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The distribution of receptor subtypes for endothelin (ET) in the kidney of the freshwater turtle, Amyda japonica, was examined by quantitative in vitro receptor autoradiography using iodinated mammalian type ET‐1 (125I‐ET‐1) as a radiolabeled ligand. Specific 125I‐ET‐1 bindings were localized to renal tubules, renal arteries and ureter with binding densities of 111.21 ± 19.14, 182.13 ± 10.57 and 219.46 ± 12.83 amol/mm2, respectively. Binding dissociation constants in renal tubules, renal arteries and ureter were 1.05 ± 0.63, 2.03±0.56 and 1.70±0.47nM, respectively. Receptor subtypes for ET in the kidney were characterized by competition with BQ 123 and BQ 788 as specific antagonists for ET receptors, type A (ETA), and type B (ETB) subtypes, respectively. Specific 125I‐ET‐1 bindings in renal arteries and ureter were potently inhibited by BQ 123 in a dose‐dependent manner, whereas BQ 788 was not in competing for specific 125I‐ET‐1 bindings in this structure. However, specific 125I‐ET‐1 bindings in renal tub...

The role of endothelin receptor subtypes, i.e., ET(A) and ET(B) receptors, in the behavioral effects of the intracerebroventricular (ICV) administration of endothelin-1 were examined in conscious rats. ICV administration of endothelin-1 (1-9 pmol/rat) dose dependently produced barrel rolling and other convulsive behaviors including bodily twitching, rigidity, back crawling, fore/hindlimb dystonia, fore/hindlimb clonus, tail extension, and facial clonus. Moreover, a marked increase in spontaneous locomotor activity was observed in animals that were treated with a low dose of endothelin-1 (1 pmol/rat, ICV). Endothelin-1 (9 pmol/rat, ICV)-induced barrel rolling and other convulsive behaviors were completely suppressed by the coadministration of BQ-123 (15 nmol, ICV), a specific endothelin ET(A) receptor antagonist, but not of BQ-788 (15 nmol/rat, ICV), a specific endothelin ET(B) receptor antagonist. In contrast, increased locomotor activity produced by treatment with a low dose of endothelin-1 (1 pmol/rat, ICV) was antagonized by coadministration of BQ-788, but not of BQ123. These results indicate that endothelin-1, which has affinity for both endothelin ET(A) and ET(B) receptors, most likely acts on central ET(A) receptors to evoke barrel rolling and other convulsive behaviors. In addition, activation of central ET(B) receptors may be involved in the increase in spontaneous locomotor activity. These results suggest that brain endothelin receptor subtypes may be involved in the regulation of various physiological functions.

In isolated guinea-pig bronchial preparations the selective endothelin ETB agonist, IRL 1620 caused a concentration-dependent contraction. The pD2 value (7.16 +/- 0.09, n = 6) was significantly increased in the presence of peptidase inhibitors (thiorfan 1 microM, captopril 1 microM, bestatin 1 microM) (pD2 = 7.75 +/- 0.09, n = 6). Indomethacin (5 microM) did not appear to influence the ETB-agonist pD2 value (6.92 + 0.11, n = 6) but potentiated its maximal response significantly (67.23 +/- 4.81% vs. 53.37 +/- 4.80%). The concentration-response curve for the contractile response to IRL 1620 (pD2=7.83 +/- 0.01, n=16); was reproducible, although not completely, since the second curve to this selective ETB agonist was shifted significantly to the right (pD2 = 7.34 +/- 0.09, n = 16) and a decrease in the maximal response was observed (20.0 +/- 2.0%). BQ 788, a selective antagonist for ETB receptors, employed in concentrations ranging from 1.5 to 150 nM, caused a dose-dependent shift to the right of the concentration-response curve to IRL 1620, with a pIC50 value of 8.11 +/- 0.03; this action was not influenced by adding enzyme inhibitors (pIC50 = 8.17 +/- 0.29). Our data show that IRL 1620 undergoes a hydrolytic metabolism in guinea-pig bronchial preparations, which could influence the calculation of the pD2. Pretreatment of the tissue with peptidase inhibitors and indomethacin is consequently significant in the evaluation of IRL 1620 activity, while it does not influence the action of the antagonist, BQ 788.

Experiments have been conducted to investigate the actions of endothelins on the guinea-pig prostate gland. Saturation experiments with [125I]-endothelin-1 (2–800 pM) in guinea-pig prostatic homogenates indicated the presence of high affinity binding sites with an equilibrium dissociation constant (KD) of 230±50 pM, a maximum number of binding sites (Bmax) of 52±16 fmol mg−1 protein or 269±61 fmol g−1 tissue and a Hill coefficient (nH) of 1.01±0.03 (n=3). Competition experiments revealed that binding of [125I]-endothelin-1 (20 pM) was inhibited with the following order of potency: endothelin-1 >>BQ-788 (N-cis-2,6-dimethylpiperidinocarbonyl-L-γ-methyl-Leu-D-Trp[1-CO2CH3-D-Nle-ONa])>BQ-123 (cyclo-D-Asp-L-Pro-D-Val-Leu-D-Trp) ⩾sarafotoxin S6c. At concentrations with negligible influence on smooth muscle tone, endothelin-1, endothelin-2 and sarafotoxin S6b (1 nM–0.1 μM) produced concentration-dependent potentiation of the contractions evoked by electrical field stimulation with trains of 20 pulses at 10 Hz every 50 s, 0.5 ms pulse width and a dial setting of 60 V. In contrast, the endothelin ETB receptor-preferring agonist endothelin-3 (1 nM–1 μM) was much less potent, and the endothelin ETB receptor-selective agonists sarafotoxin S6c and BQ-3020 (Ac-[Ala11,15]-endothelin-1 (6-21)), up to 1 μM, were without effect. The endothelin ETA receptor antagonist BQ-123 (1 μM) markedly inhibited the potentiation induced by endothelin-1, endothelin-2 and sarafotoxin S6b while the endothelin ETB receptor antagonist BQ-788 (1 μM) was less effective. While our binding data indicates the presence of ETA and ETB binding sites in the guinea-pig prostate, the endothelin-induced facilitation of neurotransmission to the prostatic smooth muscle is mediated largely via activation of endothelin receptors of the ETA subtype. Keywords: Endothelin ETA and ETB receptors, endothelin-1, -2 and -3, sarafotoxins S6b and S6c, BQ-123, BQ-788, BQ-3020, smooth muscle contraction, field stimulation, guinea-pig prostate Introduction Since the discovery of endothelin in the porcine vascular endothelial cell cultures in 1988 by Yanagisawa et al. (1988), the presence of this peptide and its receptors has been demonstrated in a variety of tissues and organs (see reviews by Huggins et al., 1993; Rubanyi & Polokoff, 1994; Sokolovsky, 1995). The existence of three genes encoding a family of structurally similar peptides, endothelin-1, -2 and -3, has been described (Inoue et al., 1989). Endothelins have been reported to have roles not only in smooth muscle contraction of vascular and non-vascular tissues, but also in promoting cell growth (see reviews by Battistini et al., 1993; Hay, 1995) and in the modulation of neuroeffector transmission in the airways (Takimoto et al., 1993; Henry & Goldie, 1995; Fernandes et al., 1996) and in the urogenital tract (urinary bladder: Saenz de Tejada et al., 1992; Donoso et al., 1994; vas deferens: Wiklund et al., 1989; Donoso et al., 1992; Lau et al., 1995; Maas et al., 1995). There is now a growing body of evidence that indicates the presence of endothelins in the prostate. Endothelin precursors and the endothelin converting enzyme (ECE-1) have been reported to be expressed in the human prostate (Rossi et al., 1995; Prayer-Galetti et al., 1997; Walden et al., 1998). To date, two major endothelin receptor subtypes have been identified (Masaki et al., 1994), and both endothelin ETA and ETB receptors are detected in human cultured prostatic smooth muscle cells (Saita et al., 1997), but the ETA receptors are predominantly expressed in prostatic membrane homogenates (Le Brun et al., 1996) and slide-mounted prostatic sections (Kobayashi et al., 1994a; Imajo et al., 1997). Quantitative receptor autoradiographic studies by Kobayashi et al. (1994b), have, however, shown a preferential localization of endothelin ETA and ETB receptors in stroma and epithelium, respectively. Endothelin-1 immunoreactivity is prominent in the glandular epithelium (Langenstroer et al., 1993). It is possible that endothelins play a physiological role in modulation of secretion in the gland, or a physiological or pathophysiological role to attenuate apoptosis (Wu-Wong et al., 1997a) or to exert mitogenic effects in human prostatic smooth muscle cells (Saita et al., 1998). In addition, endothelin-1 potently contracts the human prostate in vitro. These contractions are shown to be mediated by both endothelin receptor subtypes (Kobayashi et al., 1994a) although it has also been proposed that in prostatic stromal cells, contraction is mediated by ETB receptors (Webb et al., 1995). Endothelins may thus play an important role in the regulation of the contractile function of smooth muscle cells associated with prostatic tension and secretory processes. Recent reports of upregulated endothelin binding sites in the benign hyperplastic prostate (Kondo et al., 1995; Moriyama et al., 1996) and of increased levels of endothelin-1 with decreased expression of endothelin ETB binding sites in prostate cancer (Nelson et al., 1996) suggest a possible involvement of endothelins in prostatic disorders. Prayer-Galetti et al. (1997) have proposed that zonal-specific distribution of endothelin receptors in the human prostate may be of relevance in the pathogenesis of benign prostatic hyperplasia and prostate cancer. The main aim of the present study was to examine the actions of endothelins and to characterize endothelin receptor subtype/s in the prostate, using the guinea-pig as an experimental model. The guinea-pig prostate has a substantial stromal component, which comprises predominantly smooth muscle cells (Ricciardelli et al., 1989). With age, the guinea-pig prostate develops a stromal hyperplasia which is histologically indistinguishable from that observed in ageing men (Horsfall et al., 1994). The influences of endothelins and related sarafotoxins on prostatic smooth muscle contractility and on neurotransmission in the guinea-pig isolated prostate were examined. Pharmacological tools including the endothelin ETB receptor agonists sarafotoxin S6c (Williams et al., 1991) and BQ-3020 (Ihara et al., 1992b), and the endothelin ETA and ETB receptor-selective antagonists, BQ-123 (Ihara et al., 1992a) and BQ-788 (Ishikawa et al., 1994), respectively, have been employed in the characterization of endothelin receptor subtype/s in the guinea-pig prostate gland. Preliminary accounts of some of the results of this study have been communicated to the Australian Neuroscience Society (Pennefather et al., 1998; Lau et al., 1999).

The pharmacological properties of endothelin receptors (ETR) were investigated in guinea-pig bronchus by comparing binding and functional results. In binding assays, both the ETB agonists, endothelin-3 (ET-3) and N-suc-[Glu9,Ala11,15]ET-1(8–21) (IRL 1620), and the antagonist, N-cis-2,6-dimethylpiperidinocarbonyl-L-γ-methylleucyl-D-1-methoxycarbonyltryptophanyl-D-norleucine (BQ 788), showed biphasic inhibition curves of [125I]-endothelin-1 (ET-1) binding to bronchus membranes prepared from intact or epithelium-deprived tissue. IRL 1620 did not completely displace specifically [125I]-ET-1 bound to these tissue preparations. In the presence of the ETA-selective antagonist, cyclo(-D-Trp-D-Asp-L-Pro-D-Val-L-Leu) (BQ 123, 1 μM), IRL 1620 displacement curves were shallow but a complete inhibition was reached at a concentration of 1 μM. Both curves were better represented by two-site models. In addition, BQ 788 competition curves became monophasic when binding experiments were performed in the presence of 1 μM BQ 123. The non-selective agonist, ET-1, and BQ 123 inhibited [125I]-ET binding to bronchus membranes in dose-dependent fashions with monophasic curves. The contracting activity of IRL 1620 (0.55 nM–1.6 μM) was tested on multiple-ring bronchial preparations pretreated with peptidase and cyclo-oxygenase inhibitors. BQ 788 shifted IRL1620 concentration-response curves to the right while BQ 123 did not influence bronchial responsiveness. In addition, a potentiation of the maximal response to the agonist was observed in BQ 788 treated bronchial rings. This effect was abolished by tissue pretreatment with Nω-nitro-L-argininemethylester (L-NAME) or epithelium removal but not by pretreatment with atropine or iberiotoxin. Our results demonstrate that guinea-pig bronchus contains two populations of ETB receptors with different affinities for the ETB-selective agonist, IRL 1620. One ETB receptor population appears to activate bronchial muscle contraction while another on epithelial cells causes muscle relaxation through the release of nitric oxide (NO). Keywords: Endothelin receptors, ETB receptors, endothelins, guinea-pig bronchus Introduction Endothelins (ETs)1, a family of three closely related peptide hormones, endothelin-1, -2, and -3 (ET-1, ET-2, and ET-3), were originally recognized as potent modulators of vascular functions participating in both vasoconstriction and vasodilatation. Thereafter, the presence of ETs was demonstrated in several other tissues of different species including humans, where they exert various physiological effects (for a review see: Rubanyi & Polokoff, 1994; Webb et al., 1998). All activities of ETs are mediated through the interaction with specific cell surface receptors (for a review see: Bax & Saxena, 1994; Ohlstein et al., 1996; Pollock et al., 1995). Two ETR subtypes, termed ETA and ETB, have been identified, cloned and expressed from rat (Lin et al., 1991; Sakurai et al., 1990; Sugiura et al., 1989), bovine (Arai et al., 1990; Saito et al., 1991), and human tissues (Adachi et al., 1991; Cyr et al., 1991; Ogawa et al., 1991; Sakamoto et al., 1991). Both receptor subtypes belong to the large receptor family characterized by seven transmembrane-spanning regions which regulate distinct intracellular signalling pathways through the activation of G proteins. The two subtypes can be distinguished by different rank orders of affinity toward the three ET isopeptides. The ETA receptor binds ET-1, ET-2 and ET-3 with decreasing affinities whereas the ETB receptor shows a similar affinity for all three isopeptides. An additional classification of both receptor subtypes as ETA1, ETA2, ETB1, and ETB2 has been proposed on the basis of their affinities for agonists or antagonists (Karaki et al., 1994; Sudjarwo et al., 1994). The ETA receptor mediates contraction of arterial smooth muscles, while the ETB receptor exhibits either contractile or relaxing effects depending on the vascular bed examined (Pollock et al., 1995). Selective activation of ETB receptors on endothelial cells causes relaxation of rat aorta through the release of nitric oxide (Karaki et al., 1993). However, activation of ETB receptors on venous smooth muscles gives rise to vasoconstriction (Moreland et al., 1994). ETs modulate airway activities of different species including humans (Hay et al., 1993; Knott et al., 1995). These peptides are potent constrictors of airways both in vivo and in vitro (Hay et al., 1993; Touvay et al., 1990). In guinea-pig trachea and bronchus, the contraction induced by ET-1 appears to involve a mixed population of ETRs (Hay et al., 1993). In addition, regional differences of ETR subtype distribution seem to exist in guinea-pig airways, with bronchus expressing mainly ETB receptor subtypes (Hay et al., 1993). In guinea-pig trachea, functional (Battistini et al., 1993) and autoradiographic studies (Tschirhart et al., 1991) have shown the presence of ETRs on the epithelium. Functional data reported by Battistini et al. (1993) have also indicated that in guinea-pig trachea, ET-1 activates ETA receptors on epithelial cells, inducing the release of prostanoids, while airway contraction is mediated via ETB receptors probably present on smooth muscles. Recently, it has been suggested (Yoneyama et al., 1995b) that ETs modulate the airway tone by acting not only directly on airway smooth muscle, but also on the cholinergic nerve, to modulate acetylcholine (ACh) release. The pathophysiological role of ETs in asthma and other respiratory disorders has received some attention (Hay et al., 1993). The presence of ETB receptors in guinea-pig and human bronchus mediating smooth muscle contraction is well established (Battistini et al., 1994; Hay et al., 1993; 1998; Hay & Luttman, 1997). However, in guinea-pig this receptor population has not been characterized for its biochemical and pharmacological features. Two subtypes of ETA and ETB receptors seem to mediate smooth muscle contraction of rabbit trachea (Yoneyama et al., 1995a). Similarly, the ETB receptor population in guinea-pig bronchus cannot be homogeneous. As guinea-pig bronchus is frequently used as an experimental model to test the activity of new anti-asthmatic drugs, characterization of its ETB receptor population is pivotal for elucidating the pathophysiological significance of ETs in asthma. Thus, we investigated the biochemical and pharmacological properties of ETRs in guinea-pig bronchus using selective agonist and antagonist ligands.

Contractile responses to endothelin-1 (ET-1) were investigated in isolated trachea from rats previously exposed to chronic hypoxia (10% O(2)) or room air for 14 days. Concentration-response curves were constructed to ET-1 (10(-11)-3x10(-7)m ) in the presence and absence of the ET(A)receptor antagonist FR 139317 (10(-8), 10(-7)and 10(-6)m ), the ET(B)receptor antagonist BQ 788 (10(-6)and 3x10(-6)m ), the non-selective ET receptor antagonist SB 209670 (10(-7)and 10(-6)m ) and a combination of FR 139317 (10(-6)m ) and BQ 788 (10(-6)m ). Concentration-response curves were also conducted to the ET(B)receptor agonist sarafotoxin S6c (10(-11)-3x10(-7)m ). In addition, responses to ET-1 (10(-11)-3x10(-7)m ) were examined in the presence and absence of the nitric oxide synthase inhibitor, L-NAME. In control rat trachea, both FR 139317 and BQ 788 failed to inhibit ET-1-induced contractions and, indeed, FR 139317 (10(-8)m ) and BQ 788 actually potentiated responses. In trachea from chronic hypoxic rats, FR 139317 did not alter ET-1 responses whereas BQ 788 again potentiated ET-1-induced contractions. The non-selective ET receptor antagonist SB 209670 attenuated ET-1-evoked contractions in trachea from control and chronically hypoxic rats. A combination of FR 139317 (10(-6)m ) and BQ 788 (10(-6)m ) also attenuated ET-1 responses in control rat trachea, but not trachea from chronically hypoxic rats. In trachea from both control and chronically hypoxic rats, L-NAME significantly potentiated responses to ET-1. To investigate ET receptor-mediated relaxation, tissues were preconstricted with methacholine and concentration-response curves were conducted to ET-1 (10(-13)-10(-8)m ) in the presence and absence of BQ 788 (10(-6)m ) and to the ET(B)receptor agonist sarafotoxin S6c (10(-13)-10(-8)m ). In trachea from control and chronic hypoxic rats, ET-1 and sarafotoxin S6c evoked only very small, non-reproducible relaxatory responses.

The purpose of this work was to investigate whether endothelin-1 (ET-1) was able to induce the release of an inhibitory factor from the airway epithelium in isolated human bronchi and to identify this mediator as well as the endothelin receptor involved in this phenomenon. In intact bronchi, ET-1 induced a concentration-dependent contraction (−logEC50=7.92±0.09, n=18) which was potentiated by epithelium removal (−logEC50=8.65±0.11, n=17). BQ-123, an ETA receptor antagonist, induced a significant leftward shift of the ET-1 concentration-response curve (CRC). This leftward shift was abolished after epithelium removal. L-NAME (3×10−3M), an inhibitor of nitric oxide (NO) synthase, induced a significant leftward shift of the ET-1 CRC, and abolished the potentiation by BQ-123 (10−8 M) of ET-1-induced contraction. In intact preparations, the ETB receptor antagonist BQ-788 induced only at 10−5 M a slight rightward shift of the ET-1 CRC. In contrast, in epithelium-denuded bronchi or in intact preparations in the presence of L-NAME, BQ-788 displayed a non-competitive antagonism toward ET-1-induced contraction. IRL 1620, a selective ETB receptor agonist, induced a contraction of the isolated bronchus (−logEC50=7.94±0.11, n=19). This effect was not modified by epithelium removal or by BQ-123. BQ-788 exerted a competitive antagonism against IRL 1620 which was similar in the presence or absence of epithelium. These results show that ET-1 exerts two opposite effects on the human airway smooth muscle. One is contractile via ETB-receptor activation, the other is inhibitory and responsible of NO release which counteracts via ETA-receptor activation the contraction. British Journal of Pharmacology (1999) 126, 529–535; doi:10.1038/sj.bjp.0702327

The purpose of this study was to determine whether vascular endothelial and vascular smooth-muscle endothelin ET B receptors could be quantitatively differentiated by PD 142893 (PD), SB 209670 (SB), and BQ 788 (BQ) in the same species by using closely matched experimental conditions. The isolated perfused rat kidney (vascular smooth muscle) and isolated perfused rat mesentery (vascular endothelium) were challenged with increasing bolus doses of sarafotoxin S6c in the absence and presence of antagonist. PD. SB, and BQ produced parallel concentration-dependent rightward shifts in the S6c dose-response curve in the kidney. PD and SB also produced parallel concentration-dependent rightward shifts in the S6c dose-response curve in the mesentery. In contrast, BQ produced an insurmountable antagonism. Schild-derived pA 2 values for PD and SB were significantly greater for inhibiting endothelial versus smooth-muscle ET B receptors. Furthermore, PD and SB differed in their relative potency between the two assays. Because BQ produced an insurmountable antagonism in the mesentery, it was not possible quantitatively to compare the antagonist activity in the two assays. These results indicate that PD, SB, and BQ selectively antagonize endothelial ET B -receptor activity over smooth-muscle ET B -receptor activity.

1. The influence of endothelin receptor antagonists on febrile responses to E. coli lipopolysaccharide (LPS), interleukin-1beta (IL-1beta), tumour necrosis factor-alpha (TNF-alpha) and endothelin-1 (ET-1) was assessed in conscious rats. 2. Intravenous (i.v.) LPS (5.0 microg kg(-1)) markedly increased rectal temperature to a peak of 1.30 degrees C over baseline at 2.5 h. Pretreatment with the mixed endothelin ET(A)/ET(B) receptor antagonist bosentan (10 mg kg(-1), i.v.) or the selective endothelin ET(B) receptor antagonist BQ-788 (N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma-methylleucyl-D -1-methoxycarboyl-D-norleucine; 3 pmol, into a lateral cerebral ventricle-i.c.v.) reduced the peak response to LPS to 0.90 and 0.75 degrees C, respectively. The selective endothelin ET(A) receptor antagonist BQ-123 (cyclo[D-Trp-D-Asp-Pro-D-Val-Leu]; 3 pmol, i.c.v.) was ineffective. 3. Increases in temperature caused by IL-1beta (180 fmol, i.c.v.), TNF-alpha (14.4 pmol, i.c.v.) or IL-1beta (150 pmol kg(-1), i.v.) were unaffected by BQ-788 (3 pmol, i.c.v.). 4. Central injection of endothelin-1 (0.1 to 3 fmol, i.c.v.) caused slowly-developing and long-lasting increases in rectal temperature (starting 2 h after administration and peaking at 4-6 h between 0.90 and 1.15 degrees C) which were not clearly dose-dependent. The response to endothelin-1 (1 fmol, i.c.v.) was prevented by BQ-788, but not by BQ-123 (each at 3 pmol, i.c.v.). Intraperitoneal pretreatment with the cyclo-oxygenase inhibitor indomethacin (2 mg kg(-1)), which partially reduced LPS-induced fever, did not modify the hyperthermic response to endothelin-1 (3 fmol, i.c.v.). 5. Therefore, central endothelin(s) participates importantly in the development of LPS-induced fever, via activation of a prostanoid-independent endothelin ET(B) receptor-mediated mechanism possibly not situated downstream from IL-1beta or TNF-alpha in the fever cascade.

Objective: Although a number of pharmacologic agents have been shown to reduce intimal hyperplasia in animal models of restenosis, to date no systemic agent has conclusively been shown to be effective in humans. Recently, considerable attention has been directed towards endothelin (ET), a potent vasoconstrictor and a powerful mitogen for vascular smooth muscle cells, as a mediator of intimal hyperplasia. Endothelin-1 has been shown to be mitogenic for human saphenous vein smooth muscle cells, and expression also is elevated in human vein graft stenosis. The aim of this study was the investigation of whether ET receptor antagonists can attenuate neointima formation in a laboratory model of vein graft intimal hyperplasia and the determination of whether the effects are mediated by a specific ET receptor subtype. Methods: We used an organ culture of human saphenous vein, a well-validated model of vein graft intimal hyperplasia. Paired segments of human long saphenous vein were cultured with and without the following antagonists: bosentan, a nonselective ET receptor antagonist; BQ 123, a specific endothelin-A antagonist; or BQ 788, a specific endothelin-B (ET B ) antagonist. After 14 days in the culture, the segments were fixed and processed and the sections were immunostained to facilitate the measurements of neointimal thickness with a computerized image analysis system. Results: The nonselective antagonist bosentan and the ET B selective antagonist BQ 788 significantly reduced neointima formation by 70% ( P = .001) and 50% ( P = .03), respectively, but the ET A antagonist BQ 123 had no significant effect on the reduction of neointima formation ( P = 1.0). Conclusion: The results of this study imply an important role for ET as a mediator of human vein graft intimal hyperplasia and imply further that a specific ET B antagonist may have a therapeutic potential for the prevention of vein graft stenosis. (J Vasc Surg 1998;28:695-701.)

Endothelin-1, unlike the selective endothelin ETB receptor agonist sarafotoxin S6c, causes nociception in the rat when injected intraarticularly into the naive knee-joint. By using selective antagonists, the present study further characterizes the receptors underlying the articular nociceptive actions of endothelin-1, as well as the possible contribution of endogenous endothelins towards nociception induced by carrageenan or E. coli lipopolysaccharide (LPS) in this tissue. Nociception was evaluated by placing the animal for 1 min each hour on a revolving (3 rpm) cylinder and measuring the increase in time the hindpaw of the limb affected by the intra-articular (i.a.) injection of the nociceptive agent, failed to touch its metallic surface (i.e. paw elevation time, PET). In naive joints, endothelin-1 (120 pmol) increased the area under the PET curve (AUC 0-6 h, in arbitrary units) from 61+/-3 (control) to 156+/-12. This nociceptive effect was reduced by prior intravenous (i.v.) injection of the mixed ET(A)/ET(B)receptor antagonist bosentan (by 54 and 73% with 10 and 30 mg/kg) or i.a. administration of the selective ETA receptor antagonist BQ-123 (cyclo [D-Asp-Pro-D-Val-Leu]; by approximately/= 45% with 10 or 30 nmol), but was unaffected by the selective ET(B) receptor antagonist BQ-788 (N-cis-2,6-dimethyl-piperidinocarbonyl-L-gamma-methoxycarbonyl- tryptophanil-D-norleucine; 10 nmol). Prior joint challenge with carrageenan (300 microg) 72 h beforehand (i.e. priming) rendered the joint more sensitive to nociception induced by either endothelin-1 or sarafotoxin S6c (15, 30 and 60 pmol). Responses elicited by endothelin (30 pmol) in the primed joint were sensitive to inhibition by either BQ-123 or BQ-788 (each causing approximately/= 80% inhibition at 10 nmol). Priming also enhanced PET responses to carrageenan itself and to LPS (1 microg) markedly and persistently, increasing the area under the curve (AUC 0-12 h, in arbitrary units) from 241+/-19 to 409+/-50 and from 312+/-40 to 466+/-25, respectively (P < 0.05), without changing that measured following vehicle injection (from 121+/-3 to 117+/-4). Bosentan (up to 30 mg/kg, i.v.) failed to modify nociception caused by carrageenan or LPS in naive joints, by carrageenan in the primed joint, or control PET responses. LPS-induced nociception in the primed joint, however, was inhibited by 52 to 56% by bosentan (3 or 10 mg/kg) or 59% by local injection of the selective endothelin ET(B) receptor antagonist BQ-788 (10 nmol, i.a.), but was unaffected by the selective endothelin ETA receptor antagonist BQ-123. Thus, nociception induced by endothelin-1 in the naive joint is mediated largely via endothelin ETA receptors, whereas both ET(A)and ET(B) receptors contribute to its action in the carrageenan-primed joint. Furthermore, LPS-induced nociception in the primed joint is mediated to a large extent via endothelin release and activation of ET(B) receptors within the joint itself. These findings may be relevant to the etiology of pain underlying chronic arthritic disease in humans.

Contractions induced by endothelin-1, endothelin-3 and the selective ET B agonist, sarafotoxin S6c, were studied in segments of human temporal artery. The results in fresh arteries were compared with those obtained after 1 or 4 days in organ culture, and with the specific ET A antagonist FR 139317, the specific, mixed antagonist bosentan, or the specific ET B antagonist, BQ 788. Sarafotoxin S6c induced no contractile activity in fresh artery segments, but elicited marked contractions after culture. This contraction was only slightly inhibited by FR 139317, but was abolished by BQ 788. Contractions induced by endothelin-1 were antagonized by FR 139317 and bosentan, but not by BQ 788. Organ culture did not change the overall pattern, but all concentration-response curves were shifted leftwards. Contractions induced by endothelin-3 were abolished by all antagonists in fresh arteries, but some activity was restored after organ culture. Sensitivity to endothelin-3 was markedly increased. The results suggest a change in endothelin receptors during organ culture, resulting in a marked increase in contractile ET B activity, and possibly some increase in ET A activity. Such changes illustrate the complexity of endothelin responses in this vascular bed.

1 Contractile responses to endothelin-1 (ET-1) and sarafotoxin S6c (S6c) were studied in pulmonary resistance arteries (∼320 μm i.d.) from fetal, 0–24 h, 4 day and 7 day rabbits. The effects of the ETA-selective antagonist FR139317, the selective ETB receptor antagonist BQ-788 and the non-selective ETA/ETB receptor antagonist SB 209670, on these responses, were determined. Acetylcholine-induced vasodilation and noradrenaline-evoked contractions were also examined. 2 ET-1 potency was in the following order (pEC50 values): fetal (8.7) = 0–24 h (8.8) = 4 day (8.6) > 7 day (8.0). The order of potency for S6c was 7 days (11.1) = 4 days (10.8) >0–24 h (9.7) > fetal (8.6). Hence, S6c and ET-1 were equipotent in the fetus but S6c was increasingly more potent than ET-1 with increasing age, being some 1000 times more potent by 7 days. By 7 days, responses to ET-1 were also resistant to both FR139317 and BQ-788. FR139317 inhibited responses to ET-1 in vessels from 0–24 h and 4 day, but not fetal, rabbits (pKb: 6.4 in 4 day rabbits). BQ-788 inhibited responses to ET-1 at all age points except for 7 days (pKb: 6.7 at 0–24 h; 6.2 at 4 days). BQ-788 inhibited responses to S6c at all age points (pKb: 8.5 at 4 days). SB 209670 inhibited responses to ET-1 and S6c at 0–24 h and 4 days (pKb for ET-1: 8.3 and 8.0 respectively; pKb for S6c: 9.2 and 10.2 respectively). 3 Acetylcholine (1 μm) induced vasodilation at all age points (inhibited by 100 μm l-Nω-nitroarginine methylester) although the degree of vasodilation was significantly reduced (∼75%) at 0–24 h. Noradrenaline induced contraction at all age points except 7 days and its response was significantly enhanced at 0–24 h. 4 Over the first week of life, the potency of S6c increases whilst that to ET-1 decreases suggesting differential development of responses to ET-1 and S6c and heterogeneity of ETA- or ‘ETB-like’ receptor-mediated responses. There is no synergism between ETA and ETB receptors at birth but this is established by 7 days. Immediately after birth rabbit Pulmonary Resistance Arteries are hyperresponsive to ET-1 and noradrenaline but exhibit impaired nitric-oxide dependent vasodilation. British Journal of Pharmacology (1998) 124, 1165–1174; doi:10.1038/sj.bjp.0701950

We measured the activity of mitogen-activated protein (MAP) kinases, enzymes believed to be involved in the pathway for cell proliferation, in rat aortic strips with or without endothelium, and examined effects of angiotensin receptor antagonists, endothelin receptor antagonists and nitric oxide (NO)-related agents. Endothelium removal produced an activation of MAP kinase activity in the strips, whereas the enzyme activity was not affected in the adventitia. The MAP kinase activation was inhibited by either the angiotensin AT1 receptor antagonist losartan or the endothelin ETA receptor antagonist BQ 123. The combination of both antagonists caused an additive inhibition. The angiotensin AT2 receptor antagonist PD 123,319 and the endothelin ETB receptor antagonist BQ 788 did not affect the MAP kinase activation. The NO synthase inhibitor NG-nitro- l -arginine methyl ester ( l -NAME) caused an activation of MAP kinase in the endothelium-intact aorta and the MAP kinase activation was inhibited by losartan or BQ123. The NO releaser nitroprusside inhibited the MAP kinase activation induced by endothelium removal or angiotensin II. These results suggest that even in isolated arteries, NO of endothelial origin tonically exert MAP kinase-inhibiting effects and endogenous angiotensin II and endothelins in the media are tonically released to cause MAP kinase-stimulating effects in medial smooth muscle.

BQ-123 is extensively used as an antagonist at endothelin (ET) receptors, having selectivity at the ET(A) receptor subtype. In this study, the effects of BQ-123 per se on action potentials, L-type calcium currents, and potassium currents, were examined in ventricular cardiomyocytes isolated from adult, male, New Zealand White rabbits, using the patch-clamp technique. BQ-123 (1 microM) increased (P0.02) the duration of the action potential to 267 +/- 36 ms from a control duration of 228 +/- 30 ms. BQ-123 did not have any effect on the inward rectifier or transient outward potassium currents, but increased (P0.02) the L-type Ca2+ current to -2.76 +/- 0.3 nA from a control value of -2.45 +/- 0.28 nA. The increases in both duration of the action potential and L-type Ca2+ current were reversed upon washout (233 +/- 28 ms and -2.32 +/- 0.31 nA, respectively) and were not different from the control values in the absence of BQ-123. In contrast, the endothelin receptor antagonists, BQ-788, PD155080 and PD145065 (1-10 microM) did not affect the L-type Ca2+ current. These results indicate that, unlike PD155080, BQ-788 and PD145065, the conventional ET(A) receptor-selective antagonist, BQ-123, exerts a unique positive effect on the L-type Ca2+ current in ventricular cardiomyocytes isolated from rabbit myocardium. The mechanism of action of BQ-123, therefore, is not confined to ET receptor antagonism.

The aim of this investigation was to determine whether the endothelin receptor subtype of a megakaryoblastic cell line (MEG-01) changes during culture passages as cells undergo maturation and differentiation. On early-passage cells, binding of [ 125 I ]endothelin-1 was completely inhibited by 1 μM BQ 123 (cyclo-[ d -tryptophanyl- d -aspartyl–prolyl- d -valyl–leucyl]), but not by sarafotoxin 6C. Also the endothelin-1-enhancing effect on [Ca2+]i was prevented by BQ 123, whereas sarafotoxin 6C had no effect on [Ca2+]i. In late-passage cells, endothelin ETB analogs, unlike endothelin ETA analogs, competed with binding of [ 125 I ]endothelin-1. Endothelin ETB receptor agonists increased [Ca2+]i while the endothelin-1-induced response was inhibited by BQ 788 ([N-[(2R,6S)-2,6-dimethyl-piperidinocarbonyl]-4-methyl- d -leucyl]-[Nω-(methoxycarbonyl)- d -tryptophanyl]- d -norleucine), but not by BQ 123, although both endothelin ETA and ETB receptor mRNAs were expressed, as shown by reverse transcriptase–polymerase chain reaction. These results demonstrate that in MEG-01 cells switch from expression of endothelin ETA to expression of ETB receptors during culture. The data also suggest that late-passage MEG-01 cells look like platelets, in terms of endothelin receptor subtype.

BACKGROUND Endothelin-1 (ET-1) interacts with specific G-protein-coupled receptors to initiate short-term (contraction) and long-term (mitogenesis) events in target cells. ET-1 is an abundant prostate secretory protein that, in its biologically active form, elicits prostatic smooth muscle contraction. The present study was designed to determine the effects of ET-1 on prostate cell growth and to examine the regulation of endogenous ET-1 activity and bioavailability. METHODS Primary cultures of prostate secretory epithelial (PE) and prostate fibromuscular stromal (PS) cells were established from benign human prostate tissue. RESULTS In culture, PE cells secrete immunoreactive ET-1 (38.5 ± 1.6 pg/ml/106 cells/24 hr) into the conditioned medium. Levels of immunoreactive ET-1 produced by PS cells were more than 10-fold lower. Endothelin-converting enzyme-1 (ECE-1) mRNA was detected in PE cells and not in PS cells; however, big ET-1 was the predominant immunoreactive ET-1 secretory product of PE cells. The ETB endothelin receptor was the predominant subtype in both PE and PS cells. In PS cells, but not PE cells, ET-1 induced significant inositol phosphate accumulation and [3H]-thymidine uptake. Agonist activity was inhibited by the ETB receptor selective antagonist, BQ 788. Intact PE cell monolayers secrete ET-1 through the apical surface, consistent with secretion of ET-1 into the glandular lumen in vivo. CONCLUSIONS On the basis of these findings, regulation of ET-1 activity and bioavailability appears to be tightly regulated. Such findings have important implications in the pathophysiology of prostate disease. Prostate 34:241–250, 1998. © 1998 Wiley-Liss, Inc.

We investigated effects of the endopeptidase 24.11 inhibitor, SCH 39370, on uterotonic effects of endothelins (ETs) and sarafotoxin S6b. Responses of uteri from non-pregnant rats were inhibited by the ETA receptor antagonist, BQ123 (1 microM) but not the ETB receptor antagonist, BQ 788 (1 microM). ET-1, sarafotoxin S6b and ET-2 were more potent than ET-3 in tissues from non-pregnant and pregnant rats. SCH 39370 (10 microM) did not affect uterotonic responses to these peptides in either group, but inhibited those of big ET-1 in non-pregnant rat tissues, indicating inhibition of conversion of big ET-1 to ET-1. These data indicate that endopeptidase 24.11 does not inactivate the endothelin peptides in the rat uterus.

The role of endogenous endothelin-1 in variant angina was investigated using two endothelin receptor antagonists: LU 135252 (ETA) and BQ 788 (ETB). Cyclic flow reductions were induced in a coronary artery of mongrel dogs by combining critical stenosis with endothelial injury. One hour after induction of cyclic coronary flow reductions the dogs were randomized to intravenous treatment with either saline, or LU 135252 (10 mg kg−1), or BQ 788 (0.1 mg kg−1). Cyclic coronary flow reductions were monitored for two hours after drug and remained constant in controls as well as after BQ 788. LU 135252 reduced the number of cyclic coronary flow reductions significantly (about 50%) without effects on hemodynamics or hemostasis.

Endothelin (ET) is a potent vasoconstrictor peptide which has been shown to have an important role in the regulation of systemic and renal haemodynamics. In order to elucidate the role of endogenous ET in the kidney, we examined the effects of ET receptor antagonists on systemic and renal vasculature in normotensive anaesthetized rats. Intravenous injection of a selective ETA receptor antagonist, FR139317 (0.5 μmol kg−1, for 20 min) induced a very small fall in blood pressure. Similarly, a non-selective ETA/ETB receptor antagonist, TAK-044 (12.5 μmol kg−1, for 20 min) slightly decreased blood pressure. A selective ETB receptor antagonist, BQ-788 (0.5 μmol kg−1, for 20 min) had no effect on blood pressure. FR139317 and TAK-044 did not affect renal blood flow or calculated renal vascular resistance. In contrast, BQ-788 significantly reduced renal blood flow by 18.2±2.4% and increased renal vascular resistance. Furthermore, the renal vascular action of BQ-788 was not observed when combined with FR139317. Pretreatment with a nitric oxide (NO) synthase inhibitor Nω-nitro-L-arginine methyl ester (L-NAME, 37 μmol kg−1, i.v.) and a cyclo-oxygenase inhibitor ibuprofen (44 μmol kg−1, i.v.) completely abolished the BQ-788-mediated renal vasoconstriction. These results indicate that activation of ETB receptors by endogenous ET acts as a physiological brake for the ETA-mediated renal vasoconstriction; this effect appears to be mediated by stimulation of NO and/or vasodilator prostaglandin(s) release. British Journal of Pharmacology (1997) 122, 81–86; doi:10.1038/sj.bjp.0701349

Nagase, Takahide, Tomoko Aoki, Teruaki Oka, Yoshinosuke Fukuchi, and Yasuyoshi Ouchi. ET-1-induced bronchoconstriction is mediated via ETB receptor in mice. J. Appl. Physiol. 83(1): 46–51, 1997.—Endothelin (ET)-1 is one of the most potent agonists of airway smooth muscle and can act via two different ET receptor subtypes, i.e., ETA and ETB. To determine the effects of ET-1 on in vivo pulmonary function and which ET receptors are involved in murine lungs, we investigated 1) the effects of ET and sarafotoxin S6c (S6c), a selective ETB agonist, on pulmonary function and 2) the effects of BQ-123 and BQ-788, specific ETA- and ETB-receptor antagonists, on ET-1-induced bronchoconstriction. ICR mice were anesthetized and mechanically ventilated (frequency = 2.5 Hz, tidal volume = 8 ml/kg, positive end-expiratory pressure = 3 cmH2O). Intravenous ET-1, ET-2, and ET-3 increased lung resistance similarly and equipotently, whereas S6c elicited a greater degree of bronchoconstriction. Mice were then pretreated with saline (Sal), BQ-123 (0.2, 1, and 5 mg/kg), or BQ-788 (0.2, 1, and 5 mg/kg) before administration of ET-1 (10−7 mol/kg iv). No dose of BQ-123 blocked ET-1-induced constriction, whereas pretreatment with each dose of BQ-788 significantly inhibited ET-1-induced responses. There were significant differences in morphometrically assessed airway constriction between Sal and BQ-788 and between BQ-123 and BQ-788, whereas no significant difference was observed between Sal and BQ-123. There were no significant morphometric differences in the airway wall area among the three groups. These observations suggest that the ETB- but not ETA-receptor subtype may mediate the changes in murine pulmonary function in response to ET-1. In addition, the ETB-receptor antagonist reduces ET-1-induced airway narrowing by affecting airway smooth muscle contraction in mice.

We investigated which endothelin receptors mediate the vasoconstrictive effects of endothelin-1 on liver circulation.An isolated perfused rat liver model in recirculation was used.The perfusion of 10(-10) M endothelin-1 had no significant influence on the liver flow, whereas 10(-9) M endothelin-1 induced significant vasoconstriction, with flow dropping from 3.20 +/- 0.34 to 1.48 +/- 0.28 ml. min-1.g-1 liver tissue (p0.01 vs controls). The liver flow was interrupted following the perfusion of 10(-8) M endothelin-1. Sarafatoxin C and BQ 3020, two agonists of ETB receptor, had vasoconstrictive effects in this model. Sarafatoxin C decreased the liver flow in a dose-dependent manner, from 3.32 +/- 0.21 to 2.18 +/- 0.20, 1.60 +/- 0.09, and 1.01 +/- 0.06 ml.min-1. g-1, respectively, with 10(-9) M, 10(-8) M, and 10(-7) M. While BQ 123, an antagonist of ETA receptor, or BQ 788, an antagonist of ETB receptor, partially reversed the effect of 10(-9) M endothelin-1, the simultaneous administration of BQ 123 and BQ 788 completely reversed these effects.These results indicate that the vasoconstrictive effects of endothelin-1 on the liver circulation are mediated through both ETA and ETB receptors.

1. Endothelin-1 (ET-1) produces constriction of the rat mesenteric vascular bed in vivo via ETA and ETB receptor subtypes. The aim of this study was to investigate the relative roles of these receptor subtypes in rat isolated, endothelium-denuded, small mesenteric arteries, under pressure, by use of ET-1; the ETA receptor antagonist, BQ-123; the ETB receptor selective agonist, sarafotoxin S6c (SRTX S6c); the ETB receptor selective antagonist, BQ-788; and the ETA/ETB antagonist, TAK-044. 2. In 3rd generation mesenteric arteries, ET-1 (10(-13)-10(-7) M) produced concentration-dependent contractions (pD2 9.86). SRTX S6c (10(-12)-10(-7) M) also induced concentration-dependent contractions in 53% of arteries studied, although the Emax was much less than that obtained with ET-1 (10.7 +/- 2.9% vs 101.9 +/- 2.6% of the 60 mM KCl-induced contraction). 3. Neither ETB receptor desensitization, by a supra-maximal concentration of SRTX S6c (10(-7) M), nor incubation with BQ-788 (3 x 10(-8) M), had any significant effect on the ET-1 concentration-response curve, although both treatments tended to enhance rather than inhibit responses to ET-1. 4. In the presence of BQ-123 (10(-6) M), responses to low concentrations of ET-1 (up to 10(-10) M) were unaffected but responses to concentrations of ET-1 above 10(-10) M were significantly inhibited. 5. SRTX S6c desensitization followed by incubation with BQ-123 (10(-6) M) or co-incubation with BQ-788 (3 x 10(-8) M) and BQ-123 caused inhibition of responses to all concentrations of ET-1, resulting in a rightward shift of the ET-1 concentration-response curve. The same effect was obtained by incubation with TAK-044 (10(-8) M and 3 x 10(-7) M). 6. Thus, responses of rat small mesenteric arteries to ET-1 are mediated by both ETA and ETB receptors. The relative role of ETB receptors is greater than that predicted by the small responses to SRTX S6c or by resistance of ET-1-induced contraction to ETB receptor desensitization or BQ-788. The effect of ETB receptor desensitization or blockade is only revealed in the presence of ETA receptor blockade, suggesting the presence of a 'crosstalk' mechanism between the receptors. These results support the concept that dual receptor antagonists, like TAK-044, may be required to inhibit completely constrictor responses to ET-1.

Pigs were subjected to acute, intermittent hypoxia (fraction of inhaled O2 0.1, n = 10). The increase in mean pulmonary artery pressure during hypoxia was not altered after i.v. administration of the selective endothelin ETB receptor antagonist BQ-788 (N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma-methylleucyl-D -1- methoxycarbonyltryptophanyl-D-norleucine) (1 mg). However, the vasodilatory effect of endothelin-1 (25 ng/kg per min) infused into the pulmonary artery during hypoxia was attenuated by BQ-788. The present results suggest that the pulmonary vasodilator effect of exogenously administered endothelin-1 during acute hypoxia is mediated by endothelin ETB receptors in the pig. Furthermore, endothelin ETB receptor antagonism with BQ-788 does not influence the pulmonary vascular response to acute hypoxia.

We investigated the effect of endothelins (ETs) on receptor-mediated phosphoinositide turnover in whole adrenal medulla. Endothelin -1, -2, -3 increased phosphoinositide (PI) turnover by 30% in whole adrenal medulla prelabeled with (3H)myoinositol. ET-stimulation of PI hydrolysis was almost completely dependent on the presence of the extracellular calcium since its chelation resulted in a blockade of ETs induced InsP1 accumulation. Addition of cadmium increased basal and ETs-stimulated InsP1 accumulation. ETs induced InsP1 accumulation was inhibited by BQ 123, a selective antagonist of the ETA receptor, while BQ 788, a selective antagonist of the ETB receptor, was ineffective. The selective agonist at the endothelin ETB receptor, IRL 1620, was ineffective to induce changes in inositide metabolism. Our data indicate that stimulation of PI turnover constitutes one of the signalling pathways of ETs in rat adrenal medulla and that this action is mediated through ETA receptor activation. These results suggest that endothelin could play a role in the regulation of adrenal medulla function.

This study assessed whether endothelin-1 (ET1) helps mediate post-ischemic acute kidney injury (AKI) progression to chronic kidney disease (CKD). The impact(s) of potent ETA or ETB receptor-specific antagonists (Atrasentan and BQ-788, respectively) on disease progression were assessed 24 hours or 2 weeks following 30 minutes of unilateral ischemia in CD-1 mice. Unilateral ischemia caused progressive renal ET-1 protein/mRNA increases with concomitant ETA, but not ETB, mRNA elevations. Extensive histone remodeling consistent with gene activation and increased RNA polymerase II binding occurred at the ET-1 gene. Unilateral ischemia produced progressive renal injury as indicated by severe histologic injury and a 40% loss of renal mass. Pre- and post-ischemia or just post-ischemic treatment with Atrasentan conferred dramatic protective effects such as decreased tubule/microvascular injury, normalized tissue lactate, and total preservation of renal mass. Nuclear KI-67 staining was not increased by Atrasentan, implying that increased tubule proliferation was not involved. Conversely, ETB blockade had no protective effect. Thus, our findings provide the first evidence that ET-1 operating through ETA can play a critical role in ischemic AKI progression to CKD. Blockade of ETA provided dramatic protection, indicating the functional significance of these results.

Objective: We investigated both the effects of the endothelin type B (ETB) receptor antagonist, BQ-788, on amitriptyline-induced cardiotoxicity and the role of ETB receptors on amitriptyline-induced cardiovascular depression. Methods: Male Wistar rats were anaesthetized with urethane/chloralose. Mean arterial pressure (MAP), heart rate (HR) and QRS duration were recorded. Toxicity was induced by amitriptyline infusion (0.94 mg/kg per min) until the 50% inhibition of MAP. In the first protocol, 5% dextrose or BQ-788 bolus was administered to control or experimental group animals, respectively. In the second protocol, after incubation with BQ-788 or 5% dextrose, amitriptyline was infused. Results: Amitriptyline caused a significant decrease in MAP, prolonged QRS duration and decreased HR for both the groups. BQ-788 administration improved MAP (5, 10 and 15 min), shortened the prolonged QRS (5 and 10 minutes) and increased HR (5, 10 and 15 min) compared with dextrose group. While all the amitriptyline-infused rats survived in BQ-788 group, all the amitriptyline-infused rats died within 20 min in dextrose group. In the second protocol, BQ-788 incubation did not cause any statistically significant change in amitriptyline-induced cardiovascular depression. Conclusion: BQ-788 may have beneficial effects in amitriptyline-induced cardiovascular changes via a physiologic antagonism. ETB receptor antagonists may be the promising antidotes for the cardiovascular toxicity with hypotension and bradycardia.

1. The effect of calcitonin gene-related peptide (CGRP) on airway smooth muscle is controversial. The aim of this study was to determine whether the action of CGRP on tracheal strips of guinea-pigs is modulated by epithelium and whether this peptide-induced action involves other mediators including nitric oxide (NO) and endothelin (ET)-1. 2. CGRP produced a weak dose-dependent increase in guinea-pig tracheal tension in vitro (-logEC50 = 8.5 +/- 0.1, maximum contraction = 8.3 +/- 1.2% of 50 mM KCl-induced contraction, n = 6). In epithelium-depleted preparations, CGRP (10(-7) M)-induced contraction was significantly potentiated from 9.0 +/- 1.9% to 41.1 +/- 6.0% (n = 6). 3. L-NG-nitro-arginine methyl ester (L-NAME, 10(-4) M), which inhibits NO synthesis, enhanced the contractile response to CGRP from 9.0 +/- 1.9% to 31.2 +/- 1.1% (n = 6). Indomethacin (10(-5) M) also enhanced the response to CGRP, although the effect was weak (13.4 +/- 3.2%, n = 6). 4. Anti-ET-1 serum changed the CGRP-induced contraction into a relaxation. After incubation of the trachea with ET-1 (10(-7) M) to attenuate ET-1-induced responses, the CGRP-induced contraction also changed into a relaxation. BQ-123 (an ETA receptor antagonist) and BQ-788 (an ETB receptor antagonist) caused the same conversion of the CGRP response, from contraction to relaxation, although the relaxing effect elicited by BQ-788 was more potent than that by BQ-123. Maximum inhibitory responses were -31.0 +/- 3.3% and -13.0 +/- 2.3% of 50 mM KCl-induced contraction, respectively (n = 6). 5. In primary culture, guinea-pig tracheal epithelial cells released ET-1, and CGRP (10(-5) M) significantly increased the release of ET-1. 6. These data suggest that the action of CGRP is modulated by airway epithelium and this mechanism involves the release of NO and ET-1. Especially, the majority of contractile action elicited by CGRP consists of an action of ET-1 via the predominant ETB receptor.

1. The pharmacology and mRNA expression of endothelin (ET) receptors in human omental arteries were characterized by use of functional contractile assays and the reverse transcriptase-polymerase chain reaction (RT-PCR). 2. In freshly obtained segments of human omental arteries, ET-1 and ET-3 induced concentration-dependent contractions which were normalized to the response produced by 60 mM K+. ET-1 produced a maximum contraction (Emax) amounting to 151 +/- 17% of the K+ response. The pEC50 for this agonist was 8.64 +/- 0.17. The effect of ET-3 was less pronounced (Emax: 71 +/- 22% and pEC50: 6.69 +/- 0.17) than that of ET-1. The ET receptors involved were characterized with FR139317 (a selective ETA receptor antagonist), PD 145065 (a mixed ETA and ETB receptor antagonist) and BQ 788 (an ETB receptor antagonist). A high concentration of these antagonists (10 microM) abolished the contractile responses to ET-3, and produced a parallel rightward shift of the ET-1 concentration-response curve without changing the maximal effect. FR139317 and PD 145065 were equally effective while BQ 788 was much less effective. This is consistent with ETA receptors mediating contraction in human omental arteries. 3. Arterial segments cultured for 5 days in serum-free Dulbecco's medium at 37 degrees C under sterile and humidified conditions retained contractility although responses to 60 mM K+ were somewhat reduced. ET-3 was significantly more potent in the cultured arteries (pEC50: 8.56 +/- 0.15) and achieved a greater maximum effect (Emax: 116 +/- 19%). Responses were not antagonised by FR139317 but were competitively blocked by PD 145065 and BQ 788 with the latter antagonist being the more potent. In contrast Emax (179 +/- 17%) and pEC50 (8.66 +/- 0.23) values for ET-1 were not significantly different from those obtained with fresh arteries. PD 145065 still demonstrated a rightward shift of the ET-1-induced concentration-response curve, whereas FR139317 and BQ 788 caused non-significant shifts. These findings suggest that functional ETB receptors contribute significantly to the endothelin contractile response in cultured arteries. 4. Two-site analysis of the ET-1 induced concentration-response curve from cultured arteries suggests that ETB receptors, at the high potency component, and ETA receptors, at the low potency component, contribute both to the contractile response in relative proportion of 70% and 30%, respectively. Further analysis suggested that the ETA receptor would be capable of evoking at least 75% of the ET-1 contraction in the absence of ETB receptors, although with a lower potency as compared to fresh arteries. 5. Electrophoresis of RT-PCR products from the smooth muscle layer of freshly obtained human arteries indicated the presence of mRNA for both ETA and ETB receptors. Arteries cultured for 1 and 5 days demonstrated an increase of mRNA for the ETB receptor as compared to the ETA receptor. The identities of the PCR products were verified by restriction enzyme digestion. 6. In freshly obtained human omental arteries, the contractile effects of endothelins appear to be mediated predominantly by the ETA receptor subtype, with a negligible contribution by ETB receptors. Cultured arterial segments, however, exhibited a substantial ETB receptor mediated contractile response and an increase in ETB receptor mRNA content, consistent with an upregulation of functional ETB receptors. These in vitro data suggest plasticity in the smooth muscle cell expression of contractile ETB receptors.

1. This study examined the influence of respiratory tract infection with influenza A/PR-8/34 virus on endothelin receptor-mediated modulation of contraction induced by stimulation of cholinergic nerves in mouse isolated trachea. 2. The ETB receptor-selective agonist, sarafotoxin S6c (30 nM) induced large transient contractions (118 +/- 5% Cmax, n = 13; where Cmax is the contraction induced by 10 microM carbachol) of isolated tracheal segments from control mice. The peak contractile response to 30 nM sarafotoxin S6c was significantly lower in preparations from virus-inoculated mice at day 2 (57 +/- 8% Cmax, n = 3, P < 0.05) and 4 post-inoculation (90 +/- 8% Cmax, n = 9, P < 0.05), consistent with virus-induced attentuation of the ETB receptor-effector system linked to airway smooth muscle contraction. The mean peak contraction to 30 nM sarafotoxin S6c of preparations from virus-inoculated mice at day 8 post-inoculation (94 +/- 17% Cmax, n = 4) was not significantly different from that of control. 3. Electrical field stimulation (EFS; 90 V, 0.5 ms duration, 10 s train, 0.1-30 Hz) of preparations from control and virus-inoculated mice, caused contractions that were abolished by 0.1 microM atropine or 3 microM tetrodotoxin, indicating that these responses were mediated by neuronally released acetylcholine. Sarafotoxin S6c markedly potentiated contractions induced by a standard stimulus (0.3 Hz, every 3 min) in tracheal segments from control and virus-inoculated mice. In tracheal tissue from control mice, 30 nM sarafotoxin S6c significantly increased a standard EFS-induced contraction of 24 +/- 4% Cmax by a further 24 +/- 3% Cmax (i.e. 2 fold increase, n = 11). Sarafotoxin S6c (30 nM) also markedly potentiated standard EFS-induced contractions in preparations from virus-inoculated mice at day 2 (17 +/- 2% Cmax, n = 3), day 4 (17 +/- 5% Cmax, n = 9) and day 8 (26 +/- 5% Cmax, n = 4) post-inoculation. The level of potentiation of EFS-induced contractions in preparations from virus-inoculated mice was similar to that in tissue from control mice at days, 2, 4 and 8 post-inoculation. In contrast, sarafotoxin S6c (30 nM) did not enhance contractile responses of tracheal segments from control and virus-inoculated mice to exogenously applied acetylcholine (n = 3). 4. Endothelin-1 (1 nM) caused similar potentiations of standard EFS-induced contractions in tracheal segments from control (13 +/- 2% Cmax, n = 23) and virus-inoculated mice at day 2 (13 +/- 1% Cmax, n = 5), day 4 (16 +/- 5% Cmax, n = 6), and day 8 (13 +/- 3% Cmax, n = 8) post-inoculation. In contrast, 1 nM endothelin-1 did not enhance contractile responses of tracheal segments from control and virus-inoculated mice to exogenously applied acetylcholine (n = 4). Neither the ETA receptor-selective antagonist, BQ-123 (3 microM) nor the ETB receptor-selective antagonist, BQ-788 (1 microM) alone had any significant inhibitory effect on endothelin-1-induced potentiations of tracheal segments from control or virus-inoculated mice at days 2, 4 and 8 post-inoculation. However, simultaneous pre-incubation with BQ-123 (3 microM) and BQ-788 (1 microM) prevented endothelin-1-evoked potentiations, indicative of a role for both ETA and ETB receptors in this system. 5. These data clearly demonstrate that respiratory tract viral infection attenuated the function of the postjunctional ETB receptor-effector system linked directly to airway smooth muscle contraction. However, the function of prejunctional ETA and ETB receptor-effector systems linked to augmentation of cholinergic nerve-mediated airway smooth muscle contraction remained unaffected during respiratory tract viral infection in mice.

1. We investigated the effects of the selective endothelin (ET)A receptor antagonist BQ-485 and the selective ETB receptor antagonist BQ-788 on circulatory failure, multiple organ dysfunction syndrome (MODS) and the alterations in acid base balance caused by endotoxaemia in the anaesthetized rat. 2. Male Wistar rats were anaesthetized (thiopentone sodium; 120 mg kg-1, i.p.) and received a continuous infusion of vehicle (saline, 0.6 ml kg-1h-1, i.v.), BQ-485 (10 nmol kg-1 min-1, i.v.) or BQ-788 (10 nmol kg-1 min-1, i.v.). Fifteen min later, animals received a bolus injection of either saline (0.9% NaCl, 1 ml kg-1, i.v.) or E. coli lipopolysaccharide (LPS, 10 mg kg-1, i.v.). 3. Injection of LPS resulted in a fall in blood pressure from 115 +/- 4 mmHg (time 0) to 82 +/- 4 mmHg at 360 min (n = 15) as well as a hyporeactivity to the pressor responses to noradrenaline (NA, 1 microgram kg-1, i.v.). Infusion of BQ-788 attenuated the delayed hypotension (at 360 min: 100 +/- 4 mmHg, n = 7; P < 0.05) and significantly enhanced the pressor responses elicited by NA (at 60 to 240 min). In contrast, treatment of LPS-rats with BQ-485 augmented the hypotension (at 360 min), but did not affect the vascular hyporeactivity elicited by endotoxaemia. 4. Endotoxaemia for 360 min resulted in rises in the serum levels of urea and creatinine (indicators of renal failure), glutamate-oxalate-transferase (GOT) and glutamate-pyruvate-transferase (GPT) (indicators of hepatocellular injury), and bilirubin and gamma-glutamyl transferase (gamma GT) (indicators of liver failure) as well as nitrite (indicator of the induction of nitric oxide synthase; iNOS). Treatment of LPS-rats with BQ-788, but not with BQ-485, attenuated the degree of liver injury and failure, while neither BQ-788 nor BQ-485 affected the acute renal failure or the induction of iNOS caused by endotoxin. 5. Endotoxaemia also caused (within 15 min) an acute metabolic acidosis (falls in pH, HCO3-and base excess) which was compensated by hyperventilation (fall in PaCO2). Treatment of LPS-rats with BQ-788 or BQ-485 did not affect the metabolic acidosis caused by LPS. 6. Thus, the selective ETB receptor antagonist BQ-788 attenuated (i) the delayed hypotension, (ii) the vascular hyporeactivity to NA as well as (iii) the degree of hepatocellular injury and dysfunction caused by endotoxin in the anaesthetized rat. In contrast, the selective ETA receptor antagonist did neither attenuate the circulatory failure nor the liver or renal dysfunction associated with endotoxaemia. We propose that the prevention of the hepatocellular dysfunction and injury caused BQ-788 in endotoxaemia is due to an improvement in oxygen delivery to the liver secondary to (i) inhibition of pre-sinusoidal constriction, (ii) inhibition of sinusoidal constriction, and (iii) improvement in perfusion pressure.

1. The type of endothelin (ET) receptor located on the myenteric neurones of guinea-pig ileum was determined by receptor autoradiography and function of the receptor was examined by release experiments of acetylcholine (ACh) from the longitudinal muscle myenteric plexus (LM-MP) preparations. 2. Specific [125I]-ET-1 binding sites were distributed in muscle layers, myenteric and submucous plexuses, and mucosa layers. High-grain densities were detected in both myenteric and submucous plexuses. 3. Binding in the myenteric plexus was abolished by incubation with either IRL 1620 (endothelin ETB receptor agonist) or BQ 788 (endothelin ETB receptor antagonist), but not with BQ 123 (endothelin ETA receptor antagonist). The [125I]-IRL 1620 binding sites were evident in the myenteric plexus. Thus, the endothelin receptor located on the myenteric neurones is of the ETB type. 4. ET-1 (10(-10)-3 x 10(-8) M) and ET-3 (10(-10)-3 x 10(-8) M) evoked 3H outflow from LM-MP preparations of ileum preloaded with [3H]-choline, in a concentration-dependent manner. There was no significant difference between maximum amounts of ET-1-evoked and ET-3-evoked 3H outflow. 5. ET-1 and ET-3 evoked outflow of 3H was BQ 788-sensitive, but BQ 123-insensitive. Both evoked outflows of 3H were Ca(2+)-dependent and tetrodotoxin-sensitive. 6. These results indicate that the endothelin ETB receptor is located on the enteric cholinergic neurones and that stimulation evokes the release of ACh.

1. IRL 1620 (0.01-0.1 mg kg-1, i.v.), a selective endothelin B (ETB) receptor agonist, induced a dose-dependent biphasic increase in total lung resistance and a decrease in dynamic compliance in anaesthetized and artificially ventilated guinea-pigs. After intravenous injection of IRL 1620 (0.03 mg kg-1), the first phase was observed within 2 min whereas the second phase started between 5 and 10 min after injection and was long lasting. 2. In order to characterize which endothelin receptors are involved in both phases of bronchoconstriction, we studied the effect of ETA and ETB receptor antagonists (BQ 123 and BQ 788, respectively). BQ 788 (0.1-1 mg kg-1, i.v.) inhibited, in a dose-dependent manner, both phases of bronchoconstriction. BQ 123 (3 mg kg-1, i.v.) markedly inhibited (by 76%) the second phase of bronchoconstriction but had no effect on the early component of the response. 3. The effect of atropine, neurokinin-I (NK1) and neurokinin-2 (NK2) receptor antagonists (SR140333 and SR48968, respectively) were tested to investigate the possible involvement of cholinergic and sensory nerve activation, respectively, in the response to IRL 1620. Likewise, the role of arachidonic acid metabolites (leukotriene D4 antagonist, ONO-1078 and thromboxane A2 (TXA2) inhibitor, OKY-046) in this response was also investigated. OKY-046 (1 mg kg-1, i.v.) and atropine (1 mg kg-1, i.v.) partially inhibited the first phase (by 80% and 20%, respectively) without affecting the late phase of bronchoconstriction. Neither ONO-1078 (1 mg kg-1, i.v.) nor the combination of SR140333 (0.2 mg kg-1, i.v.) and SR 48968 (0.2 mg kg-1, i.v.) modified IRL 1620-induced bronchoconstriction. 4. A low dose of IRL 1620 (0.005 mg kg-1, i.v.) induced a monophasic bronchoconstriction. Pretreatment by phosphoramidon (100 mumol kg-1, i.v.) restored the second phase of bronchoconstriction. In this condition, BQ 123 (3 mg kg-1, i.v.) was able to inhibit partially the second phase of bronchoconstriction. 5. These results suggest that both phases of bronchoconstriction induced by IRL 1620 were mediated primarily by ETB receptor activation, the first phase being a consequence of TXA2 and acetylcholine release. The inhibition by an ETA receptor antagonist and the restoration by a neutral endopeptidase (NEP) inhibitor of the second phase of bronchoconstriction suggests that primary activation of ETB receptors leads to autocrine/paracrine endothelin-1 (ET-1) release that would subsequently cause profound bronchoconstriction through both ETA and ETB receptor activation.

Summary The actions of the endothelins (endothelin-I. endothelin-2 and endothelin-3) are mediated via endothelin-A (EA) and endothelin-B (ETB) receptors. the former generally mediating vasoconstriction and the latter vasodilation. Peptide antagonists selective for either receptor sUbtype [e.g. BQ 123 (ETA) and BQ 788 (ETB)] and combined ETA/ETB receptor antagonists (e.g. PD 145065 and TAK 044) have been developed. More recently. small molecule non-peptide antagonists have also been synthesised. ETA receptor-selective agents include PD 155080 and BMS 182874. while Ro 46-2005 and bosentan are combined ETA/ETB receptor antagonists. The role of the endothelin family of vasoconstrictor peptides in the pathophysiology of cerebrovascular disease has been speculated upon. Increases in plasma and CSF levels of endothelin-I in delayed vasospasm following subarachnoid haemorrhage and acute ischaemic stroke have implicated the endothelins in these cerebrovascular diseases. The development of non-peptide endothelin receptor antagonists has facilitated investigations into the role of the endothelins in cerebrovascular disease. The endothelin receptor antagonists have been demonstrated to attenuate cerebral vasospasm following experimental subarachnoid haemorrhage in a variety of species. Additionally, the endothelin receptor antagonists ameliorate neuronal damage following eXIkrimental focal and global cerebral ischaemia. These actions have highlighted the therapeutic potential of endothelin receptor antagonists in cerebrovascular disease.

1. Endothelin (ET)-1 has been postulated to be involved in the development of obstructive airway diseases in man. In the present study, we attempted to characterize ET receptor subtypes mediating ET-1-induced contraction in human isolated bronchi. The ET receptor antagonists used in the present study were BQ-123 (ETA receptor-selective), BQ-788 (ETB receptor-selective) and BQ-928 (ETA/ETB dual). Sarafotoxin S6c (S6c) was also used as an ETB receptor-selective agonist. 2. In human bronchi, ET-1 and S6c (10(-12)M to 10(-7) M) produced concentration-dependent contraction with almost equal potency (pD2: 8.88 +/- 0.16 for ET-1 and 9.42 +/- 0.15 for S6c). The contraction induced by S6c was competitively antagonized by BQ-788 alone (1 and 10 microM) with a pKB value of 7.49 +/- 0.21, suggesting that the stimulation of ETB receptors causes a contraction of human bronchi. However, contrary to expectation, the concentration-response curves for ET-1 were not affected by BQ-788. The ET-1- and S6c-induced contractions were not affected by BQ-123 (10 microM). Thus, ET-1-induced contraction of human bronchi is not antagonized by BQ-123 alone or by BQ-788 alone. 3. Combined treatment with 10 microM BQ-123 and 10 microM BQ-788 significantly antagonized the contraction induced by ET-1 with a dose-ratio of 11. BQ-928 also significantly antagonized ET-1-induced contraction with a pKB value of 6.32 +/- 0.24. 4. The specific binding of [125I]-ET-1 to human bronchial membrane preparations was inhibited by BQ-123 (100 pM to 1 microM) by approximately 40%. Combination treatment with BQ-788 (100 pM to 1 microM) completely inhibited the BQ-123-resistant component of [125I]-ET-1 specific binding. 5. In conclusion, the present study demonstrates that BQ-788 alone cannot inhibit ET-1-induced contractions in human bronchi, although human bronchial ETB receptors are BQ-788-sensitive. Furthermore, it was shown that blockade of both receptor subtypes antagonizes ET-1-induced contraction, and that both receptor subtypes co-exist in human bronchial smooth muscles. These findings suggest that ETA receptors as well as ETB receptors are involved in ET-1-induced contraction in human bronchi. If ET-1 is involved in human airway diseases, dual blockade of ETA and ETB receptors may be necessary to treat the diseases.

1. We investigated the characteristics of endothelin (ET)-induced contraction and changes in intracellular Ca2+ concentration ([Ca2+]i) using the fura-2-loaded and non-loaded rabbit iris dilator. ET-1 and ET-2 (3-100 nM) and ET-3 (30-100 nM) caused contraction in a concentration-dependent fashion. 2. The selective ETB-receptor agonists, IRL1620 and sarafotoxin S6c produced only a small contraction or no contraction at a concentration of 1 microM. The rank order of potencies for the contraction (pD2 value) was ET-1 = ET-2 > ET-3 >> sarafotoxin S6c = IRL1620. 3. The contractile response to ET-3 was antagonized by pretreatment with BQ-123 (10 nM), a selective ETA receptor antagonist. The contractile responses to ET-1 and ET-2 were antagonized by pretreatment with BQ-123 (10 microM), but not at a concentration of 10 nM. 4. ETs increased [Ca2+]i and sustained muscle contraction. ET-1 (100 nM), ET-2 (100 nM), and ET-3 (1 microM) induced an elevation of [Ca2+]i consisting of two components: first a rapid and transient elevation to reach a peak, followed by a second, sustained elevation; a sustained contraction was produced without a transient contraction. The ETB receptor-selective agonist, IRL1620 (1 microM) and sarafotoxin S6c (1 microM) also induced a rapid and transient elevation of [Ca2+]i to reach a peak and a sustained elevation, together with only a small contraction or no contraction. 5. ET-1 (100 nM) induced a transient increase in [Ca2+]i in a Ca(2+)-free, 2 mM EGTA-containing physiological saline solution (Ca(2+)-free PSS), and a small sustained contraction which was significantly different from that induced by ET-1 (100 nM) in normal PSS. The ET-1-induced increase in [Ca2+]i and sustained contraction were not affected by the voltage-dependent Ca2+ channel blocker, nicardipine (10 microM). The ET-1-induced transient increase in [Ca2+]i was significantly reduced by the sarcoplasmic reticulum (SR) Ca(2+)-ATPase inhibitor, cyclopiazonic acid (30 microM); however, the ET-1-induced sustained contraction was not affected by this agent. 6. The selective ETA receptor antagonist, BQ-123 (100 nM) reduced the ET-3 (100 nM)-induced contraction, but did not affect the transient increase or elevation of the second phase of [Ca2+]i. However, this antagonist at 1 microM did not affect the ET-1 (100 nM)- and ET-2 (100 nM)-induced elevation of [Ca2+]i and contractile response, or the IRL1620-induced elevation of [Ca2+]i. 7. The selective ETB receptor antagonist, BQ-788 (1 microM) reduced the transient increase in [Ca2+]i induced by ET-1 (30 nM), ET-2 (30 nM), ET-3 (100 nM) and IRL1620 (1 microM), but did not affect the sustained elevation of [Ca2+]i and contractile responses produced by ET-1, ET-2 and ET-3. 8. Pretreatment with IRL1620 (1 microM) reduced the increase in [Ca2+]i induced by IRL1620 (1 microM) and sarafotoxin S6c (1 microM), as well as the ET-1 (100 nM)-, ET-2 (100 nM)- and ET-3 (1 microM)-induced elevation of [Ca2+]i, whereas in the presence of IRL1620, ET-1-, ET-2- and ET-3-induced contractions were unaltered. 9. These results suggest that ETA and ETB receptor subtypes exist in the rabbit iris dilator muscle, and that the ETA receptor is divided into: (1) BQ-123-sensitive ETA subtypes activated by ET-1, ET-2 and ET-3, and (2) BQ-123-insensitive ETA subtypes activated by ET-1 and ET-2, which cause the sustained increase of [Ca2+]i and contraction; in contrast, ETB receptor subtypes are activated by ET-1, ET-2, ET-3, IRL1620 and sarafotoxin S6c and cause the transient and sustained increase in [Ca2+]i which is not able to contract the smooth muscle.

1. In the current study, the density and function of ETA and ETB receptors in mouse tracheal airway smooth muscle were determined over the time course of respiratory tract infection with influenza A/PR-8/34 virus. 2. Quantitative autoradiographic studies using [125I]-endothelin-1 revealed that the tracheal airway smooth muscle from control mice contained ETA and ETB sites in the ratio of 49%:51% (+/- 2%, n = 29 mice). Respiratory tract viral infection was associated with increases in the density of ETA sites and decreases in the density of ETB sites at days 1, 2 and 4 post-inoculation which were reversible by day 19. For example, at day 4 post-inoculation, a time when the manifestations of viral infection were at or near their peak, the ratio of ETA:ETB sites was 72%:28% (+/- 4%, n = 6 mice, P < 0.05). In contrast, at day 19 post-inoculation, by which time viral infection had essentially resolved, the ratio of ETA:ETB sites was similar to control (51%:49% (+/- 3%), n = 6 mice). 3. Endothelin-1 was a potent spasmogen in isolated tracheal airway smooth muscle preparations from control mice (ED70 = concentration producing 70% of contraction induced by 10 microM carbachol = 6.3 nM (95% confidence limits, 4.0-10; n = 6 mice)). Neither the ETA receptor-selective antagonist, BQ-123 (3 microM), nor the ETB receptor-selective antagonist, BQ-788 (1 microM) alone had any significant inhibitory effect on endothelin-1-induced contractions of mouse isolated tracheal smooth muscle. However, simultaneous treatment with BQ-123 (3 microM) and BQ-788 (1 microM) resulted in a 10 fold rightward shift in the concentration-effect curve to endothelin-1 (ED70 = 60 nM, (44-90; n = 6 mice, P < 0.05)), indicating that contraction was mediated via both ETA and ETB receptors. 4. Endothelin-1 evoked similar concentration-dependent contractions of tracheal smooth muscle isolated from control and virus-inoculated mice. In the presence of the ETB receptor-selective-antagonist, BQ-788 (1 microM), the potency and maximum response to endothelin-1 were similar in preparations from control and virus-inoculated mice at all time points investigated. However, unlike control responses, endothelin-1-induced contractions in preparations from virus-infected mice were significantly inhibited by the ETA receptor-selective antagonist, BQ-123. For example, at day 4 post-inoculation, the contractile response to 30 nM endothelin-1, in the presence of BQ-123 (3 microM), was only 20 +/- 12% (n = 6 mice, P < 0.05) of that produced in control preparations under similar conditions. However, at day 19 post-inoculation, contraction evoked by 30 nM endothelin-1 in the presence of BQ-123 (3 microM), was similar to that in preparations from control mice. 5. In summary, during the early stages (days 1-8 post-inoculation) of respiratory tract infection with influenza A/PR-8/34 virus, we observed decreases in the density of tracheal airway smooth muscle ETB receptors which were reflected in decreases in ETB receptor-mediated airway smooth muscle contraction. In addition, during the same period of viral infection we observed increases in the density of tracheal airway smooth muscle ETA receptors which were not associated with increased function of the ETA receptor-effector system linked to contraction. Virus-associated modulation of ETA and ETB receptor density and function was reversible with recovery from infection.

Cloned rat and human endothelin-B receptors (ETBR) were utilized to determine if there are significant pharmacological differences between highly homologous ETBR from different species. Recombinant rat and human ETBR were expressed in CHO-K1 cells, and radioligand binding studies were carried out with [125I]-ET-3 to determine the affinities of various ET receptor agonists and antagonists for rat and human ETBR. These receptors had similar affinities for a number of ETBR agonists (ET-1, ET-3, S6C, BQ 3020) and antagonists (Ro 47-0203, PD 142893). However, several peptide (PD 147452, PD 151583, BQ 788) and non-peptide (PD 156707, SE 209670) antagonists had different affinities for rat and human ETBR, with differences in Ki values between species ranging from 4.1- to 53.4-fold. The ETBR-selective agonist IRL 1620 also had a 5.7-fold higher affinity for rat ETBR than human ETBR. Thus despite their high degree of homology, rat and human ETBR show significant pharmacological differences with respect to both antagonist and agonist binding.

IRL 1620 (Suc-[Glu9, Ala11,15]ET-1(8-21), a selective ETB receptor agonist) contracted rat trachea and bronchus. The maximum response to IRL 1620 was less than that to endothelin-1 (ET-1) and, compared with ET-1 responses, IRL 1620 responses reached equilibrium more quickly. IRL 1620 responses were antagonized by the selective ETB antagonist, BQ-788 (3 microM), but not by the selective ETA antagonist, BQ-123 (3 microM). ET-1 concentration-response curves were shifted only in the presence of both BQ-123 and BQ-788 (3 microM). In the presence of BQ-123, the time course of ET-1 responses changed from being slow and sustained (ETA, see below) to being more like that to IRL 1620 (ETB). IRL 1620 did not contract pulmonary artery preparations from rats but ET-1 produced slow and sustained contractile responses which were antagonized by BQ-123 but not by BQ-788. Thus, ET-1 contracts rat pulmonary artery and aorta predominantly via ETA receptors (responses blocked by BQ-123) and functional ETB receptors are unlikely to be present (no responses to IRL 1620). In contrast, ET-1 contracts rat trachea and bronchus via ETA and ETB receptors (tissues contracted to IRL 1620, ET-1 responses were blocked by a combination of BQ-123 and BQ-788 and ET-1 responses resembled ETB in character when ETA receptors were blocked.

The receptor subtypes mediating tension responses to endothelin-1 (ET-1) in isolated rat and rabbit aortic preparations were examined with various ET receptor antagonists. In the presence of 3 μM IRL 2500 (an ET B -selective antagonist), SB 209670 (an ET A /ET B antagonist) induced monophasic inhibitions of the ET-1 dose-response curves of both rat (pK B 9.1) and rabbit aortas (pK B 8.9). In the presence of IRL 2500, PD 156707 (an ET A antagonist) also caused a similar monophasic inhibition of the agonist-induced dose-response curve of the rat aorta (pK B 7.7) but produced a biphasic inhibition of the curve obtained with the rabbit aorta. PD 156707, however, produced monophasic inhibitions of the ET-I-induced dose-response curve of rabbit aortas pretreated either with BQ 788 (an ET B -selective antagonist) or desensitized with sarafotoxin S6c (an ET B -seBecsive agonist). The receptor subtypes mediating the contractile responses to ET-I were further characterized by examining the effects of the antagonists on the binding of [ 125 I]ET-1 to these two tissues. In the presence of 1 nM IRL 2500, SB 209670 displaced [ 125 I]ET-1 binding in both rat and rabbit aortic membrane preparations in a monophasic manner, with similar potencies (IC 50 1.2-1.6 nM). Similar results were also obtained with PD 156707 in rat aortic membrane (IC 50 0.27 nM). In contrast, the data obtained with PD 156707 using rabbit aortic membrane were best-fitted with a two-site model (site 1, IC 50 0.21 nM, 67% of B max ; site 2, 100 nM and 33%, respectively). Therefore, the constrictor response to ET-1 in the rat aorta is mediated exclusively by the ET A receptor, whereas that in the rabbit aorta is additionally mediated by an ET B receptor subtype that is sensitive to SB 209670 and BQ 788 but insensitive to IRL 2500.

1. The present study characterizes the receptors responsible for endothelin-1-induced release of thromboxane A2 from the guinea pig lung and of endothelium-derived nitric oxide from the rabbit perfused kidney, by the use of the selective ETA receptor antagonist, BQ-123, and a novel selective ETB receptor antagonist, BQ-788. 2. In the guinea pig perfused lung, endothelin-1 (ET-1) (5 nM) induced a marked increase of thromboxane A2 which was reduced by 17 +/- 5.0, 70 +/- 1.0 and 93 +/- 1.2% by BQ-788 infused at concentrations of 1, 5 and 10 nM respectively. In contrast, BQ-123 (0.1 and 1.0 microM) had little or no effect on the ET-1-induced release of thromboxane A2. 3. In the same perfused model, the selective ETB agonist, IRL 1620 (50 nM), stimulated the release of thromboxane A2, but not prostacyclin. The eicosanoid-releasing properties of IRL 1620 were abolished by BQ-788 at 10 nM, yet were unaffected by BQ-123 (1 microM). 4. In the rabbit perfused kidney, BQ-788 (10 nM) potentiated the increase of perfusion pressure induced by endothelin-1 (1, 5 and 10 nM) by approximately 90%, but not that induced by angiotensin II (1 microM). Furthermore, the selective ETB receptor antagonist did not reduce the release of prostacyclin triggered by either peptide. 5. In another series of experiments, pretreatment of the perfused kidney with a nitric oxide synthase inhibitor, L-NAME (100 microM), potentiated the pressor responses to both endothelin-1 and angiotensin II. Under L-NAME treatment, BQ-788 did not further potentiate the pressor response to endothelin-1. 6 Our results illustrate the predominant role of ETB receptor activation in the release of thromboxane A2 and nitric oxide triggered by endothelin-l in the guinea pig perfused lung and rabbit kidney respectively.

Exogenous endothelin (ET) is rapidly cleared from the circulation. We investigated which ET receptor subtypes (ETA and ETB) participate in ET-1 clearance. Following an intravenous (i.v.) bolus dose of [125I]ET-1 in anesthetized rats, radioactivity was rapidly cleared from the circulation and trapped by the lungs, kidneys and liver. Tissue distribution of the radioactivity was significantly inhibited in the lungs and kidneys, but not in the liver by infusion of the ETB antagonist BQ-788 (0.1 mg/kg/min i.v.), and the ET-1 clearance rate was reduced, while the ETA antagonist BQ-123 had no such effect. Furthermore, in isolated perfused rat lungs, about 80% of bolus-injected [125I]ET-1 was retained by the lungs after one passage. The retention of ET-1 was significantly inhibited by infusion of 1 microM BQ-788, but not BQ-123. These results suggest that ETB receptors play an important role in the clearance of ET-1.

1. We have compared the receptors mediating the contractions of rings of rat thoracic aorta or rabbit pulmonary artery and rat stomach strips in response to the endothelin/sarafotoxin (ET/SX) family of peptides and to those mediating endothelium-dependent vasodilations within the isolated perfused mesentery of the rat. To discriminate ETA receptors from ETB receptors we have used the criteria that ET-1 is more active than SX6c on ETA receptors, and that the ET/SX peptides are equiactive on ETB receptors. We have also assessed the effects of the ETA receptor-selective antagonist BQ-123, and the non-selective ET receptor antagonist PD 142893 on the responses of each preparation to the ET/SX peptides. 2. ET-1-induced constrictions of the rat thoracic aorta (EC50 3 x 10(-10) M), a prototypic ETA receptor-mediated response, or isolated perfused mesentery of the rat were antagonized by BQ-123 (10(-5) M) or PD 142893 (10(-5) M). SX6c did not constrict either the rat isolated perfused mesentery or the rat thoracic aorta. Thus, ETA receptors mediate these constrictions. 3. ET-1 and SX6c were approximately equipotent in constricting rabbit pulmonary artery rings (EC50S 3-6 x 10(-10) M). Neither BQ-123 (10(-5) M) nor PD 142893 antagonized the contractions induced by ET-1. These effects suggest mediation by ETB receptors but PD 142893 (10(-5) M) did give a 3 fold antagonism of constrictions induced by SX6c. 4. SX6c was more potent than ET-1 in contracting the rat stomach strip (threshold concentrations 10(-10) and 3 x 10(-10) M). Contractions to ET-1 or SX6c were unaffected by BQ-123 (10-5 M), again indicative of ETB receptor-mediated events. PD 142893 (10-5 M) was ineffective against ET-1 but produced a 3 fold antagonism of SX6c.5. In the rat isolated perfused mesentery ET-1 or SX6c (0.3-300pmol) were equipotent in producing dose-related vasodilatations that were unaffected by BQ-123 (10-6 M), indicative of an ETB receptor mediated response. In contrast to the other ETB-mediated responses, PD 142893 (10-6 M) strongly antagonized these vasodilatations.6. Thus, ETA receptors mediate constrictions of the rat thoracic aorta and rat isolated perfused mesentery whereas ETB receptors mediate constrictions of the rabbit pulmonary artery and rat stomach strip and endothelium-dependent dilatations within the mesentery. However, within the group of ETB receptor-mediated responses, endothelium-dependent vasodilatations are sensitive to PD 142893, whereas contractions of the isolated smooth muscle preparations are not. Thus, the receptor present on the endothelium responsible for the release of nitric oxide in response to the ET/SX peptides is most probably different from that present on smooth muscle that mediates BQ-123-insensitive contractions.

Clonidine decreases mean arterial pressure (MAP) by acting as an α(2)-adrenergic receptor (AR) agonist in the central nervous system; it also acts on peripheral α-ARs to produce vasoconstriction. Endothelin (ET) has been shown to modulate the action of ARs. The present study was conducted to determine the involvement of ET in cardiovascular effects of clonidine. Intravenous administration of clonidine (10, 30 and 90μgkg(-1)) produced a dose-dependent decrease in MAP and heart rate (HR). Treatment with ET-1 (100, 300 and 900ngkg(-1)) significantly attenuated clonidine (10μgkg(-1)) induced fall in MAP and HR. Rats treated with ET-1 (900ngkg(-1)) showed an increase in MAP and HR after clonidine administration compared to untreated rats, while ET(A/B) antagonist, TAK-044 (1mgkg(-1)) and ET(A) antagonist, BMS-182874 (9mgkg(-1)) potentiated the hypotensive effect of clonidine. ET(B) receptor agonist, IRL-1620 (5μgkg(-1)) produced significant attenuation of clonidine induced fall in MAP and HR, while ET(B) receptor antagonist, BQ-788 (0.3mgkg(-1)), potentiated the hypotensive effect of clonidine. Prazosin (0.1mgkg(-1)) completely blocked ET-1 induced changes in cardiovascular effects of clonidine. Clonidine-induced contraction of rat abdominal aortic ring was potentiated by ET-1, which was completely blocked by prazosin. Clonidine produced an increase in ET(A) receptor expression in the brain and abdominal aorta while ET(B) receptors were not affected. It is concluded that ET enhances the responsiveness of vascular ARs to the constrictor effect of clonidine and ET antagonists potentiate the hypotensive effect of clonidine suggesting that a combination of ET antagonist with clonidine may be a useful option to treat hypertension.

It has been suggested that the endothelin (ET) ETB receptor could mediate endothelium-dependent vasodilation to ET-1 or ET-3, but its in vivo role is still largely unknown. We used sarafotoxin S6C, a selective agonist of the ETB receptor, to study the in vivo effects of ETB stimulation. SRTX S6C induced a transient decrease in blood pressure, followed by a long-lasting pressor response accompanied by a marked renal and mesenteric vasoconstriction. No constriction was observed in isolated mesenteric arteries in vitro, indicating that the in vivo vasoconstrictor effect is most likely indirect. The pressor effect of SRTX S6C was not dependent on central stimulation of ETB receptors and was not mediated by catecholamines from the adrenal medulla, prostanoids or ET-1.

In contrast to endothelin-1 (ET-1) and several of its analogues, sarafotoxin S6c (S6c) was a much more potent inhibitor of [125I]-ET-1 binding in rat hippocampus and cerebellum (Ki approximately 20 pM) than in rat atria and aorta (Ki approximately 4500 nM), suggesting the existence of ET-1 receptor subtypes (aorta/atria, ETA; hippocampus/cerebellum, ETB). S6c was a potent activator of PI turnover in hippocampus (EC50 approximately 10 nM) but not atria (EC50 greater than 1 microM), unlike ET-1 which was active in both tissues. S6c, therefore, is a highly selective ETB agonist. Furthermore, S6c was a potent pressor agent in the pithed rat (ED25 mm Hg approximately 0.1 nmoles/kg, i.v.), suggesting that the ETB receptor subtype may be important in cardiovascular function.

Levels of endothelins are particularly high in the lung, and there is evidence that these peptides are involved in asthma. Asthma is a chronic inflammatory disease associated with lymphocyte infiltration. In the present study, we used a murine model of asthma to investigate the role of endothelins in lymphocyte and eosinophil infiltration into the airway hyperreactivity and mucus secretion. Sensitized C57Bl/6 mice were treated with endothelin ETA receptor antagonist (BQ123) or endothelin ETB receptor antagonist (BQ788) 30 min before an antigen aerosol challenge. After 24 h, dose response curves to methacholine were performed in isolated lungs, FACS analysis of lymphocytes and eosinophil counts were performed in bronchoalveolar lavage fluid and mucus index was determined by histopathology. In sensitized and antigen-challenged mice there is a marked increase in the T CD4+, T CD8+, B220+, Tgammadelta+ and NK1.1+ lymphocyte subsets. Treatment with BQ123 further increased these cell populations. The number of eosinophils, airway hyperreactivity and mucus were all reduced by BQ123 treatment. The BQ 788 had no significant effect on the parameters analyzed. Treatment with BQ123 reduced the endothelin concentration in lung homogenates, suggesting that endothelins exert a positive feedback on their synthesis. We show here that in murine asthma the ETA receptor antagonist up-regulates lymphocyte infiltration and reduces eosinophils, hyperreactivity and mucus.