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
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.
Keywords: 3-nitrotyrosine; S-nitrosocysteine; S-nitrosothiols; nitric
oxide; chronic lung disease; prematurity; baboon
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
Animal Model
(Received in original form May 13, 2005 and in final form August 16, 2005)
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: [email protected]
This article has an online supplement, which is accessible from this issue’s table
of contents at www.atsjournals.org.
Am J Respir Cell Mol Biol Vol 33. pp 582–588, 2005
Originally Published in Press as DOI: 10.1165/rcmb.2005-0182OC on September 15, 2005
Internet address: www.atsjournals.org
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),
Munson, Grubb, Kerecman, et al.: Nitric Oxide Metabolites in Premature Baboons
583
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 ␮m.
NOS Enzymatic Activity
Measurement of Nitrate and Nitrite
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.
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 ␮M and 5 mM, respectively. Protein SNO
and low-molecular-weight SNO were measured by chemiluminescencebased 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 ⫻ 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;
␮mol/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.
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
RESULTS
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).
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 ␮M
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 ␮mole 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
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2005
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).
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).
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 ␮M (Figure 5B), which
represents a tripling of the 125-d gestation level of 17 ␮M. Plasma
nitrate and nitrite rose over time during the 2 wk of ventilation,
reaching a level of 88 ␮M 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 ␮mol
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).
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 ␮mol 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)
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.
Munson, Grubb, Kerecman, et al.: Nitric Oxide Metabolites in Premature Baboons
585
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.
(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 ␮M. 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).
DISCUSSION
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.
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 33
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, oneway 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).
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 ␮M) compared with late thirdtrimester levels (mean of 44 ␮M). 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,
2005
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).
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.
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).
Munson, Grubb, Kerecman, et al.: Nitric Oxide Metabolites in Premature Baboons
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.
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
587
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
588
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 33
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.
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.
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