Introduction

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Endothelin receptors, members of the rhodopsin superfamily of heptahelical receptors, mediate the wide-ranging actions of the endothelin and sarafotoxin families of peptides. Two major types of endothelin receptor, termed ET_A and ET_B, have been cloned, sequenced, and characterized (Masaki et al., 1994). ET_A receptors exhibit a higher affinity for endothelin-1 and endothelin 2 than for endothelin 3, and they are selectively blocked by BQ-123 (Ihara et al., 1991) and FR139317 (Sogabe et al., 1993). In contrast, ET_B receptors display equal affinity for each of the endothelin-1, -2, and -3 isoforms, are selectively activated by sarafotoxin S6c (Williams et al., 1991), and are selectively blocked by BQ-788 (Ishikawa et al., 1994). Binding of endothelin-1 to ET_A and ET_B receptors can also be inhibited by nonselective antagonists including bosentan (Clozel et al., 1993) and SB209670 (Ohlstein et al., 1994).

However, not all responses induced by endothelin-1 and related peptides can be readily classified as having been mediated via these conventional ET_A and ET_B receptors. For example, endothelin-1-induced contractions in some ET_A receptor-containing vascular preparations are less sensitive to inhibition by BQ-123 than are contractions induced by sarafotoxin S6b or ET-3 (Bax et al., 1993; Salom et al., 1993; Clark and Pierre, 1995; Maguire et al., 1996; Devadason and Henry, 1997). One possible explanation for these findings is that endothelin-1 stimulated two types of ET_A receptor, only one of which was sensitive to the actions of BQ-123, ET-3, or sarafotoxin S6b. Consistent with this, Maguire and coworkers (1996) reported that endothelin-1 bound to a larger population of binding sites than did sarafotoxin S6b, although competition binding studies failed to identify two subtypes of ET_A receptor. Recent studies using rat renal artery also failed to identify atypical ET_A receptors in competition binding studies despite strong evidence from functional contractile studies of atypical ET_A receptor-mediated responses (Devadason and Henry, 1997). Thus, although functional studies have provided considerable data on the existence of atypical ET_A receptor-mediated responses (i.e., agonist dependence of antagonist potency), evidence from radioligand-binding studies supporting the existence of atypical ET_A receptors has been less forthcoming.

The existence of atypical ET_A receptor-mediated responses does not seem to be unique to vascular ET_A receptors, having been reported in studies using smooth muscle preparations from the rat vas deferens (Eglezos et al., 1993) and rabbit iris (Ishikawa et al., 1996). However, it is not known whether atypical ET_A receptors or atypical ET_A receptor-mediated responses exist in airway smooth muscle. This is particularly relevant in light of a recent study by Hay et al. (1998), which reported that contractions induced by endothelin-1, endothelin-3, or sarafotoxin S6c in the ET_B receptor-containing human isolated bronchus were not sensitive to classical ET_B receptor antagonists such as BQ-788, and which suggested the presence of a novel ET_B receptor subtype. Thus, the aim of the current study was to characterize the ET_A receptor population on airway smooth muscle using isometric tension recording and radioligand-binding techniques and to investigate the possibility that atypical ET_A receptor-induced responses were mediated via typical ET_A receptors but reflect kinetic differences in the binding characteristics of the ligands to the ET_A receptor. These studies were performed on sheep isolated tracheal smooth muscle, a preparation that contains high densities of ET_A receptors (Goldie et al., 1994).

	Experimental Procedures

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Materials. ¹²⁵I-Endothelin-1 (2000 Ci/mmol), ¹²⁵I-sarafotoxin S6b (2000 Ci/mmol), endothelin-1, sarafotoxin S6b, sarafotoxin S6c, BQ-123 (cyclo[D-Trp-D-Asp-L-Pro-D-Val-L-Leu]) (Auspep, Melbourne, Australia), phenylmethylsulfonyl fluoride (Calbiochem, La Jolla, CA), and phosphoramidon, indomethacin, and BSA (Sigma Chemical Company, St. Louis, MO) were used. FR139317 was a generous gift from Fujisawa Pharmaceutical Company (Osaka, Japan). Endothelin-1, sarafotoxin S6b, and sarafotoxin S6c were prepared as 50 µM stock solutions in 0.1 M acetic acid and stored at 20°C. Dilutions were made in 0.9% NaCl and kept on ice.

Preparation of Sheep Isolated Tracheal Smooth Muscle. Tracheal tissue was obtained from 6-month-old lambs that had been sacrificed with a bolt to the head (Murdoch Veterinary School, Perth, Australia). The trachea was placed in chilled Krebs' bicarbonate solution and cut into rings. The tracheal smooth muscle band was isolated from associated connective tissue and epithelium under a dissecting microscope. Krebs' bicarbonate solution (pH 7.4) contained: 117 mM NaCl, 5.36 mM KCl, 25 mM NaHCO₃, 1.03 mM KH₂PO₄, 7 mM MgSO₄, 0.57 mM H₂O, 2.5 mM CaCl₂·H₂O, 11.1 mM glucose, and 0.0025 mM indomethacin.

Functional Studies. Strips of tracheal smooth muscle, approximately 2 mm wide by 4 mm in length, were suspended in 3-ml organ baths containing Krebs' bicarbonate solution at 37°C and bubbled with 5% CO₂ in O₂. Preparations were washed twice at 15-min intervals during a 40-min equilibration period and then exposed to KCl solution at a final bath concentration of 40 mM. Following a further 15-min washout period, all preparations were incubated with a leukotriene receptor antagonist SKF 104353 (1 µM) and with BQ-123 (0.1, 0.5, or 3 µM), FR139317 (0.05 or 0.3 µM), or vehicle (control) for another 20 min. Cumulative concentration-response curves to either ET-1 or Stx-S6b were then constructed using 0.5-log concentration increments. Only one concentration-effect curve was constructed in each preparation.

Radioligand-Binding Studies. Frozen blocks were prepared by placing a stack of six tracheal smooth muscle bands (each 1-mm thick and 7-mm square) in Macrodex (6% dextran 70, in 5% glucose solution) and freezing in isopentane cooled by liquid nitrogen. Sections (10-µm thick) were cut on a cryostat at 20°C and mounted on gelatin chrom-alum-coated glass slides. Each slide contained three sections from different blocks (animals) and were stored at 85°C before use. Thawed slide-mounted sections were washed twice for 10 min in binding medium containing 50 mM Tris-HCl, 100 mM NaCl, 0.25% BSA, and 10 µM phenylmethylsulfonyl fluoride at pH 7.4. Typically, tissue sections were incubated with ¹²⁵I-endothelin-1 or ¹²⁵I-sarafotoxin S6b at 22°C in binding medium containing 100 nM sarafotoxin S6c (to block ET_B receptor binding) for a designated incubation time, washed twice for 15 min in binding medium, and wiped from the slide with glass fiber filter paper; radioactivity was then measured using a gamma counter. Levels of nonspecific binding were determined by incubating with 1 µM BQ-123 and separately with 30 nM endothelin-1. The levels of nonspecific binding obtained with these two methods were not significantly different in any experiment.

Data and Statistical Analyses. In functional studies, contractile responses to endothelin-1 and sarafotoxin S6b were expressed as a percentage of the response induced by 40 mM KCl (100%). Grouped responses were expressed as the arithmetic mean ± S.E.M. The concentration of endothelin-1 or sarafotoxin S6b that produced 40% KCl response (EC₄₀) was estimated from linear interpolation of individual concentration-effect curves and used as an estimate of agonist potency. The 40% level of response (rather than the more conventional 50% level) was selected post hoc because it permitted the calculation of concentration ratios for the lowest concentrations of BQ-123 and FR139317 against the agonist sarafotoxin S6b. The geometric mean of EC₄₀ values (and associated 95% confidence limits) was calculated for each agonist in preparations from n animals. Concentration ratios were calculated as the EC₄₀ (in the presence of antagonist)/EC₄₀ (in the absence of antagonist). Differences between concentration-ratio values were determined from Student's t test analysis of log (EC₄₀) data.

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	Results

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Functional Studies. Endothelin-1 and sarafotoxin S6b induced concentration-dependent contractions of sheep tracheal smooth muscle (Figs. 1 and 2). The mean concentrations of endothelin-1 and sarafotoxin S6b that produced 40% of the contraction induced by 40 mM KCl were 13.1 nM (95% CLs, 11.7-14.8 nM, n = 15) and 34.0 nM (31.5-37 nM, n = 18), respectively. Thus, endothelin-1 was on average 2.6-fold more potent than sarafotoxin S6b. Both endothelin-1- and sarafotoxin S6b-induced contractions were inhibited by the ET_A receptor antagonists BQ-123 (Fig. 1) and FR139317 (Fig. 2) in a concentration-dependent manner. However, when determined at the 40% response level, both antagonists were significantly more potent at inhibiting contractions induced by sarafotoxin S6b than by endothelin-1 (Figs. 1 and 2). For example, 0.1 µM BQ-123 shifted the concentration-effect curve to sarafotoxin S6b by 2.6-fold (95% CLs, 1.1-6.2-fold, n = 6) but caused no significant shift of the curve to endothelin-1 (1.05-fold; 95% CLs, 0.62-1.8-fold, n = 7; P < .05). Similarly, when determined at the 40% response level, 0.3 µM FR139317 shifted the concentration-effect curve to sarafotoxin S6b by 8.3-fold (95% CLs, 2.9-24-fold, n = 8) and to endothelin-1 by only 1.8-fold (95% CLs, 1.1-2.9-fold, n = 8; P < .01).

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Mean cumulative concentration-effect curves to sarafotoxin S6b (A) and endothelin-1 (B) determined in the absence () and presence of 0.1 µM (), 0.5 µM (), and 3 µM () BQ-123. Data are expressed as mean ± S.E.M. of 6 to 15 observations.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Mean cumulative concentration-effect curves to sarafotoxin S6b (A) and endothelin-1 (B) determined in the absence () and presence of 0.05 µM () and 0.3 µM () FR139317. Data are expressed as mean ± S.E. M. of eight observations.

Radioligand-Binding Studies. Specific binding of [¹²⁵I]endothelin-1 to sheep tracheal smooth muscle sections increased in a time-dependent manner (Fig. 3A). The rate of association of specific ¹²⁵I-endothelin-1 binding was concentration-dependent, with the higher concentration binding reaching a plateau level of binding more quickly (Fig. 3A). The plateau levels of specific binding obtained at 3 h with 0.2 and 1.0 nM ¹²⁵I-endothelin-1 were not significantly different and were not significantly different from the levels obtained with 0.5 nM ¹²⁵I-endothelin-1 (100%, data not shown). The levels of specific ¹²⁵I-endothelin-1 binding to sheep isolated tracheal smooth muscle sections did not decrease significantly during a 5-h period following the replacement of unbound ¹²⁵I-endothelin-1 with an excess of unlabeled endothelin-1 (Fig. 3B). In competition-binding experiments, the binding of ¹²⁵I-endothelin-1 was concentration-dependently inhibited by endothelin-1, BQ-123, and sarafotoxin S6b (Fig. 3C). Each of the competing ligands produced a similar extent of inhibition of ¹²⁵I-endothelin-1 binding, and each of the competition binding curves was best fitted to a one-site model.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Association- (A), dissociation- (B), and competition- (C) binding curves for 125I-endothelin-1 binding to slide-mounted sections of sheep isolated tracheal smooth muscle. A, specific binding of 0.03 nM (), 0.2 nM (), and 1.0 nM () 125I-endothelin-1 determined after 10-, 20-, 40-, 80-, 120-, 180-, and 240-min incubation periods. Levels of specific binding were expressed as a percentage of the specific binding obtained with 0.5 nM 125I-endothelin-1 in the same experiment. B, percentage of specific 125I-endothelin-1 binding (0.2 nM) remaining associated with the tissue sections following various washout periods (30, 60, 120, 180, 240, and 300 min), illustrating the irreversible manner of 125I-endothelin-1 binding. C, competition binding curves for endothelin-1 (), BQ-123 (), and sarafotoxin S6b () against 0.15 nM 125I-endothelin-1. Each datum point is the mean ± S.E.M. of triplicate values obtained from two to three experiments.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Association- (A), dissociation- (B), saturation- (C), and competition- (D) binding curves for 125I-sarafotoxin S6b binding to slide-mounted sections of sheep isolated tracheal smooth muscle. A, specific binding of 0.2 nM () 125I-sarafotoxin S6b determined after 10-, 20-, 40-, 80-, 120-, 180-, and 240-min incubation periods. B, percentage of specific 125I-sarafotoxin S6b binding (0.2 nM) remaining associated with the tissue sections following various washout periods (30, 60, 120, 180, 240, and 300 min), illustrating the reversible manner of 125I-sarafotoxin S6b binding. C, levels of specific binding for 0.1, 0.4, 0.8, 1.5, 3, and 5 nM 125I-sarafotoxin S6b determined at equilibrium (3 h). Levels of specific binding were expressed as a percentage of the specific binding obtained with 0.5 nM 125I-endothelin-1 in the same experiment to define maximum binding. D, competition binding curves for endothelin-1 (), BQ-123 (), and sarafotoxin S6b () against 0.15 nM 125I-sarafotoxin S6b. Each datum point is the mean ± S.E.M. of triplicate values obtained from two to three experiments.

	Discussion

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The principle aim of this study was to pharmacologically characterize the population of ET_A receptors that mediate endothelin-1-induced contraction in sheep isolated tracheal smooth muscle. In isometric tension-recording studies, the ET_A receptor antagonists BQ-123 and FR139317 were significantly more potent in inhibiting contractions induced by sarafotoxin S6b than endothelin-1. Although agonist dependence of antagonist potency may be indicative of the existence of multiple ET_A receptor subtypes, subsequent radioligand binding experiments failed to identify more than a homogeneous population of ET_A receptors to which endothelin-1, sarafotoxin S6b, and BQ-123 each bound. Additional kinetic studies suggest that this apparent paradox may be explained by consideration of the markedly different kinetic binding profiles of these ligands.

Previous studies have demonstrated that endothelin-1-induced contractions in sheep isolated tracheal smooth muscle were mediated via stimulation of ET_A receptors, a conclusion based on the findings that endothelin-1-induced contractions were inhibited by an ET_A receptor antagonist BQ-123 and that the ET_B receptor-selective agonists sarafotoxin S6c and BQ3020 were inactive as spasmogens (Goldie et al., 1994). The current study has extended these findings by investigating the influence of the ET_A receptor-selective antagonists BQ-123 and FR139317 on contractions induced by endothelin-1 and sarafotoxin S6b. Of particular interest was the finding that, when determined at the 40% response level, both antagonists were significantly more potent at inhibiting contractions induced by sarafotoxin S6b than by endothelin-1. One possible explanation for the agonist dependence of antagonist potency is the existence of multiple ET_A receptors. For example, if endothelin-1 could stimulate an atypical ET_A receptor subtype, in addition to the typical ET_A receptor stimulated by sarafotoxin S6b and blocked by BQ-123 and FR139317, then it might be expected to be less effectively inhibited by the antagonists than sarafotoxin S6b. Similar mechanisms have been proposed to explain this phenomenon in vascular (Bax et al., 1993; Salom et al., 1993; Clark and Pierre, 1995; Maguire et al., 1996) and nonvascular preparations (Eglezos et al., 1993; Ishikawa et al., 1996). Subtypes of ET_A receptors, designated ET_A1 (BQ-123 sensitive) and ET_A2 (BQ-123 insensitive), have been proposed to mediate rabbit isolated tracheal smooth muscle contraction (Yoneyama et al., 1995), although these studies were complicated by the existence of a large functional population of ET_B receptors.

Radioligand-binding studies were used to determine whether endothelin-1 bound to a population of atypical ET_A receptors in addition to the typical ET_A receptors bound by BQ-123 or sarafotoxin S6b. If so, ¹²⁵I-endothelin-1 would be expected to have bound to a significantly larger population of ET_A-binding sites (typical and atypical ET_A receptors) than the number of binding sites identified by ¹²⁵I-sarafotoxin S6b (typical ET_A receptors only). However, the estimated B_max values for ¹²⁵I-endothelin-1 and ¹²⁵I-sarafotoxin S6b were not significantly different; furthermore, in competition-binding studies both BQ-123 and sarafotoxin S6b were able to fully compete for specific ¹²⁵I-endothelin-1-binding sites. Specific ¹²⁵I-sarafotoxin S6b binding was also monophasically and completely inhibited by increasing concentrations of endothelin-1, sarafotoxin S6b, and BQ-123. Together, these findings suggest that binding of endothelin-1, BQ-123, and sarafotoxin S6b was to a single common site. An important assumption made in these studies is that ¹²⁵I-endothelin-1 will label all existing ET_A subtypes and not exclusively a subtype to which BQ-123 and sarafotoxin S6b preferentially bind. At present, we cannot exclude the possibility that the low concentrations of ¹²⁵I-endothelin-1 used in these binding studies have failed to label a lower affinity ET_A receptor subtype (atypical receptor) that mediates contraction induced by higher concentrations of endothelin-1 but not sarafotoxin S6b. However, all endothelin receptors investigated to date have exhibited very high affinity for endothelin-1, and it is thus unlikely that ¹²⁵I-endothelin-1 has failed to label all existing endothelin receptors in the current study. On the balance of evidence, it is improbable that the existence of an atypical ET_A receptor subtype has contributed significantly to the atypical ET_A receptor-mediated responses observed in sheep isolated tracheal smooth muscle. These findings concur with several recent studies in rat isolated renal artery (Devadason and Henry, 1997) and rabbit iris (Nosaka et al., 1998).

In the absence of any supporting evidence for the existence of atypical ET_A receptors in sheep isolated tracheal smooth muscle, alternative mechanisms need to be considered to explain the agonist dependence of antagonist potency. In the current study, specific ¹²⁵I-endothelin-1 binding to ET_A receptors was essentially irreversible, consistent with many previously published findings (Hemsen et al., 1991; Waggoner et al., 1992; Ihara et al., 1992, 1995; Devadason and Henry, 1997). In contrast, the binding of ¹²⁵I-sarafotoxin S6b to ET_A receptors was readily reversible, with about 80% of specific binding having dissociated within 5 h. A similar rate of reversibility for ¹²⁵I-sarafotoxin S6b binding from ET_A receptors was observed in rat renal artery (Devadason and Henry, 1997). The binding of BQ-123 is also rapidly reversible (Ihara et al., 1995). Thus, it is possible that these marked differences in binding characteristics may explain, at least in part, the appearance of atypical responses (i.e., agonist dependence of antagonist potency) despite the presence of typical receptors. It is conceivable that differences in the rates of dissociation of endothelin-1 and sarafotoxin S6b from the ET_A receptor contribute significantly to the potency of BQ-123 in inhibiting contractile responses. Indeed, if endothelin-1 does not dissociate from its receptor, then the ability of a reversibly binding antagonist such as BQ-123 to compete for the receptor will be severely reduced. In support of this postulate, the ability of BQ-123 to inhibit endothelin-1 binding was found to decrease significantly with longer periods of coincubation (Wu-Wong et al., 1994a,b; 1995), presumably because of the greater reversibility of antagonist binding than endothelin-1 binding. Furthermore, Gresser et al. (1996) reported that the agonist dependence of BQ-123 potency is an intrinsic property of ET_A receptors, rather than indicative of the presence of atypical receptors.

In conclusion, radioligand-binding studies have demonstrated significant differences in the binding characteristics of ¹²⁵I-endothelin-1 and ¹²⁵I-sarafotoxin S6b to a single subtype of ET_A receptor in sheep isolated tracheal smooth muscle. Thus, the observed greater potency of BQ-123 for inhibiting contractile responses to sarafotoxin S6b than endothelin-1 may be due to the significantly greater reversibility of ET_A receptor binding to sarafotoxin S6b and BQ-123 than to endothelin-1, rather than to the presence of multiple ET_A receptor subtypes.