Time course of changes in ETB receptor density and function in tracheal airway smooth muscle during respiratory tract viral infection in mice.

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.     

Influence of respiratory tract viral infection on endothelin-1-induced potentiation of cholinergic nerve-mediated contraction in mouse trachea.

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.     

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)     

Inhibition by endothelin-1 of cholinergic nerve-mediated acetylcholine release and contraction in sheep isolated trachea.

1. The relative roles of ETA and ETB receptor activation on cholinergic nerve-mediated contraction and acetylcholine (ACh) release were examined in sheep isolated tracheal smooth muscle. 2. Electrical field stimulation (EFS; 90 V, 0.5 ms duration, 1 Hz, 10 s train) applied to sheep isolated tracheal smooth muscle strips induced monophasic contractile responses that were abolished by either 1 microM tetrodotoxin or 0.1 microM atropine, but were insensitive to 10 microM hexamethonium and 100 microM L-NAME. Thus, EFS-induced contractions resulted from the spasmogenic actions of ACh released from parasympathetic, postganglionic nerves. 3. As expected, sheep isolated tracheal smooth muscle preparations did not contract in response to the ETB receptor-selective agonist, sarafotoxin S6c (0.1-100 nM). However, sarafotoxin S6c caused a concentration-dependent and transient inhibition of EFS-induced contractions. The inhibitory effect induced by a maximally effective concentration of sarafotoxin S6c (10 nM; 72.1 +/- 5.7%, n = 6) was abolished in the presence of the ETB receptor-selective antagonist BQ-788 (1 microM). Contractile responses to exogenously administered ACh (10 nM-0.3 mM) were not inhibited by sarafotoxin S6c (1 or 10 nM; n = 7). 4. In contrast to sarafotoxin S6c, endothelin-1 induced marked contractions in sheep isolated tracheal smooth muscle. These contractions were inhibited by BQ-123, consistent with an ETA receptor-mediated response. In the presence of BQ-123 (3 microM), endothelin-1 produced a concentration-dependent inhibition of EFS-induced contractions (30 nM endothelin-1, 68.9 +/- 10.2% inhibition, n = 5). These responses were inhibited by 1 microM BQ-788, indicative of an ETB receptor-mediated process. Endothelin-1 was about 3 fold less potent than sarafotoxin S6c. 5. EFS (90 V, 0.5 ms duration, 1 Hz, 15 min train) induced the release of endogenous ACh (1.94 +/- 0.28 pmol mg-1 tissue, n = 12), as assayed by h.p.l.c. with electrochemical detection. EFS-induced release of ACh was inhibited to a similar extent by 100 nM endothelin-1 (47 +/- 4%, n = 9) and 10 nM sarafotoxin S6c (46 +/- 9%, n = 3). These effects of endothelin-1 on ACh release were inhibited by 1 microM BQ-788 alone (n = 4), by BQ-788 in the presence of 3 microM BQ-123 (n = 4), but not by 3 microM BQ-123 alone (n = 5). 6. In summary, sheep isolated tracheal smooth muscle contains two anatomically and functionally distinct endothelin receptor populations. ETA receptors located on airway smooth muscle mediate contraction, whereas ETB receptors appear to exist on cholinergic nerves that innervate tracheal smooth muscle cells and mediate inhibition of ACh release. The inhibitory effect of ETB receptor stimulation on cholinergic neurotransmission is in stark contrast to the enhancing effects hitherto described in the airways.     

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)     

Influence of regional differences in ETA and ETB receptor subtype proportions on endothelin-1-induced contractions in porcine isolated trachea and bronchus.

1. Quantitative autoradiographic studies were conducted to determine the distributions and densities of ETA and ETB binding site subtypes in porcine tracheal and bronchial smooth muscle. In addition, the roles of ETA and ETB receptors in endothelin-1-mediated contraction of these tissues were assessed. 2. Quantitative autoradiographic studies revealed that both ETA and ETB binding sites for [125I]-endothelin-1 were present in both bronchial and tracheal airway smooth muscle. However, the proportions of these sites were markedly different at these two levels within the respiratory tract. In tracheal smooth muscle, the proportions of ETA and ETB sites were 30 +/- 1% and 70 +/- 1% respectively, whereas in bronchial smooth muscle, these proportions were virtually reversed, being 73 +/- 2% and 32 +/- 8% respectively. 3. Endothelin-1 induced concentration-dependent contraction of porcine tracheal and bronchial airway smooth muscle. Endothelin-1 had similar potency (concentration producing 30% of the maximum carbachol contraction, Cmax) in trachea (22 nM; 95% confidence limits (c.l.), 9-55 nM; n = 9) and bronchus (22 nM; c.l., 9-55 nM; n = 6). Endothelin-1 also produced comparable maximal contractions in trachea (59 +/- 5% Cmax; n = 9) and bronchus (65 +/- 4% Cmax, n = 6). 4. In trachea, endothelin-1 induced contractions were not significantly inhibited by either the ETA receptor-selective antagonist, BQ-123 (3 microM) or the ETB receptor-selective antagonist, BQ-788 (1 microM). However, in the combined presence of BQ-123 and BQ-788, the concentration-effect curve to endothelin-1 was shifted to the right by 3.7 fold (n = 8; P = 0.01). 5. In bronchus, concentration-effect curves to endothelin-1 were shifted to the right by BQ-123 (3 microM; 4.3 fold; P < 0.05), but not by BQ-788 (1 microM). In the presence of both antagonists, concentration-effect curves to endothelin-1 were shifted by at least 6.7 fold (n = 6; P = 0.01). 6. Sarafotoxin S6c induced contraction in both tissue types, although the maximum contraction was greater in trachea (53 +/- 7% Cmax; n = 6) than in bronchus (21 +/- 5% Cmax; n = 6). BQ-788 (1 microM) markedly reduced sarafotoxin S6c potency in both trachea and bronchus (e.g. by 50 fold in trachea; c.l., 14-180; n = 6; P < 0.05). 7. These data demonstrate that the proportions of functional endothelin receptor subtypes mediating contraction of airway smooth muscle to endothelin-1, vary significantly at different levels in the porcine respiratory tract.     

Potentiation by endothelin-1 of cholinergic nerve-mediated contractions in mouse trachea via activation of ETB receptors.

1. We have previously shown that endothelin-1-induced contraction of mouse isolated tracheal smooth muscle was mediated via both ETA and ETB receptors. In the current study, we have investigated endothelin-1-induced potentiation of cholinergic nerve-mediated contractions in mouse isolated trachea and have characterized pharmacologically the endothelin receptors mediating this response. 2. Electrical field stimulation (EFS; 70 V, 0.5 ms duration, 10s train, 0.1-60 Hz) of mouse isolated trachea caused frequency-dependent, monophasic contractions (magnitude of contraction of 60 Hz was 56 +/- 4% Cmax (n = 6), where Cmax is the contractile response to 10 microM carbachol). EFS-induced contractions were abolished by either 0.1 microM atropine or 3 microM tetrodotoxin, but were not affected by 1 microM hexamethonium, indicating that they were induced by stimulation of postganglionic cholinergic nerves. In contrast, contractions induced by exogenously applied acetylcholine were inhibited by atropine, but not by either tetrodotoxin or hexamethonium. 3. The ETB receptor-selective agonist, sarafotoxin S6c, caused marked concentration-dependent potentiation of EFS-induced contractions in mouse isolated tracheal segments. At 0.1 nM, sarafotoxin S6c exerted no direct contractile effect, but significantly increased a standard EFS-induced contraction of 20% Cmax by 8 +/- 2% Cmax (i.e. 1.4 fold, n = 5, P < 0.05). At higher concentrations, 10 nM sarafotoxin S6c induced a large, transient contraction (peak response of 74 +/- 2% Cmax at 10 min; 3 +/- 2% Cmax at 45 min) and enhanced the standard EFS-induced contraction by 30 +/- 4% Cmax (i.e. 2.5 fold, n = 5, P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of respiratory tract viral infection on murine airway beta-adrenoceptor function, distribution and density.

1. The effects of a respiratory tract viral infection on beta-adrenoceptor density, distribution and function were investigated in murine airways. 2. Following intranasal inoculation of CBA/CaH mice with influenza A/PR-8/34 virus, the virus proliferated rapidly in trachea (peak titres 2 days post-inoculation) and lung (peak titres 4-6 days post-inoculation). Respiratory tract viral infection was associated with a significant increase in lung weight (88% higher than control mice at day 6 post-inoculation) that was related temporally to the development of peripheral lung inflammation and consolidation. 3. Analysis of specific binding of [125I]-cyanopindolol to beta-adrenoceptors revealed that on days 2, 4 and 8 post-inoculation with virus, mouse isolated tracheal sections contained, on average, 40% more beta-adrenoceptors than tracheal sections from time matched control mice. Subsequent quantitative autoradiographic studies demonstrated that this increase in total tracheal beta-adrenoceptors was due primarily to a 90% increase in the density of beta-adrenoceptors in the tracheal epithelium in virus-infected mice. 4. In contrast, virus-infection had no significant effect on the density of beta-adrenoceptors in tracheal airway smooth muscle, although within 2 days of inoculation with virus, mouse tracheal smooth muscle segments were approximately 2 fold less sensitive to the beta-adrenoceptor agonist, noradrenaline (mean pD2 = 6.57 +/- 0.04, n = 24) and to the adenylyl cyclase-activator forskolin (mean pD2 = 6.78 +/- 0.04, n = 12) compared to segments from control mice (mean pD2 = 6.84 +/- 0.06 for noradrenaline; mean pD2 = 7.03 +/- 0.07 for forskolin). Similar values were obtained 8 days post-inoculation.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Receptors for endothelin-1 in asthmatic human peripheral lung.

[125I]-endothelin-1 ([125I]-ET-1) binding was assessed by autoradiography in peripheral airway smooth muscle and alveolar wall tissue in human non-asthmatic and asthmatic peripheral lung. Levels of specific binding to these structures were similar in both non-asthmatic and asthmatic lung. The use of the receptor subtype-selective ligands, BQ-123 (ETA) and sarafotoxin S6c (ETB), demonstrated the existence of both ETA and ETB sites in airway smooth muscle and in alveoli. In airway smooth muscle from both sources, the great majority of sites were of the ETB subtype. Quantitative analyses of asthmatic and non-asthmatic alveolar wall tissue demonstrated that 29-32% of specific [125I]-ET-1 binding was to ETA sites and 68-71% was to ETB sites. Thus, asthma was not associated with any significant alteration in the densities of ETA and ETB receptors in peripheral human lung.     

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.     

Inhibitory effects of nordihydroguaiaretic acid on ETA-receptor-mediated contractions to endothelin-1 in rat trachea.

1. It has been shown previously that nordihydroguaiaretic acid (NDGA) inhibits endothelin-1 (ET-1)-induced contractions in rat isolated tracheal smooth muscle. To investigate the underlying mechanisms, this study examined the effects of NDGA on various aspects of the ETA and ETB receptor-effector systems which mediate ET-1-induced contractions in this preparation. 2. NDGA inhibited contractions induced by each of the isoforms of ET (ET-1, ET-2 and ET-3) but not those induced by the ETB receptor-selective agonist, sarafotoxin S6c, the cholinoceptor agonist, carbachol or the depolarizing spasmogen, KCl. 3. Quantitative autoradiographic studies of [125I]-ET-1 binding to rat tracheal smooth muscle indicated that NDGA was not an ET receptor antagonist. 4. NDGA inhibited the ETA receptor-mediated, intracellular Ca(2+)-dependent contractions induced by 100 nM ET-1 in Ca(2+)-free solution (by 75%, P < 0.01). Furthermore, NDGA markedly inhibited the contractions induced by ryanodine and cyclopiazonic acid; contractions purportedly due to Ca2+ release from intracellular stores. 5. Like NDGA, the sarcoplasmic reticulum Ca(2+)-ATPase inhibitors cyclopiazonic acid and thapsigargin inhibited contractions to ET-1, but not carbachol or KCl. However, cyclopiazonic acid, but not NDGA, also (a) induced transient contractions in rat trachea, (b) potentiated contractions induced by KCl, and (c) potentiated the extracellular Ca(2+)-dependent phase of ET-1-induced contractions, indicating that NDGA did not inhibit ET-1-induced contractions through Ca(2+)-ATPase inhibition and depletion of sarcoplasmic reticular Ca2+. 6. In control preparations, ET-1 induced a slowly developing, sustained contraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

EndothelinB (ETB) receptor-activated potentiation of cholinergic nerve-mediated contraction in human bronchus.

In human isolated bronchial preparations, the endothelinB (ETB) receptor-selective agonist, sarafotoxin S6c (Stx6c; 1 nM) increased nerve-mediated contraction in response to electrical field stimulation (EFS) at 0.5-1 Hz from 19 +/- 4% to 42 +/- 7% (n = 9). This effect was blocked in the presence of the ETB receptor-selective antagonist, BQ-788 (10 microM). These data are consistent with findings in some animal species that ET-1 and related peptides have marked neuromodulatory influences on the cholinergic system. Furthermore, they provide additional support for the concept that ET-1 may have a mediator role in bronchial obstruction in asthma.     

Evidence for a differential location of vasoconstrictor endothelin receptors in the vasculature.

1. There are at least two subtypes of vascular endothelin (ET) receptors. Stimulation of the ETA receptors on vascular smooth muscle cells leads to vasoconstriction, whereas activation of the ETB receptors on endothelial cells elicits vasodilatation. Several reports in the literature have suggested the presence of a vasoconstrictor non-ETA receptor on vascular smooth muscle which has pharmacological similarities to the ETB receptor. The present study was undertaken to determine the location of this ETB-like receptor within the vascular system. 2. Fourteen vascular smooth muscle preparations from six species were used to determine the effect of the ETA receptor antagonist, BQ-123, on concentration-response curves elicited by ET-1 and the ability of the ETB receptor agonist, sarafotoxin S6c, to cause contraction. The vessels fell into two categories. One group was sensitive to BQ-123 and insensitive to sarafotoxin S6c and, thus, probably contained ETA receptors. The other group, with vasoconstrictor ETB-like receptors, was insensitive to BQ-123 and sensitive to sarafotoxin S6c. 3. Vessels from cynomolgus monkeys, when studied in vitro, appeared to contain primarily ETA receptors, although the potency of BQ-123 was quite variable, suggesting the possibility of ETA receptor subtypes. In contrast, both ET-1 and sarafotoxin S6c, given as intravenous injections in conscious monkeys, produced transient, equipotent, and dose-related increases in blood pressure. The highest dose of sarafotoxin S6c (1 nmol kg-1, i.v.) also caused a marked secondary depressor response (-80 +/- 6 mmHg) that lasted approximately 10 min. The pressor responses suggest that the vasoconstrictor ETB-like receptors are present in cynomolgus monkeys.(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)     

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.     

Identification of endothelin receptor subtypes in rat kidney cortex using subtype-selective ligands.

125I-Endothelin (ET)-1 and 125I-ET-3 displayed specific, saturable, and high affinity binding to membranes prepared from rat kidney cortex. Saturation binding experiments using 125I-ET-1 and 125I-ET-3 revealed that 125I-ET-3 binding sites were 40-50% less abundant than 125I-ET-1 binding sites. The dissociation constants (Kd) and maximum binding (Bmax) for 125I-ET-1 and 125I-ET-3 with these membranes were 218 +/- 23 pM and 275 +/- 20 fmol/mg of protein and 207 +/- 19 pM and 113 +/- 17 fmol/mg of protein, respectively. In the presence of 10 nM sarafotoxin 6c, a selective agonist for ETb receptors, 125I-ET-1 binding was decreased by 45-50% and 125I-ET-3 binding was totally abolished, suggesting that approximately 40-50% of kidney cortex ET receptors are of the ETB subtype and that 125I-ET-1 binds to both ETA and ETB receptors with the same high affinity, whereas 125I-ET-3 binds to only ETB receptors with high affinity. In addition, in the presence of BQ123 [cyclo(D-Trp,D-Asp,L-Pro,D-Val,L-Leu)], a selective antagonist for ETA receptors, 125I-ET-1 binding was decreased by 50%, whereas 125I-ET-3 binding was unaffected. Our results strongly suggest that rat kidney cortex contains ETA and ETB receptors in a 50:50 ratio and that sarafotoxin 6c and BQ123 are valuable tools in identifying the subtypes of ET receptors in various tissues.     

Endothelin receptors in human coronary artery and aorta.

1. ETA and ETB-selective and non-selective ligands were used to define the endothelin receptors in the media (vascular smooth muscle layer) of human aorta and coronary artery. Saturation experiments with iodinated endothelin-1 (ET-1), endothelin-2 and sarafotoxin 6b (S6b) identified high affinity binding sites in aorta (KD [125I]-ET-1 0.33 +/- 0.02 nM (n = 9), KD [125I]-ET-2 1.04 +/- 0.23 nM (n = 5), KD [125I]-S6b 0.15 +/- 0.01 nM (n = 9 +/- s.e.mean)) and coronary artery (KD [125I]-ET-1 0.43 +/- 0.10 nM, KD [125I]-ET-2 0.71 +/- 0.17 nM, KD [125I]-S6b 0.27 +/- 0.03 nM (n = 3 +/- s.e.mean)). Hill coefficients (nH) approached unity in each case. 2. No specific binding was detectable with [125I]-ET-3 (4 pM-4 nM) in aorta. Unlabelled ET-3 competed monophasically with [125I]-ET-1 in aorta (KD, 8.21 +/- 1.62 nM, compared to unlabelled ET-1 KD, 0.60 +/- 0.20 nM) (n = 3 +/- s.e.mean). In coronary artery, the KD and Bmax values calculated from [125I]-ET-3 saturation experiments were 2.13 +/- 1.39 nM and 20.6 +/- 12.9 fmol mg-1 protein, respectively (n = 3 +/- s.e.mean). 3. ETA antagonists competed monophasically for [125I]-ET-1 (100 pM) binding sites with nanomolar or subnanomolar affinity in the aorta (KD BQ123, 0.47 +/- 0.13 nM; KD FR139317, 0.40 +/- 0.10 nM; KD PD151242, 2.09 +/- 0.48 nM) and coronary artery (KD FR139317, 0.41 +/- 0.13 nM; KD PD151242, 3.60 +/- 0.74 nM) (n = 3 +/- s.e.mean). However, two site fits were preferred on analysis of competition experiments with ETB-selective agonists versus [125I]-ET-1 in coronary artery (BQ3020: KDETA 0.96 +/- 0.14 microM, KD ETB 1.34 +/- 1.08 nM and sarafotoxin 6c: KD ETA 1.15 +/- 0.14 microM, KD ETB 1.77 +/- 0.72 nM) (n = 3 +/- s.e.mean). The selectivity of the agonists for ETB receptors (700 fold) was lower than reported in other species. 4. Sarafotoxin 6b (2 pM-2 microM) completely inhibited [125I]-ET-1 (100 pM) binding in aorta (KD 1.36 +/- 0.22 nM) (n = 3 +/- s.e.mean). The non-peptide compounds Ro462005 and bosentan, competed with [125I]-ET-1 binding in coronary artery with KD values of 0.19 +/- 0.04 microM and 2.94 +/- 0.95 nM, respectively (n = 3 +/- s.e.mean). 5. Inhibition of [125I]-ET-2 and [125I]-S6b binding by FR139317 was similar to the inhibition of [125I]-ET-1 binding in both arteries, being monophasic with KD values in the same range. 6. ETA receptors in coronary artery media were detected by [125I]-PD151242 (KD 0.23 +/- 0.04 nM, Bmax 10.1 +/- 1.2 fmol mg-1 protein) (n = 3 +/- s.e.mean). [125I]-BQ3020, an ETB-selective radioligand, indicated the presence of a smaller population of ETB receptors in this tissue (KD 0.60 +/- 0.31 nM, Bmax 4.5 +/- 2.1 fmol mg-1 protein) (n = 3 +/- s.e.mean). 7. Autoradiography with [125I]-PD151242 and [125I]-BQ3020 confirmed the predominance of ETA receptors in the media of both arteries. 8. The results of this study indicate that ETA receptors predominate in the vascular smooth muscle of human cardiac arteries, with a small and variable population of ETB receptors detectable in the coronary artery.     

Endothelin-1 and the periaqueductal gray area of the rat: an autoradiographic and functional pharmacological study.

Endothelin-1 (ET-1) injected centrally induces pressor effects and associated haemodynamic changes. Here we have evaluated the effects on systemic and regional cardiovascular parameters of injection of ET-1 into the periaqueductal gray (PAG) area of anaesthetized rats. In addition, we have used the ETA receptor-selective antagonist, FR 139317, the ETB receptor-selective antagonist, BQ-788, and the ETA/ ETB receptor non-selective antagonist, SB 209670, to identify the receptor(s) mediating these effects. We have also used in vitro autoradiography to identify binding sites for ET-1 in the PAG. 2. In vitro autoradiography showed dense binding of [125I]-PD 151242 (for ETA receptors) in the PAG area, with the binding sites being homogeneously distributed within the dorsal, lateral and ventral subregions. Tissues incubated with [125I]-BQ 3020 (for ETB receptors) had little binding. 3. Injection of ET-1 (0.1, 1 and 10 pmol per rat) in the dorsolateral PAG area significantly increased, in a dose-dependent manner the mean arterial blood pressure (MAP). The highest dose of ET-1 (10 pmol) also decreased the heart rate by 18 +/- 1%, n = 6 (P < 0.05). Increases in blood pressure induced by ET-1 (1 pmol; 31 +/- 6.6 mmHg, n = 6) were greatly reduced by pre-administration to the PAG area of FR 139317 (5 nmol per rat) or SB 209670 (3 nmol per rat) (97 and 94%, respectively), but were unaffected by BQ-788 (5 nmol per rat). Similarly, FR 139317 and SB 209670 prevented the decrease in heart rate induced by ET-1 while BQ-788 did not affect it. 4. Injection of ET-1 to the PAG area caused falls in renal blood flow (RBF) as measured by an ultrasonic flow probe, and increased renal vascular resistance (RVR). Pre-treatment of the PAG with FR 139317 or SB 209670, but not with BQ-788, prevented this ET-1-induced effect. 5. Injection of ET-1 (10 pmol) also increased total peripheral resistance (TPR; control, 2.39 +/- 0.2 mmHg ml-1 min 100 g body weight) by 100 +/- 9% (n = 5) and reduced the cardiac output (CO; control, 94.7 +/- 3.1 ml min-1) by 30 +/- 3% (n = 5), as determined by radioactive microspheres. Vascular resistances were increased in other organs, such as skeletal muscle (88 +/- 5%, n = 4), the colon (55 +/- 7%, n = 4) and the stomach (47 +/- 3%, n = 4). Pretreatment of the PAG area with FR 139317 or SB 209670 reduced the increases in TPR and vascular resistance, and the reduction in CO caused by ET-1. BQ-788 did not effect the responses to ET-1. 6. Thus, there are predominantly ETA binding sites within the PAG area and injection of ET-1 into the PAG area causes complex haemodynamic changes which are sensitive to ETA receptor antagonism. ETA receptors are, therefore, the predominant mediators of the actions of ET-1 in the PAG of the rat.