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PPHN is a clinical syndrome characterized by elevated pulmonary vascular resistance resulting in right-to-left shunting across the foramen ovale and ductus arteriosus with severe hypoxemia(1). Although mechanisms contributing to PPHN are poorly understood, clinical and experimental studies suggest that chronic pulmonary hypertension in utero leads to failure of the normal transition at birth(1–4). Autopsy studies have demonstrated extensive hypertensive structural pulmonary vascular lesions even in neonates with PPHN who died shortly after birth(4). Chronic intrauterine pulmonary hypertension due to ligation of the ductus arteriosus in fetal lambs causes abnormal pulmonary vasoreactivity, structural remodeling, and the failure to achieve the normal decline in pulmonary resistance at birth(2–5). Ligation of the ductus arteriosus in late-gestation fetal lambs has provided an experimental model for studying mechanisms contributing to structural and functional changes associated with perinatal pulmonary hypertension(2, 3, 5).

Past studies of this experimental model of PPHN suggest that high pulmonary vascular resistance is partly due to structural changes and an imbalance in production or responsiveness to vasodilator and vasoconstrictor stimuli(2–6). ET-1 is a potent vasoconstrictor peptide produced primarily by the vascular endothelium(7, 8) which has been implicated in some models of chronic pulmonary hypertension in adult animals(9, 10). In addition, ET-1 is present and contributes to maintenance of high pulmonary vascular resistance in the normal fetal lung(11–14). Although the role of ET-1 in clinical pulmonary hypertension is uncertain, high circulating levels of immunoreactive ET-1 have been reported in human neonates with severe PPHN(15). Whether ET-1 is simply a marker or directly contributes to high pulmonary vascular resistance in PPHN is unknown, and has not been studied in experimental models of chronic perinatal pulmonary hypertension.

The effects of ET-1 on pulmonary vascular tone are mediated by at least two ET receptors, ETA and ETB(16). In the normal ovine fetal lung, the ETA receptor is present on vascular smooth muscle and mediates vasoconstriction(13, 16), whereas the ETB receptor mediates vasodilation(13). Agonists and antagonists for the ETA and ETB receptors have been used in studies of pulmonary hypertension in some animal models(9, 10, 17), but their effects in chronic pulmonary hypertension in the perinatal period are unknown.

Therefore, we performed several experiments to study the role of ET-1 and its receptors in the development of chronic pulmonary hypertension caused by partial ligation of the ductus arteriosus in fetal lambs. We hypothesized that high pulmonary vascular resistance in experimental PPHN may be due to increased lung ET-1 production and changes in ETA and ETB receptor activities, favoring vasoconstriction. We first sought to determine changes in ETB receptor activity by examining the hemodynamic response to ET-3, a selective ETB agonist(18, 19), during chronic intrauterine pulmonary hypertension. We also studied the hemodynamic effects of ET-3 before and after selective ETB blockade with RES-701 in the normal fetus. The hemodynamic effects of selective ETB blockade with RES-701(20) have not been studied in the normal ovine fetal lung. We studied the activities of the ETA and ETB receptors in experimental pulmonary hypertension by determining the effects of the ETB receptor agonist, ET-3, the ETA and ETB receptor agonist ET-1, and the ETA receptor antagonist, BQ 123(21), in partial ligation of the ductus arteriosus in the late gestation fetal lamb. Finally, ET-1 levels were measured to determine whether endogenous production of ET-1 is increased with chronic partial ductus arteriosus ligation.

METHODS

Surgical Preparation

All procedures and protocols were previously reviewed and approved by the Animal Care and Use Committee at the University of Colorado Health Sciences Center. Mixed breed (Columbia-Rambouillet) pregnant ewes between 125 and 130 d of gestation (term = 147 d) were fasted 24 h before surgery. Ewes were sedated with i.v. pentobarbital sodium (2-4 g) and anesthetized with 1% tetracaine hydrochloride (3 mg) by lumbar puncture. Ewes were kept sedated but breathed spontaneously throughout the surgery. Penicillin 500 mg and streptomycin 1 g were administered to the ewe at surgery. Under sterile conditions, the fetal lamb's left forelimb was delivered through a uterine incision. A skin incision was made under the left forelimb after local infiltration with lidocaine (2-3 mL, 1% solution). Polyvinyl catheters were advanced into the ascending aorta and superior vena cava after insertion into the axillary artery and vein. A left thoracotomy exposed the heart and great vessels. Catheters were inserted into the left pulmonary artery and main pulmonary artery by direct puncture through purse string sutures. Catheters were guided into position with a 14 or 16 gauge i.v. placement unit (Angiocath; Travenol, Deerfield, IL). These catheters were secured by tightening the purse string suture as the introducer was withdrawn. The left pulmonary artery catheter was inserted at the bifurcation of the main pulmonary artery and the ductus arteriosus and guided through the common pulmonary artery into the left pulmonary artery. The main pulmonary artery catheter was inserted between the ductus arteriosus and the pulmonic valve. A 6.0-mm ultrasonic flow probe (Transonics, Ithaca, NY) was placed around the left pulmonary artery to measure left pulmonary artery blood flow. The ductus arteriosus was isolated and partially ligated with umbilical tape. The chest was closed with the uteroplacental circulation intact and the fetuses were gently placed in the uterus. Ampicillin 500 mg was added to the amniotic cavity before closure of the hysterotomy. The catheters and flow transducer were externalized through a skin incision in the lateral abdominal wall of the ewe and placed in a pouch until studies were performed. Ampicillin 250 mg was infused daily in the fetus and amniotic cavity during the first 3 d after surgery. In four animals, surgery was performed as above with placement of aortic and pulmonary catheters as well as a flow probe around the left pulmonary artery. In these four animals, the ductus arteriosus was left intact and left atrial catheters were placed using purse string sutures as described above. Whole lung tissue was removed for measurement of immunoreactive ET-1 levels in 10 separate control animals and seven separate animals with pulmonary hypertension caused by complete ligation of the ductus arteriosus for 7-10 d.

Physiologic Measurements

Flow transducer cables were attached to an internally calibrated flowmeter(Transonics, Ithaca, NY) for continuous measurements of left pulmonary artery flow. The main pulmonary artery, aortic, left atrial, and amniotic cavity catheters were connected to a Gould-Statham P23 ID pressure transducer. Pressures were referenced to the amniotic cavity pressure. Calibration of the pressure transducer was performed with a mercury column manometer. Measurements were continuously recorded on a Gould chart recorder. Calculation of resistances are reported as TPR (mm Hg/mL/min = main pulmonary artery pressure/left pulmonary artery flow). Before and after infusions, blood samples for pH, Paco2, and Pao2 were drawn from the main pulmonary artery catheter and were measured at 39.5°C with a Radiometer OSM-3 blood gas analyzer and hemoximeter (Radiometer, Copenhagen).

Experimental Design

Protocol 1: Effects of chronic partial ductus arteriosus ligation at 1-3 and 7-10 d (n = 6 animals). Study variables in the late-gestation fetal lamb were compared after 1-3 and 7-10 d of partial ductus arteriosus ligation. Cardiac weights (right ventricle, left ventricle + septum, and the ratio of right ventricle/left ventricle + septum) were compared between six hypertensive animals after 7-10 d of partial ductus arteriosus ligation and six control animals of similar gestational ages. Three of the control weights were obtained in twins of the hypertensive animals.

Protocol 2: Hemodynamic effects of intrapulmonary infusion of ET-3 in the normal fetus (n = 4 animals). To determine the dose related effects of ET-3 in the normal fetus, we studied the effects of ETB receptor stimulation with ET-3 before and after RES-701, a selective ETB receptor antagonist(20). After 30 min of baseline measurements in the normal fetus, ET-3 (Peptides International, Louisville, KY) was infused in the left pulmonary artery (50 and 500 ng/min for 10 min; in saline). Hemodynamic measurements were recorded for 20 min after infusion. As ET-3 (500 ng/min for 10 min) increased left pulmonary artery flow 2-fold, this dose of ET-3 was used for subsequent protocols. RES-701 (1, 5, and 10μg/min for 10 min in the left pulmonary artery; in 0.5% DMSO; Peptides International) was infused after infusion of ET-3 (500 ng/min for 10 min). On separate days with return to baseline between infusions, ET-3 (500 ng/min for 10 min in the left pulmonary artery) was infused before and after RES-701 (10μg/min for 10 min in the left pulmonary artery). Hemodynamic measurements were recorded for 20 min.

Protocol 3: Hemodynamic effects of intrapulmonary infusion of ET-3 at 7-10 d of chronic hypertension (n = 4 animals). To determine whether ETB receptor vasodilation with ET-3 is altered during chronic pulmonary hypertension, we studied the hemodynamic effects of ET-3 (500 ng/min for 10 min in the left pulmonary artery) at 7-10 d of chronic pulmonary hypertension. After 30 min of baseline measurements, ET-3 was infused in the left pulmonary artery and hemodynamic measurements were recorded for 20 min.

Protocol 4: Hemodynamic effects of intrapulmonary infusion of ET-1 at 1-3 and 7-10 d of chronic hypertension (n = 6 animals). To determine the hemodynamic effects of ETA and ETB receptor stimulation in the hypertensive fetus, we studied the serial effects of ET-1, a nonselective ETA and ETB receptor agonist (Peptides International) during early and late pulmonary hypertension. After 30 min of baseline hemodynamic measurements, ET-1 (50 ng/min; in saline) was infused for 30 min in the left pulmonary artery. The dose of ET-1 used (50 ng/min for 30 min) was based on previous experiments performed in the normal ovine fetal lung(22). Hemodynamic measurements were recorded for 60 min after the infusion.

Protocol 5: Hemodynamic effects of intrapulmonary infusion of BQ 123 at 1-3 and 7-10 d of chronic hypertension (n = 6 animals). To determine the hemodynamic effects of ETA receptor blockade in the hypertensive fetus, we studied the serial effects of BQ 123, a selective ETA receptor antagonist (Peptides International) during early and late pulmonary hypertension. After 30 min of baseline hemodynamic measurements, BQ 123 (1, 10, and 100 μg/min; in saline) was infused for 10 min in the left pulmonary artery. Hemodynamic measurements were recorded for 30 min after the infusion. The doses of BQ 123 used were based on previous experiments in the normal ovine fetal lung(13).

Protocol 6: Measurement of ET-1 immunoreactivity in normal and pulmonary hypertensive fetal lungs. ET-1 was measured in separate normal(n = 10) and hypertensive (n = 7) ovine lung homogenates of both lungs using a RIA kit from Peninsula Labs (Belmont, CA). Whole ovine lungs were isolated and weighed before homogenization in 100% cold methanol. The homogenates were placed on ice and stored at -20°C. Aliquots of the homogenate from normal or hypertensive lung were diluted 1/10 in assay buffer and SEP-packed with a C18 cartridge (Waters). The sample was eluted with 60% acetonitrile and 0.1% trifluoroacetic acid. The samples were incubated for 1 h at 4°C with 100 μL of diluted ET-1 antibody before addition of (10,000 cpm/100 μL) 125labeled-ET-1 for an overnight incubation. A charcoal solution (1.25 g of Norit A, 0.12 g of BSA in 50 mL of assay buffer) was added at room temperature for 15 min, vortexed, and centrifuged in a microcentrifuge for 2.5 min at 4°C to separate antibody and free tracer peptide. The supernatants were then removed and counted in a gamma-counter. ET-1 in the homogenates was expressed in picograms of ET-1/g wet lung weight. The antibody used for this assay cross-reacts with the precursor to ET-1, big-ET-1 (35%), and to ET-3 (7%).

Data Analysis

Data are presented as means ± 1 SEM. Statistical analysis was performed with the Statview SE software package (Abacus Concepts, Berkeley, CA). Statistical comparisons of responses between treatment periods were performed using the paired t test (protocols 2 and 3). Comparisons of continuous variables were performed using one-way repeated measures analysis of variance. Where significant differences were identified,post hoc analysis was performed using Fisher's protected least significant difference test (protocols 4 and 5). An unpaired t test was used to compare cardiac weights and immunoreactive ET-1 measurements(protocols 1 and 5). p < 0.05 was considered significant.

RESULTS

Protocol 1: Hemodynamic effects of chronic partial ductus arteriosus ligation at 1-3 and 7-10 d. Partial ductus arteriosus ligation created progressive pulmonary hypertension. Left pulmonary artery flow fell from 94 ± 11 mL/min in the normal fetus to 58 ± 7 mL/min during early pulmonary hypertension (p < 0.05) and further fell to 45± 4 mL/min during late pulmonary hypertension (p < 0.05; early versus late). Mean pulmonary artery pressure increased from 43± 4 mm Hg in the normal fetus to 60 ± 4 mm Hg during early pulmonary hypertension (p < 0.05) and further increased to 78± 3 mm Hg during late pulmonary hypertension (p < 0.05; early versus late). TPR increased from 0.52 ± 0.10 mm Hg/ml/min in the normal fetus to 1.2 ± 0.3 mm Hg/mL/min during early pulmonary hypertension (p < 0.05) and further increased to 1.9± 0.2 mm Hg/mL/min during late pulmonary hypertension (p < 0.05; early versus late) (Fig. 1). Baseline values during early pulmonary hypertension for pH (7.37 ± 0.01), Paco2 (6.9 ± 0.1 kPa) and oxygen saturation (51 ± 3%) did not change during late pulmonary hypertension. Pao2 decreased from 2.4± 0.1 to 2.1 ± 0.1 kPa (p < 0.05; earlyversus late).

Figure 1
figure 1

Partial ductus arteriosus ligation caused progressive pulmonary hypertension. TPR increased 2-fold above normal (NL) values at 1-3 d (EARLY) of partial ductus arteriosus ligation (* =p < 0.05). TPR doubled again at 7-10 d of ligation(LATE) (** = p < 0.05, EARLY vs LATE).

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Partial ductus arteriosus ligation increased right ventricular weight from 6.0 ± 0.5 g in the normal fetus to 9.1 ± 0.7 g (p < 0.05) in the hypertensive fetus after 7-10 d of partial ductus arteriosus ligation. The left ventricle + septum weight did not change with chronic pulmonary hypertension (10.8 ± 1.1 versus 10.7 ± 0.2 g). The ratio of right ventricle/left ventricle + septum increased from 0.56± 0.02 in the normal fetus to 0.85 ± 0.05 g in the hypertensive fetus (p < 0.05). There was no difference between gestational ages of the normal and hypertensive animals (135 ± 2 versus 136 ± 2 d).

Protocol 2: Hemodynamic effects of intrapulmonary infusion of ET-3 in the normal fetus. Intrapulmonary infusion of ET-3 (500 ng/min for 10 min) in the normal fetus increased left pulmonary artery flow and decreased TPR (Fig. 2) without changing pulmonary artery pressure, aortic pressure, or left atrial pressure (Table 1). A lower dose ET-3 (50 ng/min) had no hemodynamic effect. RES-701, a selective ETB receptor antagonist at a dose of 10 μg/min for 10 min, blocked the increase in left pulmonary artery flow and fall in TPR from ET-3(Table 1). RES-701 (10 μg/min for 10 min) did not change basal pulmonary tone in the ovine fetal lung (Table 1). Lower doses of RES-701 (1 and 5 μg/min for 10 min) did not block ET-3 induced vasodilation. The RES-701 carrier solution (DMSO, 0.5%) did not change pulmonary tone. Baseline values for heart rate (179 ± 7 beats per min), pH (7.35 ± 0.01), Paco2 (6.9 ± 0.1 kPa), Pao2 (2.3 ± 0.1 kPa), Hb (64 ± 4 g/L), and oxygen saturation (50 ± 5%) did not change during any infusions.

Figure 2
figure 2

In the normal ovine fetal lung (CONTROL), ETB receptor stimulation with ET-3 (500 ng/min for 10 min) decreased TPR (n = 4); however, after 7-10 d of partial ductus arteriosus ligation (HYPERTENSION), no vasodilation was seen (n = 4)(upper). The fall in TPR to ET-3 was greater in the normal fetus than during late pulmonary hypertension as the percent change in TPR was greater in the normal than the hypertensive fetal lung (lower).

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Table 1 Hemodynamic effects of ETB receptor stimulation with intrapulmonary infusion of ET-3 (500 ng/min for 10 min) with and without pretreatment with a selective ETB receptor antagonist, RES-701 (10 μg/min for 10 min), in the normal fetus
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Protocol 3: Hemodynamic effects of intrapulmonary infusion of ET-3 at 7-10 d of chronic hypertension. Intrapulmonary infusion of ET-3 (500 ng/min for 10 min) did not cause vasodilation in the hypertensive fetus during late pulmonary hypertension (Fig. 2). Baseline values for left pulmonary artery flow (45 ± 4 mL/min), pulmonary artery pressure(81 ± 4 mm Hg) aortic pressure (44 ± 3 mm Hg) and TPR (1.8± 0.1 mm Hg/ml/min) (Fig. 2) did not change with infusion of ET-3. Baseline values for heart rate (179 ± 7 beats/min), pH (7.35 ± 0.01), Paco2 (6.9 ± 0.1 kPa), Pao2 (2.3± 0.1 kPa), and Hb (64 ± 4 g/L) did not change during infusion of ET-3 in the hypertensive fetus.

Protocol 4: Hemodynamic effects of intrapulmonary infusion of ET-1 at 1-3 and 7-10 d of chronic hypertension. ET-1 decreased TPR during early but not late pulmonary hypertension (Table 2, Fig. 3). Infusion of ET-1 did not cause vasodilation during late pulmonary hypertension (Table 2, Fig. 3). ET-1 increased TPR during late pulmonary hypertension (Table 2, Fig. 3).

Table 2 Hemodynamic effects of intrapulmonary infusion of ET-1 (50 ng/min for 30 min) at 1-3 and 7-10 d of partial ductus arteriosus ligation
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Figure 3
figure 3

Nonselective ETA and ETB receptor stimulation with ET-1 (50 ng/min for 30 min) decreased TPR at 1-3 d(EARLY) but not 7-10 d (LATE) of pulmonary hypertension caused by partial ductus arteriosus ligation (n = 6). ET-1 increased TPR during late pulmonary hypertension (upper). The early vasodilator response to ET-1 was greater during early than late pulmonary hypertension as the percent change in TPR was greater during early than late pulmonary hypertension (lower).

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Protocol 5: Hemodynamic effects of intrapulmonary infusion of BQ 123 at 1-3 and 7-10 d of chronic hypertension. BQ 123 (100 μg/min for 10 min) caused greater pulmonary vasodilation during late than early pulmonary hypertension (Fig. 4). BQ 123 infusion increased left pulmonary artery flow and decreased pulmonary artery pressure during late but not early pulmonary hypertension (Table 3). TPR decreased with BQ 123 infusion (100 μg/min for 10 min) during late but not early pulmonary hypertension (Table 3). Infusion of BQ 123 at 1 and 10 μg/min for 10 min did not change baseline tone.

Figure 4
figure 4

ETA receptor blockade with BQ 123 (100 μg/min for 10 min) decreased TPR at 7-10 d (LATE) of pulmonary hypertension caused by partial ductus arteriosus ligation (n = 6) (upper). The fall in TPR to BQ 123 was greater during late than early pulmonary hypertension as the percent change in TPR was greater during late than early pulmonary hypertension (lower).

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Table 3 Hemodynamic effects of ETA receptor blockade with intrapulmonary infusion of BQ 123 (100 μg/min for 10 min) at 1-3 and 7-10 d of partial ductus arteriosus ligation
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Protocol 6: Measurement of ET-1 immunoreactivity in normal and pulmonary hypertensive fetal lungs. Chronic ductus arteriosus ligation increased tissue immunoreactive ET-1 levels from 2010 ± 28 to 5900± 50 pg ET-1/g wet lung weight (p < 0.05).

DISCUSSION

We report that chronic pulmonary hypertension caused by partial ligation of the ductus arteriosus increases lung ET-1 content and alters ET-1 receptor activity in the late gestation ovine fetus. Specifically, chronic hypertension decreases ETB-mediated pulmonary vasodilation and increases ETA-mediated vasoconstriction, which would shift receptor activity in favor of vasoconstriction. Evidence of diminished ETB activity has been demonstrated in two ways. First, vasodilation to ET-3 was markedly attenuated during chronic pulmonary hypertension. Second, although ET-1-induced pulmonary vasodilation was maintained during the first few days after partial ductus arteriosus ligation, ET-1-mediated pulmonary vasodilation decreased after prolonged hypertension, and ET-1 increased pulmonary resistance during late hypertension. Enhanced ETA-mediated vasoconstriction during partial ductus arteriosus ligation was demonstrated by serial studies using BQ 123, a selective ETA receptor antagonist. BQ 123 caused greater pulmonary vasodilation during late than early pulmonary hypertension, suggesting increased ETA receptor activity. In addition, lung ET-1 content increased 3-fold after ductus arteriosus ligation, further demonstrating that increased ET-1 production contributes to pulmonary hypertension in this experimental model. These findings suggest that chronic fetal pulmonary hypertension leads to diminished ETB receptor activity and increased ETA receptor activity.

These findings are interesting as little is known of the role of ET-1 in development of pulmonary hypertensive states. Blockade of ETA-mediated vasoconstriction attenuates the development of pulmonary hypertension and right ventricular hypertrophy in adult experimental models caused by hypoxia or monocrotaline(9, 10). Furthermore, adult animal models of pulmonary hypertension demonstrate increased expression of the ETA receptor(23) and decreased expression of the ETB receptor(24). Increased production of ET-1 has also been shown in adult pulmonary hypertension models(24–26). We have shown that pulmonary hypertension due to partial ductus arteriosus compression increases ET-1 production and leads to diminished ETB-mediated vasodilation and enhanced ETA-mediated vasoconstriction, suggesting that increased ET-1 and changes in its receptor activity at least in part mediate the altered reactivity in this model of pulmonary hypertension. It is unknown whether changes in expression of the ETA or ETB receptors are associated with these changes in ET receptor activity in the fetal lung during pulmonary hypertension.

The physiologic role of ET in the normal ovine fetal pulmonary circulation remains controversial(13, 17, 22, 27–30). Brief infusion of ET-1 in the normal ovine fetal lung acutely causes fetal pulmonary vasodilation(22, 29); however, hypertension develops during prolonged infusion(22). Although ET-1 infusions cause vasodilation in the presence of high pulmonary vascular tone, similar infusions cause vasoconstriction when the pulmonary vascular tone is decreased during acute ventilation(27). Several studies suggest that the predominant role of endogenous ET-1 in the normal ovine fetus is stimulation of the ETA receptor mediating vasoconstriction(13, 14, 17). Infusion of big-ET-1, the precursor of ET-1, causes only hypertension without vasodilation(13, 28). Furthermore, selective ETA receptor blockade with BQ 123 causes only vasodilation in the normal ovine fetus(13). Selective ETB receptor blockade with RES-701 does not change normal fetal basal tone in the ovine fetal lung, suggesting that ETA receptor-mediated vasoconstriction is more important than ETB receptor-mediated vasodilation in the ovine fetal lung. However, others suggest that the primary physiologic role of ET-1 in the ovine perinatal lung is vasodilation(30). We speculate that endogenous ET-1 production in the normal ovine fetal lung appears to predominantly cause vasoconstriction, not vasodilation.

Mechanisms which maintain high pulmonary vascular resistance in neonatal pulmonary hypertension remain incompletely understood, but are likely to involve an imbalance of endogenous vasodilator and vasoconstrictor activities. Findings from this study and others(14, 17, 22, 27, 28) suggest that ET-1 may contribute to vasoconstriction and altered vasoreactivity in experimental PPHN. An imbalance in the nitric oxide-cGMP system and ET system may also contribute to pulmonary hypertensive states. Diminished nitric oxide production and altered smooth muscle cell responsiveness are known to contribute to pulmonary hypertension(6, 31–34). The decrease in nitric oxide production may lead to increased ET-1 production(35). Increased ET-1 activity also causes smooth muscle proliferation, which may further increase pulmonary resistance in this model(36). Although ET-1 has been shown to contribute to the development of pulmonary hypertension due to chronic hypoxia or monocrotaline-induced hypertension(9, 10), the role of ET-1 in development of pulmonary hypertension during partial ductus arteriosus ligation remains unclear. It appears that ET-induced vasoconstriction is important in the development of pulmonary hypertension in this model as ETA blockade caused greater vasodilation during late than early pulmonary hypertension.

Several mechanisms may explain the attenuation of ETB receptor vasodilation during chronic pulmonary hypertension. ET-induced vasodilation may be mediated by nitric oxide production(13, 37), ATP sensitive potassium channels(37), or prostacyclin production(38). Chronic pulmonary hypertension may decrease ET-1 induced production of these vasodilating stimuli by decreasing nitric oxide synthase or prostacyclin synthesis. Furthermore, chronic pulmonary hypertension may alter production of receptors leading to altered vasoreactivity. Monocrotaline-induced pulmonary hypertension causes decreased ETB receptor number as measured by Northern analysis(24), but this has not been shown in pulmonary hypertension due to partial ductus arteriosus ligation. Enhanced ETA activity has been shown in pulmonary hypertension due to hypoxia(10, 23, 39) and monocrotaline(9), but has not been shown in pulmonary hypertension from partial ductus arteriosus ligation. Finally, diminished ETB receptor numbers may lead to decreased clearance of ET-1 in the lung leading to increased circulating ET-1(40). The mechanism of diminished ETB mediated vasodilation in this model must be further examined.

ET-1 levels are increased in many human disorders of pulmonary hypertension. Elevated immunoreactive ET-1 levels have been found in primary pulmonary hypertension, the Eisenmenger syndrome(41), PPHN(15), and children with pulmonary hypertension associated with congenital heart disease and bronchopulmonary dysplasia(42). Increased expression of endothelin-1 in vascular endothelial cells has been reported in adult patients with primary pulmonary hypertension, suggesting that the local production of endothelin-1 may contribute to the altered vascular reactivity and structural changes seen in pulmonary hypertension(43). Whether ET-1 contributes to high pulmonary vascular resistance or is simply a marker of disease in patients with severe pulmonary hypertension is uncertain.

In summary, chronic intrauterine pulmonary hypertension decreases vasodilation to ET-1 and ET-3, suggesting that ETB receptor activity is diminished. ETA-mediated vasoconstriction significantly contributes to pulmonary hypertension in partial ductus arteriosus ligation as ETA blockade caused a greater fall in pulmonary resistance during late than early pulmonary hypertension. Chronic ductus arteriosus ligation increased lung ET-1 content 3-fold higher than the normal late-gestation fetus. We conclude that chronic intrauterine pulmonary hypertension causes the loss of ETB-mediated vasodilation, persistent ETA-mediated vasoconstriction, and increased lung ET-1 content. We speculate that diminished ETB receptor-mediated vasodilation in combination with enhanced ETA receptor-mediated vasoconstriction contributes to high pulmonary resistance in perinatal pulmonary hypertension.