INTRODUCTION

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DESPITE THE PROBABILITY that the pathophysiology of pulmonary hypertension may differ between newborns and adults, newborn models of this disease have received relatively little study compared with adult models. To improve the understanding of the pathogenesis of neonatal pulmonary hypertension, our laboratory (4-6) developed a model of chronic hypoxia-induced pulmonary hypertension in newborn piglets. In addition to developing pulmonary hypertension, newborn piglets exposed to 10-12 days of chronic hypoxia exhibit blunted pulmonary arterial responses to the endothelium-dependent dilator acetylcholine and have reduced lung endothelial nitric oxide synthase (eNOS) amounts (5, 6). These latter findings suggest that impaired endothelial function might contribute to the pathogenesis of pulmonary hypertension in the newborn.

Devising therapies that might prevent the onset or progression of pulmonary hypertension has received particularly little attention in newborns in comparison to studies with adult animals. For example, treatment with calcium-channel blockers such as nifedipine has been shown to ameliorate the development of chronic hypoxia-induced pulmonary hypertension in adult animals (17), but the efficacy of these agents in preventing pulmonary hypertension in neonates has not been evaluated. Moreover, the mechanisms by which nifedipine attenuates the development of pulmonary hypertension in adults remain unknown. Because endothelial dysfunction is one potential cause of pulmonary hypertension (11, 20, 22), it is possible that nifedipine treatment ameliorates pulmonary hypertension, at least in part, by improving endothelial function. The purposes of the present study were to determine whether treatment with nifedipine would ameliorate the development of pulmonary hypertension in chronically hypoxic newborn piglets and prevent the altered endothelium-dependent responses that we have found in this newborn model. In addition, because reduced eNOS amounts might underlie altered endothelium-dependent responses in the lungs of chronically hypoxic newborn piglets (6), we evaluated eNOS amounts in whole lung homogenates from both nifedipine-treated and untreated control and chronically hypoxic piglets.

	METHODS

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Animals. Newborn piglets (1-3 days old) were placed in chambers with either a room air environment (control; n = 13) or a hypoxic normobaric environment (chronic hypoxia; n = 24) for 10-12 days. Except for the case of one control piglet, two piglets were placed in each chamber. For the chronically hypoxic piglets, the normobaric hypoxic environment was produced by delivering compressed air and nitrogen to an incubator (Thermocare). The O₂ content was regulated at 8-10% (PO₂ 60-72 Torr), and PCO₂ was maintained at 3-6 Torr by absorption with soda lime. The chamber was opened two to three times a 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. Piglets were randomly chosen from both groups (n = 5 control and 10 hypoxic) for nifedipine treatment (3-5 mg/kg sublingually three times a day). The dose of nifedipine was chosen because in pilot studies with chronically hypoxic piglets, we found that lower doses had no effect on pulmonary vascular resistance and that larger doses were associated with a high incidence of unexpected death. Some nifedipine-treated piglets were raised in the same chamber with an untreated animal from the same group (n = 3 control and 5 hypoxic). Because Fike et al. (6) previously found no differences in endothelium-dependent responses or lung eNOS amounts between control piglets raised on the farm and control piglets raised in a normoxic chamber, 11 untreated 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, all piglets 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 into the right femoral artery for monitoring systemic blood pressure and arterial blood gases. Next, for most piglets (n = 16 untreated control, 5 nifedipine-treated control, 13 untreated hypoxic, and 11 nifedipine-treated hypoxic), another catheter was placed through the right external jugular vein into the pulmonary artery to monitor pulmonary arterial pressure. To obtain 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 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 blood gases were measured, all animals were given heparin (1,000 IU/kg iv) and additional anesthesia (3-5 mg/kg of pentobarbital sodium iv) and then exsanguinated. Most lungs were left in situ and used for perfusion as described in Measurements in isolated perfused lungs. A few lungs (n = 1 untreated control and 1 untreated hypoxic) were immediately frozen in liquid nitrogen and stored at 80°C for later analysis of eNOS as described in Immunoblot analysis.

Measurements in isolated perfused lungs. For lung 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), with a tidal volume of 15-20 ml/kg and a respiratory rate of 15-20 breaths/min (mean airway pressure of 4-6 cmH₂O). 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. The vascular cannulas were connected to the perfusion circuit as previously described (4, 5). The perfusion circuit was filled with 100-200 ml of the animal's own blood collected during exsanguination (as described in Measurements in anesthetized animals) and mixed with 50-100 ml of 3% albumin-saline. The perfusion circuit included a rotary pump (model 7523-00, Cole Palmer Masterflex) that 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.

After connection to the perfusion circuit, the lungs were perfused for 30-60 min until a stable pulmonary arterial pressure was achieved. The perfusate flow and left atrial pressure were adjusted to respective levels of 50 ml · min¹ · kg¹ and 0 cmH₂O and were maintained constant for the remainder of the study.

Changes in pulmonary arterial pressure in response to the endothelium-dependent agent acetylcholine were then measured in some lungs. Because baseline pulmonary arterial pressure is greater in chronically hypoxic than in control animals (see Refs. 5 and 6 and RESULTS), the pulmonary arterial pressure in the control animals was elevated to a level comparable to that in the chronically hypoxic animals by adding 1 M KCl to the perfusate of the control lungs. Acetylcholine was added cumulatively to the reservoir to achieve concentrations of 10⁹ to 10⁶ M. The acetylcholine responses were transient so that the pulmonary arterial pressure was allowed to return to baseline between doses. After the response to acetylcholine was measured, papaverine was added to the reservoir (300 µg/ml of perfusate) of most lungs and allowed to circulate for 20 min to determine the contribution of vasomotor tone to pulmonary arterial pressure. In a few other lungs (n = 5 untreated control and 2 untreated hypoxic), a similar protocol was followed except that the responses to acetylcholine were not measured before papaverine was added to the perfusate.

At the end of perfusion, some lungs from each group (n = 4 untreated control, 5 nifedipine-treated control, 4 untreated hypoxic, and 4 nifedipine-treated hypoxic) were then immediately frozen in liquid nitrogen and stored at 80°C for later analysis of eNOS as described in Immunoblot analysis.

Immunoblot analysis. Tissue pieces that did not contain large airways or large vessels were selected from frozen perfused and unperfused lungs from treated and untreated piglets from both the control (n = 4 perfused and 1 unperfused untreated and 5 perfused nifedipine treated) and chronically hypoxic (n = 4 perfused and 1 unperfused untreated and 4 perfused nifedipine treated) groups. The tissue pieces were homogenized in 10 mM HEPES buffer containing 250 mM sucrose, 3 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride, pH 7.4, on ice with 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 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 buffer [Novex; 0.25 M Tris · HCl, 5% (wt/vol) SDS, 2.5% (vol/vol) -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 two Tris-glycine precast 8% polyacrylamide gels (Novex) so that 10 µg of protein lung homogenate samples were loaded on both gels. We used 10-µg protein samples because Fike et al. (6) previously found that this amount of protein was within the dynamic range of our immunoblot analysis. Each gel contained 10-µg protein samples from at least one nifedipine-treated and one untreated lung from both the control and chronically hypoxic groups. Furthermore, to normalize the results between the two different blots, at least one lane of each gel was loaded with 10 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 10 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 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 ABC 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 with enhanced chemiluminescence reagents (Amersham), and the chemiluminescent signal was captured on X-Ray film (Bio-Max MR, Kodak) with 1-min exposures. The bands for eNOS were quantified with 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 the gels.

Statistics. Data are presented as means ± SE. A one-way ANOVA with a multiple comparison test was used to compare the data between nifedipine-treated and untreated control and chronically hypoxic animals. P < 0.05 was indicative of significance.

	RESULTS

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After 10-12 days of hypoxia, nifedipine-treated and untreated chronically hypoxic piglets had higher hematocrits than both groups of control piglets (Table 1). The measured values of blood pH, PO₂, and PCO₂ obtained during hemodynamic measurements in anesthetized piglets breathing room air were similar between all groups of piglets except for a slightly higher PCO₂ in the nifedipine-treated control group (Table 1). For the perfused lungs, the hematocrits in both groups of chronically hypoxic piglets were slightly higher than the hematocrits in both groups of control piglets. The measured values of pH, PO₂, and PCO₂ in the perfusate of isolated lungs from both the nifedipine-treated and untreated chronically hypoxic piglets were not different from those in the control piglets (Table 1).

Measurements of pulmonary arterial pressure, pulmonary wedge pressure, left ventricular end-diastolic pressure, cardiac output, and aortic pressure in the anesthetized piglets are shown in Table 2. Pulmonary arterial pressure was greater in the untreated chronically hypoxic piglets than in the other three groups of piglets. The pulmonary arterial pressure in the nifedipine-treated group of chronically hypoxic piglets was intermediate in value between that in both groups of control piglets and that in untreated chronically hypoxic piglets. Pulmonary wedge pressure did not differ among nifedipine-treated chronically hypoxic, untreated control, and nifedipine-treated control piglets but was less in these three groups than in the untreated chronically hypoxic piglets (Table 2). Measurements of left ventricular end-diastolic pressure did not differ significantly from measurements of pulmonary wedge pressure (Table 2) and were used to calculate pulmonary vascular resistance if we were unable to obtain a pulmonary wedge pressure. Cardiac output did not differ between the untreated groups of control and hypoxic piglets but was greater in the nifedipine-treated piglets than in their corresponding group of untreated piglets (Table 2). The calculated value of pulmonary vascular resistance [(pulmonary arterial pressure  pulmonary wedge pressure)/cardiac output] was greater for the untreated chronically hypoxic piglets than for all the other groups of piglets (Table 2, Fig. 1). Most importantly, the pulmonary vascular resistance was the same in the nifedipine-treated chronically hypoxic piglets as in the untreated group of control piglets (Table 2, Fig. 1).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Pulmonary vascular resistance in anesthetized control (n = 16 untreated and 5 nifedipine-treated) and chronically hypoxic (n = 13 untreated and 11 nifedipine-treated) piglets. Values are means ± SE. Significantly different (P < 0.05) from: * untreated control piglets; + nifedipine-treated control piglets; # nifedipine-treated chronically hypoxic piglets (by 1-way ANOVA with multiple comparison test).

In the perfused lungs, left atrial pressure and perfusate flow were the same in all groups and were maintained constant throughout the study so that changes and differences in pulmonary arterial pressure, respectively, represent changes and differences in pulmonary vascular resistance. Note that the baseline pulmonary arterial pressure did not differ between the nifedipine-treated and the corresponding group of untreated piglets (Table 3) and that the baseline pulmonary arterial pressure was greater in both groups of hypoxic piglets than in either group of control piglets (Table 3). After the addition of KCl, the pulmonary arterial pressure in the control groups was raised to a level comparable to that in the hypoxic groups (Table 3). Addition of all doses of acetylcholine resulted in similar decreases in pulmonary arterial pressure in the nifedipine-treated piglets as in the corresponding groups of untreated piglets (Table 3, Fig. 2). The response to all but the lowest dose of acetylcholine was less in both the nifedipine-treated and untreated groups of hypoxic lungs compared with either group of control lungs (Table 3, Fig. 2). The addition of papaverine and removal of vasomotor tone resulted in a pulmonary arterial pressure in the nifedipine-treated group of chronically hypoxic piglets that was less than the pulmonary arterial pressure in the untreated group of chronically hypoxic piglets and that did not differ from the pulmonary arterial pressure in either the treated or untreated group of control piglets (Fig. 3).

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Change in pulmonary arterial pressure with acetylcholine in isolated, perfused lungs from control (n = 8 untreated and 5 nifedipine-treated) and chronically hypoxic (n = 5 untreated and 10 nifedipine-treated) piglets. Values are means ± SE. Significantly different (P < 0.05) from: * untreated control piglets; + nifedipine-treated control piglets (by 1-way ANOVA with multiple comparison test).

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Pulmonary arterial pressure after papaverine in isolated, perfused lungs from control (n = 13 untreated and 5 nifedipine-treated) and chronically hypoxic (n = 7 untreated and 8 nifedipine-treated) piglets. Values are means ± SE. Significantly different (P < 0.05) from: * untreated control piglets; + nifedipine-treated control piglets; # nifedipine-treated chronically hypoxic piglets (by 1-way ANOVA with multiple comparison test).

One of the immunoblot analyses for eNOS in the whole lung homogenates is shown in Fig. 4. In both the nifedipine-treated and untreated groups of control and chronically hypoxic lungs, the antibody to eNOS detected eNOS at an apparent molecular mass of 135 kDa as determined from an exponential fit of molecular-mass standards. Figure 5 shows the mean data for the absorbance of the eNOS bands as determined by laser densitometry for the whole lung homogenates from the untreated and nifedipine-treated control and the untreated and nifedipine-treated chronically hypoxic lungs and shows that absorbance of the eNOS bands was less for the lungs from both the nifedipine-treated and untreated chronically hypoxic piglets compared with that for the lungs from the untreated control piglets.

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Immunoblot for endothelial nitric oxide synthase (eNOS) in homogenates of lungs from untreated control (C), nifedipine-treated control (NC), untreated chronically hypoxic (H), and nifedipine-treated chronically hypoxic (NH) piglets. Standard, 10-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 that intensity of eNOS band is greater for 10-µg sample of untreated control lung homogenates than for 10-µg sample of either untreated or nifedipine-treated chronically hypoxic lung homogenates. Nos. on right, molecular mass.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Absorbance of eNOS bands as determined by laser densitometry for whole lung homogenate samples from control (n = 5 untreated and 5 nifedipine-treated) and chronically hypoxic (n = 5 untreated and 4 nifedipine-treated) piglets. Values are means ± SE. Note that absorbance of eNOS bands does not differ between untreated and treated lungs of corresponding control and chronically hypoxic groups and that absorbance of eNOS bands is less in lungs from both untreated and nifedipine-treated chronically hypoxic groups compared with lungs from untreated control group. * Significantly different from untreated control piglets, P < 0.05 by 1-way ANOVA with multiple comparison test.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

In agreement with the findings of previous studies by Fike and colleagues (4-6), in this study we show that when exposed to 10-12 days of chronic hypoxia, newborn piglets develop pulmonary hypertension. Also in agreement with previous studies by Fike and colleagues (5, 6), the chronically hypoxic piglets exhibit blunted pulmonary vascular responses to the endothelium-dependent dilator acetylcholine and have reduced amounts of lung eNOS. A new finding is that the pulmonary vascular resistance in newborn piglets treated with nifedipine during chronic hypoxia did not differ from that in control piglets. This finding is consistent with the results of a study in adult models of pulmonary hypertension (17) and indicates that treatment with nifedipine ameliorates the development of pulmonary hypertension in our newborn model. Another new finding in this study is that the pulmonary arterial responses to acetylcholine were blunted and eNOS amounts were reduced in the lungs from chronically hypoxic piglets regardless of whether or not they were treated with nifedipine. This latter finding indicates that mechanisms other than an improvement in endothelial function or restoration of lung eNOS amounts underlie the ameliorative effect of nifedipine.

Although pulmonary vascular resistances in nifedipine-treated hypoxic and untreated control piglets did not differ in this study, the in vivo pulmonary arterial pressure was greater in the nifedipine-treated chronically hypoxic piglets than in the control piglets (Table 2). In addition, in the perfused lungs, the baseline pulmonary arterial pressure was greater in the nifedipine-treated hypoxic piglets than in the control piglets. This latter finding can be attributed to the influence of vasomotor tone in the isolated lung preparation because there was no diffference between pulmonary arterial pressures in nifedipine-treated hypoxic and untreated control lungs once the vasomotor tone was removed (Fig. 3). In regard to the in vivo measurements, it is probable that the measurement of pulmonary arterial pressure was affected by differences in left atrial pressure and cardiac output. Moreover, even though not as low as in the control piglets, the pulmonary arterial pressure in the nifedipine-treated hypoxic piglets was less than that in the untreated hypoxic piglets. This latter finding clearly indicates that although pulmonary hypertension was not completely prevented, nifedipine treatment inhibited the development of the disorder.

Had we used higher doses of nifedipine as in a study with chronically hypoxic adult rats (17), we might have achieved even greater reductions in pulmonary vascular resistance in the hypoxic piglets. However, the dose that we used is high compared with common clinical usage in newborns (1, 7), and in pilot studies, we found that larger doses than these were not well tolerated by the newborn piglets. The mechanism by which nifedipine treatment inhibits the development of pulmonary hypertension in either our newborn model or adult models remains unknown. Of course, part of the difficulty in determining the mechanism underlying the ameliorative effect of nifedipine is the lack of understanding of the pathogenesis of pulmonary hypertension in either newborns or adults. Endothelial dysfunction is one potential cause of pulmonary hypertension (11, 20, 22). Another possibility is that either in association with or separate from endothelial dysfunction, decreased production of endothelium-derived vasodilators such as nitric oxide could contribute to the development of pulmonary hypertension (11, 20, 22). Although the findings of Fike and colleagues (5, 6) and those of others (16, 19, 21) are supportive of the preceding mechanisms as a cause of neonatal pulmonary hypertension, our findings in the present study are also notable for providing evidence that mechanisms other than an improvement in endothelial function or restoration of lung eNOS amounts underlie the ameliorative influence of nifedipine. Indeed, one logical possibility is that nifedipine provided the reduction in smooth muscle tone, i.e., vasodilatory effect, that the dysfunctional pulmonary vascular endothelium and/or reduced lung eNOS could not.

The ability of nifedipine to reduce pulmonary arterial pressure and inhibit hypoxic pulmonary vasoconstriction from a smooth muscle cell vasorelaxant effect has been previously proposed as a mechanism for preventing pulmonary hypertension in chronically hypoxic adult animals (9, 17). That nifedipine can be an effective pulmonary vasodilator even in the nonhypoxic newborn pulmonary circulation is suggested by the slightly lower pulmonary vascular resistance in the nifedipine-treated than in the untreated control piglets (Fig. 1, Table 2). In addition to blockade of smooth muscle cell calcium channels, the vasodilatory effect of nifedipine could result from altered production of vasoactive agents. For example, nifedipine inhibited the production of the vasoconstrictor thromboxane by cultured rabbit aortic endothelial cells (13). Yet, it is important to note that despite effectively lowering pulmonary arterial pressure, not all smooth muscle cell vasodilators have been shown to inhibit the development of chronic hypoxia-induced pulmonary hypertension (17). The ability of nifedipine to ameliorate pulmonary hypertension might be more complex than can be explained by smooth muscle cell dilation.

One possibility is that nifedipine has an inhibitory effect on smooth muscle cell proliferation (12). Another possibility is that nifedipine modulates metabolism of collagens within the extracellular matrix (15). Both of these effects would help maintain a lower pulmonary vascular resistance by preventing the pulmonary vascular remodeling associated with chronic hypoxia-induced pulmonary hypertension (17, 18). The mechanism for these effects could involve a decrease in intracellular calcium concentration due to the inhibition of voltage-gated L-type calcium channels. That is, nifedipine might ameliorate pulmonary vascular wall changes by inhibiting the calcium-triggered transcription of a myriad of genes (14). Additional pathways independent of calcium may also be involved (3).

Supportive of the potential for the above-noted protective effects of nifedipine, other investigators (17) found that nifedipine treatment inhibited pulmonary vascular remodeling in chronically hypoxic adult animals. Because we did not perform morphometry, we do not know with certainty that nifedipine prevented the pulmonary vascular remodeling that we (4) and others (8) have found in chronically hypoxic newborn piglets. However, our finding that with removal of vasomotor tone, the pulmonary vascular resistance in isolated lungs from nifedipine-treated hypoxic piglets did not differ from that in untreated control piglets provides evidence that the pulmonary vascular bed does not differ structurally in these two groups of piglets (Fig. 3). In addition, consistent with our previous findings (4), structural remodeling is the most likely explanation for the persistently greater pulmonary arterial pressure in the untreated hypoxic lungs than in all other groups of lungs even with removal of vasomotor tone (Fig. 3).

Our finding that lung eNOS amounts were reduced in the lungs of hypoxic piglets regardless of nifedipine treatment merits some additional comments. Oxygen tension and shear stress are two stimuli that have been shown to regulate eNOS expression (2, 10). In the chronically hypoxic piglet model, oxygen tension changes but so do a number of determinants of shear stress including pulmonary arterial pressure, cardiac output, and hematocrit. Whether any or all of these stimuli are responsible for the reduced eNOS observed in the lungs of chronically hypoxic piglets remains unknown. Of these stimuli, it is of interest that findings in fetal lambs with pulmonary hypertension due to in utero closure of the ductus arteriosus support the notion that prolonged in vivo exposure to elevated pulmonary arterial pressure decreases lung eNOS in immature animals (16, 21). In this regard, the reason that lung eNOS amounts remained reduced despite nifedipine treatment could be due to the failure of nifedipine to completely reduce pulmonary arterial pressure in the hypoxic piglets to the level of control animals. Other possibilities are that the potential to restore lung eNOS by lowering pulmonary arterial pressure was offset by a direct effect of hypoxia or by some other effect of the nifedipine treatment such as increased cardiac output.

In summary, we found that nifedipine-treatment attenuated the development of pulmonary hypertension in a newborn model and that the effectiveness of the treatment was not due to an improvement in endothelial dysfunction nor to preventing a reduction in lung eNOS amounts. It seems logical that the presence of endothelial dysfunction and decreased lung eNOS amounts increase the likelihood that there will be worsening of the pulmonary hypertensive process so that counteracting, reversing, or preventing these alterations remains a reasonable therapeutic target that requires further exploration.