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
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.
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 (Po 2 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 (seeMeasurements 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 · min−1 · kg−1and 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 inPerfusate accumulation of NOx− in isolated lungs. Throughout the perfusate sampling period, exhaled NO was continuously monitored for determination of exhaled NO output as described inExhaled 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.
The perfusate NOx− concentration (nmol/ml) was determined for each collection time as described above. The amount of NOx− in the perfusate (nmol) at each collection time was calculated by multiplying the perfusate concentration of NOx− at that sample collection time by the volume of the system (perfusion circuit plus reservoir) at the sample collection time plus the amount of NOx− removed with all previous samples. The amount of NOx− accumulated (nmol) was the amount of NOx− at each collection time minus the amount of NOx− attime 0. The amount of NOx− at time 0 was determined from the y-intercept of a linear regression line fit to the amount of NOx− in the perfusate vs. time for the first 90 min of perfusion.
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.
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, seeresults), 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 5,600g 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 unpairedt-test. A value of P < 0.05 was used as indicative of statistical significance.
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, Po 2, and Pco 2 obtained with the animals breathing room air did not differ significantly between control and hypoxic piglets (Table1). 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). Figure3 A 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, Po 2, and Pco 2 measured in the perfusate did not differ between control and hypoxic piglets (Table2). 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. 3 A), exhaled NO output in isolated perfused lungs of hypoxic piglets was significantly reduced compared with that of control piglets (Fig. 3 B). Moreover, NOx− in the airway incubation solution from hypoxic piglets was reduced compared with the value measured in control piglets (Fig.4 A). 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. 4 B).
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. 6 A, left; for pulmonary artery homogenates in Fig. 6 B, left; and for airway homogenates in Fig. 6 C, left). There was no difference in the absorbance of the bands for the eNOS isoform for either distal lung homogenates (Fig.6 A, right) or pulmonary artery homogenates (Fig. 6 B, 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. 6 C, 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.7 A, right and 7 B, right) from control compared with hypoxic piglets, whereas the mean absorbance of the bands for nNOS was greater for airway homogenates (Fig. 7 C, 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, Aand 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.
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 inl-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.
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).
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:).
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First published November 8, 2002;10.1152/ajplung.00246.2002
- Copyright © 2003 the American Physiological Society