Chronic hypoxia decreases nitric oxide production and endothelial nitric oxide synthase in newborn pig lungs

Candice D. Fike, Mark R. Kaplowitz, Carol J. Thomas, and Leif D. Nelin

Department of Pediatrics, Medical College of Wisconsin, Milwaukee 53226; and Research Services, Zablocki Veterans Affairs Medical Center, Milwaukee, Wisconsin 53295

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

To examine the effect of chronic hypoxia on nitric oxide (NO) production and the amount of the endothelial isoform of nitric oxide synthase (eNOS) in lungs of newborn piglets, studies were performed using 1- to 3-day-old piglets raised in room air (control) or 10% O2 (chronic hypoxia) for 10-12 days. Exhaled NO output and plasma nitrites and nitrates (collectively termed NO<SUP>−</SUP><SUB>x</SUB>) were measured in anesthetized animals. NO<SUP>−</SUP><SUB>x</SUB> concentrations were measured in the perfusate of isolated lungs. eNOS amounts were assessed in whole lung homogenates. In the intact piglets, exhaled NO outputs and plasma NO<SUP>−</SUP><SUB>x</SUB> were lower in the chronically hypoxic (exhaled NO output = 0.2 ± 0.1 nmol/min; plasma NO<SUP>−</SUP><SUB>x</SUB> = 10.3 ± 3.7 nmol/ml) than in control animals (exhaled NO output = 0.8 ± 0.2 nmol/min; plasma NO<SUP>−</SUP><SUB>x</SUB> = 22.3 ± 4.3 nmol/ml). In perfused lungs, the perfusate accumulation of NO<SUP>−</SUP><SUB>x</SUB> was lower in chronic hypoxia (1.0 ± 0.3 nmol/min) than in control (2.6 ± 0.6 nmol/min) piglets. The amount of whole lung homogenate eNOS from the chronic hypoxia piglets was 40 ± 8% less than that from the control piglets. The reduced NO production observed in anesthetized animals or perfused lungs of chronically hypoxic newborn piglets is consistent with the finding of reduced lung eNOS protein amounts. Decreased NO production might contribute to the development of chronic hypoxia-induced pulmonary hypertension in newborns.

isolated perfused lungs; neonatal pulmonary hypertension; plasma levels

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

IT HAS BEEN SUGGESTED that decreased production of or sensitivity to endogenously produced vasodilators such as nitric oxide (NO) could contribute to the development of chronic hypoxia-induced pulmonary hypertension. In support of this notion, adult humans with end-stage chronic obstructive pulmonary disease (10) and adult rats with chronic hypoxia-induced pulmonary hypertension exhibit reduced pulmonary vasodilator responses to acetylcholine (2, 6, 20, 28), an agent thought to cause vasodilation by stimulating NO production by the pulmonary vascular endothelium. In addition, acetylcholine-induced elevations in NO, as measured by pulmonary vascular smooth muscle cell cGMP, were found to be less in lungs of chronically hypoxic than control rats (35). However, not all evidence supports the idea that chronic hypoxia decreases NO production in lungs of adult rats. For example, some investigators found that pulmonary vascular responses to acetylcholine or other endothelium-dependent vasodilators were either not reduced or enhanced in lungs of mature rats with chronic hypoxia-induced pulmonary hypertension (11, 16, 29, 30). In addition, NO synthesis inhibitors caused either the same or greater pulmonary vasoconstriction in lungs of chronically hypoxic compared with control rats (5, 25, 29). Moreover, the perfusate concentrations of nitrites and nitrates (collectively termed NO<SUP>−</SUP><SUB>x</SUB>), the stable metabolic products of NO (16, 22), and the amounts of the endothelial isoform of nitric oxide synthase (eNOS) and mRNA (18, 33) were higher in lungs of chronically hypoxic rats than control rats. Hence, the role of NO production in chronic hypoxia-induced pulmonary hypertension in adults remains unclear.

The effect of chronic hypoxia on the neonatal lung may differ from the adult lung in that the contribution of NO to the regulation of pulmonary vasomotor tone changes with postnatal age (32, 44). We have shown that newborn piglets exposed to chronic hypoxia developed pulmonary hypertension and that their pulmonary circulation exhibited reduced responses to agents that stimulate or inhibit the release of NO (13, 14). These findings support the notion that NO production is decreased in lungs of chronically hypoxic newborn piglets. To further evaluate this hypothesis, we measured the exhaled NO output and the plasma NO<SUP>−</SUP><SUB>x</SUB> concentration in intact control and chronically hypoxic newborn piglets. We also measured the basal and acetylcholine-stimulated accumulation of NO<SUP>−</SUP><SUB>x</SUB> in the perfusate of isolated lungs from control and chronically hypoxic newborn piglets. In addition, we measured the amount of eNOS in whole lung homogenates from control and chronically hypoxic piglets.

    METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Animals

Newborn pigs (1-3 days old) were placed in either a room-air environment (control; n = 7 pigs) or a hypoxic normobaric environment (chronic hypoxia; n = 14 pigs) for 10-12 days. Normobaric hypoxia was produced by delivering compressed air and N2 to an incubator (Thermocare). The O2 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 3 times/day for cleaning and to weigh the animals. The animals were fed ad libitum with an artificial sow milk replacer from a feeding device attached to the chamber. We previously found no differences in pulmonary vascular pressure responses to agents that stimulate or inhibit NO production between control piglets raised on the farm and control piglets raised in a normoxic chamber (14), so that for these studies, eight additional control animals were studied on the day of arrival from the farm at 11-15 days of age.

Measurements in Anesthetized Animals

On the day of study, the animals were weighed and anesthetized with ketamine (30 mg/kg im) and pentobarbital sodium (10 mg/kg iv). Additional intravenous pentobarbital sodium was given as needed via an ear vein to maintain anesthesia during placement of the catheters. First, the trachea of the piglet was cannulated so that the animal could be ventilated if necessary. Then a catheter was placed in the right femoral artery for monitoring systemic blood pressure and arterial blood gases. Another catheter was placed through the right external jugular vein in 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), a thermistor was placed in the aortic arch via the left femoral artery, and a catheter that served as an injection port was placed in 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). Eight control piglets and five chronically hypoxic piglets were then given additional anesthesia, their tracheal cannula was attached to a piston-type ventilator, and their lungs were ventilated with a normoxic gas mixture (21% O2 and balance N2; Matheson, Chicago, IL) using a tidal volume of 15-20 ml/kg and a respiratory rate of 15-20 breaths/min for measurement of exhaled NO output as described in Exhaled NO Measurement. After measurement of blood gases, pulmonary arterial pressure, pulmonary wedge pressure, left ventricular end-diastolic pressure, and cardiac output, all the animals were given heparin (1,000 IU/kg iv), and 10-ml samples of blood were drawn from the pulmonary arterial and left ventricular catheters. The blood was centrifuged, and the plasma was stored at -70°C for determination of NO<SUP>−</SUP><SUB>x</SUB> concentration as described in <IT>NO</IT><SUP><IT>−</IT></SUP><SUB><IT>x</IT></SUB> Assay. Three control piglets and five chronically hypoxic piglets were then given additional anesthesia (3-5 mg/kg pentobarbital sodium iv) and exsanguinated, after which their lungs were excised, immediately frozen in liquid N2, and stored at -70°C for later analysis of eNOS as described in Unperfused lungs.

Measurements in Isolated Perfused Lungs

Ten control piglets and eight chronically hypoxic piglets were given additional anesthesia (3-5 mg/kg pentobarbital sodium iv) and were exsanguinated, and the lungs were excised for perfusion. The tracheal cannula 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 and a respiratory rate of 15-20 breaths/min (mean airway pressure of 3-5 mmHg). A midline sternotomy was performed, and a clamp was placed across the ductus arteriosus. Saline-filled cannulas, which included electrodes for use in measuring vascular volumes as described below, were placed in the pulmonary artery and left atrium through incisions in the right and left ventricles. The diaphragm and all abdominal contents were removed. The vascular cannulas were connected to the perfusion circuit described previously (13, 14). Briefly, in the perfusion circuit, a rotary pump (model 7523-00, Cole Palmer Masterflex) continuously circulated the perfusate from the reservoir through a bubble trap into the pulmonary arterial cannula, through the lungs to the left atrial cannula, and back to the reservoir. The perfusion circuit also included an injection valve (9) located a few centimeters proximal to the pulmonary arterial cannula for use in measuring vascular volumes as described below. 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.

A Krebs-Ringer bicarbonate solution containing 5% dextran (molecular weight 70,000) at 37°C was used as the perfusate. Initially, lung perfusion was nonrecirculating. When the effluent from the lung was nearly free of blood, recirculating perfusion was initiated such that the perfusate had a hematocrit <1%. The volume of recirculating perfusate was adjusted to 130-170 ml, and the flow rate was adjusted to 50 ml · min-1 · kg-1. The lungs were perfused for 30-60 min until a stable pulmonary arterial pressure was achieved.

In six control and seven chronically hypoxic lungs, the vascular volume of the perfused lungs was measured. To do this, a 2-ml bolus of 1.2% saline was injected via the injection port into the lungs, and the change in electrical conductivity with time was measured at the pulmonary arterial (inflow) and left atrial (outflow) cannulas by use of impedance bridges (model AMPP87, General Electric) as previously described (9). The inflow and outflow conductance curves were used to calculate the mean transit time through the lungs, and the vascular volume (ml) was calculated by multiplying the mean transit time (seconds) by the flow rate (ml/s) (9). During these measurements, the ventilator was stopped at end expiration.

For all control and chronically hypoxic lungs, perfusate samples (3 ml) were then removed from the left atrial cannula every 15 min for 90 min (baseline period). Next, for eight control piglet lungs and for eight chronically hypoxic piglet lungs, acetylcholine (10 µg/min) was infused into the pulmonary arterial cannula, and perfusate samples were collected every 10 min for 30 min (acetylcholine infusion period). The perfusate samples were centrifuged, and the supernatant was stored at -70°C for future analysis of NO<SUP>−</SUP><SUB>x</SUB> concentrations. At the end of the perfusion, the volume of perfusate remaining in the circuit and reservoir was measured. The chronically hypoxic and control lungs were then immediately frozen in liquid N2 and stored at -70°C for later analysis of eNOS as described in Perfused lungs.

Exhaled NO Measurement

A chemiluminescence technique, which has been previously described (24), was used for determination of exhaled NO output in eight control and five chronically hypoxic anesthetized piglets. Expiratory gas was sampled continuously for a 10- to 15-min period from the exhalation limb of the ventilator at a rate of 4 ml/s and was passed through a chemiluminescence analyzer (model 270B NOA; Sievers, Boulder, CO). The analyzer was calibrated daily with authentic NO (1 part/million in N2; Matheson) mixed with O2-free N2 using precision flowmeters to obtain concentrations ranging from 0 to 60 parts/billion (ppb). The NO detection limit was 0.5 ppb (vol/vol). The exhaled NO output was calculated from the measured NO concentration, the ventilation rate, and the tidal volume.

NO<SUP>−</SUP><SUB>x</SUB> Assay

A spectrophotometric analysis, which has been described previously (24), was used for determination of plasma and lung perfusate NO<SUP>−</SUP><SUB>x</SUB> concentrations. All reagents were obtained from Sigma. Plasma samples were diluted 1:10 with fresh perfusate. Fifty microliters of a stock NADPH solution (0.8 µg NADPH/ml phosphate buffer) and 10 µl of a stock nitrate reductase solution (5 units nitrate reductase/ml phosphate buffer) were added to 500 µl of lung perfusate or diluted plasma. After incubation 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 or plasma 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 and plasma samples was used as a blank. Duplicate assays were carried out for each sample of plasma or lung perfusate.

The perfusate NO<SUP>−</SUP><SUB>x</SUB> concentration (nmol/ml) was determined for each collection time as described above. The amount of NO<SUP>−</SUP><SUB>x</SUB> in the perfusate (nmol) at each collection time was calculated by multiplying the perfusate concentration of NO<SUP>−</SUP><SUB>x</SUB> at that sample collection time by the volume of the system (perfusion circuit plus reservoir) at the sample collection time plus the amount of NO<SUP>−</SUP><SUB>x</SUB> removed with all previous samples. The amount of NO<SUP>−</SUP><SUB>x</SUB> accumulated (nmol) was the amount of NO<SUP>−</SUP><SUB>x</SUB> at each collection time minus the amount of NO<SUP>−</SUP><SUB>x</SUB> at zero time. The amount of NO<SUP>−</SUP><SUB>x</SUB> at zero time was determined from the y-intercept of a linear regression line fit to the amount of NO<SUP>−</SUP><SUB>x</SUB> in the perfusate vs. time for the first 90 min of perfusion.

Immunoblot Analysis

Perfused lungs. Tissue pieces that did not contain large airways or large vessels were selected from frozen perfused lungs of control (n = 8) and chronically hypoxic (n = 8) 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 lung homogenate was determined by the Bio-Rad protein assay. Each lung homogenate was diluted with PBS to obtain protein concentrations of 0.5, 1.0, 1.5, 2.5, 3.0, and 4.0 mg/ml. Forty microliters of each protein concentration were solubilized in 40 µl of denaturing, reducing buffer [Novex; 0.25 M Tris · HCl, 5% (wt /vol) SDS, 2.5% (vol/vol) beta -mercaptoethanol, 10% glycerol, and 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 (20 µl) of these supernatants were then applied to Tris-glycine precast 8% polyacrylamide gels (Novex) so that each gel was loaded with 5, 10, 15, 25, 30, and 40 µg of protein from the same lung homogenate of either a control or chronically hypoxic piglet. In addition, so that every gel contained 15 µg of protein samples from both a control and a chronically hypoxic lung, each gel was loaded with 15 µg of protein of lung homogenate from a piglet of the other experimental group. Furthermore, to normalize the results between all the different blots, one lane of every gel was loaded with 15 µg of protein from a lung homogenate that was designated as the standard. 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 the eNOS, the nitrocellulose membrane was incubated for 1 h at room temperature with mouse anti-human eNOS (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 anti-mouse antibody (Vector Elite, avidin-biotinylated horseradish peroxidase complex kit, 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 PBS containing 0.1% Tween 20 after the final incubation. To visualize the biotinylated antibody, the membranes were developed using enhanced chemiluminescence reagents (Amersham), and the chemiluminescent signal was captured on X-ray film (Bio-Max MR, Kodak) using 30-s to 5-min exposures. The bands for eNOS were quantified using laser densitometry. The absorbance of the eNOS band for the standard lung homogenate was used to normalize the peak absorbance of the eNOS bands on all gels.

Unperfused lungs. We performed two additional immunoblot analyses to assess findings in unperfused lungs. For the first of these analyses, we selected tissue pieces that did not contain large airways or large vessels from frozen unperfused lungs of three control and five chronically hypoxic piglets. Following the methods described above, we loaded one lane each of a gel with whole lung homogenate samples containing 15 µg of total protein from the control and chronically hypoxic lungs. In addition, one lane of the gel was loaded with 15 µg of protein from the standard lung homogenate.

For the second of these analyses, we anesthetized and exsanguinated two additional control piglets. The left lung of each piglet was excised and immediately frozen, and the right lung was isolated and perfused with Krebs-Ringer bicarbonate solution containing 5% dextran for 2 h and then frozen. Tissue pieces that did not contain large airways or large vessels were selected from perfused and unperfused lungs of each piglet. Following the methods described above, we loaded one lane each of a gel with lung homogenate samples containing 15 µg of protein from perfused and unperfused lungs of both piglets. In addition, one lane of the gel was loaded with 15 µg of protein from the standard lung homogenate.

Calculations and Statistics

Data are presented as means ± SE. Unpaired t-tests were used to compare the hemodynamic and immunoblot data between control and chronically hypoxic animals. The NO<SUP>−</SUP><SUB>x</SUB> accumulation data were analyzed in two different ways. For one analysis, the mean of the amount of perfusate NO<SUP>−</SUP><SUB>x</SUB> accumulated (nmol) at each collection time was calculated for each group. The baseline rate of NO<SUP>−</SUP><SUB>x</SUB> accumulation (nmol/min) for the group was then determined by linear regression of the group mean values of accumulated amounts of perfusate NO<SUP>−</SUP><SUB>x</SUB> (nmol) vs. time (min). The amount of NO<SUP>−</SUP><SUB>x</SUB> accumulated during acetylcholine infusion was corrected for the baseline amount of NO<SUP>−</SUP><SUB>x</SUB> accumulation by subtraction of the baseline NO<SUP>−</SUP><SUB>x</SUB> accumulation rate from the acetylcholine infusion NO<SUP>−</SUP><SUB>x</SUB> accumulation rate. The linear regression fits for both the baseline and acetylcholine perfusion periods for each group were then compared using an analysis of covariance, with a Newman-Keuls post hoc test to determine the significant differences between the two groups and the two perfusion periods. For the other analysis, the rate of NO<SUP>−</SUP><SUB>x</SUB> accumulation (nmol/min) for each lung was determined by linear regression of the accumulated NO<SUP>−</SUP><SUB>x</SUB> (nmol) vs. time (min) data for each individual lung. Then, the means of the individual rates of NO<SUP>−</SUP><SUB>x</SUB> accumulation (nmol/min) were calculated and compared between control and chronic hypoxic groups for both baseline and acetylcholine perfusion periods using a one-way analysis of variance and a Newman-Keuls post hoc test to determine the significant differences between the two groups and the two perfusion periods. P < 0.05 was indicative of statistical significance.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

After 10-12 days of hypoxia, the chronically hypoxic piglets weighed less and had a higher hematocrit than the corresponding control piglets (Table 1). The measured values of blood pH, PO2, and PCO2 obtained during hemodynamic measurements in anesthetized piglets breathing room air did not differ significantly between control and chronically hypoxic piglets (Table 1). Likewise, measured values of pH, PO2, and PCO2 in the perfusate of isolated lungs from chronically hypoxic piglets were not significantly different from those of control piglets (Table 1).

                              
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Table 1.   Physiological measurements for control and chronically hypoxic piglets

Measurements of pulmonary arterial pressure, pulmonary wedge pressure, left ventricular end-diastolic pressure, cardiac output, aortic pressure, and calculated pulmonary vascular resistance [(pulmonary arterial pressure - wedge pressure) / cardiac output] in the anesthetized piglets are shown in Table 2. Measurements of left ventricular end-diastolic pressures did not differ significantly from measurements of wedge pressures (Table 2). Thus left ventricular end-diastolic pressure was used for calculation of pulmonary vascular resistance when we were unable to obtain a wedge pressure. Pulmonary arterial pressures and pulmonary vascular resistances were significantly greater in the chronically hypoxic than in the control piglets (Table 2).

                              
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Table 2.   Hemodynamic measurements in anesthetized piglets

The calculated vascular volumes for perfused lungs of chronically hypoxic piglets did not differ significantly from those of the control group (Table 3). Although the baseline pulmonary arterial pressures were significantly higher in perfused lungs of chronically hypoxic than control piglets (Table 3), the mean percent change in pulmonary arterial pressure in response to acetylcholine was significantly less in the chronically hypoxic than in the control lungs (Table 3).

                              
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Table 3.   Vascular volumes and pulmonary arterial responses to infusion of ACh (10 µg/min) in perfused lungs of control and chronically hypoxic piglets

For the anesthetized animals, exhaled NO output (Fig. 1) and left ventricular plasma NO<SUP>−</SUP><SUB>x</SUB> concentrations (Fig. 2) were significantly lower in chronically hypoxic than in control piglets. Pulmonary arterial plasma NO<SUP>−</SUP><SUB>x</SUB> concentrations were also significantly lower in chronically hypoxic (10 ± 4 nmol/ml) than in control piglets (22 ± 4 nmol/ml) but are not included in Fig. 2 because they did not differ significantly from their respective left ventricular plasma values.


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Fig. 1.   Exhaled NO output for control (n = 8) and chronically hypoxic (n = 5) piglets. All values are means ± SE. * Significantly different from corresponding control, unpaired t-test.


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Fig. 2.   Left ventricular plasma nitrite and nitrate (collectively termed NO<SUP>−</SUP><SUB>x</SUB>) concentrations from control (n = 15) and chronically hypoxic (n = 15) piglets. All values are means ± SE. * Significantly different from corresponding control, unpaired t-test.

In the perfused lungs, the NO<SUP>−</SUP><SUB>x</SUB> concentration measured in the chronically hypoxic group at the first collection time was not significantly different from that measured in the control group (Table 4). However, the perfusate NO<SUP>−</SUP><SUB>x</SUB> concentration measured at the last collection time was lower for chronically hypoxic than for control lungs (Table 4). Figure 3 shows the group mean amounts of NO<SUP>−</SUP><SUB>x</SUB> accumulated (nmol) for control and chronically hypoxic animals at each collection time for the 90-min baseline period and the 30-min acetylcholine infusion period. Comparison of the linear regression of these group mean values showed that the mean rate of perfusate NO<SUP>−</SUP><SUB>x</SUB> accumulation for the 90-min baseline period was 2.5 ± 0.3 nmol/min for control lungs (Fig. 3), which was greater than the 0.8 ± 0.4 nmol/min NO<SUP>−</SUP><SUB>x</SUB> accumulation rate found in chronically hypoxic lungs. For the control lungs, the infusion of acetylcholine resulted in an NO<SUP>−</SUP><SUB>x</SUB> accumulation rate that was 5.1 ± 0.6 nmol/ml above the baseline accumulation rate. For the chronically hypoxic lungs, the infusion of acetylcholine did not result in an NO<SUP>−</SUP><SUB>x</SUB> accumulation rate that was significantly above the baseline rate (accumulation rate of 2.8 ± 0.3 nmol/min above the baseline rate). However, when corrected for the baseline accumulation rates, the rates of NO<SUP>−</SUP><SUB>x</SUB> accumulation during the acetylcholine infusion period did not differ between control and hypoxic lungs.

                              
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Table 4.   Perfusate NO<SUP>−</SUP><SUB>x</SUB> concentrations at first collection time (0 min) and after 90 and 120 min of perfusion for control and chronically hypoxic piglets


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Fig. 3.   Group mean amounts of NOx- accumulated in perfusate for control (open circle ) and chronically hypoxic (black-square) lungs at each collection time. Group mean amounts differed between control and chronically hypoxic animals at time points indicated by asterisks. Vertical line denotes time that acetylcholine (ACh) infusion was started. Solid and dotted lines represent linear regression ( y = mx + b ) for 90-min baseline period and 30-min acetylcholine infusion period, respectively. Linear regressions differed between control and chronically hypoxic animals during both baseline and acetylcholine perfusion periods (analysis of covariance with a Newman-Keuls post hoc test; see METHODS for details). However, when corrected for baseline accumulation rates, there was no difference between control and chronically hypoxic animals during acetylcholine infusion period. Values are means ± SE.

Figure 4 illustrates the perfusate NO<SUP>−</SUP><SUB>x</SUB> accumulation data for control and chronically hypoxic animals when the NO<SUP>−</SUP><SUB>x</SUB> accumulation rates (nmol/min) for each group were calculated as means of individual lung NO<SUP>−</SUP><SUB>x</SUB> accumulation rates (nmol/min). Comparison of these NO<SUP>−</SUP><SUB>x</SUB> accumulation rates showed that the rate of NO<SUP>−</SUP><SUB>x</SUB> accumulation was 2.6 ± 0.6 nmol for the control lungs and was 62% less in the chronically hypoxic lungs (1.0 ± 0.3 nmol/min; Fig. 4). The mean rate of accumulation during the 30-min acetylcholine infusion period was 7.4 ± 1.4 nmol/min in the control lungs and was 53% less for the chronically hypoxic lungs (3.3 ± 0.9 nmol/min). When the mean rates of accumulation during the 30-min acetylcholine infusion period were corrected for the baseline accumulation, there was no statistically significant difference between the resultant increase in rate caused by acetylcholine infusion in the control lungs (4.8 ± 1.4 nmol/min) and the resultant increase in rate caused by acetylcholine infusion in the chronically hypoxic lungs (2.3 ± 0.7 nmol/min).


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Fig. 4.   NOx- accumulation rates for control (hatched bars) and chronically hypoxic (solid bars) groups calculated from means of individual lung NOx- accumulation rates for 90-min baseline period and 30-min acetylcholine infusion period (control piglets: n = 10 for baseline, n = 8 for acetylcholine; chronically hypoxic piglets: n = 8 for both baseline and acetylcholine). All values are means ± SE. * Significantly different from corresponding control value, ANOVA with a Newman-Keuls post hoc test.

A typical immunoblot analysis for eNOS in the perfused lung homogenates is shown in Fig. 5. In both control and chronically hypoxic lungs, the apparent molecular mass of eNOS was 135 kDa, as determined from linear regression of molecular-mass standards. Figure 6 shows the mean data for the absorbance of the eNOS bands as determined by laser densitometry for the six different amounts of total whole lung homogenate protein for the control (n = 8) and chronically hypoxic (n = 8) perfused lungs and shows that for amounts of total whole lung homogenate protein within the dynamic range of the immunoblot assay (5-25 µg total protein), the absorbance of the eNOS bands was less for the chronically hypoxic lungs than for the control lungs. Moreover, comparison of the absorbance of the eNOS bands for the 15-µg whole lung homogenate protein samples from the two experimental groups located on the same blots (e.g., control 15-µg protein and chronically hypoxic 15-µg protein in Fig. 5) showed that the absorbance of the eNOS bands was decreased for the chronically hypoxic lungs when expressed either as a percentage of the absorbance of control lungs or as a fraction of the absorbance of the standard lung (peak absorbance of the sample/peak absorbance of the standard lung was 0.82 ± 0.14 vs. 1.43 ± 0.17 for 8 chronically hypoxic vs. 8 control lungs, respectively).


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Fig. 5.   An example of an immunoblot for endothelial isoform of nitric oxide synthase (eNOS) in homogenates of perfused lungs from control (C) and chronically hypoxic (H) piglets. Standard denotes a 15-µg sample of whole lung homogenate from a standard lung that was applied to each gel and used to normalize intensity of eNOS bands between blots. Note greater intensity of eNOS bands for larger amounts of total protein. Also note that intensity of eNOS band is greater for 15-µg sample of control lung homogenate than for 15-µg sample of chronically hypoxic lung homogenate.


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Fig. 6.   Mean data for absorbance of eNOS bands as determined by laser densitometry for whole lung homogenate samples containing different amounts of total protein for 8 control (open circle ) and 8 chronically hypoxic (bullet ) perfused lungs. For amounts of total protein within dynamic range of assay (between 5 and 25 µg of total protein), absorbance of eNOS bands is less for chronically hypoxic than for control lungs. All values are means ± SE. * Significantly different from corresponding chronically hypoxic value, unpaired t-test.

Results of the immunoblot analysis in unperfused lungs were similar to those in perfused lungs. Specifically, the absorbance of the eNOS bands as determined by laser densitometry was less for the unperfused lungs of chronically hypoxic piglets than for the unperfused lungs of control piglets (peak absorbance of the sample/peak absorbance of the standard lung was 0.68 ± 0.17 vs. 1.39 ± 0.05 for 5 chronically hypoxic vs. 3 control unperfused lungs, respectively; unpaired t-test). In addition, the absorbance of the eNOS bands did not differ between perfused and unperfused lungs from the same control piglets (peak absorbance of the sample/peak absorbance of the standard lung was 1.43 ± 0.05 vs. 1.35 ± 0.01 for 2 unperfused vs. 2 perfused lungs, respectively).

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

In agreement with the findings of previous studies (13, 14), in this study, we show that pulmonary vascular resistance was elevated in chronically hypoxic newborn piglets, indicating that pulmonary hypertension developed after 10-12 days of exposure to chronic hypoxia. More importantly, in this study, we found that exhaled NO output and plasma concentrations of NO<SUP>−</SUP><SUB>x</SUB> were significantly decreased in piglets with chronic hypoxia-induced pulmonary hypertension and that the rate of perfusate NO<SUP>−</SUP><SUB>x</SUB> accumulation both before and after acetylcholine addition was significantly reduced in isolated, perfused lungs of chronically hypoxic newborn piglets. In this study, we also found significantly lower levels of eNOS for equivalent amounts of total protein in whole lung homogenates from chronically hypoxic piglets compared with control piglets. Together, these findings indicate that pulmonary NO production was decreased when newborn pigs were exposed to chronic hypoxia and suggest that a reduction in eNOS protein levels may be one mechanism for the decreased NO production.

Another finding in this study was that pulmonary vascular responses to the NO synthesis stimulator acetylcholine were decreased in lungs of chronically hypoxic piglets. This is consistent with our previous finding of blunted pulmonary vascular responses to both acetylcholine and the NO synthesis inhibitor Nomega -nitro-L-arginine methyl ester in lungs of piglets exposed to chronic hypoxia for 10-12 days (14). Our finding in this study that the rate of perfusate NO<SUP>−</SUP><SUB>x</SUB> accumulation was reduced both before and after acetylcholine addition in perfused lungs of chronically hypoxic newborn piglets might suggest that decreased NO production underlies these impaired pulmonary vascular responses. However, when corrected for the lower baseline accumulation rate, the NO<SUP>−</SUP><SUB>x</SUB> accumulation rate resultant from acetylcholine was not less in hypoxic than in control piglets. Thus, although our findings suggest that the basal production of NO was impaired, acetylcholine-stimulated NO release does not appear to be altered during chronic hypoxia. Similar dissociations between impairments in basal and stimulated NO production have been described by others (39). Instead of an impairment in the amount of NO released with stimulation, it is possible that altered sensitivity to NO might contribute to impaired NO-dependent pulmonary vascular responses. This possibility is supported by findings of other investigators who showed that responses to NO were impaired in intrapulmonary arteries from newborn piglets exposed to hypobaric hypoxia for 3 days (40). Another explanation for the blunted acetylcholine responses shown in chronically hypoxic piglets (14) could be that chronic hypoxia affects muscarinic receptors. However, this explanation does not seem likely because relaxant responses to the calcium ionophore A-23187, a nonreceptor-dependent agonist, were recently shown to be blunted in intrapulmonary arteries of chronically hypoxic piglets (40).

In this study, we did not detect a difference in the plasma concentration of NO<SUP>−</SUP><SUB>x</SUB> when we compared samples collected from the left ventricle with those from the pulmonary artery for either the control or chronically hypoxic piglets. This inability to measure a significant increase in the amount of NO metabolites leaving the lung is consistent with findings of other investigators (42, 43) and is at least partly due to the fact that the half-life of NO metabolites is long so that these metabolites accumulate in the plasma (43). Regardless of sampling site, the plasma levels of NO<SUP>−</SUP><SUB>x</SUB> measured in the control piglets in this study are consistent with both our previously reported values in 2-wk-old piglets (24) and those of others in adult pigs (31) and human infants (12).

Although we could not detect a transpulmonary production of NO<SUP>−</SUP><SUB>x</SUB> in either control or chronically hypoxic piglets, the plasma levels of NO<SUP>−</SUP><SUB>x</SUB> differed between the two groups. There are many in vivo sources of NO production that could be impaired in the chronically hypoxic piglets and contribute to the altered plasma NO<SUP>−</SUP><SUB>x</SUB>. Differences in extracellular volume might also contribute to differences in plasma NO<SUP>−</SUP><SUB>x</SUB> (43). The volume of distribution of nitrate in the conscious dog was calculated to most closely approximate the extracellular fluid volume, not the plasma volume, so that if extracellular volume is markedly increased, the volume of distribution may be larger, making the measurements of plasma nitrate a greater underestimate of the real production rate of NO (43). We do not know whether extracellular fluid volume increases when newborn pigs are exposed to chronic hypoxia. However, based on the body weights shown in Table 1, it is highly unlikely that an increase in extracellular volume alone would account for the decreased plasma NO<SUP>−</SUP><SUB>x</SUB> found in the chronically hypoxic piglets.

As with the plasma NO<SUP>−</SUP><SUB>x</SUB> findings, the in vivo source of NO production responsible for the decreased exhaled NO output in the chronically hypoxic piglets is not known. The respiratory system is the most likely source of the reduced exhaled NO output because the limited diffusion distance of NO in biological tissue and the high affinity of hemoglobin for NO make it unlikely that NO is transported from peripheral tissues via the blood stream to the lungs. Tracheostomies obviated the upper airways as a source of differences in exhaled NO output between control and chronically hypoxic piglets. However, nitric oxide synthase (NOS) immunoreactivity has been localized in pulmonary vascular endothelium, nerves, and airway epithelium (17), so that any of these tissues in the lower respiratory system could be a source of impaired NO production and contribute to the decreased exhaled NO output found in the chronically hypoxic piglets.

Consistent with the direction of change in NO production found in the anesthetized piglets, NO<SUP>−</SUP><SUB>x</SUB> production in the perfusate of perfused lungs of the chronically hypoxic piglets was decreased. The influence of experimental conditions on NO<SUP>−</SUP><SUB>x</SUB> production merits some comment. For example, because of the difference in body weight between control and chronically hypoxic animals and the use of perfusion flows based on body weight, the absolute flow used for perfusion in this study was slightly less for the chronically hypoxic (148 ± 10 ml/min) than for the control lungs (192 ± 16 ml/min). Thus differences in pulmonary vascular endothelial shear stress (7) might be questioned as a source of the different NO<SUP>−</SUP><SUB>x</SUB> accumulation rates measured in perfused lungs of control compared with chronically hypoxic piglets. However, based on our previous finding that lung dry weight-to-body weight ratios are the same for control and chronically hypoxic piglets (13), the perfusion rates that we used ensured that the flow per gram of dry lung weight was the same in the two groups. In addition, differences in perfused surface areas are not a likely explanation for the markedly different NO<SUP>−</SUP><SUB>x</SUB> accumulation rates measured in lungs of control compared with chronically hypoxic piglets in this study because we found no difference in vascular volume between perfused lungs of the two groups.

One possible mechanism for the decreased NO<SUP>−</SUP><SUB>x</SUB> accumulation rate that we show in lungs of chronically hypoxic piglets could be that the amount of enzyme is reduced during hypoxia. This possibility is supported by our findings of decreased eNOS in lung homogenates of chronically hypoxic piglets. A contributing mechanism could be that the activity of the enzyme eNOS is reduced during chronic hypoxia. O2 is the electron acceptor in the NOS-mediated synthesis of NO from L-arginine (23), so that enzyme activity could be impaired by O2 depletion during chronic hypoxia. However, the recent finding that the Michaelis constant for O2 for the NOS activity of aortic endothelial cells was ~6 Torr (26) suggests that eNOS activity should not have been affected by very much with the reduction in inspired PO2 from 150 to 70 Torr that was experienced by the chronically hypoxic piglets in our study. It is also possible that other enzyme substrates or cofactors, such as arginine and tetrahydrobiopterin, are depleted during chronic hypoxia and thereby limit eNOS activity (38).

Potential mechanisms for the reduction in eNOS protein amounts in lungs of chronically hypoxic piglets should also be discussed. One possibility is that eNOS gene expression was impaired during chronic hypoxia. In support of this possibility, eNOS protein and mRNA levels were shown to decrease when bovine pulmonary vascular endothelial cells were cultured under hypoxic conditions for up to 72 h (19, 21). However, the effect of low O2 tension on the regulation of eNOS expression remains uncertain because other investigators (4) found that levels of eNOS protein increased when bovine aortic endothelial cells were cultured under hypoxic conditions for 24 h. Another potential explanation for the reduction in eNOS lung protein amounts is that hypoxia-induced changes in lung tissue composition, including changes in the extracellular matrix composition of vascular walls (15, 37) and/or changes in lung endothelial cell density, reduced the relative contribution from endothelial cell protein to total protein in whole lung homogenate samples from chronically hypoxic piglets compared with those from control piglets. We minimized the potential contribution from changes in vascular wall matrix composition by using peripheral lung tissues for our whole lung homogenate samples. As for endothelial cell densities, our findings of no significant differences between vascular volumes of perfused lungs of control and chronically hypoxic piglets indicate that endothelial cell surface areas did not differ between the two groups. However, it should be noted that endothelial cells may not be the sole cell type responsible for expression of eNOS in lungs (34).

It is notable that in contrast to our findings in newborn piglet lungs, an increased perfusate NO<SUP>−</SUP><SUB>x</SUB> concentration was found in lungs from chronically hypoxic adult rats (16, 22). Similarly, whereas we show decreased amounts of eNOS protein in lung homogenates of chronically hypoxic piglets, the amount of eNOS was shown to be increased in lung homogenates of chronically hypoxic adult rats (18, 33). There are a number of factors that could contribute to the differences between our results in newborn piglets and results of these latter studies in adult rats. One possibility is that the magnitude of changes in pulmonary arterial pressure, cardiac output, or hematocrit, which are all determinants of shear stress, differed between studies. In other words, it is possible that differences in the magnitude of change in shear stress, a stimulus which has been shown to alter NO production (7), could explain why chronic hypoxia altered lung NO differently in newborn pigs and adult rats. Another possibility is that the effect of either decreased O2 tension or alterations in shear stress on NO production differs with the age at which the animal is exposed to it. There is evidence from in vitro studies that O2 regulation of pulmonary NO production changes with development (32). Maturational differences in the influence of shear stress on pulmonary vascular NO production have yet to be determined. Species effects must also be considered as a source of the difference in results between studies with newborn pigs and newborn rats, particularly in light of evidence that the role of NO in the regulation of pulmonary vascular tone and reactivity may not be the same in all species (1). Additionally, differences in experimental conditions and techniques for NO<SUP>−</SUP><SUB>x</SUB> and eNOS measurement (3) could influence the results.

Although both shear stress and O2 tension have been shown to regulate NO production, it is not clear which or whether either of these stimuli are responsible for the decreased NO production found in lungs of chronically hypoxic newborn piglets. In this regard, fetal lambs with chronic pulmonary hypertension due to in utero closure of the ductus arteriosus have been found to have decreased lung eNOS (36, 41). These results support the notion that prolonged in vivo exposure to elevated pulmonary vascular resistance decreases NO production in the immature lung. On the other hand, some investigators studying chronically hypoxic adult rats have attributed the finding of increased lung eNOS to hemodynamic changes (27). In fact, the effect of hemodynamic changes on lung eNOS may be determined by species and/or postnatal age. Thus it may be that the hemodynamic changes caused by chronic hypoxia and not the low O2 tension per se are responsible for the decreased NO production found in our studies with chronically hypoxic newborn piglets.

In summary, findings in both this and our previous study (14) suggest that NO production is impaired in lungs of piglets exposed to chronic hypoxia. Findings in this study also suggest that one mechanism for the decreased NO production in lungs of chronically hypoxic newborn piglets is a reduction in eNOS protein amounts. Unlike the studies (16, 18, 22, 33) that indicate that NO production might increase with chronic hypoxia in lungs of adult rats and thereby counteract the development of pulmonary hypertension, our findings consistently support the notion that, in newborn pigs, NO production is decreased during chronic hypoxia. The decreased NO production may contribute to the development of neonatal pulmonary hypertension.

    ACKNOWLEDGEMENTS

We wish to thank Christopher Dawson and Marilyn Merker for advice and helpful comments.

    FOOTNOTES

This work was supported in part by an American Heart Association Grant-in-Aid (to C. D. Fike), a Children's Hospital of Wisconsin Foundation Grant (to C. D. Fike), and a March of Dimes Birth Defects Foundation Research Grant (to L. D. Nelin and C. D. Fike).

This work was done during the tenure of an American Heart Association Clinician Scientist Award (to L. D. Nelin).

Address for reprint requests: C. D. Fike, Research Services 151, Zablocki VAMC, 5000 W. National Ave., Milwaukee, WI 53295.

Received 25 July 1997; accepted in final form 19 December 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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