increased between 125 and 140 d of gestation in the large airways. The changes in SNO observed in the large airways are not reflected by increases in lavage or plasma SNO levels, suggesting that they are specific to the locations of elevated NOS expression and activity (8, 10). Staining for 3-nitrotyrosine demonstrated essentially the same pattern as that of S-nitrosocysteine. Lavage and plasma levels of 3-nitrotyrosine measured by LC/ESI/MS/MS were not different between the 125- and 140-d gestational control animals, but levels in lung homogenate are consistent with the observed immunohistochemistry. Although the biological significance of proteins modified by NO in the airway during development is unclear, they represent a portion of the biologically active NO and nitrite and as such could report on changes in local production and bioavailability of NO.

Pulmonary NO production is critical to a normal decrease in pulmonary vascular resistance after delivery, contributing to a dramatic increase in pulmonary blood flow during transition to air breathing (20, 21). We would, therefore, expect to see a corresponding rise in NO metabolites after delivery. In fact, data show a tripling of systemic nitrate and nitrite in full-term postnatal animals (mean of 134 µM) compared with late third-trimester levels (mean of 44 µM). However, previous data in this model indicate that the expression levels of eNOS and nNOS were suppressed by 90% compared with animals allowed to develop in utero over a similar time course. The same study demonstrated attenuated levels of exhaled NO over the first few days of life in the CLD model (10). It was, therefore, surprising that with preterm delivery and mechanical ventilation, plasma and tracheal aspirate levels of nitrate and nitrite increased compared with their respective developmental control animals. One possible explanation is that the low levels of constitutive NOS, and perhaps the inducible isoform (iNOS), which is unchanged in this model, are capable of increasing production of NO in response to the stimuli associated with the transition to air breathing. Alternatively, a systemic increase in NO production may be responsible for the observed changes.

We did not observe any changes in NO-mediated protein modifications in lung parenchyma, tracheal aspirate, or plasma in the animals in the CLD group compared with gestational control animals. This finding was surprising for a number of reasons. First, based on the nitrate and nitrite data, there seems to be an increase in NO production in these animals and thus an expectation for increased levels of protein modifications. Second, 3-nitrotyrosine is a marker of inflammation and oxidative stress, which are considered to play a significant role in the pathogenesis of CLD of prematurity (21–26). These animals are exposed to positive-pressure ventilation and increased fractions of inspired oxygen, both of which would be expected to contribute to inflammatory conditions. Elevated levels of 3-nitrotyrosine have been documented in several models of hyperoxia (27, 28), and interference with inflammation has been shown to attenuate this response (29). Contrary to these expectations, there was no significant immunoreactivity for 3-nitrotyrosine in the alveoli or distal airways in this model of CLD. It is possible that an inflammatory state occurs early in the course of mechanical ventilation and that this marker is largely cleared by the 2-wk time point when the animals were killed for histopathology (a number of potential removal pathways have been proposed) (30, 31). More likely, the low-volume ventilator strategy and strict oxygen parameters used in this model result in a disease that truly reflects modern CLD, a disease of impaired alveolarization and pulmonary development rather than one of extensive inflammation and fibrosis. Although we did not measure any direct markers of global inflammation aside from 3-nitrotyrosine, there is no difference in cell counts from terminal lavage fluid between the CLD and CLD plus iNO animals. This was the case even though there was a greater than twofold increase in DNA present in the lungs of animals exposed to inhaled NO (11).

Inhaled NO led to an increase in plasma levels of nitrate and nitrite but did not affect tracheal aspirate levels of these same metabolites. Here, as during development, the products of NO oxidation do not change in the pulmonary fluid but increase in the systemic circulation consistent with the observations that inhaled NO and nitrite and nitrate rapidly cross into the bloodstream (19). There was no difference in urine output between animals receiving inhaled NO and those that did not. Therefore, the systemic levels are a consequence of increased NO supply and not a consequence of changes in metabolite clearance. Furthermore, there was no difference in oxygenation index between the CLD and the CLD plus inhaled NO groups (11). The oxidative metabolites are unlikely to be related to any differences in oxygen exposure. The only additional difference noted with exposure to inhaled NO was an increase in plasma SNO, again demonstrating systemic effects of the inhaled gas. Recently, the clinical effects of inhaled NO in the baboon model were reported by McCurnin and colleagues (11). They noted that the animals exposed to inhaled NO required greater vasopressor support. The increased pool of plasma SNO and systemic nitrate and nitrite may contribute to systemic vasodilation in these premature animals with an already tenuous cardiovascular status.

McCurnin and colleagues also reported structural and functional changes in the lungs of baboons in the CLD plus inhaled NO model. The most dramatic changes were an increase in lung weight by 19% and an increase in lung volume by 45%. These changes were accompanied by evidence of increased cell proliferation. In addition, inhaled NO resulted in an attenuation of the disordered elastin deposition seen in the lungs of the CLD model (11). Such findings reflect the role of NO in the regulation of cell signaling and show effects beyond what might be expected from transient improvements in resistance and compliance mediated by smooth muscle relaxation. NO is known to be involved in regulation of many cellular processes through S-nitrosation of proteins (32). Therefore, we hypothesized that supplemental NO in the form of inhaled NO might result in an increase in the pulmonary pool of SNO. We were unable to detect any such change. NO is not mediating its effects in this disease model through S-nitrosation of proteins, or the protein modifications are dynamic, as might be expected in a mechanism modulating cellular activity and signal transduction. SNO are transient species, and although their biological lifetime is undetermined, measurement of steady-state levels is unsuited for discovering dynamic changes. Cell models evaluating the role of SNO in the proliferation of pulmonary epithelium or in the induction of tropoelastin in myofibroblasts would serve to clarify the role of NO in regulating these processes.

Finally, we have shown for the first time that inhaled NO does not lead to changes in the levels or activity of pulmonary eNOS or nNOS in this model of prematurity. Supplying exogenous NO has been shown to decrease NOS activity in lambs (33). More recently, inhaled NO was shown to decrease eNOS protein levels in a lamb model of pulmonary hypertension, but there was a compensatory increase in eNOS activity (34). In both systems, the animals were exposed to 40 ppm of NO. In the model of premature lung disease presented here where animals were exposed to 5 ppm of inhaled NO for 14 d, neither NOS activity nor NOS protein levels were affected.

In summary, in the present studies pulmonary and systemic metabolites of NO reflected known increases in the pulmonary isoforms of NOS over the course of late fetal development. In spite of the previously demonstrated decreases in eNOS and nNOS in the CLD model, systemic and lung fluid levels of nitrate and nitrite increased above levels seen in normal fetal development. A deficit in total NO production in the premature baboon was not evident in the surrogate markers measured in this study. Mechanical ventilation in the premature baboon also did not result in an elevation in levels of 3-nitrotyrosine in any of the compartments measured. With inhaled NO there were no demonstrable changes in steady-state levels of SNO in the pulmonary parenchyma, but there were marked increases in systemic nitrate, nitrite, and SNO, which may have important systemic consequences. The potential biological consequences of these circulating NO-derived metabolites should be considered in infants receiving inhaled NO after preterm birth.


   Footnotes
 
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org.

Originally Published in Press as DOI: 10.1165/rcmb.2005-0182OC on September 15, 2005

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form May 13, 2005

Accepted in final form August 16, 2005


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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