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), plays a critical role in multiple processes in the lung during the perinatal period. To better understand the regulation of pulmonary NO production in the developing primate, we determined the cell specificity and developmental changes in NOS isoform expression and action in the lungs of third-trimester fetal baboons. Immunohistochemistry in lungs obtained at 175 days (d) of gestation (term = 185 d) revealed that all three NOS isoforms, neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS), are primarily expressed in proximal airway epithelium. In proximal lung, there was a marked increase in total NOS enzymatic activity from 125 to 140 d gestation due to elevations in nNOS and eNOS, whereas iNOS expression and activity were minimal. Total NOS activity was constant from 140 to 175 d gestation, and during the latter stage (160-175 d gestation), a dramatic fall in nNOS and eNOS was replaced by a rise in iNOS. Studies done within 1 h of delivery at 125 or 140 d gestation revealed that the principal increase in NOS during the third trimester is associated with an elevation in exhaled NO levels, a decline in expiratory resistance, and greater pulmonary compliance. Thus, there are developmental increases in pulmonary NOS expression and NO production during the early third trimester in the primate that may enhance airway and parenchymal function in the immediate postnatal period.
airway epithelium; compliance; expiratory resistance; primate
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INTRODUCTION |
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THE SIGNALING MOLECULE nitric oxide (NO), produced by nitric oxide synthase (NOS), plays a critical role in physiological processes in the pulmonary epithelium (1, 10, 17). NO is detectable in expired gas (9), and studies in both animals and humans suggest that the principle source of expired NO is the lung epithelium rather than the pulmonary vasculature (7, 12). The functions of NO in the mature airway include neurotransmission, smooth muscle relaxation, and bacteriostasis, and also the modulation of mucin secretion, ciliary motility, and plasma exudation (1, 10).
There is mounting evidence that NO is of great importance to lung epithelial function in the perinatal period. The stimulation of NO synthesis by acetylcholine or bradykinin causes marked decreases in lung liquid production in late-gestation fetal lambs (5), and the instillation of NO or cGMP, the second messenger for NO, into the fetal lung liquid has the same effect (4, 6). The decrease in lung liquid production by the respiratory epithelium at the time of birth is an essential component of the transition of the fetus from a liquid-breathing to an air-breathing status. Epithelium-derived NO is also critical to the regulation of bronchomotor tone in the early newborn period, playing an important role in the opposition of airway contraction (14). In addition, NOS antagonism causes increased tissue resistance in the newborn lung, suggesting that endogenous NO may regulate peripheral contractile elements (22).
We and others have shown that the pulmonary expression of the neuronal and endothelial isoforms of NOS (nNOS and eNOS) in nonprimate mammalian species increases during fetal life (19, 20, 29). In the developing sheep, lung nNOS is primarily expressed in the proximal airway epithelium; the inducible isoform of the enzyme (iNOS) is also expressed and most abundant in proximal airway epithelium, and eNOS is found in both proximal airway epithelium and vascular endothelium (31). Experiments with nonselective and isoform-selective NOS antagonism in sheep have shown that all three NOS isoforms contribute to the regulation of pulmonary vasomotor tone during late fetal and early postnatal life (23-25). However, little is known about the cell specificity, the developmental regulation, and function of the three NOS isoforms in the lungs of primates and higher species.
To better understand the regulation of pulmonary NO production during late gestation and in the perinatal period, we designed the present studies to delineate the cellular distribution of NOS isoform expression in the respiratory epithelium of the baboon fetus. Additional experiments were performed to evaluate maturational changes in NOS enzymatic activity in the proximal lung during the third trimester. Quantitative changes in nNOS, eNOS, and iNOS protein abundance were also assessed. Furthermore, to determine the impact of developmental changes in NOS abundance on epithelial NO production and action, we compared exhaled NO levels and associated differences in airway resistance and lung compliance immediately after the delivery of premature baboon fetuses at two different gestational ages. We tested the overall hypothesis that there are maturational increases in NOS expression and NO production during the third trimester that have an impact on early pulmonary function.
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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 days (d) of estimated fetal gestation. Experiments evaluating NOS isoform cellular distribution, activity, and expression were done in proximal lung specimens taken at the time of hysterotomy from fetuses at 125, 140, 160, and 175 d gestational age (term = 185 d). The studies performed at 125 d gestation in the baboon are at a developmental stage that mimics that of an extremely premature human infant (68% of gestation, equivalent to 27 wk of gestation in the human). The specimens obtained at the later time points reveal the changes in NOS expression and function during the third trimester.
In separate experiments, measurements of fractional excretion of NO (FENO) and pulmonary function were performed shortly after stabilization at birth in animals delivered by hysterotomy at either 125 or 140 d gestation. These ages were selected for study on the basis of the findings for the maturational changes in NOS activity and expression (see RESULTS). Due to methodological considerations, the measurements of exhaled NO levels and pulmonary function were done in separate groups of animals. Details of animal care have been published elsewhere (3). At birth, the baboons were weighed, sedated, and intubated, and the animals delivered at 125 d gestation were also given 4 ml/kg of surfactant (Survanta; courtesy of Ross Laboratories, Columbus, OH) before initiation of ventilator support. Ventilation was provided 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. Exhaled NO levels and pulmonary function were evaluated at 1 h of life.Immunohistochemistry. Immunohistochemistry to determine NOS isoform cellular distribution was performed by methods previously employed in studies of ovine fetal lung (31). Specimens of proximal lung from 175-d-gestation baboon fetuses were fixed in 2% paraformaldehyde in PBS for 4 h (4°C), immersed in an increasing sucrose-PBS gradient (10% sucrose, 90 min; 15%, 60 min; 20%, 60 min; 4°C), fixed further for 2 h in 10% neutral buffered formalin (Richard-Allan Scientific, Kalamazoo, MI) at 40°C, processed through graded alcohols, and embedded in paraffin. For all immunohistochemical and biochemical studies, the lung samples were obtained from the most proximal one-third of the lung parenchyma adjacent to the bronchus, which is a region rich in large airways. Deparaffinized sections of 4 µm were incubated for 18 h with primary antisera specific for eNOS at 1:100 dilution (mouse monoclonal against human eNOS; Transduction Laboratories, Lexington, KY), iNOS at 1:1,500 (rabbit polyclonal against murine iNOS; Upstate Biotechnology, Lake Placid, NY), or nNOS at 1:3,000 (rabbit polyclonal against rat nNOS; Upstate Biotechnology) at 4°C. In studies of eNOS and iNOS, the incubation with primary antisera was preceded by heat-induced epitope retrieval in basic buffer (AR10 buffer; Biogenex, San Ramon, CA). After quenching endogenous peroxidases with 3% H2O2 in H2O, we performed immunostaining using standard streptavidin-biotin horseradish peroxidase detection methodology and hematoxylin counterstaining. All three NOS isoforms were evaluated simultaneously along with negative controls consisting of lung tissue incubated in the absence of primary antisera. Immunostaining of ovine fetal pulmonary artery endothelial cells, lipopolysaccharide (LPS)-treated bovine trachealis, and ovine cerebellum provided positive controls for eNOS, iNOS, and nNOS, respectively. Preparation of bovine trachealis involved culturing individual strips (0.8 × 3.0 × 0.5 mm) in 1 ml of DMEM with 10 µg/ml LPS (serotype 0127:B8; Sigma, St. Louis, MO) for 24 h. Findings were confirmed in three independent experiments.
NOS enzymatic activity.
NOS enzymatic activity was determined in proximal specimens of fetal
baboon lung obtained at 125, 140, 160, and 175 d gestation using
previously described methods (30). 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 (2), and NOS activity was determined in the
supernatant by measuring the conversion of
[3H]L-arginine to
[3H]L-citrulline (18). Fifty
microliters of tissue preparation were 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 (L-NAME). The calcium dependence of NOS activity was
evaluated by the addition of 2.5 mM EGTA to the incubation mixture.
Immunoblot analysis. The methods for immunoblot analysis were similar to those previously described (30). Proximal lung samples from 125-, 140-, 160-, and 175-d-gestation fetal baboons were thawed on ice and homogenized in ice-cold 50 mM Tris buffer and processed as described above for enzymatic activity analysis. SDS-polyacrylamide gel electrophoresis 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 primary antisera generated either to the unique midmolecule peptide PYNSSPRPEQHKSYK of eNOS, which corresponds to a conserved epitope identical in sequence between bovine and human, or to the COOH-terminal peptide ESKKDTDEVFSS of human nNOS. iNOS protein abundance was quantitated using monoclonal antibody (Transduction Laboratories). After incubation with primary antiserum, the nitrocellulose filters were washed with 150 mM NaCl buffer and 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. NO concentration in tracheal gas was measured in premature baboons at 125 d (n = 4) or 140 d (n = 4) gestation at 1 h of life after delivery by hysterotomy, intubation, and stabilization, using a chemiluminescence technique (NOA 280; Sievers, Boulder, CO). We drew samples continuously from a distal endotracheal tube sampling port at constant flow (40 ml/min) by placing a section of polyethyl ethyl ketone tubing in the sampling line proximal to the reaction chamber. All analytic connections and lines were made with Teflon tubing and fittings. Calibration was performed using serial flow dilutions monitored by NIST digital flowmeters (Fischer, Pittsburgh, PA) with reference-grade NO in N2 (10 and 25 parts per million; Scott Gas, Houston, TX) with a certified NO zero gas as a diluent (UZAM; Scott Gas). Concentration response was linear from 1.8 to 28 parts per billion (ppb; correlation coefficient = 0.969). The response time of the instrument to 90% full strength was 0.4 s. The NO signal in ppb was recorded at 20 Hz for later analysis.
At the time of measurement, a set of three gas samples was obtained for each animal: 1) flowing zero gas to verify baseline, 2) proximal ventilator circuit to ascertain ambient inhaled NO, and 3) endotracheal gas from the sampling lumen of the endotracheal tube to measure exhaled NO. Each sampling set was evaluated for baseline drift in the zero gas signal and for elevation of ambient inhaled NO in the proximal ventilator signal. NO peaks recorded from the endotracheal sampling lumen in excess of the ambient inhaled NO level measured in the proximal ventilator circuit were considered to be from exhaled gas. Peak NO levels were obtained by inspection of the peaks from the recorded waveform. FENO (nl/l) was obtained by averaging the recorded signal over time.Pulmonary function testing. Pulmonary function testing was performed at 1 h of life, immediately after intubation and stabilization, using the VT1000 body plethysmograph (Vitaltrends Technology, New York, NY). This system is a flow-through whole body plethysmograph for the continuous measurement of gas exchange and ventilation in infants during assisted ventilation (13, 27). The system uses a differential piezoresistive pressure transducer interfaced with a single-screen pneumotachometer to detect airflow in and out of the sealed plethysmograph. Designed specifically for neonatal use, it has a tidal volume range from 1.0 to 50.0 ml (resolution 0.1 ml), frequency response to 5 Hz, and flow range of ±175 ml/s. The system interfaces with two dedicated microcomputers capable of pattern recognition, data storage, data analysis, and real-time presentation of flow-volume and pressure-volume curves. Resistance and compliance measurements were of the respiratory system as a whole, and compliance was corrected for body weight.
Statistical analysis. Analysis of variance with Newman-Keuls post hoc testing was used to compare mean values between more than two groups. Single comparisons between two groups were performed with nonpaired Student's t-tests. 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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Immunohistochemistry for NOS isoforms.
Immunohistochemistry was performed in lungs obtained at 175 d
gestation to delineate the cell specificity of NOS isoform expression (Fig. 1). Robust signal for nNOS protein
was detected in the bronchial and bronchiolar epithelium (Fig.
1A). There was also faint detection of nNOS in airway smooth
muscle. In addition, nNOS protein was readily detectable in the
alveolar wall (Fig. 1C). Negative control studies for the
proximal airway and alveolar walls are shown in Fig. 1, B
and D. Signal for eNOS was seen in bronchial and bronchiolar epithelium as well as in vascular endothelium (Fig. 1E).
iNOS protein was also detected in the airway epithelium (Fig.
1F).
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NOS enzymatic activity.
Maturational changes in NOS enzymatic activity in the proximal lung are
shown in Fig. 2. There was a doubling of
total NOS activity between 125 and 140 d gestation, and the levels
of activity were similar at 140, 160, and 175 d gestation (Fig.
2A). Calcium-dependent NOS enzymatic activity, indicative of
nNOS or eNOS activity, also doubled between 125 and 140 d
gestation. Calcium-dependent activity was similar at 140 and 160 d
gestation, and it then declined at 175 d gestation to levels
comparable with 125 d gestation (Fig. 2B). The findings
for calcium-independent NOS activity are provided in Fig.
2C. Calcium-independent activity was detectable at all ages
tested. However, the contribution of calcium-independent activity to
total activity was negligible until it increased over fourfold between
160 and 175 d gestation to achieve levels that were one-third of
total enzymatic activity.
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NOS isoform expression.
Whereas changes in NOS enzymatic activity in the presence of excess
amounts of substrate, calcium, and cofactors may reflect differences in
the levels of NOS expression, specific alterations in levels of isoform
abundance are best revealed by immunoblot analysis. Developmental
changes in nNOS protein expression are provided in Fig.
3. In the representative immunoblot shown
(Fig. 3A), nNOS protein was detectable at 125 d
gestation, it increased markedly and progressively in abundance from
125 to 140 d and to 160 d gestation, and abundance fell from
160 to 175 d gestation. These observations were confirmed in four
independent experiments that revealed a 2.5-fold increase in nNOS
protein levels from 125 to 140 d gestation, a further 70%
increase from 140 to 160 d gestation, and then a 50% decline from
160 to 175 d gestation.
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Exhaled NO levels.
To determine the impact of changes in NOS expression on the capacity
for epithelial NO production during fetal lung maturation, we measured
the FENO within 1 h of delivery by hysterotomy at either 125 or
140 d gestation. These ages were studied because they represent
the time points at which the most dramatic changes in NOS activity were
evident. In parallel with the maturational changes observed in NOS
activity and abundance, FENO at 140 d gestation was twice that
found at 125 d gestation (Fig. 6).
The estimated minute ventilation of NO (ENO) can be
calculated from the tidal volume measurements in comparable animals
(32), which were 5 ± 0.3 and 7.1 ± 0.3 ml/kg,
respectively, in 125- and 140-d-gestation groups (P < 0.05). Using this approach, we found
ENO estimates to be 0.35 and 0.94 nl · kg
1 · min
1
in 125- and 140-d-gestation animals, respectively.
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Pulmonary function tests.
To determine the potential impact of developmental changes in NOS
expression and epithelial NO production on early lung function, we
compared expiratory resistance and pulmonary compliance shortly after
stabilizing animals after birth at either 125 or 140 d. Expiratory
resistance was decreased by 38% in 140-d animals compared with 125-d
animals (Fig. 7A). Pulmonary
compliance was increased by 80% in the older animals vs. the younger
animals (Fig. 7B).
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DISCUSSION |
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Investigations in both animal models and humans indicate that NO is critically involved in pulmonary function in the perinatal period (28). In an effort to ultimately better understand the regulation of pulmonary NO production during human fetal development, the present study evaluated the expression and function of the three NOS isoforms in fetal baboon lung during the third trimester. We observed that there are marked increases in pulmonary NOS expression and activity at the onset of the third trimester in the baboon, thereby enhancing the capacity for lung NO production as term approaches.
Immunohistochemistry was performed to reveal the cellular distribution of the three NOS isoforms in fetal baboon lung. nNOS protein was primarily expressed in the proximal airway epithelium, but it was also found more distally in the respiratory tree down to the level of the alveolar wall. eNOS protein was also quite abundant in proximal airway epithelium, as well as being found predictably in pulmonary endothelial cells. iNOS was additionally abundant in the proximal airway epithelium. These observations in fetal baboon lung are essentially identical to our previous findings in fetal, newborn, and adult sheep lung, in which we also demonstrated that the cellular distributions of the NOS isoforms are comparable at the different ages studied and confirmed in these findings by RT-PCR (31). In addition, the present findings for nNOS in proximal airway epithelium are consistent with previous observations in the adult rat (16, 26, 35). Those for eNOS are similar to prior results in the adult human and adult and newborn rat lung (11, 36), and the abundant iNOS expression in the fetal baboon airway epithelium agrees with previous findings in adult and fetal rat trachea and fetal and adult human lung (16, 33, 34, 36). As such, there are consistencies between certain current observations for airway NOS distribution in the fetal baboon and findings in other species. However, our observations for nNOS and eNOS provide the first information regarding the cellular distribution of these isoforms in the lungs of fetal primates.
In addition to revealing the cellular distribution of pulmonary NOS in the primate, we also determined maturational changes in the activity and expression of the enzyme in the proximal lung. We found that there is a marked increase in nNOS and eNOS protein expression and related enzymatic activity from 125 to 140 d gestation. Total NOS enzymatic activity is then maintained at this elevated level from 140 to 175 d gestation. However, during the latter stage of this developmental period (160-175 d gestation), there is a dramatic fall in nNOS and eNOS expression and activity that is replaced by a rise in iNOS expression and activity. As such, there is dynamic regulation of NOS isoform expression in the fetal primate lung that results in the initiation and then maintenance of an elevated capacity for NO production during the third trimester, and the latter process entails a NOS isoform switch as term approaches. We postulate that the observed upregulation in the capacity for pulmonary NO production occurring in the early third trimester plays an important role in successful respiratory function at term. We additionally postulate that the interruption in this developmental process that occurs with premature birth contributes to the respiratory abnormalities that are often observed in preterm infants.
To determine whether the maturational alterations observed in pulmonary
NOS expression and activity lead to changes in pulmonary NO production,
exhaled NO levels were evaluated shortly after stabilization in baboons
delivered by hysterotomy at 125 or 140 d gestation. These ages
were chosen for study because they border the developmental stage over
which the major increase occurs in lung NOS expression and activity. We
observed a doubling in FENO and an almost threefold increase in
estimated ENO from 125 to 140 d gestation.
Because exhaled NO is primarily derived from proximal epithelial
sources (7, 12), these findings indicate that epithelial
NO production normally increases with fetal development during the
early third trimester in parallel with the rise in nNOS and eNOS
expression in the proximal lung.
The functional roles of epithelium-derived NO in the developing lung can be categorized into those impacting on airway function, those mediating distal respiratory function, and those impacting on the pulmonary circulation. Within the developing airway, there is evidence that NO mediates bronchomotor tone. In studies of isolated tracheae from newborn piglets, the nonspecific NOS antagonist L-NAME caused an increase in the contractile response of the airway to acetylcholine, and similar observations were obtained with removal of the epithelium. The latter cell type is the most likely major source of endogenous NO because L-NAME did not alter the contractile response of epithelium-denuded airways (14). Similarly, in studies of ventilated, open-chest newborn piglets, L-NAME caused an increase in airway resistance (22). The current investigation adds a new dimension to our understanding of these processes, because the differences in exhaled NO production observed shortly after birth in the 125- and 140-d-gestation baboons were associated with complementary differences in expiratory resistance, with greater NO production and lower resistance in the older animals. Although the findings are correlative in nature and possibly influenced by other maturational changes such as increasing airway size, they provide potential evidence of a role for epithelium-derived NO in airway function in the developing primate and suggest that the processes are developmentally regulated to enhance bronchodilation during maturation in the third trimester. Detailed studies of the effects of NOS antagonism are now warranted to assess the relative contribution of NO to maturational changes in early airway function.
Epithelium-derived NO may also affect distal lung function. There is evidence of a role for NO in the regulation of lung liquid production around the time of birth. The infusion of the NO agonists acetylcholine or bradykinin causes a dramatic fall in lung liquid production in late-gestation fetal lambs (5), as does the instillation of NO or cGMP directly into the fetal lung liquid (4, 6). Lung liquid production represents a balance between chloride secretion and sodium reabsorption by the alveolar epithelium (15). The effect of NO to decrease lung liquid production may be through changes in chloride flux because NO alters chloride transport in epithelial cells (8, 21). In addition, NO may be involved in the regulation of lung compliance, since L-NAME administration causes a decline in tissue resistance in newborn piglet lungs, suggesting a physiological role for endogenous NO in the regulation of peripheral contractile elements (22). Although correlative in nature and potentially explained by other processes, the present findings of greater respiratory compliance at 1 h of life in association with greater epithelial NO production at 140 d compared with 125 d gestation may be related to maturational changes in NO-mediated mechanisms in the distal lung. The observed differences in compliance are potentially not related to disparities in surfactant deficiency, because the 125-d-gestation group received surfactant replacement whereas the 140-d-gestation group did not. Now that we know NO production is markedly altered during the early third trimester in the baboon and that it is associated with differences in compliance despite surfactant administration, further well-controlled experiments with NOS antagonism are indicated in both age groups to test this possibility.
The present findings may also provide insights into the cellular sources of NO mediating pulmonary vasomotor tone in the perinatal period. Experiments with nonselective and isoform-selective NOS antagonism in sheep have shown that both nNOS and iNOS contribute to the regulation of pulmonary blood flow during late fetal and early postnatal life (23-25). In the current investigation we observed that the vast majority of both nNOS and iNOS protein is found within the airway epithelium of the baboon. Thus, isoforms of NOS other than eNOS expressed in the respiratory epithelium may be an important source of NO mediating vascular processes in the developing lung of the primate.
In summary, the present studies demonstrate marked increases in NOS enzymatic activity and NOS isoform expression in the proximal lung during the third trimester of fetal development in the baboon. Immunohistochemistry further reveals that all three NOS isoforms are primarily expressed in the proximal airway epithelium of the primate. The most dramatic maturational increase in enzyme abundance and activity occurs during the early third trimester, and it is associated with an elevation in airway NO production, a decline in expiratory resistance, and greater pulmonary compliance during very early postnatal life. These cumulative observations indicate that there is a developmental increase in pulmonary NOS expression and NO production during the early third trimester in the primate that may enhance airway and parenchymal function in the immediate postnatal period.
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ACKNOWLEDGEMENTS |
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The authors thank all the personnel who support the BPD Resource Center: the animal husbandry group led by Drs. D. Carey and M. Leland, the NICU staff (H. Martin, D. Correll, W. Cox, L. Kalisky, L. Nicley, R. Degan, S. Salazar), the Wilford Hall Medical Center neonatal fellows who assist in the care of the animals, and the University of Texas Health Science Center, San Antonio pathology staff (V. Winter, L. Buchanan, H. Dixon, 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 Institutes of Health 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.
July 3, 2002;10.1152/ajplung.00112.2002
Received 15 April 2002; accepted in final form 28 June 2002.
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