INTRODUCTION

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PULMONARY VASCULAR RESISTANCE (PVR) is elevated in the normal fetal lung because pulmonary blood flow accounts for <8-10% of the combined ventricular output of blood from the heart (15). Mechanisms responsible for the maintenance of high PVR in the fetus may include physical factors such as lack of an air-liquid interface or ventilation, relative low oxygen tension, decreased vasodilator activity, or perhaps increased vasoconstrictor activity (3, 12, 33). Endothelium-derived products, including vasodilator stimuli such as nitric oxide and prostacyclin, and vasoconstrictor stimuli, such as leukotrienes and endothelin (ET)-1, contribute to vascular tone in the fetal lung (3, 8, 10, 12, 17, 33, 37). These endothelial products may contribute not only to basal tone in the fetal lung but may also modulate responses to physiological stimuli, such as increases in pressure and shear stress (1, 8, 18). Intrauterine mechanisms of altered pulmonary vascular structure and function may be an important determinant of successful postnatal adaptation.

The ETs are a family of isopeptides consisting of ET-1, ET-2, and ET-3. ET-1 is the best-characterized member of the ET family, is a potent vasoactive peptide with comitogenic effects on vascular smooth muscle, and is produced primarily by the vascular endothelium in the normal lung circulation (7, 42). ET-1 or ET-2 is 10 times more abundant than ET-3 in the adult rat lung (27), and ET-1 is 2.5 times more potent than ET-2 in the pulmonary vasculature (28). The actions of ET-1 depend on a complex cascade of signal transduction pathways. ET-1 is initially synthesized as a 203-amino acid prepropeptide (preproET-1) that is then cleaved by an endopeptidase to proET-1 ("Big ET-1"). Big ET-1, a 38-amino acid peptide, is then converted to ET-1 by an ET-converting enzyme, which is a membrane-bound metalloprotease present in endothelial and smooth muscle cells (41). The activity of ET-1 depends on activation of at lease two distinct ET receptors (ET_A and ET_B) that may mediate different ET activities in endothelium and vascular smooth muscle cells (2). The ET_A receptors are located on smooth muscle cells and mediate vasoconstriction in most vascular beds. ET_A receptors have a high affinity for ET-1 and ET-2, with much lower affinity for ET-3 (16). ET_B receptors have equal affinity for ET-1, ET-2, and ET-3; are mostly present on endothelial cells; and may release nitric oxide with stimulation (17). Some studies have shown ET_B receptors on vascular smooth muscle mediating vasoconstriction (31); however, ET_B receptors are present only on the endothelium in the ovine fetal lung (17). Furthermore, recent studies suggest that the ET_B receptor may act as a clearance receptor for circulating ET-1 (13).

In the normal fetal lung at 1-2 wk before birth, ET-1 is present and contributes to high PVR (17, 26, 30, 37). The effects of ET-1 in the ovine fetal lung are dependent on stimulation of ET_A and ET_B receptors, which mediate vasoconstriction and vasodilation, respectively (17). However, the primary role of ET-1 and its receptors in regulation of vascular tone in the ovine fetus remains incompletely understood. Several studies have suggested that the predominant role of endogenous ET-1 in the normal ovine fetus is vasodilation (4, 40), whereas other studies have suggested that the predominant role of ET-1 is vasoconstriction (17, 37, 38). We hypothesized that from mid to late gestation ET levels increase and that the balance of ET receptor activity favors vasoconstriction. To test this hypothesis, we performed a series of molecular and physiological studies in the fetal lamb, newborn lamb, and ewe. Northern blot analysis of the mRNA for ppET-1, ET peptide levels, and cellular localization of ET-1 expression by immunohistochemistry was performed on lung tissue from the fetus, newborn lamb, and ewe. Analysis of the mRNA for the ET_A or ET_B receptors was performed at similar ages. Physiological studies utilizing selective antagonists to the ET_A or ET_B receptors were performed in fetal lambs at 120, 130, and 140 days gestation.

	METHODS

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Northern blot analysis. To determine the ontogeny of gene expression of ppET-1 and the ET_A and ET_B receptors in the ovine fetal lung, we performed Northern analysis of whole lung homogenates. At different ages, the ewe was sedated with pentobarbital sodium. Distal pieces of whole lung were rapidly frozen in liquid nitrogen (19). Total RNA was purified from 48 lung samples from separate animals with TRI Reagent (Molecular Research Center, Cincinnati, OH) and the method of Chomczynski and Sacchi (6). Lung samples were obtained from separate animals of the following gestational ages: 70 days (n = 3), 90 days (n = 5), 113 days (n = 4), 118 days (n = 4), 125 days (n = 4), 130 days (n = 4), 135 days (n = 4), and 140 days (n = 4). Samples from newborn lungs were obtained at 1 day (n = 4), 5 days (n = 4), and 13 days (n = 4) as well as samples of lung from postpartum ewes (n = 4). The RNA was quantified by measuring the absorbance at 260 nm. Twenty micrograms of total RNA per lung were analyzed with standard Northern blot and hybridization techniques using cDNA probes as previously described (19). Ovine ppET-1, human ET_A, and human ET_B cDNA probes were labeled with [-³²P]dCTP with random-primer labeling (RTS Random Primer DNA Labeling System; GIBCO BRL, Life Technologies, Gaithersburg, MD). Dr. Thomas Quertermous (Vanderbilt University School of Medicine, Nashville, TN) kindly provided the 522-bp sheep-specific ppET-1 cDNA probe (11). Dr. Scott Magnuson (Abbott Laboratories, Abbott Park, IL) kindly provided the 1,350-bp human ET_A and human ET_B cDNA probes. An 18S rRNA oligonucleotide was labeled with terminal deoxytransferase and [-³²P]dCTP. After hybridization, blots were washed at room temperature in 1× saline-sodium citrate (SSC) and 0.1% SDS (low stringency) and then at 65°C in 0.4× SSC and 0.1% SDS (high stringency). Imaging and quantification of mRNA signals was performed with a Molecular Dynamics Storm 860 phosphorimager. Normalization to 18S rRNA levels was used in quantification of mRNA signals.

ET peptide ELISA methods. To determine the ontogeny of ET protein expression in the fetal lung, we performed analysis of whole lung homogenates. Twenty-four lung samples were studied from the following ages: 70-90 days (n = 4), 118 days (n = 4), 130 days (n = 4), and 140 days gestation (n = 4) as well as the 1 day newborn (n = 4) and postpartum ewe (n = 4). Frozen tissue (100 mg) was homogenized in 1 ml of 1 M acetic acid-0.1% Triton X-100 (Sigma, St. Louis, MO) and immediately boiled for 7 min as previously reported (25). C2 columns (Amersham, Arlington Heights, IL) were equilibrated with 2 ml of methanol followed by 2 ml of deionized water. Forty microliters of homogenate were applied to the column and washed with 5 ml of 0.1% trifluoroacetic acid (TFA). The sample was eluted with 2 ml of 80% methanol-0.1% TFA, and the volume was reduced for 5 h (to near dryness) on a Speed-Vac concentrator (Savant, Farmingdale, NY). Samples were reconstituted to 250 µl, and 100 µl of sample and standard were applied to replicate wells of the ET-1 peptide ELISA kit (Cayman Chemical, Ann Arbor, MI) according to the manufacturer's instructions. The antibody used in this assay cross-reacts with ET-2 and ET-3. Briefly, samples and an acetylcholinesterase-linked ET antibody were incubated overnight at 4°C in a 96-well plate. After the samples were washed and Ellman's reagent was applied, enzyme activity was quantified by determination of optical density units at 410 nm with a DynaTech MR 700 plate reader (Bio-Tek, Winooski, VT).

Immunolocalization of ET-1 protein. To determine localization of ET-1 protein in the fetal lung, we performed immunohistochemistry. Sheep lung tissue was prepared, and immunolocalization was performed as previously described (14, 35). Three to five sections were studied from the following ages: 70 days gestation, 114 days gestation, 130 days gestation, 140 days gestation, and 1 day newborn. At autopsy, the lung was inflated by slow infusion of agarose. The pulmonary vasculature was perfused with saline, followed by buffered Formalin at 30-40 cmH₂O pressure. Paraffin sections (5 µm) were serially mounted onto Superfrost Plus slides (Fisher Scientific, Fair Lawn, NJ). The slides were deparaffinized in 100% Hemo-De and then rehydrated by immersion in 100% ethanol, 95% ethanol-5% water, 70% ethanol-30% water, and then 100% deionized water. Endogenous peroxidase activity in the tissue sections was blocked by 2% hydrogen peroxidase-60% methanol. The slides were washed in 1× PBS. Sections were blocked with Super Block (diluted 1:10 vol/vol in 1× PBS; Sky Tek, Logan, UT), washed in 1× PBS (once for 5 min), and then incubated with ET-1 antibody (QED Bioscience) diluted 1:300 or mouse IgG, negative control (Jackson Laboratories, West Grove, PA), in horse serum for 60 min. After the unbound antibody was washed off (3 times for 5 min in 1× PBS), specific ET-1 antibody binding was detected with a biotin-labeled anti-mouse secondary antibody (Vector Laboratories, Burlingame, CA) diluted at 1:200 in 2.5 ml of 1× PBS plus 50 µl of horse serum. After the excess secondary antibody was washed off in 1× PBS, the slides were incubated in streptavidin-peroxidase solution and developed with diaminobenzidine (DAB) and hydrogen peroxide, with nickel chloride for enhancement (Vector). The NiCl₂ enhancement DAB color development reaction was stopped by rinsing in water, then the slides were dehydrated in 70% ethanol-30% water; 95% ethanol-5% water; 100% ethanol; and finally 100% Hemo-De before coverslips were applied. An observer blinded to the gestational age (T. D. Le Cras) determined intensity of staining. The staining intensity was evaluated as absent, strong, or weak.

Surgical preparation . To determine whether activity of the ET_A and ET_B receptors changes during mid to late gestation, we infused selective antagonists to the ET_A and ET_B receptors at 120, 130, and 140 days gestation. All procedures and protocols were previously reviewed and approved by the Animal Care and Use Committee at the University of Colorado Health Sciences Center. Eleven mixed breed (Columbia-Rambouillet) pregnant ewes between 116 and 118 days gestation (term = 145 days) underwent surgery with methods previously described (17). Ewes were sedated with intravenous pentobarbital sodium (2-4 g) and anesthetized with 1% tetracaine hydrochloride (3 mg) by lumbar puncture. Ewes remained sedated with pentobarbital sodium but breathed spontaneously throughout the 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). A left axillary to sternal thoracotomy exposed the heart and great arteries. A catheter was inserted into the main pulmonary artery (MPA) by direct puncture through purse-string sutures. The MPA catheter was inserted between the ductus arteriosus and the pulmonic valve. A left atrial (LA) catheter was inserted in the medial portion of the left atrial appendage. An ultrasonic flow transducer (6 mm; Transonic Systems, Ithaca, NY) was placed around the left pulmonary artery (LPA) to measure blood flow to the left lung. The thoracotomy incision was closed in layers. The ewe was allowed to recover. The flow transducer cables were attached to an internally calibrated flowmeter (Transonics) for continuous measurements of LPA flow. The absolute values of flows were determined from phasic blood flow signals obtained during baseline periods as previously described (17, 21). A correction factor between end-diastolic flow and the internally calibrated zero point on the Transonics flowmeter was added to the mean flow on the Transonics flowmeter. The value obtained from this method correlates with previously determined measures of LPA flow in the late-gestation ovine fetal lung (24). Calculation of resistances is reported as left lung PVR [PVR (mmHg · ml¹ · min) = (mean MPA pressure  LA pressure)/LPA flow]. Study measurements included pulmonary arterial pressure, aortic pressure, LA pressure, LPA flow, and arterial blood gas tensions. The aortic, MPA, and LA catheters were connected to a computer-driven system to record pressures and flow (Biopac, Santa Barbara, CA). The pressure transducer was calibrated with a mercury column manometer. Blood samples for pH, arterial PCO₂, and arterial PO₂ were drawn from the aorta and measured at 39.5°C with a Radiometer OSM-3 blood gas analyzer and hemoximeter (Radiometer, Copenhagen, Denmark).

Statistical analysis. Data are presented as means ± SE. For hemodynamic parameters, statistical comparisons were made with ANOVA for repeated measures and Fisher's least significant difference test for post hoc comparisons. For all other comparisons, nonparametric analysis was performed. The Kruskal-Wallis test was performed to detect overall group differences in Northern blot analysis for preproET-1 mRNA, ET_A receptor mRNA, and ET_B receptor mRNA as well as ET lung protein content. Because a group difference was found with each variable, pairwise comparisons were made with Dunn's procedure, with an experimentwise type I error rate of 0.05.

	RESULTS

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ET-1 . Northern blot analysis of the mRNA for ppET-1 revealed a single 2.3-kb transcript as previously reported (19). We found that lung ppET-1 mRNA levels increased from 113 to 130 days gestation (Fig. 1). Lung ppET-1 levels were higher in the 13-day newborn than in the 113-day fetal lung. ET peptide levels in whole lung homogenates increased from 118 to 130 days gestation but fell at 140 days gestation before birth. ET peptide levels were greater at 130 days gestation than in the newborn lamb (Fig. 2).

View larger version (2917K): [in this window] [in a new window]   Fig. 1.   Maturation-related changes in steady-state preproendothelin-1 (ppET-1) mRNA expression in the normal ovine fetal lung. A: representative Northern blots. Nos. on top, days gestation; NB, newborn; M, maternal. B: expression of ppET-1 mRNA was greater at 130 days gestation and 13 days after birth than in the 113-day fetal lung.

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Whole lung endothelin (ET) peptide content in the normal ovine fetal lung. ET peptide content was higher at 130 days fetal gestation than at 118 and 140 days gestation as well as in the newborn lamb.

View larger version (83K): [in this window] [in a new window]   Fig. 3.   Immunolocalization of ET-1 peptide in the normal ovine fetal lung. ET-1 peptide immunostaining is noted in the vascular endothelium (open arrows) and bronchial epithelium (solid arrows). At 70 and 114 days, strong staining was seen in the bronchial epithelium, with weaker staining of isolated pneumocytes and the vasculature. At 130 days, 140 days, and in the newborn lamb, strong staining was seen in the bronchial epithelium, vascular endothelium, and isolated pneumocytes, with weaker staining in the vascular media.

ET_A receptor . Northern blot analysis of the mRNA for the ET_A receptor revealed a 5.2- and 4.2-kb transcript as previously described (25). We found that ET_A receptor mRNA levels were greater at 125 and 135 days than in the 90-day fetal lung (Fig. 4).

View larger version (2721K): [in this window] [in a new window]   Fig. 4.   Maturation-related changes in steady-state endothelin A receptor (ETA) mRNA expression in the normal ovine fetal lung. A: representative Northern blots. Nos. on top, days gestation. B: expression of ETA receptor mRNA was higher at 125 and 135 days than at 90 days in the fetal lamb.

ET_B receptor. Northern blot analysis of the mRNA for the ET_B receptor revealed a single 5.0-kb transcript as previously described (25). We found that ET_B receptor mRNA levels were greater at 125 and 135 days than in the 90-day fetal lung (Fig. 5). Lung ET_B receptor mRNA levels were greater in the 13-day newborn lung than at 90 days gestation.

View larger version (2622K): [in this window] [in a new window]   Fig. 5.   Maturation-related changes in steady-state endothelin B receptor (ETB) mRNA expression in the normal ovine fetal lung. A: representative Northern blots. Nos. on top, days gestation. B: expression of ETB receptor mRNA was higher at 125 and 135 days gestation than at 90 days in the fetal lamb. ETB receptor mRNA was also higher in the 13-day newborn than the 90-day fetus.

Hemodynamic data . Baseline values for hemodynamic and blood gas variables at 120, 130, and 140 days are shown in Table 1. Mean pulmonary arterial pressure was greater at 140 than at 120 or 130 days. LPA blood flow was greater at 140 than at 120 days. PVR was lower at 140 than at 120 days. Aortic pressure was greater at 140 than at 120 days. There was no difference in baseline values for pH, PCO₂, or PO₂ at 120, 130, or 140 days.

View larger version (11K): [in this window] [in a new window]   Fig. 6.   Dose-response study of the hemodynamic effect of BQ-788 in the normal ovine fetal lung at 135 ± 3 days. BQ-788 (1, 10, and 100 µg) did not change basal fetal pulmonary tone (data not shown). Vasodilation to SFX-S6c (1 µg over 10 min), a selective ETB receptor agonist, was blocked by BQ-788 at doses of 10 and 100 µg. PVR, pulmonary vascular resistance.

View larger version (15K): [in this window] [in a new window]   Fig. 7.   Maturation-related changes in hemodynamic responses to ET receptor antagonists in the normal ovine fetal lung at 120, 130, and 140 days gestation. The percent change in PVR to the selective ETA receptor antagonist BQ-123 was greater at 130 and 140 days gestation than at 120 days. Selective ETB receptor blockade with BQ-788 did not change basal pulmonary tone at 120, 130, or 140 days gestation.

	DISCUSSION

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Although past studies have demonstrated that ET is present in the fetal lung and contributes to the regulation of vascular tone during late gestation, the physiological roles of ET and its regulation in the normal ovine fetal pulmonary circulation are complex and poorly understood (4, 5, 17, 22, 38). In this study, we found that ET_A receptor mRNA expression and ET_B receptor mRNA increased from 90 days to 125 and 135 days gestation. These changes in ET_A receptor mRNA expression correlated with findings from physiological studies, demonstrating that ET_A receptor blockade caused pulmonary vasodilation at 130 and 140 days gestation but not at 120 days. In contrast, ET_B receptor blockade did not change basal PVR. In addition, lung ppET-1 mRNA expression increased before birth, and ET peptide expression was greatest at 130 days gestation. We conclude that the ET system is developmentally regulated and that the increase in ET_A receptor gene expression correlates with the onset of the vasodilator response to ET_A receptor blockade. Although ET_B receptor gene expression increases during late gestation, the balance of ET receptor activity favors vasoconstriction under basal conditions. These findings suggest that changes in ET receptor expression and activities play important roles in regulation of the fetal pulmonary circulation.

Previous studies have shown that ET-1 is present in the perinatal lung (26) and is vasoactive in the fetus (5, 17, 22, 34). Some investigators have suggested that the predominant physiological role of endogenous ET-1 in the normal ovine fetus is vasodilation (4, 40), whereas other studies have suggested that the predominant role of ET-1 is vasoconstriction (17, 37, 38). Many of these conclusions are based on observations based on the effects of infusions of ET-1 in the fetal lung. However, exogenous infusion of ET-1 may not accurately describe the hemodynamic effects of endogenous production of ET-1 in the fetal lung. Circulating plasma concentrations of ET-1 are lower than those reported to be biologically active (9), and secretion of ET-1 by endothelial cells is polar and directed in an abluminal direction toward the interstitial region (36). Brief infusion of ET-1 in the normal ovine fetal lung causes acute fetal pulmonary vasodilation (5); however, hypertension develops during prolonged infusion (5). 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 (4). Infusion of Big ET-1, the precursor of ET-1, causes only hypertension without vasodilation (17, 22), suggesting that stimulation of endogenous ET-1 may have very different effects than brief exogenous infusions of ET-1. We found that blockade of the ET_A receptor caused vasodilation, whereas blockade of the ET_B receptor did not change basal pulmonary tone. The present study suggests that under basal conditions the predominant role of endogenous ET in the ovine fetal circulation from 130 to 140 days is vasoconstriction.

Previous studies have shown that increased pulmonary blood flow and pulmonary arterial pressure and decreased PVR with advancing gestation is due to an increase in the total number of arterial vessels. Increased vasomotor reactivity is related to an increase in the total amount of smooth muscle, whereas the thickness of muscle in individual vessels remains constant (23). Prior studies have shown that vascular reactivity increases during late gestation (24, 29). Similarly, we found that reactivity to an ET_A receptor antagonist increased during late gestation, suggesting increased ET_A receptor-mediated vasoconstriction under basal conditions.

Little is known about the role of ET during the transition from intrauterine to newborn life. Studies in humans have revealed increased circulating ET-1 levels in the fetus and newborn in comparison with maternal controls (30, 32). Bosentan, a nonselective ET_A and ET_B receptor antagonist, did not change the increase in pulmonary blood flow or decrease in PVR with in utero oxygen ventilation (39). However, more selective antagonists were not studied. Preliminary data from our laboratory suggest that selective blockade of the ET_B receptor attenuates the fall in PVR at birth in the normal late-gestation ovine fetal lamb (20). We speculate that the fall in ET peptide levels before birth may facilitate the increase in pulmonary blood flow during the transition to newborn life. Nonetheless, the precise role of ET-1 at birth requires further investigation.

A potential limitation of our study is that we studied the activity of the ET receptors under basal conditions but did not evaluate the role of direct stimulation of the ET receptors. Stimulation of the ET_B receptor causes vasodilation in the ovine fetal lung (17); however, under basal conditions, activity of the ET_A receptor appears greater than that of the ET_B receptor. Because the ET-1 peptide assay used in this study cross-reacts with ET-2 and ET-3, the role of these peptides requires further study. However, studies suggest that in the lung, ET-1 activity is greater than the other ET peptides and that ET-1 is likely more abundant (27, 28)

In summary, expression of ppET-1 mRNA and ET peptide increased during late gestation in the fetal lamb lung. ET_A receptor mRNA expression and ET_B receptor mRNA increased from 90 days to 125 and 135 days gestation. ET_A receptor-mediated vasoconstriction increased from 120 to 140 days, whereas blockade of the ET_B receptor did not change fetal pulmonary tone. We conclude that the ET system is developmentally regulated and that the increase in ET_A receptor gene expression correlates with the onset of the vasodilator response to ET_A receptor blockade. Although ET_B receptor gene expression increases during late gestation, the balance of ET receptor activity favors vasoconstriction under basal conditions.