1 Department of Pediatrics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226; 2 University of New Mexico School of Medicine, Albuquerque, New Mexico 87131; and 3 Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Altered nitric oxide (NO)
production could contribute to the pathogenesis of
hypoxia-induced pulmonary hypertension. To determine whether
parameters of lung NO are altered at an early stage of hypoxia-induced
pulmonary hypertension, newborn piglets were exposed to room air
(control, n = 21) or 10% O2 (hypoxia,
n = 19) for 3-4 days. Some lungs were isolated and
perfused for measurement of exhaled NO output and the perfusate
accumulation of nitrite and nitrate (NOx), the stable metabolites of
NO. Pulmonary arteries (20-600-µm diameter) and their
accompanying airways were dissected from other lungs and incubated for
NOx
determination. Abundances of the nitric oxide synthase (NOS)
isoforms endothelial NOS and neural NOS were assessed in homogenates of
PAs and airways. The perfusate NOx
accumulation was similar, whereas
exhaled NO output was lower for isolated lungs of hypoxic, compared
with control, piglets. The incubation solution NOx
did not differ
between pulmonary arteries (PAs) of the two groups but was lower for
airways of hypoxic, compared with control, piglets. Abundances of both
eNOS and nNOS proteins were similar for PA homogenates from the two groups of piglets but were increased in airway homogenates of hypoxic
compared with controls. The NO pathway is altered in airways, but not
in PAs, at an early stage of hypoxia-induced pulmonary hypertension in
newborn piglets.
nitric oxide synthase isoforms; pulmonary vascular nitric oxide; airway nitric oxide; chronic hypoxia
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
PULMONARY HYPERTENSION DEVELOPS when newborn piglets are exposed to chronic hypoxia (2, 10, 17, 18, 34). Our laboratory has provided evidence that the evolution of pulmonary hypertension in chronically hypoxic newborn piglets consists of at least two biologically distinct stages (10). The earlier stage, which occurs over the first 3-5 days of hypoxic exposure (referred to, in this paper, as shorter hypoxia), is characterized by increased pulmonary vascular tone (10). The later stage, which is manifest after 10-12 days of hypoxia (referred to, in this paper, as longer hypoxia), is characterized by more pronounced pulmonary vascular wall thickening (10), decreased nitric oxide (NO) production (13), and by reduced abundance of endothelial nitric oxide synthase (eNOS) protein in distal lung homogenates (13).
NO is an endogenously produced pulmonary vasodilator, made by several types of pulmonary cells, including vascular endothelium and airway epithelial cells (29, 32, 37). NO production can be detected both in the perfusate of isolated lungs and in exhaled air (21, 26). Previous studies provide evidence that NO production is altered in newborn animals, with pulmonary hypertension characterized by an extensively remodeled pulmonary circulation (3, 13, 31, 35). Little information is available regarding NO production at early stages of neonatal pulmonary hypertension (2, 11).
The purpose of this study was to determine whether lung NO production
is decreased in piglets exposed to shorter hypoxia, i.e., an earlier
stage of pulmonary hypertension, as it is with longer hypoxia, i.e., a
later stage of hypoxia-induced pulmonary hypertension
(13). Because we previously found that pulmonary vascular
responses to a NO inhibitor were not altered in piglets exposed to
shorter hypoxia (11), we hypothesized that NO production would be preserved in this earlier stage of pulmonary hypertension. To
test this hypothesis, we assessed NO production in control piglets and
in piglets exposed to shorter hypoxia by measuring exhaled NO output in
living piglets and by measuring exhaled NO output and the perfusate
accumulation of nitrite and nitrate (NOx), the stable metabolites of
NO, in isolated lungs. In addition, we incubated both pulmonary
arteries, 20-600-µm diameter, and their associated airways for
NOx
determination. Furthermore, we assessed the localization and
abundances of eNOS and neural nitric oxide synthase (nNOS) protein in
lungs from both groups of piglets.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals. A total of 19 hypoxic piglets and a total of 21 control piglets were studied. For the hypoxic piglets, newborn pigs (2-3 days old) were placed in a normobaric hypoxic chamber for 3-4 days. Normobaric hypoxia was produced by delivering compressed air and N2 to an incubator (Thermocare). Oxygen content was regulated at 8-10% O2 (PO2 60-72 Torr), and CO2 was maintained at 3-6 Torr by absorption with soda lime. The chamber was opened two times/day for cleaning and to weigh the piglets. The animals were fed ad libitum with an artificial sow milk replacer from a feeding device attached to the chamber. Some (n = 6) of the control pigs were raised by placing newborn pigs (2-3 days old) in a room air environment for 3-4 days and maintaining them as described for the hypoxic piglets. Some (n = 15) of the control piglets were studied on the day of arrival from the farm at 5-7 days of age.
Measurements in anesthetized animals. On the day of study, some of the control (n = 10) and some of the hypoxic (n = 9) animals were weighed and preanesthetized with ketamine (30 mg/kg im) and then anesthetized with pentobarbital sodium (10 mg/kg iv) for determination of in vivo exhaled NO output. During the placement of catheters, additional intravenous pentobarbital sodium was given as needed via an ear vein to maintain anesthesia. For each piglet, the trachea was cannulated so that the animal could be ventilated for measurement of exhaled NO output, and a catheter was placed into the right femoral artery for monitoring systemic blood pressure and arterial blood gases. The piglet was then given additional anesthesia, its tracheal cannula was attached to a piston-type ventilator, and its lungs were ventilated with a normoxic NO free gas mixture (21% oxygen and balance nitrogen; Matheson, Chicago, IL) at a tidal volume of 15-20 ml/kg, end-expiratory pressure of 2 mmHg, and a respiratory rate of 15-20 breaths/min for measurement of exhaled NO output as described in Exhaled NO output measurement.
Next, while continuing mechanical ventilation, in some of the control (n = 8) and some of the hypoxic (n = 8) animals, we placed additional catheters for determination of in vivo pulmonary vascular resistance. One catheter was placed through the right external jugular vein into the pulmonary artery to monitor pulmonary arterial pressure. To obtain the pulmonary wedge pressure, the pulmonary arterial catheter was advanced into a distal pulmonary vessel. The zero reference for the vascular pressures was the midthorax. To measure cardiac output by the thermodilution technique (model 9520 thermodilution cardiac output computer, Edwards Laboratory, Irvine, CA), a thermistor was placed into the aortic arch via the left femoral artery, and a catheter that served as an injection port was placed into the left ventricle via the left carotid artery. Cardiac output was measured at end expiration as the mean of three injections of 3 ml of 0.9% saline (0°C). After measuring exhaled NO output, hematocrit, blood gases, as well as pulmonary arterial pressure and pulmonary wedge pressure, we gave the animals additional anesthesia (3-5 ml/kg iv of pentobarbital sodium) and exsanguinated them for lung isolation and perfusion as described below.Lung isolation and perfusion.
In addition to the 10 hypoxic piglets and 9 control piglets in which in
vivo measurements in anesthetized animals were obtained (see
Measurements in anesthetized animals), one more hypoxic and four more control piglets were preanesthetized with ketamine (30 mg/kg
im) and then anesthetized with pentobarbital sodium (10 mg/kg iv) for
lung isolation and perfusion. All animals were given heparin (1,000 IU/kg iv) and were then exsanguinated. For lung isolation and
perfusion, the tracheal cannula of each piglet was attached to a large
animal piston-type ventilator, and the lungs were ventilated with a
normoxic gas mixture (17% O2, 6% CO2, and balance N2) using a tidal volume of 15-20 ml/kg,
end-expiratory pressure of 2 mmHg, and a respiratory rate of 15-20
breaths/min (peak airway pressure of 9-12 mmHg). A midline
sternotomy was performed, and a clamp was placed across the ductus
arteriosus. Saline-filled cannulas were placed into the pulmonary
artery and left atrium through incisions in the right and left
ventricles. The diaphragm and all abdominal contents were removed. For
all lungs, the vascular cannulas were connected to a perfusion circuit that was filled with a Krebs-Ringer bicarbonate (KRB) solution containing 5% dextran (mol wt 70,000 at 37°C). Briefly, in
the perfusion circuit, a rotary pump continuously circulated the
perfusate from a reservoir through a bubble trap into the pulmonary
arterial cannula, through the lungs to the left atrial cannula, and
back to the reservoir. Pulmonary arterial, left atrial, and airway pressures were continuously monitored. The most dependent edge of the
lung was used as the zero reference for vascular pressures. The height
of the reservoir was adjusted to maintain left atrial pressure at 0 Torr. After being connected to the perfusion circuit, the lungs were
perfused for 0.5-1 h to establish stability of the pulmonary
arterial pressure. The perfusion flow rate was adjusted to 50 ml · min1 · kg
1
and maintained constant throughout the study. The perfusion flow rate
was chosen to minimize edema formation (9). Perfusate samples were collected at 15-min intervals for 90 min for measurement of NOx
using a spectrophotometric technique as described in
Perfusate accumulation of NOx
in isolated lungs.
Throughout the perfusate sampling period, exhaled NO was continuously
monitored for determination of exhaled NO output as described in
Exhaled NO output measurement. Of note, although we
attempted to study perfused lungs from 11 hypoxic piglets and 13 control piglets, we successfully completed isolated, perfused studies
in lungs from 8 of the hypoxic piglets and 9 of the control piglets. At
the end of the 90-min perfusate sampling period, a lobe from some of
the perfused lungs (n = 5 hypoxic and n = 5 control) was immediately frozen in liquid nitrogen and stored at
80°C for immunoblot analysis.
Exhaled NO output measurement. For exhaled NO measurement in the anesthetized animals, expiratory gas from 10 control piglets and 9 hypoxic piglets was sampled two to three times for 5-min periods each, from the exhalation limb of the ventilator, and was passed through a chemiluminescence analyzer (model 270B NOA; Sievers, Boulder, CO) to measure NO concentration, as previously described (13). For exhaled NO measurement in the isolated lungs (n = 9 control, n = 8 hypoxic), throughout perfusion, the expiratory gas was sampled from the tracheal tube and passed through the chemiluminescence analyzer to measure NO concentration. The NO analyzer was calibrated daily with authentic NO mixed with N2 using precision flowmeters (1 part/million in N2; Matheson). The NO detection limit was 0.5 part/billion. For both anesthetized animals and perfused lungs, the exhaled NO output was calculated from the measured NO concentration, the ventilator rate, and the tidal volume.
Perfusate accumulation of NOx in isolated
lungs.
A spectrophotometric analysis, described previously (12,
13), was used to determine perfusate NOx
concentration
(nmol/ml) at each collection time. Fifty microliters of a stock NADPH
solution (0.8 mg of NADPH/ml of phosphate buffer) and 10 µl of a
stock nitrate reductase solution (5 units of nitrate reductase/ml of phosphate buffer) were added to 500 µl of lung perfusate. After being
incubated for 3 h at room temperature, Greiss reagent [300 µl;
1% sulfanilamide, 0.1% N-(1-naphthyl)ethylenediamine
dihydrochloride, and 2.5% phosphoric acid] was added to the lung
perfusate mixtures and incubated for 10 min at room temperature, and
the absorbance was measured at 546 nm. A standard curve was prepared by
adding known amounts of NaNO3 to fresh perfusate. Fresh
perfusate with added NADPH, nitrate reductase, and the Greiss reagent
as described for the lung perfusate samples was used as a blank.
Duplicate assays were carried out for each sample of lung perfusate.
Pulmonary artery and airway tissue preparation.
Control (n = 5) and hypoxic (n = 5)
piglets were preanesthetized with ketamine (30 mg/kg im), anesthetized
with pentobarbital sodium (10 mg/kg iv), given heparin (1,000 IU/kg
iv), and then exsanguinated. Next, the lungs of the piglets were
excised, and pulmonary arteries (20-600-µm diameter) and
their associated airways were dissected (Fig.
1). Some pulmonary arteries and airways
were frozen in liquid nitrogen and stored at 80°C for immunoblot
analysis. Other pulmonary arteries and airways were used for NOx
determination.
|
Pulmonary artery and airway NOx.
Pulmonary arteries and airways from control (n = 5) and
hypoxic (n = 5) piglets were stored overnight at 4°C
and then incubated in 1 ml of Hanks'-HEPES solution at 37°C, pH 7.4, for 1 h. A 1-ml sample of Hanks'-HEPES was also incubated to be
used as a blank for the subsequent NOx
determinations. At the end of
the incubation period, the blank and incubation solutions were frozen
at
80°C for later NOx
determination, and the pulmonary arteries
and airways were dried and weighed. A chemiluminescence analysis was
used for NOx
determination. One hundred microliters of blank or
pulmonary artery or airway incubation solution were injected
anaerobically into the reaction chamber of a chemiluminescence NO
analyzer (Sievers). The reaction chamber contained vanadium(III)
chloride in 1 M HCl heated to 90°C to reduce nitrite and nitrate to
NO gas. The NO gas was carried into the analyzer using a constant flow
of N2 gas via a gas bubble trap containing 1 M NaOH to
remove HCl vapor. A standard curve was generated by adding known
amounts of NaNO3 to the Hanks'-HEPES solution and assaying
as described for the artery or airway incubation samples. The amount of
NOx
was the amount of NOx
in the artery or airway incubation
solution minus the amount of NOx
in the blank divided by the dry
weight of the arteries or airways.
Immunoblot analysis.
We performed preliminary studies with different amounts of total
protein to determine the dynamic range of the immunoblot analysis for
each protein and tissue homogenate. With the exception of inducible
nitric oxide synthase (iNOS), which we were unable to detect
(regardless of the total amount of protein used, see RESULTS), an amount of protein within the dynamic range of
the immunoblot analysis was then used to compare protein abundance between homogenates from control and short hypoxic piglets as described
below. For example, Fig. 2 shows an
immunoblot for eNOS using distal lung homogenate samples containing
total protein amounts of 5, 10, 15, 25, 30, and 40 µg. On the basis
of these findings, to compare eNOS abundance in distal lung homogenates between control and short hypoxic piglets, we followed methods as
described below using 15 µg of total protein samples. Similar methods
were followed to determine the dynamic range for nNOS, and, based on
the findings, we used distal lung homogenate samples containing total
protein amounts of 30 µg.
|
Immunoblot analysis for distal lung homogenate samples. Tissue pieces that did not contain large airways or large vessels were selected from frozen perfused lungs of control (n = 5) and short hypoxic (n = 5) piglets and homogenized in 10 mM HEPES buffer containing 250 mM sucrose, 3 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride, pH 7.4, on ice using three 15-s pulses of a Polytron blender, taking care to avoid foaming of the homogenate. Protein concentration of the distal lung homogenate was determined by the Bradford protein assay. Each lung homogenate was diluted with phosphate-buffered saline (PBS) to obtain a protein concentration of 1 mg/ml. Forty microliters of each protein concentration were solubilized in 40 µl of denaturing, reducing sample buffer [Novex; 0.25 M Tris · HCl, 5% (wt/vol) SDS, 2.5% (vol/vol) 2-mercaptoethanol, 10% glycerol, 0.05% bromphenol blue, pH 6.8], heated to 80°C for 15 min, and centrifuged for 3 min at 5,600 g in a microfuge. Equal volumes of these supernatants were then applied to Tris-glycine precast 8% polyacrylamide gels (Novex) so that equal amounts of protein were loaded. We used 15-µg protein samples for eNOS. Electrophoresis was carried out in 25 mM Tris, 192 mM glycine, and 0.1% SDS (pH 8.3) at 125 V for 1.7 h. The proteins were transferred from the gel to a nitrocellulose membrane (Novex) at 100 V for 1 h in 25 mM Tris, 192 mM glycine, and 20% methanol (pH 8.3). The membrane was incubated overnight at 4°C in PBS containing 10% nonfat dried milk and 0.1% Tween 20 to block nonspecific protein binding. To detect eNOS, the nitrocellulose membrane was incubated for 1 h at room temperature with the primary antibody (Transduction Laboratories) diluted 1:500 in PBS containing 0.1% Tween 20 and 1% nonfat dried milk (carrier buffer), followed by incubation for 30 min at room temperature with a biotinylated secondary antibody (Vector Laboratories) diluted 1:5,000 in the carrier buffer, followed by incubation for 30 min at room temperature with streptavidin-horseradish peroxidase conjugate (Amersham) diluted 1:1,500 in PBS containing 0.1% Tween 20. The nitrocellulose membrane was washed three times between the first two incubations with the carrier buffer and three times with the carrier buffer plus one time with milk-free PBS containing 0.1% Tween 20 after the final incubation. To visualize the biotinylated antibody, the membranes were developed using enhanced chemiluminescence reagents (ECL, Amersham), and the chemiluminescent signal was captured on X-ray film (ECL Hyperfilm, Kodak). The bands for eNOS were quantified using densitometry.
Similar procedures were followed using primary antibodies for nNOS (Transduction Laboratories) and iNOS (Transduction Laboratories and Calbiochem).Immunoblot analysis for pulmonary artery and airway homogenate samples. For both pulmonary artery and airway homogenate samples, procedures similar to those described above for whole lung homogenate samples were applied to frozen samples of 20-600-µm-diameter arteries or airways.
Immunohistochemical localization of eNOS and nNOS proteins. Control (n = 3) and hypoxic (n = 3) piglets were preanesthetized with ketamine (10 mg/kg im), anesthetized with pentobarbital sodium (15 mg/kg iv), given heparin (1,000 IU/kg iv), and then exsanguinated. Cannulas were placed into the trachea, pulmonary artery, and left atrium of each piglet. After being perfused with normal saline to remove all blood from the pulmonary circulation, the lungs were perfused for 5 min with 10% neutral buffered formalin (4°C). Next, the lungs were fixed by instillation of the formalin into the airway, pulmonary artery, and left atrium. After 24 h of fixation, pieces of lung were embedded in paraffin and sectioned. The tissue sections were deparaffinized, rehydrated, and then microwaved in citrate buffer (Bio-Genex, San Ramon, CA) to improve antigen detection (32). Next, the tissue sections were treated with blocking serum and incubated with one of the same primary antibodies used for immunoblots, either eNOS at a dilution of 1:100, nNOS at a dilution of 1:500, or iNOS at a dilution of 1:100. Methanol-H2O2 was used to block endogenous peroxidase activity, and a standard peroxidase method was used for antigen detection (Elite ABC kit; Vector Laboratories, Burlingame, CA). The tissue sections were counterstained with hematoxylin. Immunohistochemical staining controls included omission of the primary antibody and omission of the secondary antibody.
Calculations and statistics.
Data are presented as means ± SE. Unpaired t-tests
were used to compare the data between control and hypoxic animals. For the NOx perfusate analysis, the rate of NOx
accumulation (nmol/min) for each lung was determined by linear regression of the accumulated NOx
(nmol) vs. time (minute) data for each individual lung. The means
of the individual rates of NOx
accumulation (nmol/min) were then
calculated and compared between control and hypoxic groups by unpaired
t-test. A value of P < 0.05 was used as
indicative of statistical significance.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
After 3-4 days of hypoxia, there was no significant
difference in weight in control (2,409 ± 127 g, n = 21) compared with hypoxic (2,298 ± 116 g, n = 19) piglets. For anesthetized piglets, the measured values of pH,
PO2, and PCO2 obtained
with the animals breathing room air did not differ significantly
between control and hypoxic piglets (Table
1). For those anesthetized
piglets in which the appropriate catheters were placed,
measurements of pulmonary arterial pressure, pulmonary wedge
pressure, cardiac output, and calculated pulmonary vascular resistance
[(pulmonary arterial pressure wedge pressure)
cardiac output] are shown in Table 1. Pulmonary arterial
pressures and pulmonary vascular resistances were significantly greater
in the piglets raised in short hypoxia than in the control piglets,
with pulmonary vascular resistance being doubled in hypoxic compared
with control animals (Table 1). Figure
3A shows that the exhaled NO
output in the anesthetized animals breathing a normoxic NO free gas
mixture was significantly reduced in piglets raised in hypoxia compared with control piglets.
|
|
For the isolated lungs, values of pH, PO2, and
PCO2 measured in the perfusate did not differ
between control and hypoxic piglets (Table
2). Pulmonary arterial pressures were
significantly greater in the perfused lungs of hypoxic than in the
perfused lungs of control piglets (Table 2). Perfusion flow rates did
not differ between the two groups (Table 2), and left atrial pressures
were maintained constant at 0 mmHg throughout perfusion for both
groups. Therefore, the higher pulmonary arterial pressure in hypoxic
lungs (Table 2) reflects a greater pulmonary vascular resistance in the
piglets raised in hypoxia compared with control lungs. As with in vivo
measurements (Fig. 3A), exhaled NO output in isolated perfused lungs of hypoxic piglets was significantly reduced compared with that of control piglets (Fig. 3B). Moreover, NOx in
the airway incubation solution from hypoxic piglets was reduced
compared with the value measured in control piglets (Fig.
4A). As summarized in Fig.
5, perfusate NOx
accumulation rates
did not differ significantly between the hypoxic and control piglets.
Likewise, NOx
in the pulmonary artery incubation solution did not
differ between hypoxic and control piglets (Fig. 4B).
|
|
|
We were unable to detect iNOS protein by immunoblot technique in
homogenates of any lung tissue (distal lung, pulmonary arteries or
airways) from either control or hypoxic piglets. For all tissues from
both control and hypoxic lungs, eNOS was detected at an apparent molecular mass of 135 kDa, as determined from linear regression of
molecular mass standards (immunoblot analysis for eNOS is shown for
distal lung homogenates in Fig. 6A,
left; for pulmonary artery homogenates in Fig. 6B, left; and for airway homogenates in
Fig. 6C, left). There was no difference in the absorbance of
the bands for the eNOS isoform for either distal lung homogenates (Fig. 6A, right) or pulmonary artery homogenates (Fig. 6B,
right) from control compared with short hypoxic piglets. In
contrast, the mean data for the absorbance of the eNOS bands for the
airway homogenates were greater (Fig. 6C, right) for piglets
raised in hypoxia compared with control piglets.
|
Figure 7, A-C, left,
shows that we detected nNOS in distal lung homogenates, pulmonary
artery homogenates, and airway homogenates of both control and hypoxic
piglets. The apparent molecular mass of nNOS in all tissue types was
150 kDa, as determined from linear regression of molecular mass
standards. Similar to findings for eNOS (Fig. 6, A-C,
right), there was no difference in the absorbance of the bands for
nNOS for either distal lung or pulmonary artery homogenates (Fig.
7A, right and 7B, right) from control compared with hypoxic piglets, whereas the mean absorbance of the bands for nNOS
was greater for airway homogenates (Fig. 7C, right) from hypoxic compared with control piglets.
|
As with the immunoblot technique, we were unable to detect iNOS protein
by immunohistochemical technique applied to lungs of either control or
hypoxic piglets. Immunohistochemical localization of eNOS protein in
representative lungs of hypoxic and control piglets is shown in Fig.
8. For both control (Fig. 8, A
and B) and hypoxic (Fig. 8, C and D)
lungs, staining for eNOS was readily observed in the endothelium of all
levels of the pulmonary vasculature, from hilar vessels to capillaries,
and was also detected in the endothelium of bronchial vessels. In
addition, for both control and hypoxic lungs, staining for eNOS was
observed in the epithelium of some, but not all, airways along the
entire respiratory tree, from hilar to alveolar levels. By comparison
with the endothelium, the intensity of staining for eNOS in the
epithelium was faint.
|
Figure 9 shows the immunohistochemical
localization of nNOS protein for representative lungs of both control
and hypoxic piglets. For lungs of both groups, unlike eNOS (Fig. 8),
nNOS staining was seen in airway and vascular smooth muscle (Fig. 9).
Similar to eNOS (Fig. 8), nNOS staining was seen in airway epithelial cells associated with all levels of airways, from hilum to alveoli for
both control (Fig. 9, A and B) and hypoxic (Fig.
9, C and D) lungs. However, by comparison with
eNOS (Fig. 8), the intensity of staining for nNOS in epithelial cells
of both control and hypoxic lungs was distinct and readily found.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In agreement with our previous studies on neonatal piglets exposed
to long hypoxia (10, 11), in this study we found that pulmonary vascular resistance is elevated both in vivo and in isolated,
perfused lungs when newborn piglets are exposed to short hypoxia. Thus
exposure to either 3-4 days or 10-12 days of hypoxia results
in increased pulmonary vascular resistance in the newborn piglet. We
hypothesized that the mechanisms underlying the increase in pulmonary
vascular resistance might differ between the short and long periods of
hypoxia. Our hypothesis was based, in part, on our previous findings
that pulmonary vascular responses to a NO inhibitor were unaltered in
piglets exposed to short hypoxia but were blunted in piglets exposed to
longer hypoxia (11). However, inconsistent with our
hypothesis, in this study we found that, similar to our previous
studies in neonatal piglets exposed to long hypoxia (13),
exhaled NO production was decreased both in vivo and in the isolated
lung in piglets exposed to short hypoxia. Together, these data suggest
that the increased pulmonary vascular resistance in piglets exposed to
both durations of hypoxia is at least in part caused by decreased NO
production. Yet, unlike our previous studies in neonatal piglets
exposed to long hypoxia (13), in neonatal piglets exposed
to short hypoxia, perfusate NOx accumulation and eNOS and nNOS
protein levels were unaltered. Thus it appears that both short and long
exposures to hypoxia result in decreased exhaled NO, which in turn may
be due to impaired NOS activity in the airways. In the case of piglets
exposed to short hypoxia, the decrease in NOS activity in airways could
contribute to an increase in pulmonary vascular resistance despite
maintained vascular and airway protein levels of eNOS and nNOS. By
comparison, for piglets exposed to longer hypoxia, both altered NOS
activity in airways and impairments in the NO pathway of the
vasculature (the latter reflected by the diminished perfusate
NOx
accumulation and reduction in distal lung eNOS amounts)
(13) might contribute to the increase in pulmonary
vascular resistance.
Limitations of our experimental methods need to be considered. Changes in perfused vascular surface area might lead to changes in exhaled NO production. For example, if perfused vascular surface area was significantly decreased by short hypoxic exposure, then this alone could account for a decrease in exhaled NO. The perfused vascular surface area would be influenced by the vascular pressures. Notably, in intact piglets exposed to short hypoxia, the pulmonary arterial and wedge pressure increased with no change in cardiac output. The predicted effect from these hemodynamic changes would be to increase perfused vascular surface area and thereby increase, not decrease, NO production. It does merit comment that the potential to increase NO production from increased vascular volume in intact short hypoxic piglets might be counterbalanced by an increased uptake of NO by hemoglobin, an effect that would be predicted to lower exhaled NO output (1). As to the isolated lung, pulmonary arterial pressure was increased in lungs from short hypoxic piglets, whereas pulmonary venous pressure, airway pressure, and pulmonary blood flow were held constant between groups. As with intact piglets, the change in pulmonary arterial pressure would be expected to increase perfused vascular surface area in the isolated lungs of short hypoxic piglets, and thereby increase NO. Moreover, the potential influence from differences in hemoglobin-NO uptake was negated by use of blood-free perfusate for both groups of lungs. Furthermore, we have previously demonstrated in the isolated perfused lungs from piglets exposed to longer hypoxia that pulmonary vascular volume was not significantly different from controls (13). Thus changes in perfused vascular surface area were probably not responsible for reduced exhaled NO either in vivo or in the isolated lungs from hypoxic vs. control piglets.
Another factor that might impact the exhaled NO production is lung ventilation. However, both groups were ventilated with volume ventilators delivering similar tidal volumes and rates, thus the distribution of ventilation should have been similar. Exhaled NO concentration was converted to exhaled NO production by multiplying by the minute ventilation. Thus differences in ventilation alone are unlikely to account for the difference in exhaled NO production seen.
The decrease in exhaled NO production could be secondary to a decrease in NOS expression, a decrease in NOS activity, or an increase in NO scavenging by free radicals. Our immunoblot and immunohistochemistry data demonstrate either no difference or an increase in eNOS and nNOS expression in lung tissues of piglets exposed to short hypoxia compared with controls. Thus one interpretation of these data is that NOS activity was decreased in the face of maintained NOS expression in the lungs from neonatal piglets exposed to short hypoxia compared with controls. This interpretation is consistent with studies demonstrating that prolonged hypoxia results in a decreased NOS activity due to decreased availability of necessary substrates and cofactors to NOS (4). It is unlikely that a change in L-arginine availability to NOS was responsible for the decrease in NO production, since it has been previously shown that hypoxic exposure for neither 3 nor 14 days altered pulmonary arterial or plasma levels of L-arginine compared with controls (2). However, impaired coupling of eNOS and the chaperone protein heat shock protein 90 may also occur during hypoxia, resulting in decreased NO production from NOS (14, 33). Alternatively, a decrease in exhaled NO production could be consistent with increased scavenging of NO by oxygen free radicals during hypoxia. Evidence suggests that oxygen free radical production is increased in lung tissue during prolonged exposure to hypoxia (15). In addition, due to the cleaning of the animals and cages (needed for hygiene), the hypoxic piglets were exposed to periods of reoxygenation that could have contributed to free radical production (8). Our data cannot differentiate between decreased NOS activity and increased NO scavenging as the mechanism underlying decreased exhaled NO production in piglets exposed to short hypoxia.
Perfusate NOx accumulation was unaffected by short hypoxia exposure
in this study. One interpretation of this finding might be that the
perfusate NOx
measurement lacks the sensitivity to detect changes in
lung NO production in short hypoxic exposure. However, our finding
regarding perfusate NOX
accumulation is consistent with previous
studies evaluating NO parameters in vasculature from piglets exposed to
short hypoxia (2, 34). For example, the accumulation of
cGMP was not different in pulmonary arterial rings isolated from
piglets exposed to either normoxia or hypoxia for 3 days
(34). Furthermore, calcium-dependent NOS activity did not
differ in pulmonary arterial homogenates from piglets exposed to 3 days
of either normoxia or hypoxia (2). Thus it seems likely
that pulmonary vascular NO production was maintained after exposure to
short hypoxia.
Indeed, our finding that perfusate NO production appears to be
maintained is also consistent with our findings that NOx from the
pulmonary artery incubation solution was not different between controls
and short hypoxia-exposed piglets. Moreover, our additional finding
that the NOx
from the airway incubation solution was decreased in
short hypoxia compared with control-exposed piglets is consistent with
our finding of reduced exhaled NO output. Thus, in this model, the
exhaled NO may reflect airway NO production, and the perfusate NOx
may reflect vascular NO production.
The mechanisms underlying the disparate effects on airway and vascular NO production with short hypoxia in newborn piglets are not known. In fact, data regarding the influence of chronic hypoxia on the NO pathway in airways are scarce and have, up to this time, been limited to lungs of adults (6, 36). There are data from a recent study showing that 3 wk of mechanical ventilation impaired eNOS expression in the epithelium of small airways of preterm lambs (25). Our findings by immunohistochemistry would suggest that the cellular sources and NOS isoforms involved with NO impairment in airways of piglets exposed to short hypoxia include nNOS and eNOS in epithelial cells and nNOS in smooth muscle cells. The explanation for impairments in NOS activity in these airway cell types in the face of preserved NO in the vasculature will require further study but includes the possibility that the effect of hypoxia is cell type variable.
The finding that the piglet lungs exposed to short hypoxia had an increase in pulmonary vascular resistance and a decrease in exhaled NO suggests that airway NO may be involved in the control of pulmonary vasomotor tone. This interpretation is consistent with a study demonstrating that in isolated perfused rabbit lungs, acute alveolar hypoxia decreased exhaled NO and increased pulmonary vascular resistance (19). It is also consistent with a human study wherein exhaled NO production was found to be inversely correlated with pulmonary artery pressure (7). Furthermore, it has been demonstrated that endogenous NO production may be involved in the maintenance of ventilation-perfusion matching and normally low pulmonary vascular resistance (24, 30). Together, these studies support the concept that airway-produced NO is involved in the regulation of pulmonary vascular tone. Thus the increased pulmonary vascular resistance seen in the piglets exposed to short hypoxia may, in part, be due to the decrease in airway NO production found in these animals.
The finding of decreased NO production by the airways may also have implications in airway function in hypoxic exposure. For example, there is evidence that by opposing airway contraction, endogenous epithelium-derived NO plays an important role in regulating bronchomotor tone in newborns (22). Furthermore, studies in newborn piglets have shown that endogenously produced NO, although not a determinate of larger airway resistance, is an important determinate of lung tissue resistance (28). In studies in neonatal calves, exposure to 2 wk of hypobaric hypoxia increased airway resistance compared with controls (20). Thus alterations in airway NO production may also affect bronchomotor tone.
We have previously found that in neonatal piglets exposed to long hypoxia, distal lung eNOS protein expression decreases (13). Notably, in this current study, exposure to short hypoxia had no effect on distal lung eNOS protein expression. This suggests that the effect of hypoxia on eNOS expression in the distal lung of newborn piglets is time dependent. Furthermore, we found that vascular eNOS protein expression was maintained and that airway eNOS protein expression was increased with exposure to short hypoxia. Because other isoforms of NOS are found in the lung and may be important in the regulation of pulmonary vascular tone (16, 23, 27, 32, 37), we also examined nNOS and iNOS protein expression. We were unable to detect iNOS protein in any homogenates from either control or hypoxic animals, so we are unable to make conclusions regarding iNOS. Of importance, the expression of nNOS was similar to that of eNOS, unchanged in distal lung and vascular homogenates and increased in airway homogenates. Thus it may be that the increase in airway eNOS and nNOS protein expression seen with short hypoxic exposure is an attempt to overcome the decrease in airway NO production and thereby decrease pulmonary vascular resistance. Furthermore, this may be an early response with continued exposure to hypoxia, eventually leading to a decrease in eNOS protein expression in distal lung tissue of neonatal piglets (13).
It has been demonstrated that maturation affects the abundance and cellular expression of NOS expression in the lung (16, 23, 27, 32, 37). Consistent with our findings in neonatal pigs exposed to long hypoxia (12, 13), it has recently been described that in neonatal rats, exposure to hypobaric hypoxic for 14 days resulted in decreased lung eNOS expression and NO production (5). Interestingly, the investigators found that in adult rats, the same exposure resulted in increased lung eNOS expression and NO production (5). Although maturation may affect the eNOS response to chronic hypoxia, in this study, as well as our previous long hypoxia studies (10-13), the postnatal age of the pigs was similar when exposure was begun, and results were compared with an age-matched control group. Thus our studies with newborn piglets have controlled for maturational differences in the response of eNOS to hypoxia.
In summary, not all of the findings from this study are consistent with our initial hypothesis that lung NO production is preserved in newborns exposed to shorter periods of hypoxia. Instead, our findings suggest that the influence of in vivo hypoxia may differ between NO pathways in airways and vasculature, and depend on the duration of hypoxic exposure. Further studies aimed at determining the mechanisms underlying these disparate effects on the NO pathway could provide important information regarding altered NO signaling in newborns with different stages of hypoxia-induced pulmonary hypertension.
![]() |
ACKNOWLEDGEMENTS |
---|
This work was supported in part by a March of Dimes Birth Defects Foundation Research Grant (to C. D. Fike) and by an American Heart Association Mid-Atlantic Affiliate Grant (to C. D. Fike).
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: C. D. Fike, Dept. of Pediatrics, Wake Forest Univ. School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157 (E-mail: cfike{at}wfubmc.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 November 8, 2002;10.1152/ajplung.00246.2002
Received 25 July 2002; accepted in final form 4 November 2002.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Berg, JT,
Deem S,
Kerr ME,
and
Swenson ER.
Hemoglobin and red blood cells alter the response of expired nitric oxide to mechanical forces.
Am J Physiol Heart Circ Physiol
279:
H2947-H2953,
2000
2.
Berkenbosch, JW,
Baribeau J,
and
Perreault T.
Decreased synthesis and vasodilation to nitric oxide in piglets with hypoxia-induced pulmonary hypertension.
Am J Physiol Lung Cell Mol Physiol
278:
L276-L283,
2000
3.
Black, SM,
Fineman JR,
Steinhorn RH,
Bristow J,
and
Soifer SJ.
Increased endothelial NOS in lambs with increased pulmonary blood flow and pulmonary hypertension.
Am J Physiol Heart Circ Physiol
275:
H1643-H1651,
1998
4.
Block, ER,
Herrera H,
and
Couch M.
Hypoxia inhibits L-arginine uptake by pulmonary artery endothelial cells.
Am J Physiol Lung Cell Mol Physiol
269:
L574-L580,
1995
5.
Chicoine, LG,
Avitia JW,
Deen C,
Nelin LD,
Early S,
and
Walker BR.
Developmental differences in pulmonary eNOS expression in response to chronic hypoxia in rats.
J Appl Physiol
93:
311-318,
2002
6.
Clayton, RA,
Nally JE,
MacLean MR,
Thomson NC,
and
McGrath JC.
Chronic exposure to hypoxia attenuates contractile responses in rat airways in vitro: a possible role for nitric oxide.
Eur J Pharmacol
385:
29-37,
1999[ISI][Medline].
7.
Duplain, H,
Sartori C,
Lepori M,
Egli M,
Allemann Y,
Nicod P,
and
Scherrer U.
Exhaled nitric oxide in high-altitude pulmonary edema: role in the regulation of pulmonary vascular tone and evidence for a role against inflammation.
Am J Respir Crit Care Med
162:
221-224,
2000
8.
Eddahibi, S,
Raffestin B,
Tayarani Y,
Carville C,
and
Adnot S.
Hypoxia-reoxygenation impairs NO-mediated vasodilation in rat lungs.
Am J Physiol Lung Cell Mol Physiol
271:
L441-L449,
1996
9.
Fike, CD,
and
Kaplowitz MR.
Pulmonary venous pressure increases during alveolar hypoxia in isolated lungs of newborn pigs.
J Appl Physiol
73:
552-556,
1992[ISI][Medline].
10.
Fike, CD,
and
Kaplowitz MR.
Effect of chronic hypoxia on pulmonary vascular pressures in isolated lungs of newborn pigs.
J Appl Physiol
77:
2853-2862,
1994
11.
Fike, CD,
and
Kaplowitz MR.
Chronic hypoxia alters nitric oxide dependent pulmonary vascular responses in lungs of newborn pigs.
J Appl Physiol
81:
2078-2087,
1996
12.
Fike, CD,
Kaplowitz MR,
Rehorst-Paea LA,
and
Nelin LD.
L-arginine increases nitric oxide production in isolated lungs of chronically hypoxic newborn pigs.
J Appl Physiol
88:
1797-1803,
2000
13.
Fike, CD,
Kaplowitz MR,
Thomas CJ,
and
Nelin LD.
Chronic hypoxia decreases nitric oxide production and endothelial nitric oxide synthase in newborn pig lungs.
Am J Physiol Lung Cell Mol Physiol
274:
L517-L526,
1998
14.
Fleming, I,
and
Busse R.
Signal transduction of eNOS activation.
Cardiovasc Res
43:
532-541,
1999[ISI][Medline].
15.
Frank, L.
Protection from O2 toxicity by preexposure to hypoxia: lung antioxidant enzyme role.
J Appl Physiol
53:
475-482,
1982
16.
Guembe, L,
and
Villaro AC.
Histochemical demonstration of neuronal nitric oxide synthase during development of mouse respiratory tract.
Am J Respir Cell Mol Biol
20:
342-351,
1999
17.
Haworth, SG,
and
Hislop AA.
Effect of hypoxia on adaptation of the pulmonary circulation to extra-uterine life in the pig.
Cardiovasc Res
16:
293-303,
1982[ISI][Medline].
18.
Hislop, AA,
Springall DR,
Oliveira H,
Polak JS,
and
Haworth SG.
Endothelial nitric oxide synthase in hypoxic newborn porcine pulmonary vessels.
Arch Dis Child
77:
F16-F22,
1997[ISI].
19.
Ide, H,
Nakano H,
Ogasa T,
Osanai S,
Kikuchi K,
and
Iwamoto J.
Regulation of pulmonary circulation by alveolar oxygen tension via airway nitric oxide.
J Appl Physiol
87:
1629-1636,
1999
20.
Inscore, SC,
Stenmark KR,
Orton C,
and
Irvin CG.
Neonatal calves develop airflow limitation due to chronic hypobaric hypoxia.
J Appl Physiol
70:
384-390,
1990
21.
Isaacson, TC,
Hampl V,
Weir EK,
Nelson DP,
and
Archer SL.
Increased endothelium-derived NO in hypertensive pulmonary circulation of chronically hypoxic rats.
J Appl Physiol
76:
933-940,
1994
22.
Jakupaj, M,
Martin RJ,
Dreshaj IA,
Potter CF,
Haxhiu MA,
and
Ernsberger P.
Role of endogenous NO in modulating airway contraction mediated by muscarinic receptors during development.
Am J Physiol Lung Cell Mol Physiol
273:
L531-L536,
1997
23.
Kawai, N,
Bloch DB,
Filippov G,
Rabkina D,
Suen H,
Losty PD,
Janssens SP,
Zapol WM,
Monte SDL,
and
Bloch KD.
Constitutive endothelial nitric oxide synthase.
Am J Physiol Lung Cell Mol Physiol
268:
L589-L595,
1995
24.
Lundberg, JON,
Lundberg JM,
Settergren G,
Alving K,
and
Weitzberg E.
Nitric oxide, produced in the upper airways, may act in an "aerocrine" fashion to enhance pulmonary oxygen uptake in humans.
Acta Physiol Scand
155:
467-468,
1995[ISI][Medline].
25.
MacRitchie, AN,
Albertine KH,
Sun J,
Lei PS,
Jensen SC,
Freestone AA,
Clair PM,
Dahl M,
Godfrey EA,
Carlton DP,
and
Bland RD.
Reduced endothelial nitric oxide synthase in lungs of chronically ventilated preterm lambs.
Am J Physiol Lung Cell Mol Physiol
281:
L1011-L1020,
2001
26.
Nelin, LD,
Thomas CJ,
and
Dawson CA.
Effect of hypoxia on nitric oxide production in the neonatal pig lung.
Am J Physiol Heart Circ Physiol
271:
H8-H14,
1996
27.
Parker, TA,
Le Cras TD,
Kinsella JP,
and
Abman SH.
Developmental changes in endothelial nitric oxide synthase expression and activity in ovine fetal lung.
Am J Physiol Lung Cell Mol Physiol
278:
L202-L208,
2000
28.
Potter, CF,
Dreshaj IA,
Haxhiu MA,
Stork EK,
Chatburn RL,
and
Martin RJ.
Effect of exogenous and endogenous nitric oxide on the airway and tissue components of lung resistance in the newborn piglet.
Pediatr Res
41:
886-891,
1997[Abstract].
29.
Schedin, V,
Norman M,
Gustafsson LE,
Jonsson B,
and
Frostell C.
Endogenous nitric oxide in the upper airways of premature and term infants.
Acta Paediatr
86:
1229-1235,
1997[ISI][Medline].
30.
Settergren, G,
Angdin M,
Astudillo R,
Gelinder S,
Liska J,
Lundberg JON,
and
Weitzberg E.
Decreased pulmonary vascular resistance during nasal breathing: modulation by endogenous nitric oxide from the paranasal sinuses.
Acta Physiol Scand
163:
235-239,
1998[ISI][Medline].
31.
Shaul, PW,
Yuhanna IS,
German Z,
Chen Z,
Steinhorn RH,
and
Morin FC, III.
Pulmonary endothelial NO synthase gene expression is decreased in fetal lambs with pulmonary hypertension.
Am J Physiol Lung Cell Mol Physiol
272:
L1005-L1012,
1997
32.
Sherman, TS,
Chen Z,
Yuhanna IS,
Lau KS,
Margraf LR,
and
Shaul PW.
Nitric oxide synthase isoform expression in the developing lung epithelium.
Am J Physiol Lung Cell Mol Physiol
276:
L383-L390,
1999
33.
Su, Y,
and
Block AR.
Role of calpain in hypoxic inhibition of nitric oxide synthase activity in pulmonary endothelial cells.
Am J Physiol Lung Cell Mol Physiol
278:
L1204-L1212,
2000
34.
Tulloh, RMR,
Hislop AA,
Boels PJ,
Deutsch J,
and
Haworth SG.
Chronic hypoxia inhibits postnatal maturation of porcine intrapulmonary artery relaxation.
Am J Physiol Heart Circ Physiol
272:
H2436-H2445,
1997
35.
Villamor, E,
Le Cras TD,
Horan MP,
Halbower AC,
Tuder RM,
and
Abman SH.
Chronic intrauterine pulmonary hypertension impairs endothelial nitric oxide synthase in the ovine fetus.
Am J Physiol Lung Cell Mol Physiol
272:
L1013-L1020,
1997
36.
Xue, C,
Rengasamy A,
Le Cras TC,
Koberna PA,
Dailey GC,
and
Johns RA.
Distribution of NOS in normoxic vs. hypoxic rat lung: upregulation of NOS by chronic hypoxia.
Am J Physiol Lung Cell Mol Physiol
267:
L667-L678,
1994
37.
Xue, C,
Reynolds PR,
and
Johns RA.
Developmental expression of NOS isoforms in fetal rat lung: implications for transitional circulation and pulmonary angiogenesis.
Am J Physiol Lung Cell Mol Physiol
270:
L88-L100,
1996