Characterization of two new ETB selective radioligands, [125I]-BQ3020 and [125I]-[Ala1,3,11,15]ET-1 in human heart.

Two new endothelin receptor radioligands, [125I]-BQ3020 and [125I]-[Ala1,3,11,15]ET-1, were characterized in tissue sections of human right atrium and left ventricle. Both radioligands had high affinity ([125I]-BQ3020 right atrium: KD = 0.145 +/- 0.037 nM, left ventricle: KD = 0.107 +/- 0.004 nM; [125I]-[Ala1,3,11,15]ET-1 right atrium: KD = 0.239 +/- 0.036 nM, left ventricle: KD = 0.199 +/- 0.027 nM). Competition binding experiments were performed in the left ventricle. The selective ETA receptor compound BQ123 competed with low affinity against [125I]-BQ3020 (KD = 28.7 +/- 2.7 microM) and [125I]-[Ala1,3,11,15]ET-1 (KD = 28.5 +/- 4.2 microM). The selective ETB receptor compound BQ3020 competed with high affinity against [125I]-BQ3020 (KD = 40.8 +/- 6.6 pM) and [125I]-[Ala1,3,11,15]ET-1 (KD = 0.276 +/- 0.099 nM). Another selective ETB receptor compound, [Ala1,3,11,15]ET-1 also competed with high affinity against [125I]-BQ3020 (KD = 0.663 +/- 0.120 nM) and [125I]-[Ala1,3,11,15]ET-1 (KD = 0.643 +/- 0.124 nM). These results indicate that [125I]-BQ3020 and [125I]-[Ala1,3,11,15]ET-1 are selective ETB receptor radioligands. [Ala1,3,11,15]ET-1 competed with the non-selective radioligand [125I]-ET-1 in left ventricle and revealed the presence of ETA and ETB receptors in the proportions of 76:24% respectively in the human left ventricle.     

Characterization of endothelin receptors in rat renal artery in vitro.

1. The aim of this study was to investigate the function and characteristics of endothelin receptors in rat main branch renal artery in vitro. 2. Endothelin(ET)-1 (mean EC50 = 9.8 nM) was approximately 12 fold more potent than ET-3 (mean EC50 = 120 nM) as a contractile agonist and produced a greater maximum response. In contrast, neither of the ETB receptor-selective agonists, alanine[1,3,11,15]ET-1 nor sarafotoxin S6c, (0.1 nM-1 microM), induced any contractile effect, or any relaxant effect in endothelium-intact preparations pre-contracted with the thromboxane A2 mimetic, U-46619. Sarafotoxin S6c (30 nM) also failed to induce any further contraction in tissues pre-contracted with an EC50 concentration of ET-1. 3. The ETA receptor-selective antagonist, BQ123, behaved as a weak and variable antagonist of the contractile effects of ET-1 (mean pA2 estimates in the range 5.8-6.3). In contrast, BQ123 antagonized ET-3 with a potency (mean pA2 = 7.6) consistent with its affinity for ETA receptors. Co-incubation of BQ123 (3 microM) with the putative ETB receptor-selective antagonist, IRL1038 (10 microM), produced no greater antagonism of ET-1 responses than was induced by BQ123 (3 microM) alone. 4. In conclusion, ETB receptors do not appear to be present in rat main branch renal artery. The contractile effects of ET-3 in this tissue seem to be mediated by ETA receptors. While ETA receptors partly mediate the contractile effects of ET-1, these data raise the possibility that a population of novel BQ123-insensitive endothelin receptors may also contribute to this response.     

Characterization of [125I]-endothelin-1 and [125I]-BQ3020 binding to rat cerebellar endothelin receptors.

1. We performed radioligand binding experiments on rat cerebellar homogenates using [125I]-endothelin-1 ¿[1251]-ET-1¿ and [125I]-BQ3020 to examine the pharmacology of endothelin receptors in rat brain. Saturation experiments demonstrated a single population of binding sites with high affinity for both radioligands ([125I]-ET-1, pKd = 8.94 +/- 0.17; [125I]-BQ3020, pKd = 9.18 +/- 0.14 nM; mean +/- s.e.mean). However, [125I]-BQ3020 only recognised approximately one third the number of endothelin receptors measured with [125I]-ET-1. 2. Saturation binding experiments with [125I]-PD151242 revealed high affinity binding to a single population of ETA receptors in the cerebellar homogenates (pKd = 9.95 +/- 0.14; Bmax = 30 +/- 15 fmol mg-1 protein). 3. Competition experiments were performed with ligands that are either non-selective for endothelin receptor subtypes. The rat cerebellar endothelin receptor displayed a high affinity for endothelin-1 (ET-1), endothelin-3 (ET-3) and sarafotoxin-S6c (STX-6c) although the affinity for ET-3 was slightly higher than the affinity for ET-1 using both radioligands. The selective ETA antagonists, BQ123, BMS-182,874 and JKC-301 all displayed low affinities at the endothelin receptors. In contrast the selective ETB agonists, IRL1620 and [Ala1,3,11,15]ET-1 and the selective ETB antagonist, BQ-788 had moderate affinities at the endothelin receptor, in the low nanomolar range. The ETB agonist, BQ3020, had approximately 10 fold higher affinity than IRL1620 and [Ala1,3,11,15]ET-1 at the rat cerebellar endothelin receptors. The non-selective antagonists, Ro-46,2005, Ro-47,0203 and PD-142,893 displayed moderate affinities at the cerebellar receptor. 4. Since [125I]-BQ3020 recognises only a fraction of the [125I]-ET-1 binding sites, the majority of the endothelin receptors in the cerebellum cannot be classed as ETB. Although [125I]-PD151242 was able to detect ETA receptors in the rat cerebellar homogenates, the small population of ETA receptors (2% of the total endothelin population as measured with [125I]-ET-1) could not account for the non-ETB receptor population. We conclude that the rat brain cerebellar receptor has a profile similar to the ETB1 receptor as it has a high affinity for ET-1, ET-3, STX-6c and was moderately sensitive to PD-142,893. However, as the ETB ligands BQ-788, IRL1620 and [Ala1,3,11,15]ET-1 have only a moderate affinity for the rat cerebellar endothelin receptor and since ET-3 has a higher affinity as compared to ET-1, our findings suggest that the rat cerebellum contains predominately ETc receptors.     

ETA receptor-mediated constrictor responses to endothelin peptides in human blood vessels in vitro.

1. We have characterized the constrictor endothelin receptors present in human isolated blood vessels using ETA and ETB selective agonists and antagonists. 2. Monophasic dose-response curves were obtained for ET-1 with EC50 values of 6.8 nM in coronary artery, 3.9 nM in internal mammary artery, 17.4 nM in pulmonary artery, 14.5 nM in aorta and 3.2 nM in saphenous vein. In coronary artery, ET-2 was equipotent with ET-1 with an EC50 value of 5.7 nM. The non-selective peptide, sarafotoxin 6b, was 2-3 times less potent than ET-1 but the maximum responses to these two were comparable. 3. In each vessel ET-3 was much less active than ET-1. No response was obtained to ET-3 in aorta and pulmonary artery or in up to 50% of coronary artery, mammary artery and saphenous vein preparations. In those preparations that did respond, dose-response curves were incomplete at 300 nM. Variable contractions were also obtained with the ETB-selective agonist, sarafotoxin 6c (S6c). Where responses were detected, although S6c was more potent than ET-1 (EC50 values of 0.6-1.2 nM), the maximum response produced was always less than 20% of that to ET-1. 4. The synthetic ETB agonists, BQ3020 and [1,3,11,15Ala]-ET-1, were without effect in any of the five blood vessels at concentrations up to 3 microM. 5. ET-1-induced vasoconstriction was blocked by the ETA-selective antagonists, BQ123 and FR139317.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endothelin-1 (ET-1)-induced contraction in rat isolated trachea: involvement of ETA and ETB receptors and multiple signal transduction systems.

1. Quantitative autoradiographic, biochemical and functional studies were performed to investigate the endothelin receptor subtypes and signal transduction systems that mediate endothelin-1 (ET-1)-induced contraction in rat isolated tracheal smooth muscle. 2. Specific binding of 0.5 nM [125I]-ET-1 to tracheal smooth muscle was inhibited by at least 40% in the presence of either the ETA receptor selective ligand BQ-123 (1 microM) or the ETB receptor-selective ligand sarafotoxin S6c (30 nM), indicating the presence of both ETA and ETB receptors in this tissue. 3. ET-1 and sarafotoxin S6c were both potent spasmogens of rat isolated tracheal smooth muscle preparations. Sarafotoxin S6c-induced contractions were unaffected in the presence of the ETA receptor antagonist BQ-123 (10 microM), but were markedly attenuated in tissue previously exposed to 100 nM sarafotoxin S6c to induce ETB receptor desensitization. ET-1-induced contractions were, at most, only partially attenuated either by blocking the ETA receptor-effector system (with 10 microM BQ-123) or by desensitizing the ETB receptor-effector system with sarafotoxin S6c. However, ET-1-induced contractions were markedly attenuated by blocking both receptor-effector systems simultaneously. These findings suggest that ET-1 could induce contraction by stimulating either ETA or ETB receptors. 4. ET-1 (10 microM) induced a 7 fold increase in intracellular [3H]-inositol phosphate accumulation over basal levels in rat isolated tracheal smooth muscle. In contrast, sarafotoxin S6c (2.5 microM) increased intracellular [3H]-inositol phosphate accumulation by only 2 fold. ET-1-induced accumulation of [3H]-inositol phosphates was abolished by 10 microM BQ-123.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synergistic inhibition by BQ-123 and BQ-788 of endothelin-1-induced contractions of the rabbit pulmonary artery.

In the rabbit isolated pulmonary artery, neither the ETA receptor antagonist, BQ-123 (10 microM), nor the ETB receptor antagonist, BQ-788 (10 microM), inhibited the contractions induced by 1 nM endothelin-1 (ET-1). However, the combination of BQ-123 and BQ-788 completely inhibited the ET-1-induced contraction. In contrast, the ETB-selective agonist, sarafotoxin S6c (1 nM)-induced contraction was completely inhibited by BQ-788 but not by BQ-123. In receptor binding assays, [125I]-ET-1 specific binding to pulmonary arterial membranes was inhibited by BQ-123 (1 microM) by approximately 20% and additive treatment with BQ-788 (1 microM) completely inhibited the BQ-123-resistant component of [125I]-ET-1 specific binding. The present study demonstrates synergistic inhibition by BQ-123 and BQ-788 of ET-1-induced contraction of the rabbit pulmonary artery and the coexistence of ETA and ETB receptors, suggesting that the activation of either only ETA or only ETB receptors may be sufficient to cause complete vasoconstriction. Therefore, blockade of both receptor subtypes would be necessary for the inhibition of some ETA/ETB composite types of responses.     

Different endothelin receptors involved in endothelin-1- and sarafotoxin S6B-induced contractions of the human isolated coronary artery.

1. Endothelin receptors, that mediate contraction of the human isolated coronary artery, were characterized by use of a number of agonists and antagonists. Contraction induced by the non-selective agonists, endothelin (ET)-1 and sarafotoxin S6b, was compared in endothelium-intact and endothelium-denuded ring segments. The effects of ET-1 and BQ-123 (an ETA receptor antagonist) were investigated both in ring segments and in spirally cut strips. Lastly, the effect of phosphoramidon was studied on contraction induced by big-ET-1. 2. The order of agonist potency (pD2) in endothelium-intact coronary artery ring segments was: ET-1 (8.27) approximately sarafotoxin S6b (8.16) > big-ET-1 (< 7.1) approximately ET-3 (< 6.9). [Ala1,3,11,15]ET-1 (ETB receptor agonist) caused significant contraction only at 1 microM, whereas 0.3 microM big-ET-3 had no effect. Removal of the endothelium in ring segments did not affect the contractile response to ET-1 or to sarafotoxin S6b. 3. After a full concentration-response curve had been obtained to ET-1 or sarafotoxin S6b, further contractions of the endothelium-intact coronary artery segments could only be achieved by applying ET-1 in segments exposed to sarafotoxin S6b, and not the reverse. 4. BQ-123 (0.1 microM) antagonized contractions of endothelium-intact ring segments induced by sarafotoxin S6b (pKB 7.86). Only 10 microM BQ-123 antagonized contractions induced by ET-1 (pKB 5.75). FR139317 was also more potent against sarafotoxin S6b (pKB 8.24-8.47) than against ET-1 (pKB 6.11). [Ala1,3,11,15]ET-1 (1 microM) had no effect on the contractile response to ET-1 or to sarafotoxin S6b.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for ETA and ETB receptors in rat skin and an investigation of their function in the cutaneous microvasculature.

1. The relative contribution of ETA and ETB receptors in the response of rat skin to endothelins was investigated by use of the selective ETB agonist IRL-1620 and the selective ETA antagonist BQ-123. 2. Binding data suggest the presence of ETA and ETB receptors as preincubation with [Ala3,11,18Nle7]-endothelin-1 reduced ET-1 binding by approximately 40%. 3. Intradermal injection of endothelin-1 (ET-1, 1-10 pmol/site) and ET-3 (3-100 pmol/site) induced a dose-dependent decrease in local blood flow assessed by 133Xe clearance at test sites in rat skin. 4. The endothelin analogue [Ala3,11,18Nle7]-ET-1 (30-1000 pmol/site) induced significant vasoconstriction (P < 0.05) at the highest doses used and the selective ETB receptor agonist, IRL-1620 [Suc[Glu9,Ala11,15] endothelin (8-21)], (0.01-100 pmol/site) acted in a potent manner to induce a significant (P < 0.01) dose-dependent decrease in 133Xe clearance. 5. Co-injection with the selective ETA receptor antagonist, BQ-123 (1 nmol/site), completely abolished the vasoconstriction to ET-1 and partially to ET-3, but had no effect on IRL-1620-induced vasoconstriction. In addition, IRL-1620 responses were not altered at sites treated with submaximal doses of a nitric oxide synthase inhibitor or a prostaglandin synthase inhibitor. 6. ET-1 and IRL-1620 (100 fmol-1 pmol/site) did not induce oedema formation as measured by [125I]-albumin accumulation in the presence or absence of the vasodilator, calcitonin gene-related peptide (CGRP).(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of the endothelin antagonists BQ-123 and PD 142893 to reveal three endothelin receptors mediating smooth muscle contraction and the release of EDRF.

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).(ABSTRACT TRUNCATED AT 250 WORDS)     

Necessity of dual blockade of endothelin ETA and ETB receptor subtypes for antagonism of endothelin-1-induced contraction in human bronchi.

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.     

Endothelin receptor antagonist activity of (R)-(-)-2-(benzo[1,3]dioxol-5-yl)-N-(4-isopropylphenylsulfonyl)-2-(6-methyl- 2-propylpyridin-3-yloxy)acetamide hydrochloride (PABSA) in rat aortic smooth muscle cells and isolated rat thoracic aorta

Antagonist activities of (R)-(-)-2-(benzo[1,3]dioxol-5-yl)-N-(4-isopropylphenylsulfonyl)-2-(6-methyl- 2-propylpyridin-3-yloxy) acetamide hydrochloride (CAS 188710-94-3, PABSA), a novel endothelin (ET) receptor antagonist, for ETA and ETB receptors were evaluated using rat aortic smooth muscle A7r5 cells and isolated rat thoracic aorta. PABSA concentration-dependently inhibited the ET-1-induced increase in intracellular calcium concentration ([Ca2+]i) mediated via ETA receptors in A7r5 cells with an IC50 of 0.17 nmol/l. PABSA antagonized the ETA receptor-mediated contraction induced by ET-1 in endothelium-denuded rat aorta with a Kb of 0.74 nmol/l. The potency of PABSA in inhibiting ETA receptor-mediated vasocontraction was approximately 40- and 100-fold greater than those of BQ-123, a selective ETA antagonist, and bosentan, a mixed ETA/ETB receptor antagonist, respectively. ETB receptor-mediated endothelium-dependent vasorelaxation induced by ET-3 in the aorta was also antagonized by PABSA, with a Kb of 9.8 nmol/l. In contrast, PABSA affected neither the vasocontraction induced by KCl or norepinephrine nor the vasorelaxation induced by acetylcholine or prostaglandin I2 in the aorta. These results suggest that PABSA is a highly potent and selective ETA receptor antagonist.     

Characterization of the binding of endothelin ETB selective ligands in human and rat heart.

1. We determined competition binding characteristics of endothelin ETB receptor selective ligands in human left ventricle and compared these values to those obtained with rat left ventricle. Sarafotoxin S6c, ET-3, BQ788 and IRL2500 competed against [125I]-PD151242 (ETA selective radioligand) with low affinity in human left ventricle, confirming the ETB selectivity of these compounds. 2. ET-3 competed with moderate selectivity for ETB over ETA receptors in human left ventricle and with slightly higher selectivity in rat left ventricle (460 and 1,400 fold, respectively). There was a small difference in the affinity of ETA receptors for ET-3 (KD ETA in human left ventricle = 0.07 +/- 0.02 microM; KD ETA in rat left ventricle = 0.27 +/- 0.08 microM; P = 0.05) but no difference in the affinity of ETB receptors for this ligand (KD ETB in human left ventricle = 0.15 +/- 0.06 nM; KD ETB in rat left ventricle = 0.19 +/- 0.03 nM). 3. The selectivity of sarafotoxin S6c for ETB over ETA receptors in human left ventricle was 5,900 fold compared with 59,400 fold in rat left ventricle. The affinity of ETA receptors for sarafotoxin S6c was higher in human than in rat left ventricle (KD ETA = 2.00 +/- 0.20 microM and 3.50 +/- 0.26 microM, respectively; P = 0.03), while the affinity of ETB receptors for this ligand was higher in rat left ventricle (KD ETB = 0.06 +/- 0.02 nM) than in human left ventricle (KD ETB = 0.34 +/- 0.13 nM) (P = 0.02). The affinity of ETB receptors for sarafotoxin S6c in rat left ventricle determined in the absence or presence of GTP was the same indicating that differing affinity states of ETB receptors in human and rat left ventricle do not account for the variation observed between species. 4. There was no difference in the affinity of ETA receptors for BQ788 (KD ETA = 1.01 +/- 0.20 microM and KD ETA = 1.39 +/- 0.35 microM) or for the novel ETB selective antagonist. IRL2500 (KD ETA = 30.0 +/- 20.8 microM and KD ETA = 55.6 +/- 9.93 microM) in human and rat left ventricle, respectively. ETB receptors had a significantly higher affinity for BQ788 (KD ETB = 9.8 +/- 1.3 nM and KD ETB = 31.0 +/- 5.4 nM; P = 0.02) and IRL2500 (KD ETB = 78.2 +/- 9.7 nM and KD ETB = 300.0 +/- 75.1 nM; P = 0.03) in human and rat left ventricle, respectively. The synthetically synthesized ETB selective antagonist RES-701-1 (0.1 -3 microM) failed to inhibit [125I]-ET-1 binding in either tissue. 5. In conclusion, we have compared equilibrium dissociation constants for a number of ETB selective compounds in human and rat heart. The affinity of ETB receptors for sarafotoxin S6c, BQ788 and IRL2500 differed in human and rat left ventricle. No difference in affinity was detected for ET-3 binding at ETB receptors. Sarafotoxin S6c binding was unaffected by GTP indicating that the different receptor affinities in human and rat heart cannot be explained by differing ETB receptor affinity states. This study highlights the need to consider differences in binding characteristics that may arise from the use of tissues obtained from different species.     

ETB but not ETA receptor-mediated contractions to endothelin-1 attenuated by respiratory tract viral infection in mouse airways.

1. The current study investigated the effects of respiratory tract viral infection on the density of ETA and ETB receptors in murine tracheal smooth muscle and on the contractile response to endothelin-1 mediated by these receptors. 2. Quantitative autoradiographic studies using [125I]-endothelin-1 revealed that tracheal smooth muscle from control mice contained ETA and ETB receptors in the ratio of 42%:58% (+/- 4%, n = 10 mice), respectively. In contrast, tracheal smooth muscle obtained from mice 2 days post-inoculation with Influenza A/PR-8/34 virus contained 23 +/- 2% fewer receptors for [125I]-endothelin-1 (n = 10, P < 0.01). This reflected a selective reduction in ETB receptor density and a change in the ratio of ETA and ETB receptors to 77%:23% (+/- 5%, n = 10 mice), respectively. 3. The ETB receptor-selective agonist, sarafotoxin S6c, was a potent spasmogen of murine isolated tracheal smooth muscle and the EC50 for contraction was similar in preparations from control (3.6 nM [95% confidence limits, 2.7-4.8 nM], n = 16 preparations from 8 mice) and virus-inoculated mice (3.0 nM [2.4-3.7 nM], n = 16 preparations from 8 mice). However, the maximum contractions induced by sarafotoxin S6c (100 nM) in the preparations from virus-inoculated mice (37 +/- 5% Cmax, where 100% Cmax was the response to 10 microM carbachol) were significantly smaller than those from control mice (85 +/- 4% Cmax, P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential localization of endothelin ETA and ETB binding sites in human placenta.

1. The localization and differential distribution of endothelin (ET) receptor subtypes (ETA and ETB) was investigated in sections of human placenta by use of quantitative in vitro autoradiography and receptor selective ligands. 2. Specific, high density [125I]-ET-1 binding sites were localized to the decidua and foetal membranes as well as to arteries and veins in the chorionic plate and throughout the villous tree. Moderate to low density binding was found in the extravillous and villous trophoblast respectively. 3. [125I]-ET-1 binding sites exhibited a rank order of inhibition by unlabelled peptide sequences (ET-1 > ET-3 > [Ala3,11,18Nle7]-ET-1 > BQ123 > or = sarafotoxin 6c). However, in contrast to the monophasic inhibition curve of ET-1, the other sequences produced a significantly better fit to a two component inhibition curve suggesting the presence of a heterogeneous population of ET binding sites. 4. ETA and ETB receptors were distinguished by competitive inhibition of [125]-ET-1 binding with increasing concentrations of unlabelled ET-3, [Ala3,11,18Nle7]-ET-1, sarafotoxin 6c and BQ123 and by incubating sections with the ETB agonist, [125I]-BQ3020. ET receptor subtypes exhibited a differential distribution in the placenta. ETA type binding sites predominated (approximately 80% of the total) on veins and arteries in the chorionic plate. Veins in stem villi, blood vessels in distal regions of the villous tree and decidual cells displayed a high density (approximately 60-70% of the total) of the ETB receptor subtype.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological properties of endothelin-1 in the rabbit corpus cavernosum

BACKGROUND: Endothelin (ET-1) may play a role in the regulation of erection but this has not been conclusively demonstrated. Augmented cavernosal smooth muscle (CSM) contraction in the rat occurs following exposure to both ET-1 and phenylephrine (PE; alpha-1 agonist). The aim of this study was to assess the effect of ET-1 and its possible role in the alpha1-adrenergic pathway during the erectile process. MATERIALS AND METHODS: Organ bath studies were performed on CSM strips of penises obtained from 12 age-matched New Zealand White rabbits. The effect of ET-1 and PE alone on CSM tone in the absence and presence of ETA (BQ123) and ETB (BQ788) antagonists was assessed. Tissue responses were measured as tension (newton, N). EC50 values are expressed as mean +/- S.E.M. RESULTS: PE (10(8) - 10(-4) M) and ET-1 (10(-10) - 10(-6) M) produced a concentration-dependent contraction in rabbit CSM strips. The EC50 values were 1.7 x 10(-7) M +/- 1.1 and 3.4 x 10(-9) M +/- 1.5, respectively. BQ123 10(-5) M significantly inhibited ET-1-mediated CSM contractions more than BQ788 10(-5) M (both ANOVA p<0.01). The EC50 were 1.3 x 10(-6) M +/- 2.6 and 2.0 x 10(-7) M +/- 2.1, respectively. Neither the ETA or ETB receptor antagonist had a significant influence on alpha1-adrenergic receptor-mediated CSM contraction. CONCLUSION: ETA receptors may play a greater role than ETB receptors in ET-1-induced rabbit CSM contraction and the detumescence process. The a1-adrenergic-dependent pathway does not involve the ETA or ETB receptors.     

Endothelin-1-induced potentiation of human airway smooth muscle proliferation: an ETA receptor-mediated phenomenon.

1. In this study the mitogenic effects in human cultured tracheal smooth muscle cells of endothelin-1 (ET-1), ET-3, and sarafotoxin S6c (S6c), the ETB receptor-selective agonist, were explored either alone or in combination with the potent mitogen, epidermal growth factor (EGF). 2. In confluent, growth-arrested human airway smooth, neither ET-1 (0.01 nM-1 microM) nor ET-3 (0.001 nM-1 microM) or S6c (0.01 nM-1 microM) induced cell proliferation, as assessed by [3H]-thymidine incorporation. In contrast, EGF (1.6 pM-16 nM) produced concentration-dependent stimulation of DNA synthesis (EC50 of about 0.06 nM). The maximum increase of about 60 fold above control, elicited by 16 nM EGF, was similar to that obtained with 10% foetal bovine serum (FBS). EGF (0.16-16 nM) also produced a concentration-dependent increase in cell counts, whereas ET-1 (1-100 nM) was without effect on this index of mitogenesis. 3. ET-1 (1-100 nM) potentiated EGF-induced proliferation of human tracheal smooth muscle cells. For example, ET-1 (100 nM), which alone was without significant effect, increased by 3.0 to 3.5 fold the mitogenic influence of EGF (0.16 nM). The potentiating effect of ET-1 on EGF-induced proliferation was antagonized by BQ-123 (3 microM), the ETA receptor antagonist, but was unaffected by the ETB receptor antagonist BQ-788 (10 microM). 4. Neither ET-3 (1-100 nM) nor S6c (1-100 nM) influenced the mitogenic effects of EGF (0.16-1.6 nM). 5. [125I]-ET-1 binding studies revealed that on average the ratio of ETA to ETB receptors in human cultured tracheal smooth muscle cells was 35:65 ( +/- 3; n = 4), confirming the predominance of the ETB receptor subtype in human airway smooth muscle. 6. These data indicate that ET-1 alone does not induce significant human airway smooth muscle cell proliferation. However, it potently potentiated mitogenesis induced by EGF, apparently via an ETA receptor-mediated mechanism. These findings suggest that ET-1, a mediator detected in increased amounts in patients with acute asthma, may potentiate the proliferative effects of mitogens and contribute to the airway smooth muscle hyperplasia associated with chronic severe asthma.     

Characterization of endothelin (ET) receptors in the isolated gall bladder of the guinea-pig: evidence for an additional ET receptor subtype.

1. We have characterized the receptors mediating contractions induced by endothelin-1 (ET-1), ET-2, ET-3 and the ETB-selective receptor agonists, sarafotoxin 6c (SX6c), IRL 1620, BQ-3020, [Ala1,3,11,15]ET-1 and ET (16-21) in strips of the isolated gall bladder of the guinea-pig (GPGB). We used as antagonists BQ-123 (ETA receptor selective) and PD 145065 (ETA/ETB receptor non-selective). 2. ET-1, ET-2 and ET-3 (10(-10) M to 3 x 10(-7) M) caused similar slowly-developing concentration-dependent contractions of the GPGB. Contractile effects induced by ET-1, ET-2 or ET-3 (at 3 x 10(-7) M) were also similar (230 +/- 25, 241 +/- 7 and 287 +/- 37% of that to histamine at 5 x 10(-6) M, n = 7, 6, 12, respectively). However, the threshold concentration for ET-1 or ET-2 was 10(-10) M whereas it was 3 x 10(-9) M for ET-3. 3. SX6c (10(-10) M to 3 x 10(-7) M) also caused slowly-developing concentration-dependent contractions at a threshold concentration of 10(-10) M (n = 16). However, the contraction caused by SX6c at 3 x 10(-7) M was 116 +/- 9% of that to histamine at 5 x 10(-6) M, which was half of that induced by the same concentration of the ET isopeptides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Arteries and veins desensitize differently to endothelin

Hypertension is accompanied by increased arterial endothelin-1 (ET-1) and decreased arterial contraction to ET-1. By contrast, veins remain responsive to ET-1 in hypertension. Isometric contraction was used to test the hypothesis that veins do not desensitize to ET-1 to the extent of arteries, possibly because of the presence of functional ETA and ETB receptors on veins and only functional ETA receptors on arteries. Contraction to ET-1 after exposure to ET-1 (100 nmol/L) was abolished in aortae, while in veins 36.3 +/- 0.2% of maximal contraction to ET-1 remained. Aortae were unresponsive to the ETA receptor agonist ET-1(1-31) (100 nmol/L) after ET-1 exposure, while 21.9 +/- 0.6% of maximum venous contraction to ET-1 (1-31) remained. In a similar manner, the venous ETB receptor did not lose responsiveness to the ETB receptor agonist sarafotoxin 6c (S6c, 100 nmol/L); aortae did not contract to S6c. In ET-1-desensitized veins, the ETB receptor antagonist BQ-788 (100 nmol/L) decreased maximum contraction to ET-1, but did not alter potency (-log EC50 control = 8.14 +/- 0.01 mol/L; BQ-788 = 8.13 +/- 0.04 mol/L). The ETA receptor antagonist atrasentan (100 nmol/L) blocked remaining venous contraction to ET-1 (control = 8.05 +/- 0.05 mol/L; atrasentan = unmeasurable). Maintained responsiveness to ET-1 in veins occurs primarily via the ETA receptor, while in arteries the ETA receptor is responsible for desensitization to ET-1.     

Characterization of endothelin receptor subtypes mediating Ca2+ mobilization and contractile response in rabbit iris dilator muscle.

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.