Introduction

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Numerous in vitro and in vivo studies have explored the vascular effects of ET-1 in the lung (Hay and Goldie, 1995). This 21-amino acid peptide, which is synthesized and released from various cell types in the lung including the endothelium, epithelium and macrophage (Giaid et al., 1991; Ehrenreich et al., 1990; MacCumber et al., 1989; Ohlstein et al., 1990), elicits both pulmonary vasoconstriction and pulmonary vasodilation, as well as bronchial vasoconstriction and contraction of airway smooth muscle (Barnard et al., 1991; Barman et al., 1993;, Henry et al., 1990; Lippton et al., 1989; Turner et al., 1989; Wong et al., 1993). The complex response profile in the pulmonary vasculature has been attributed primarily to differential activation of distinct subpopulations of ET receptors as well as to dependence on the segment and level of tone in the pulmonary vascular bed (MacLean et al., 1994). Based on a functional characterization (Warner et al., 1993), it appears that ET_A and ET_B2 receptor subtypes mediate vasoconstriction in some pulmonary vessels (Hay et al., 1993; Warner et al., 1993), whereas the ET_B1 receptor subtype mediates vasodilation (Pinheiro and Malik, 1993; Wong et al., 1995). In addition to vasomotor actions, ET-1 acts via ET_A receptors to elicit a potent mitogenic response in vascular smooth muscle cells obtained from human pulmonary artery (Zamora et al., 1993).

Recent evidence suggests that ET-1 may play a pathophysiological role in hypoxic regulation of pulmonary vascular resistance. In rats, acute pulmonary alveolar hypoxia is associated with elevated levels of ET-1 in the plasma and the lung (Horio et al., 1991; Shirakami et al., 1995). Increases in circulating ET-1 levels were also reported in children with hypoxic pulmonary hypertension (Allen et al., 1993). In addition, plasma ET-1 levels correlate well with pulmonary arterial pressure and arterial pO₂ in healthy mountaineers (Goerre et al., 1995). Similar correlations of plasma ET-1 with the driving pressure across the pulmonary circulation have been described in patients with chronic congestive heart failure (Cody et al., 1992).

To date, the role of ET-1 in hypoxic pulmonary hypertension has been investigated with selective and nonselective ET receptor antagonists. For example, BQ-123, a selective ET_A receptor antagonist (Ihara et al., 1991), has been shown in vivo to inhibit and reverse acute hypoxic pulmonary hypertension in the rat and lamb, respectively (Oparil et al., 1995; Wang et al., 1995). Controversy exists, however, and the role of ET in pulmonary hypertension associated with hypoxia has been disputed (Wong et al. 1993; Douglas et al., 1993).

The purpose of the present study was to characterize the role of ET-1 in the regulation of pulmonary blood flow. Specifically, the effects of a potent nonpeptide mixed ET_A and ET_B receptor antagonist, SB 209670 (Ohlstein et al., 1994), were examined in the canine isolated pulmonary artery in vitro and in a canine model of acute hypoxic pulmonary hypertension in vivo.

	Methods

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Preparation of isolated canine pulmonary arteries. The preparation of vascular segments has been described in detail elsewhere (Willette et al., 1994). Lungs were removed from healthy male mongrel dogs (10-14 kg) and placed in modified Krebs-Henseleit solution. The composition of the Krebs-Henseleit solution, which was gassed with 95% O₂, 5% CO₂ and maintained at 37°C, was (mM): NaCl, 113.0; KCl, 4.8; CaCl₂, 2.5; KH₂PO₄, 1.2; MgSO₄, 1.2; NaHCO₃, 25.0; glucose, 5.5. Secondary intralobar branches of the pulmonary artery (2-3 mm diameter) were dissected free, cleaned and cut into rings approximately 2 mm wide. In some preparations the endothelium was removed by rotating the tissues several times over the shank of a 21-gauge needle. The segments were placed in tissue baths containing Krebs-Henseleit solution and connected via silk suture and a tungsten hook to a Grass FT03C force-displacement transducers (Grass Instruments, Quincy, MA). Mechanical responses were digitized, displayed, analyzed, stored and graphed using a Biopac System (Goleta, CA). Tissues were equilibrated at a resting tension of 1.5 g for 1 hr and washed every 15 min with fresh Krebs-Henseleit solution before the start of each experiment.

In vitro experimental protocol. After the equilibration period tissues were exposed to 100 mM KCl. After plateau of this reference contraction, tissues were washed several times for 30 to 45 min to return tension to base-line level. Subsequent contractile responses were expressed as a percentage of the reference contraction.

In vivo surgical preparation. The 27 healthy male mongrel dogs (10-14 kg) used for these experiments were housed in an accredited laboratory animal facility. All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals (US Department of Health, Education, and Welfare [Dept. of Health and Human Services] publication NIH 85-23) and were approved by the Institutional Animal Care and Use Committee of SmithKline Beecham Pharmaceuticals.

In vivo experimental protocol. After the surgical procedure, artificial ventilation parameters (approximately 15 ml/kg/breath and 12 breaths/min) were adjusted during an equilibration period to obtain pO₂ and pCO₂ in the normal range (95-115 mm Hg and 34-44 mm Hg, respectively). Once stabilized, vehicle (saline) or SB 209670 (3 or 30 µg/kg/min) was infused intravenously for 90 min. At 60 min into the infusion, hemodynamic and blood gas parameters were monitored (control) and the inspired gas mixture was changed from room air to 10% O₂ in 90% N₂ for the remaining 30 min of the infusion. In a separate group, after a 60-min saline infusion, SB 209670 (1 mg/kg i.v. bolus + 30 µg/kg/min) was started 3 to 5 min after beginning the 10% O₂ challenge, i.e., during the plateau of the pulmonary hypertensive response. All parameters were monitored every 10 min in each group. At the conclusion of each experiment, the mPAP-WP response to hypoxia (30 min) was quantified by determining the AUC by digital image analysis (NIH Image, Bethesda, MD). Calculated parameters were derived as follows: CI equals the CO divided by body surface area (length × height in meters); total peripheral resistance equals MAP divided by CI; and pulmonary vascular resistance equals mPAP-WP divided by CI.

ET-1-like ELISA. Ethylenediaminetetraacetic acid-plasma blood samples (3 ml) were collected from the femoral artery at various time points before and during the hypoxic period in vehicle- and SB209670-treated animals. Plasma samples were prepared immediately and frozen (<20°C) until assayed. The plasma samples were extracted (92% ecovery) and ET-1-LI was determined with the Human Endothelin-1 Parameter Elisa Assay (R&D Systems, Minneapolis, MN). The following levels of cross-reactivity have been reported with this assay: big ET (<1%), sarafrotoxin (<2%), ET-2 (45%) and ET-3 (14%). Sample assays were performed in duplicate, and group results were expressed as the mean (picograms per milliliter) ± S.E.M.

Statistical methods. All summary values were expressed as the mean ± standard error of the mean. Agonist-induced contraction in vitro was expressed as a percentage of the reference contraction (100 mM KCl). Where appropriate, the nature of the antagonism and the potency (pA₂) of antagonists at the ET receptor(s) were determined in canine pulmonary artery by the technique of Arunlaksana and Schild (1959). Dose ratios (the concentration of ET-1 required to produce a half-maximal response [EC₅₀] in the presence of SB 209670 divided by the EC₅₀ of ET-1 obtained in untreated arteries) were calculated for several concentrations of SB 209670. The log (dose ratio  1) was plotted against the log[SB 209670] (molar) on the abscissa. The slope of the regression line will not differ significantly from 1 if the antagonism is competitive; and the x-intercept, indicative of potency, is the pA₂ (logK_B). Comparisons were made by an analysis of variance followed by post hoc analysis with the Bonferroni test where probability (P)  .05 was considered to be statistically significant (Wallenstein et al., 1980).

Drugs and solutions. SB 209670 was synthesized by colleagues in the Department of Medicinal Chemistry at SmithKline Beecham Pharmaceutics (King of Prussia, PA). SB 209670 solutions for intravenous infusion were prepared in sterile saline just before administration. ET-1, S6c and BQ-123 were purchased from American Peptide (Sunnyvale, CA). All other materials were obtained from common commercial sources.

	Results

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In vitro effects on canine isolated pulmonary artery. In isolated segments (2-3 mm width) of the canine pulmonary artery, ET-1 (1-300 nM) produced a concentration-dependent contraction. The EC₅₀ produced by ET-1 was 21.9 nM (pD₂ = 7.7 ± 0.1; n = 4), and the maximum contractile response was approximately 90.4 ± 10.2% of the reference contraction elicited by 100 mM KCl (fig. 1A). S6c, a selective ET_B receptor agonist, did not contract the canine pulmonary artery at concentrations up to 1 µM, and endothelial denudation had no effect on ET-1-induced contraction (data not shown).

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Effect of SB 209670 on concentration-related contraction produced by ET-1 (A, ) and phenylephrine (B, ) in the dog pulmonary artery (A and B, respectively). The endothelium was intact in all arteries and the results are expressed as a percentage of the response to 100 mM KCl. SB 209760 concentrations in A and B were 3 nM (), 30 nM (), 300 nM (). n = 4 in each group.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   The effects of BQ-123 (3 µM, ) and SB 209670 (300 nM, ) on relaxation produced by S6c in canine pulmonary arteries contracted with phenylephrine (10 µM, ). The endothelium was intact in all arteries and the results are expressed as a percentage of the response to 10 µM phenylephrine. n = 4 in each group.

In vivo effects on canine hypoxia-induced pulmonary hypertension. Hypoxia, induced by substituting inspired air with 10% O₂ in 90% N₂, elevated pulmonary vascular resistance (table 1) and the mPAP-WP in the anesthetized dog. In the vehicle group, receiving a 90-min saline infusion, the mPAP-WP doubled (12.6 ± 1.6 mm Hg vs. 24.7 ± 2.2 mm Hg) during the 30-min hypoxic period and did not fluctuate significantly at the 10-, 20- or 30-min time points (fig. 3A). The AUC during the hypoxic period was 267.2 ± 25.3 mm Hg·min. In the low-dose SB 209670 treatment groups (3 µg/kg/min i.v.), the increase in the pulmonary driving pressure was attenuated (AUC = 158.3 ± 22.7 mmHg·min) during the hypoxic period (fig. 3, A and B). The high dose of SB 209670 (30 µg/kg/min i.v.) essentially abolished the increase in pulmonary driving pressure (AUC = 50.1 ± 4.9 mmHg·min). Hemodynamic and blood gas parameters did change significantly during the 60-min infusion in any of the groups before the hypoxic period. In addition, the arterial pO₂ and pCO₂, MAP, WP and CO values did not differ in the vehicle and treatment groups at each time point before and during the hypoxic period (table 1).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   The effects of a 90-min infusion of saline () and SB 209670 (3 [] and 30 [] µg/kg/min) on acute hypoxia-induced pulmonary hypertension (A). Hypoxia was produced by mechanical ventilation with 10% oxygen in nitrogen, and there were no differences in the arterial partial pressure of oxygen in any of the groups (B). Saline and SB 209670 were infused for 60 min before the 30-min hypoxic period. The area under the mPAP-WP-response curve (AUC) was determined for saline (control) and SB 209670 treatments. *P < .05 (A).

View larger version (13K): [in this window] [in a new window]   Fig. 4.   The effects of SB 209670 (1 mg/kg i.v. bolus + 30 µg/kg/min i.v.) to reverse the increase in mPAP-WP produced by acute hypoxia (A). SB 209670 was administered 3 to 5 min after the onset of hypoxia (B). The area under the mPAP-WP-response curve (AUC) was determined for the saline (control) and SB 209670 groups.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Plasma ET-1-LI was determined before the hypoxic period and at 3 and 30 min during hypoxia in the saline (vehicle) and SB 209670 (1 mg/kg i.v. bolus + 30 µg/kg/min iv) groups. These samples were obtained from animals in figure 4.

	Discussion

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Oxygen tension (alveolar or blood) is an important regulator of blood flow in the lung. Thus, acute pulmonary hypertension induced by hypoxia is the result of adaptive pulmonary vasoconstriction which attempts to match blood flow to ventilation (Voelkel, 1986). The precise mechanisms of this response, including the site of the oxygen sensor and the critical mediator(s), have not been determined. However, recent evidence suggests that the pulmonary endothelium plays an essential role in hypoxia-induced contraction of pulmonary arteries (Demiryurek et al., 1993; Kovitz et al., 1993). In addition, pulmonary arterial endothelial cells produce ET-1, a potent endogenous vasoconstrictor, whose biosynthesis and release is potentiated by hypoxia (Ohlstein et al., 1990). These observations suggested an important role for ET-1 in the coupling of oxygen tension to vasomotor tone in the lung. The major findings in the present study support this hypothesis.

In isolated intrapulmonary segments of the canine pulmonary artery under basal tone, ET-1 produced concentration-dependent contraction; and S6c, a selective ET_B receptor agonist, had no effect. These results are virtually identical with those obtained from similarly sized intrapulmonary segments of human pulmonary arteries (2-3 mm) in which the contractile effects of ET-1 were mediated solely by an interaction with ET_A receptors (Hay et al., 1993). However, the present findings in canine pulmonary arteries contrast with those in the rabbit pulmonary artery in which S6c produces potent ET_B receptor-mediated vasoconstriction (Warner et al., 1993). S6c also produces ET_B receptor-mediated contraction of isolated rat pulmonary arterioles (MacLean et al., 1994) and a modest relaxation of precontracted canine pulmonary arteries (present study).

SB 209670, an ET_A and ET_B receptor antagonist (Ohlstein, et al., 1994), produced a potent and competitive antagonism of ET-1 effects in the canine pulmonary artery which were similar to its competitive effects reported in the canine basilar artery (Willette et al., 1994). The actions of SB 209670 appeared to be selective for ET receptors and did not alter phenylephrine-induced contraction of canine pulmonary arteries. Results from ligand binding and functional studies also indicate that SB 209670 is a very selective ET receptor antagonist (Ohlstein et al., 1994). SB 209670 also antagonized competitively the modest ET-dependent vasorelaxation mediated by S6c in precontracted canine pulmonary arteries.

In vivo SB 209670 administration inhibited the development of hypoxia-induced pulmonary hypertension and also reversed the fully developed acute hypoxic pulmonary hypertensive response in the anesthetized dog. Both effects were dose related. Similar results have been observed with BQ-123, a selective ET_A receptor antagonist, and bosentan, an ET_A and ET_B receptor antagonist; both inhibited acute hypoxia-induced pulmonary hypertension in the rat (Oparil et al., 1995). In addition, BQ123 produced partial reversal of hypoxic pulmonary hypertension in the lamb (Wang et al., 1995). In all of these studies the ET antagonists alone had no significant effect on systemic arterial blood pressure. These results are consistent with previously reported effects of ET antagonists in the normotensive anesthetized dog (Teerlink et al., 1995).

Thus, there is compelling evidence that ET-1 is an important mediator of hypoxic pulmonary hypertension. However, in vitro studies performed with BQ-123 and bosentan failed to inhibit hypoxia-induced vasoconstriction in canine and rat pulmonary artery segments, respectively (Douglas et al., 1993; Lazor et al., 1996). The relevance of these in vitro studies to acute hypoxia-induced pulmonary hypertension is questionable when one considers that the increase in pulmonary resistance during hypoxia may be mediated primarily by small precapillary vessels or postcapillary venules in the lung [see Voekel (1986) for review]. BQ-123 also failed to inhibit acute hypoxic pulmonary vasoconstriction in the intact newborn lamb (Wong et al., 1993). The precise explanation for this lack of efficacy is uncertain; however, the possibility that ET_B receptors or other vasocontrictor mechanisms are operative in the newborn lamb remains to be determined.

The plasma levels of ET-1-LI in the canine model of acute hypoxia-induced pulmonary hypertension were unchanged throughout the control and the 30-min hypoxic period. These results suggest that the ET mediating pulmonary hypertension is most likely produced locally along the pulmonary vasculature and/or bronchial anastamoses and is probably not reaching its site of action via the systemic circulation. It was noteworthy that the infusion of SB 209670 produced profound increases (>25-fold) in plasma ET-1-LI. The increase in ET-1-LI was not caused by interference of SB 209670 with the ET-1 ELISA assay (data not shown). In fact, similar increases in plasma levels of ET-1 and ET-3, but not big-ET-1, have been observed after the administration of bosentan in the anesthetized dog and conscious rats (Loffler, et al., 1993; MacCumber et al., 1989). In these studies, the increases in plasma ET levels were not observed after the administration of selective ET_A receptor antagonists, BQ-123 and FR-139317. Thus, ET_B or ET_B-like receptors may actively regulate plasma ET levels or may act as an ET repository. Further investigations are needed to determine the precise mechanism.

ET receptor antagonists have also been evaluated in models of chronic pulmonary hypertension. In the beagle, FR-139317 reduced both systemic and pulmonary vascular resistance in dihydromonocrotaline-treated animals (Okada et al., 1995). In contrast, the ET_B receptor antagonist, RES-701-1, tended to increase pulmonary pressure in this model. In the rat monocrotaline model of pulmonary hypertension, BQ-123 inhibited the cardiopulmonary consequences of monocrotaline treatment (Miyauchi et al., 1993). BQ-123 and bosentan also inhibited the cardiopulmonary changes associated with chronic hypoxia in rats (Bonvallet, et al., 1994; Eddahibi et al., 1995).

In summary, SB 209670, a nonpeptide mixed ET_A&B receptor antagonist, selectively inhibits ET-mediated vasoconstriction and S6c-mediated relaxation in the isolated canine pulmonary artery. In vivo SB 209670 administration prevents and partially reverses hypoxia-induced pulmonary hypertension in the dog while increasing circulating plasma levels of ET. In conclusion, ET appears to play a critical role in the regulation of pulmonary blood flow by oxygen tension; and potent and selective ET receptor antagonists, i.e., SB 209670, may have utility for the treatment of pulmonary hypertension associated with a variety of disorders.