1 Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas 75390; 2 San Antonio Military Pediatric Center, Lackland Air Force Base 78235; and 3 The Southwest Foundation for Biomedical Research, San Antonio, Texas 78245
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ABSTRACT |
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Nitric oxide (NO), produced by NO synthase
(NOS), serves multiple functions in the perinatal lung. In fetal
baboons, neuronal (nNOS), endothelial (eNOS), and inducible NOS (iNOS)
are all primarily expressed in proximal respiratory epithelium. In the
present study, NOS expression and activity in proximal lung and minute
ventilation of NO standard temperature and pressure
(ENOSTP) were evaluated in a
model of chronic lung disease (CLD) in baboons delivered at 125 days
(d) of gestation (term = 185 d) and ventilated for 14 d,
obtaining control lung samples from fetuses at 125 or 140 d of
gestation. In contrast to the normal 73% increase in total NOS
activity from 125 to 140 d of gestation, there was an 83% decline with
CLD. This was related to marked diminutions in both nNOS and eNOS
expression and enzymatic activity. nNOS accounted for the vast majority
of enzymatic activity in all groups. The normal 3.3-fold maturational
rise in iNOS protein expression was blunted in CLD, yet iNOS activity
was elevated in CLD compared with at birth. The contribution of iNOS to
total NOS activity was minimal in all groups.
ENOSTP remained stable in the range of 0.5-1.0
nl · kg
1 · min
1
from birth to day 7 of life, and it then rose by 2.5-fold.
Thus the baboon model of CLD is characterized by deficiency of the principal pulmonary isoforms, nNOS and eNOS, and enhanced iNOS activity
over the first 2 wk of postnatal life. It is postulated that these
alterations in NOS expression and activity may contribute to the
pathogenesis of CLD.
endothelial nitric oxide synthase; exhaled nitric oxide; inducible nitric oxide synthase; neuronal nitric oxide synthase; primate
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INTRODUCTION |
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THE SIGNALING MOLECULE nitric oxide (NO), generated by NO synthase (NOS), plays a critical role in physiological processes in the pulmonary epithelium (6, 16). NO is detectable in expired gas (15), and the principle source of expired NO is the lung epithelium (11, 18). The normal functions of NO in the mature airway include neurotransmission, smooth muscle relaxation, bacteriostasis, and the modulation of mucin secretion, ciliary motility, and plasma exudation (6, 16). Physiological actions of NO in the lung are most likely not mediated by free NO but rather by products of NO reactions, including S-nitrosocysteines and S-nitrosothiols (17).
There is accumulating evidence that NO is of major importance to lung epithelial function in the perinatal period. Epithelium-derived NO plays a key role in the opposition of airway contraction (19) in the modulation of lung liquid production (10) and in the regulation of peripheral contractile elements in the developing lung (19). In addition, NO has a well-recognized role in mediating pulmonary vasomotor tone in the perinatal period (33). In recent studies of lungs from fetal baboons, we have shown that all three NOS isoforms, neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS), are primarily expressed in proximal respiratory epithelium in the developing primate. In addition, there are maturational increases in the expression of the three NOS isoforms and in NO production during the early third trimester that may contribute to enhanced airway and parenchymal function in the immediate postnatal period (34).
Along with serving a role in normal lung function, there is evidence that changes in pulmonary NO production related to altered NOS expression contribute to the cellular damage and parenchymal dysfunction characteristic of many pulmonary inflammatory conditions. In multiple paradigms of lung inflammation, iNOS is upregulated to produce cytotoxic levels of NO (30). However, despite iNOS upregulation, the net effect of inflammation can be an attenuation in epithelial NO production, such as in the guinea pig model of parainfluenza infection, leading to airway hyperresponsiveness (12). In a similar manner, lung transplant recipients with obliterative bronchiolitis display greater iNOS expression and attenuated nNOS and eNOS expression compared with controls (25). Thus there is evidence in the mature lung that, concomitant with NO overproduction by iNOS during pulmonary inflammation, which potentially contributes to cellular damage, NO production by nNOS and eNOS may be diminished, resulting in airway, parenchymal, and vascular dysfunction.
Chronic lung disease (CLD) is an inflammatory process that can be initiated following the early course of hyaline membrane disease in premature infants requiring ventilatory support. Before the surfactant treatment era, CLD was characterized by airway injury, parenchymal fibrosis, and inflammation. The more recent form of CLD is notable for less fibrosis and fewer and larger alveolae and an increase in elastic tissue that correlates with the severity of the clinical disorder (40). Infants with CLD have increased pulmonary vascular and airway resistance (1), and similar functional abnormalities have been documented in chronically ventilated preterm lambs and baboons (2, 7, 9, 45). In the preterm lamb model, there is also evidence that eNOS protein abundance is attenuated in small intrapulmonary arteries and small airways compared with those from control lambs born at term; in contrast, iNOS abundance is unchanged (23). Thus attenuated eNOS expression may play a role in the pathogenesis of CLD. However, it is not known whether such changes in eNOS abundance lead to alterations in pulmonary NO production or whether nNOS is affected, and it is also unclear whether similar mechanisms occur after preterm birth and ventilatory support in primates or higher species.
To better understand the potential role of pulmonary NOS in the genesis
of CLD associated with preterm birth in humans, we evaluated lung NOS
expression and function in a model of CLD in baboon fetuses delivered
at 125 days (d) of gestation (term = 185 d) and placed on
ventilatory support for 14 d. The baboons are born at 67% of term
gestation, which is comparable to 27 wk postconceptional age in humans,
and the model closely mimics the current form of CLD in extremely
preterm human infant (9, 45). Control samples were
obtained from additional fetuses at 125 or 140 d of gestation. On
the basis of the findings in other paradigms of lung inflammation
(12, 25, 30), we tested the hypothesis that nNOS and eNOS
expression are diminished and iNOS expression is upregulated in the
baboon model of CLD. The specific contributions of each of the
three NOS isoforms to enzymatic activity were also assessed. In
addition, we determined whether changes in NOS isoform expression are
associated with alterations in lung NO output evaluated by longitudinal
measurements of the minute ventilation of NO at standard temperature
and pressure (ENOSTP).
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MATERIALS AND METHODS |
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Animal model.
All animal studies were performed at the Southwest Foundation for
Biomedical Research Primate Center in San Antonio, TX. Pregnancies in
baboons (Papio papio) were timed with cycle dates, and fetal growth parameters were obtained from prenatal ultrasound examinations performed at 70 and 100 d of estimated fetal gestation. Fetal baboons were delivered at 125 ± 2 d of gestation (term = 185 d) by cesarean section. At birth, the baboons were weighed,
sedated, intubated, and given 4 ml/kg of surfactant (Survanta; courtesy of Ross Laboratories, Columbus, OH) before initiation of ventilator support. Ventilation was provided for 14 d with a humidified, pressure-limited, time-cycled infant ventilator (InfantStar;
Infrasonics, San Diego, CA), and stabilization also included the
placement of an umbilical arterial catheter and percutaneous central
venous catheter. Details of animal care have been published elsewhere (9). Exhaled NO levels were measured at hour 1,
8, and 24 of life and daily from day
2-13 of life. At the time of necropsy on day 14 of
life, proximal lung specimens were snap-frozen for later analysis. The
samples were taken from the most proximal one-third of the lung
parenchyma adjacent to the bronchus. Maturational control specimens
were obtained at the time of hysterotomy from additional fetuses at
125 d of gestation (beginning of study) and 140 d of
gestational age (end of study). Lung samples were obtained from five
animals per group. NOS enzymatic activity was evaluated in
n = 5 per group, and immunoblot analyses were performed in n = 4 per group due to limitations in tissue
availability. Total lung wet weights were not changed in CLD animals at
14 days of life compared with 125-d gestation controls (11.1 ± 0.8 vs. 9.7 ± 0.5 g, respectively, P = not
significant). In contrast, lung wet weights in 140-d gestation
controls (12.6 ± 0.5 g) were greater than at 125 d of
gestation (P < 0.05).
ENOSTP was assessed longitudinally
in a total of eight CLD animals due to the availability of three
additional animals in the consortium.
Ventilatory management. The ventilatory approach entailed a strategy to maintain tidal volumes at 4-6 ml/kg as determined with a body plethysmograph system (VT1000; Vitaltrends Technology, New York, NY) and to generate adequate chest motion by clinical examination. Tidal volumes did not vary significantly over the course of the study. Initial positive end-expiratory pressure within the first 1-2 h of life was 4-5 cmH2O, and it was weaned thereafter to a minimum of 3 cmH2O and was not significantly changed during the remaining course of study. Bias flow was maintained at 8 l/min. Target arterial blood gas parameters were PaCO2 45-55 Torr and PaO2 55-70 Torr. In an attempt to minimize exposure to high FIO2, if the PaO2 level was above target goals, FIO2 was weaned until <0.40, and then modifiers of mean airway pressure or FIO2 were decreased as tolerated. If PaO2 was below target guidelines, a chest radiograph was obtained to evaluate lung inflation. Adjustments in mean airway pressure were made to minimize underinflation or overinflation of the lung. If lung inflation was deemed adequate, FIO2 alone was adjusted.
NOS enzymatic activity.
Determinations of NOS enzymatic activity in the presence of excess
substrate and cofactors provide a reliable quantitative assessment of
enzyme abundance. As importantly, pharmacological interventions within
the activity assay provide the only means to effectively assess the
relative abundance of the three NOS isoforms and their individual
contributions to total activity. NOS enzymatic activity was determined
in proximal specimens of lung obtained at necropsy at 14 d of
postnatal life in the baboons delivered at 125 d of gestation
(CLD) and in the 125- and 140-d gestation maturational controls by
previously described methods (36). Experiments were
performed on the proximal one-third of lung parenchyma, which is rich
in large airways. Although nonairway cell types were also included,
selected dissection of airways was not feasible when multiple aliquots
of tissue must be frozen immediately for use by the consortium. The
lung tissue, which was initially snap-frozen and stored in liquid
nitrogen, was slowly thawed and homogenized on ice in 50 mM Tris buffer
(pH 7.8) containing 10 µg/ml pepstatin A, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml
N--p-tosyl-L-lysine chloromethyl
ketone, 20 µM tetrahydrobiopterin, 3.0 mM dithiothreitol, 1.0 mM
phenylmethylsulfonyl fluoride, and 10 mM
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate using a
ground glass homogenizer. The tissue homogenate was centrifuged at 10,000 g, total protein content was determined on the
supernatant by the Bradford method using bovine serum albumin as the
standard (8), and NOS activity was determined in the
supernatant by measuring the conversion of
[3H]L-arginine to
[3H]L-citrulline (26). Tissue
preparation (50 µl) was added to 50 µl of buffer, yielding final
concentrations of reagents as follows: 2 mM
-NADPH, 2 µM
tetrahydrobiopterin, 10 µM flavin adenine dinucleotide, 10 µM
flavin mononucleotide, 0.5 mM CaCl2 in excess of EDTA, 15 nM calmodulin, 2 µM cold L-arginine, and 2.0 µCi/ml
[3H]L-arginine. After incubation at 37°C
for 30 min, the assay was terminated by the addition of 400 µl of 40 mM HEPES buffer, pH 5.5, with 2 mM EDTA and 2 mM EGTA. The terminated
reactions were applied to 1-ml columns of Dowex AG50WX-8 (Tris form)
and eluted with 1 ml of the 40 mM HEPES buffer.
[3H]L-citrulline was collected in
scintillation vials and quantified by liquid scintillation
spectroscopy. NOS activity was linear with time for up to 60 min, and
it was fully inhibited by 2.0 mM nitro-L-arginine methyl
ester. The calcium dependence of NOS activity was evaluated by the
addition of 2.5 mM EGTA to the incubation mixture.
Immunoblot analyses. Immunoblot analyses for nNOS, eNOS, and iNOS provide an additional independent means of quantifying differences in the expression of a given isoform between study groups. The methods for immunoblot analysis were similar to those previously reported (36). Proximal lung samples were thawed on ice and homogenized in ice-cold 50 mM Tris buffer and processed as described above for enzymatic activity analysis. SDS-PAGE was performed with 7% acrylamide, and the proteins were electrophoretically transferred to nitrocellulose filters. The filters were blocked for 1.5 h in buffer containing 150 mM NaCl and 10 mM Tris (pH 7.5) with 0.5% Tween 20 and 5% dried milk and incubated overnight at 4°C with polyclonal antibody generated either to the unique midmolecule peptide PYNSSPRPEQHKSYK of eNOS, which corresponds to a conserved epitope identical in sequence between mouse, canine, guinea pig, bovine, and human, or to the COOH-terminal peptide ESKKDTDEVFSS of nNOS that is identical in sequence in mouse and human. iNOS protein abundance was quantitated using monoclonal antibody reactive to both the mouse and human protein (Transduction Laboratories, San Diego, CA). After incubation with primary antiserum, the nitrocellulose filters were washed with the 150 mM NaCl buffer with Tween 20 and incubated for 1.5 h with donkey anti-rabbit Ig or rabbit anti-mouse Ig antibody-horseradish peroxidase conjugates (Amersham). The filters were washed in the 150 mM NaCl buffer with Tween 20, and the bands for NOS were visualized by chemiluminescence (ECL Western Blotting Analysis System; Amersham) and quantitated densitometrically. The antiserum to eNOS was the kind gift of Dr. Thomas Michel (Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, Boston, MA), and the antiserum to nNOS was the kind gift of Dr. Kim Lau (Department of Physiology, University of Texas Southwestern Medical Center).
Measurements of exhaled NO. The procedures employed were in accordance with the 1999 American Thoracic Society (ATS) recommendations (3, 38). A chemiluminescence analyzer (EcoPhysics CLD 77 AM; EcoPhysics, Duernten, Switzerland) was used to measure NO in exhaled breath. This device detects light produced by the reaction of ozone with NO using a charge-coupled digital detection system, with subsequent 10-Hz digital signal output. The analyzer performs an internal calibration by serial dilution of a known concentration NO gas standard [8 parts per million (ppm), National Institute of Standards and Technology (NIST) traceable cylinder of Environmental Protection Agency (EPA) reference grade; Scott Specialty Gasses, Plumsteadville, PA] using NO scrubbed (Purafil, Doraville, GA) zero gas as the diluent. The internal calibration and accuracy of the resulting signal were verified by comparison against serial dilutions of an external standard. Briefly, NO standard gas (10 ppm, NIST traceable cylinder of EPA reference grade; Scott Specialty Gasses) was diluted with air passed through an NO scavenging apparatus (Purafil) using NIST traceable mass flow controllers (Cole-Parmer, Vernon Hills, IL) to achieve five known concentrations of NO over the range of 2-80 parts per billion (ppb). The correlation coefficient (r) for measured vs. predicted values was 0.998. Response time of the device was determined by the diaphragm puncture technique (31, 32). The response time to 90% of full strength signal was <0.1 s (21).
The high ventilator rate employed in a low volume ventilation strategy hampers continuous sampling of exhaled NO from a distal endotracheal port. Additionally, single breath profiles obtained from nonintubated adult volunteers have demonstrated marked flow dependence for exhaled NO (37). We therefore devised a sampling strategy that maintains a constant flow rate and is rapid enough to minimize interference with the animal's
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Statistical analysis. Analysis of variance with Newman-Keuls or Bonferroni post hoc testing was used to compare mean values between more than two groups. Significance was accepted at the 0.05 level of probability. All results are expressed as means ± SE.
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RESULTS |
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NOS enzymatic activity.
Changes in NOS enzymatic activity in the proximal lung in the baboon
CLD model are shown in Fig. 2. In the
control groups, there was a 73% increase in total NOS activity with
normal fetal development between 125 and 140 d of gestation (Fig.
2A). In contrast, with 14 d of ventilatory support
following preterm birth at 125 d of gestation (CLD), there was an
83% decline in total NOS activity in the proximal lung vs. at birth
(125-d-gestation control), and a 90% fall compared with the
postconceptional age control (140 d of gestation). Calcium-dependent
NOS enzymatic activity, indicative of nNOS or eNOS activity, accounted
for the vast majority of total NOS activity, and it rose 65% with
normal fetal maturation from 125 to 140 d of gestation (Fig.
2B). However, in lungs from CLD animals, calcium-dependent
activity was decreased by 90% compared with that at the time of birth
and by 94% compared with an equivalent in utero postconceptional age.
The findings for calcium-independent NOS activity are provided in Fig.
2C. Calcium-independent activity was detectable at all ages
tested, but it comprised a modest proportion of total NOS enzymatic
activity. During normal fetal development from 125 to 140 d of
gestation, calcium-independent activity rose sevenfold. With CLD there
was no notable change in calcium-independent NOS activity compared with
levels observed at the same postconceptional age in utero.
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NOS isoform expression.
The effects of CLD on nNOS expression in the proximal lung are shown in
Fig. 4. In the representative immunoblot
shown (Fig. 4A), nNOS protein abundance was greater at
140 d compared with 125 d of gestation in the developmental
controls. In contrast, nNOS protein was minimally detectable in CLD
lung. These observations were confirmed in four independent
experiments, which revealed a 2.2-fold increase in nNOS protein levels
from 125 to 140 d of gestation during normal fetal
maturation, and a 42% decline in nNOS levels with CLD compared with
the 125-d-gestation control (Fig. 4B). When evaluated
relative to the comparable maturational control (140-d gestation lung),
nNOS protein expression was attenuated 73% in CLD lung.
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ENO.
In recent studies of exhaled NO levels shortly after birth at 125 d and 140 d of gestation in the baboon, estimates of
ENO were nearly threefold greater in the older
age group (34). To provide a longitudinal assessment of
airway NO production during the genesis of CLD over the same
developmental period, we measured NO output within 1 h of delivery
at 125 d of gestation and repeatedly thereafter up until the day
before the termination of study at 14 d (Fig.
7).
ENOSTP remained stable in the range
of 0.5-1.0 nl · kg
1 · min
1
from birth to day 7 of life, and it then rose to be 2.5-fold greater than values measured at birth by the end of the second week of
life.
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DISCUSSION |
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NO plays an important role in the modulation of pulmonary function in the perinatal period (33). In an effort to better understand the contribution of NO to the genesis of CLD associated with preterm birth in humans, in the present study we evaluated the expression and function of the three NOS isoforms in the proximal lung in a baboon model of CLD, which closely mimics the human condition (9, 45). We observed that there is a marked decline in pulmonary NOS expression and activity with CLD, indicative of an attenuated capacity for endogenous NO production in this disease state.
NOS enzymatic activity was evaluated in the proximal lung, where the vast majority of pulmonary nNOS, eNOS, and iNOS in the fetal primate is expressed and localized in respiratory epithelium (34). Enzymatic activity measurements in the presence of excess substrate and cofactors provide a reliable quantitative determination of enzyme abundance, and pharmacological interventions within the activity assay provide the only means to effectively evaluate the relative amounts of the three NOS isoforms and their individual contributions to total activity. We found that in contrast to the increase in total NOS enzymatic activity that occurs with normal fetal baboon development during the early third trimester, there was a dramatic decline in NOS activity with the genesis of CLD over the same maturational period. In all study groups, the total NOS activity was primarily calcium dependent, indicative of nNOS and eNOS activity, and the principal change seen with CLD was a fall in calcium-dependent activity. Further studies with selective nNOS antagonism revealed that the vast majority of calcium-dependent NOS activity was derived from that isoform. Compared with developmental controls, both nNOS- and eNOS-derived enzymatic activity was decreased by 90% or more in the CLD lung. In contrast to the declines in calcium-dependent NOS isoform activity noted with CLD, the normal severalfold maturational increase in calcium-independent activity was conserved in the CLD lung. These cumulative observations indicate that nNOS is the principal source of NO in the proximal lung during the third trimester in the primate. Furthermore, with CLD, there are dramatic declines in pulmonary nNOS enzymatic activity and also in eNOS enzymatic activity, with the fall in nNOS accounting for the majority of the decline in total activity, whereas that for iNOS is unaltered.
The abundance of NOS isoform proteins was also assessed by immunoblot
analysis. Paralleling the observations for nNOS-related enzymatic
activity, nNOS protein expression was increased with normal fetal
development from 125 to 140 d of gestation, but it was
dramatically decreased with the genesis of CLD over the same maturational period. Although the diseases do not have similar pathogeneses, the findings with CLD are comparable with those of
patients with obliterative bronchiolitis who display diminished nNOS
expression (25). In more general terms, the attenuation in
nNOS abundance is similar to that observed in multiple rat tissues
following treatment with lipopolysaccharides or interferon- (4). In addition, there is evidence from multiple
paradigms that nNOS is upregulated by estrogen (14, 43),
which normally rises markedly in the fetal circulation during the third
trimester due to increasing production by the placenta
(29). We postulate that the pulmonary nNOS downregulation
observed with CLD may be mediated by a combination of relative estrogen
deprivation following premature birth and the inflammatory processes
associated with the disorder (20).
eNOS protein abundance was also evaluated by immunoblot analysis. eNOS expression was increased with normal fetal maturation from 125 to 140 d of gestation, but this upregulation was disrupted with preterm birth and the development of CLD. These results in the preterm baboons ventilated from 68 to 76% of gestation mimic the observations made in an ovine model in which immunoblots of both intrapulmonary airways and arteries displayed diminished eNOS protein levels. However, it is important to note that the preterm sheep were ventilated from 84% of gestation to term, so the maturational periods tested in the two models are entirely different, and that the present work is the first to evaluate altered NOS expression with CLD in primates. In addition, the studies in sheep did not evaluate the abundance of nNOS, which we now know is a major source of lung NOS activity in the fetal primate, and the contributions of NOS isoforms to NOS enzymatic activity were not assessed (23).
Similar to the results obtained for nNOS, the decline observed in eNOS
expression in CLD is comparable with the findings in patients with
obliterative bronchiolitis (25). eNOS is downregulated by
inflammatory mediators such as tumor necrosis factor-
(46). Mirroring the above noted hormonal modulation of
nNOS, eNOS is also upregulated by estrogen (24). As such,
we postulate that fetal estrogen deficiency due to premature birth
and/or the inflammatory mechanisms contributing to the genesis of CLD
may underlie the diminutions in both nNOS and eNOS in this disorder.
Interestingly, in a limited study of postnatal estrogen therapy in
preterm infants aimed to improve bone mineralization, CLD was absent in
15 treated infants, whereas it occurred in 4 of 15 controls
(41). Further detailed studies will be required in the
baboon model to determine the basis of nNOS and eNOS downregulation,
whether these alterations can be prevented, and whether doing so has an
impact on anatomic and functional pulmonary outcomes.
In contrast to the observed parallel diminutions in nNOS- and
eNOS-derived enzymatic activity and nNOS and eNOS protein expression with CLD, there was conservation of the normal maturational increase in
calcium-independent NOS activity in the disease model. This was despite
observing blunted iNOS protein expression with CLD compared with levels
at an equivalent in utero postconceptional age. The basis for the
disparity between the changes in iNOS-related enzyme activity,
which provides a small fraction of total NOS activity in this paradigm,
and iNOS protein levels is unclear. NOS enzymatic activity assays were
performed in the presence of excess amounts of substrate and all
required NOS cofactors such that enzyme abundance is the limiting
factor, and the effective partitioning of calcium-dependent and
calcium-independent NOS activity with calcium chelation was confirmed
in a cell transfection system expressing each isoform individually. The
enzyme activity assays may be more reliably quantified than
densitometric analysis of immunoblots because the primary readout for
the former is numeric in nature. Thus we conclude that iNOS-derived
enzymatic activity is probably conserved during the genesis of CLD in
the baboon model, and this would be consistent with the unaltered iNOS
protein abundance observed in the preterm lamb model (23).
We postulate that, despite the maturational interruption occurring with
preterm delivery, iNOS expression is maintained following preterm birth and ventilatory support for 14 d due to upregulation by
inflammatory mechanisms related to CLD. The overall contrasting effects
of CLD on nNOS and eNOS vs. iNOS mimic the opposing directional changes seen with obliterative bronchiolitis in humans (25) and
with lipopolysaccharide and interferon- treatment of multiple rat tissues (4).
To provide a longitudinal evaluation of lung NO output during the
genesis of CLD, we measured exhaled NO levels as a surrogate, most
likely inactive, marker of bioavailable NO. In prior studies we
observed almost threefold greater ENO
immediately after birth in baboons delivered at 140 d vs. 125 d of gestation (34). As such, greater NO and NO
metabolites may be present to mediate multiple pulmonary functions in
the early postnatal period in primates born later compared with earlier
in the third trimester. In the present study following premature
delivery at 125 d of gestation,
ENOSTP remained stable from birth to
day 7 of life, and it then rose to be 2.5-fold greater than
values measured at birth by the end of the second postnatal week,
suggesting constant and then rising NO and NO metabolite abundance.
However, it is important to note that the specific cell types and NOS
isoforms responsible for NO gas production have not been determined. In addition, attempts to correlate temporal changes in NO output with the
observed alterations in NOS isoform expression and activity with CLD do
not take into account possible differences in endogenous NOS inhibition
by asymmetric dimethyl arginine or in arginine metabolism by arginases
and other mechanisms (22, 44). Despite these potential
limitations, the present work provides important new information about
alterations in NOS isoform abundance and NO production in a primate
model performed at a developmental stage equivalent to 27-wk
postconceptional age in humans that closely mimics the current form of
CLD following premature birth in humans (9, 45). Studies
of the impact of NOS isoform-selective antagonists on exhaled NO should
now be considered in the preterm baboon model.
The changes in ENOSTD demonstrated
in the current work in primates complement the information related to
NO and NO metabolites from human studies. Besides generating
S-nitrosocysteines and S-nitrosothiols, NO can
react with O2 and reactive oxygen species to yield higher
oxides of nitrogen and peroxynitrite. Peroxynitrite reacts with
proteins to form 3-nitrotyrosine (17). Banks and colleagues (5) measured plasma 3-nitrotyrosine levels
following birth in premature infants with CLD and demonstrated that
levels are greater than in preterms without CLD. In addition, in the CLD group the levels rose during the latter half of the first month,
generally paralleling the increase in
ENOSTD that we report here. Vyas and
coworkers (42) reported stable nitrate levels in
bronchoalveolar lavage (BAL) early in life, which then rose in the
second week in infants with CLD, also paralleling the present findings
for NO output in the primate CLD model. In contrast, BAL nitrate levels
fell dramatically at 14 days of life in premature infants without CLD.
The authors do not comment on how their subjects tolerated BAL. In the
premature baboon, the BAL procedure results in significant hemodynamic
and pulmonary instability. As such, BAL samples are not feasible in the
baboon model before necropsy. It should be noted that Storme et al.
(39) evaluated exhaled NO levels in infants with CLD and
demonstrated greater levels than in controls, but the studies were done
from 1 mo of postnatal age onward. At any rate, the present findings for lung NO output in the baboon model of CLD are generally consistent with related available information in premature infants studied over a
comparable developmental period.
The functional implications of decreased pulmonary nNOS and eNOS expression and diminished NO production by those isoforms in the baboon CLD model are multiple. Studies in newborn piglets, using both intact animals and isolated tracheae, indicate that epithelium-derived NO counteracts bronchoconstriction (19, 27). There is also evidence that epithelium-derived NO modifies distal lung function; studies in fetal lambs indicate that NO regulates lung liquid production (10), and further work in the newborn piglet reveals a role for NO in modulating lung compliance (27). Furthermore, the critical contribution of NO to pulmonary vasomotor regulation in the perinatal period is well established (33), and investigations in fetal lambs indicate that nNOS is an important source of endogenous NO impacting on the lung circulation (28). It is possible that proximally expressed nNOS modulates pulmonary vascular and distal airway and parenchymal function via hemocrine pathways and airway lining fluid communications. With these multiple considerations in mind, we postulate that diminished nNOS- and eNOS-derived NO and NO metabolites may contribute not only to the airway dysfunction but also to the parenchymal and pulmonary vascular abnormalities that are characteristic of CLD. Studies of the impact of NO replacement in the baboon CLD model are now warranted to clarify the contribution of relative NOS deficiency to the morphological and functional consequences of the disorder.
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ACKNOWLEDGEMENTS |
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The authors thank all the personnel that support the Bronchopulmonary Dysplasia Resource Center: the animal husbandry group led by Drs. D. Carey and M. Leland, the Neonatal Intensive Care Unit staff (H. Martin, S. Ali, D. Correll, L. Kalisky, L. Nicley, R. Degan, and S. Gamez), the Wilford Hall Medical Center neonatal fellows and D. Catland, who assist in the care of the animals, and the University of Texas Health Sciences Center, San Antonio pathology staff (V. Winter, L. Buchanan, H. Dixon, and A. Schreiner), who perform necropsies and obtain biological specimens. The authors also appreciate the assistance of D. Randle and M. Dixon, who assisted in the preparation of the manuscript.
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FOOTNOTES |
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The work was supported by National Heart, Lung, and Blood Institute Grants HL-63399 and HD-30276.
Address for reprint requests and other correspondence: P. W. Shaul, Dept. of Pediatrics, Univ. of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75390 (E-mail: pshaul{at}mednet.swmed.edu).
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. Section 1734 solely to indicate this fact.
First published January 10, 2003;10.1152/ajplung.00334.2002
Received 4 October 2002; accepted in final form 7 January 2003.
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