Pharmacological evidence for distinct endothelin receptors in guinea-pig bronchus and aorta.

The ETA receptor antagonist, BQ-123 (1 microM) potently antagonized endothelin-1 (ET-1) concentration-response curves in guinea-pig aorta (pKB = 7.1). However, 10 microM BQ-123 was without effect on ET-1-induced contractions in guinea-pig bronchus. The ETB-selective agonist, sarafotoxin S6c did not contract the aorta but was a potent and effective contractile agonist in the bronchus. BQ-123 (10 microM) was without effect on sarafotoxin S6c-induced contractions in the bronchus. These data provide evidence for distinct endothelin receptors in guinea-pig aorta and bronchus, which appear to be predominantly of the ETA and non-ETA, perhaps ETB, subtypes, respectively.     

Comparison of endothelin B (ETB) receptors in rabbit isolated pulmonary artery and bronchus.

1. To explore potential differences between endothelin (ET) receptors in airway versus vascular smooth muscle from the same species, the ETB receptors mediating contractions produced by ET-1, ET-3 and the selective ETB ligands, sarafotoxin S6c (S6c) and BQ-3020, in rabbit bronchus and pulmonary artery were investigated by use of peptide and non-peptide ET receptor antagonists. 2. In rabbit pulmonary artery SB 209670 (10 microM), a mixed ETA/ETB receptor antagonist, was a more potent antagonist of contractions produced by S6c (pKB = 7.7; n = 9; P < 0.05), than those elicited by ET-1 (pKB = 6.7; n = 6) or ET-3 (pKB = 6.7; n = 5). BQ-788 (10 microM), an ETB receptor antagonist, inhibited responses produced by ET-3 (pKB = 5.1; n = 8), BQ-3020 (pKB = 5.2; n = 4) or S6c (pKB = 6.2; n = 9; P < 0.05 compared to potency versus ET-3- or BQ-3020-induced contractions), but was without inhibitory effect on ET-1-induced contractions (n = 5). RES-701 (10 microM), another selective ETB receptor antagonist, was without effect on contractions produced by S6c (n = 4) or ET-1 (n = 4), and potentiated ET-3- (n = 5) or BQ-3020-induced responses (n = 4). 3. The combination of BQ-788 (10 microM) and BQ-123 (10 microM), an ETA-selective receptor antagonist, antagonized contractions produced by lower concentrations of ET-1 (1 and 3 nM) in rabbit pulmonary artery, but was without effect on responses elicited by higher concentrations of ET-1 (n = 5). The combination of RES-701 (10 microM) and BQ-123 (10 microM) potentiated responses elicited by ET-1, producing a 3.7 fold shift to the left in the agonist concentration-response curve (n = 5). 4. In rabbit bronchus SB 209670 (3 microM) had similar potency for antagonism of contractions produced by ET-1 (pKB = 6.3; n = 6), ET-3 (pKB = 6.5; n = 6) or S6c (pKB = 6.1; n = 8). BQ-788 (3 microM) was without effect on responses elicited by ET-1, ET-3 or S6c (n = 6) but antagonized BQ-3020-induced contractions (pKB = 6.4; n = 4). RES-701 (3 microM) was without effect on contractions produced by S6c (n = 6) or BQ-3020 (n = 4), and potentiated rather than antagonized ET-1- or ET-3-induced responses (n = 6), reflected by a significant (about 6 fold) shift to the left in ET-1 or ET-3 concentration-response curves. The combination of BQ-788 (3 microM) and BQ-123 (3 microM) was without effect on contractions produced by ET-1 in rabbit bronchus (n = 6). The combination of RES-701 (3 microM) and BQ-123 (3 microM) potentiated responses elicited by ET-1, producing a 5.2 fold shift to the left in the agonist concentration-response curve (n = 5). 5. BQ-123 (3 or 10 microM), an ETA-selective receptor antagonist, was without effect on ET-1, ET-3 or S6c concentration-response curves (n = 3-6) in rabbit pulmonary artery or rabbit bronchus. 6. These data indicate that contractions induced by ET-1, ET-3, S6c and BQ-3020 in rabbit pulmonary artery or rabbit bronchus appear to be mediated predominantly via stimulation of ETB receptors. However, the qualitative and quantitative differences in the relative profiles of the various structurally diverse peptide and non-peptide antagonists examined suggests that responses produced by the ET ligands may not be mediated by a homogeneous ETB receptor population. In addition, the results suggest that differences exist in the ETB receptors mediating contraction in pulmonary vascular versus airway tissues in the same species. These receptors are not very sensitive to the standard ETB receptor antagonists, BQ-788 and RES-701. Furthermore, the results also provide further evidence that the potencies of ET receptor antagonists depend upon the ET agonist.     

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.     

Pharmacological characterization of endothelin receptor subtypes in the guinea-pig prostate gland.

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-gamma-methyl-Leu-D-Trp[1-+ ++CO2CH3-D-Nle-ONa])> BQ-123 (cyclo-D-Asp-L-Pro-D-Val-Leu-D-Trp) > or = sarafotoxin S6c. At concentrations with negligible influence on smooth muscle tone, endothelin-1, endothelin-2 and sarafotoxin S6b (1 nM-0.1 microM) 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 ET(B) receptor-preferring agonist endothelin-3 (1 nM- 1 microM) was much less potent, and the endothelin ET(B) receptor-selective agonists sarafotoxin S6c and BQ-3020 (Ac-[Ala11,15]-endothelin-1 (6-21)), up to 1 microM, were without effect. The endothelin ET(A) receptor antagonist BQ-123 (1 microM) markedly inhibited the potentiation induced by endothelin-1, endothelin-2 and sarafotoxin S6b while the endothelin ET(B) receptor antagonist BQ-788 (1 microM) was less effective. While our binding data indicates the presence of ET(A) and ET(B) 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 ET(A) subtype.     

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.     

Prostanoid receptors involved in the relaxation of human pulmonary vessels

1. To characterize the prostanoid receptors on human pulmonary smooth muscle involved in vasodilatations, isolated arteries and veins were contracted with norepinephrine (10 microM) and vessels were subsequently challenged with different prostanoid-receptor agonists in the absence or presence of selective antagonists. 2. Prostaglandin D2 (PGD2) and the selective DP-receptor agonist, BW245C, induced relaxations in the contracted human pulmonary venous preparations. The pD2 values were: 6.88+/-0.11 (n=17) and 7.31+/-0.12 (n=5), respectively. The relaxant responses induced by PGD2 were reduced by the selective DP-receptor antagonist, BWA868C, and the estimated pA2 value was 7.84+/-0.16 (n=4). PGD2 and BW245C did not relax contracted human pulmonary arteries. 3. The selective IP-receptor agonists, iloprost and cicaprost, both induced relaxations in the contracted human vascular preparations. The pD2 values for iloprost were: 7.84+/-0.08 (n=6) and 8.25+/-0.06 (n=4) and for cicaprost: 8.06+/-0.12 (n=5) and 8.11+/-0.09 (n=5) in arteries and veins respectively. 4. Prostaglandin E2 (PGE2) and the EP2/EP3-receptor agonist, misoprostol, partially relaxed the contracted venous preparations and the pD2 values were: 8.10+/-0.15 (n=15) and 6.24+/-0.33 (n=3), respectively. These relaxations suggest the presence of an EP receptor in the human pulmonary veins. The contracted human pulmonary arteries did not relax when challenged with PGE2. 5. In human pulmonary venous preparations, the PGE2-induced relaxations were neither modified by treatment with TP/EP4-receptor antagonist, AH23848B (10 and 30 microM, n=6), nor by the DP/EP1/EP2-receptor antagonist, AH6809 (3 microM, n=6). 6. These data suggest that the relaxation induced by prostanoids involved DP-, IP-receptors and to a lesser extent an EP-receptor on human pulmonary venous smooth muscle. In contrast, only the IP-receptor is involved in the prostanoid induced relaxations on human pulmonary arterial smooth muscle.     

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)     

Effect of the vascular endothelium on noradrenaline-induced contractions in non-pregnant and pregnant guinea-pig uterine arteries.

1. The effect of pregnancy on noradrenaline-mediated contraction of guinea-pig uterine artery rings with both intact and denuded endothelium was investigated. 2. Noradrenaline (25 nM-100 microM) induced concentration-dependent contraction of non-pregnant and pregnant guinea-pig uterine arterial rings with intact endothelium with similar pD2 and maximal response values (non-pregnant: pD2 = 5.85 +/- 0.02, maximal response = 121 +/- 8.2%; pregnant: pD2 = 5.81 +/- 0.04, maximal response = 122 +/- 9.1%). Removal of endothelium did not affect noradrenaline-induced contractions in non-pregnant guinea-pig uterine artery (pD2 = 5.97 +/- 0.02, maximal response = 119 +/- 8.6%). In contrast, in arteries from pregnant guinea-pigs, removal of endothelium shifted concentration-response curve for noradrenaline to the left, without affecting maximal response value (pD2 = 6.36 +/- 0.03, maximal response = 120 +/- 9.0%). 3. The pKA values for noradrenaline were: 5.76 +/- 0.09 and 5.82 +/- 0.10 for non-pregnant guinea-pig uterine artery with intact and denuded endothelium, respectively and 5.74 +/- 0.09 and 5.72 +/- 0.07 for pregnant guinea-pig uterine artery with intact and denuded endothelium, respectively. 4. The receptor occupancy-response relationship for noradrenaline was linear for all types of vessels, except for pregnant guinea-pig uterine artery with denuded endothelium, since half-maximal response to noradrenaline was obtained with 44.8 +/- 6.9% (non-pregnant guinea-pig uterine artery with intact endothelium), 43.3 +/- 6.1% (non-pregnant guinea-pig uterine artery with denuded endothelium) and 44.3 +/- 6.3% (pregnant guinea-pig uterine artery with intact endothelium) receptor occupancy. In pregnant guinea-pig uterine artery with denuded endothelium, occupancy-response relationship for noradrenaline was non-linear since half-maximal response to noradrenaline was obtained with 19.7 +/- 3.3% receptor occupancy. 5. NG-monomethyl-L-arginine (100 microM) and indomethacin (10 microM) did not affect concentration-response curve for noradrenaline in guinea-pig uterine arteries, regardless of pregnancy status or endothelial condition. 6. In quiescent preparations, the alpha-adrenoceptor antagonists, prazosin (5-50 nM) and yohimbine (1-10 microM) produced parallel rightward shifts of the curves for noradrenaline and the slopes of the Schild plots were not significantly different from unity.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effects of calcitonin gene-related peptide on tracheal smooth muscle of guinea-pigs in vitro.

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.     

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.     

Modulation of ET-1-induced contraction of human bronchi by airway epithelium-dependent nitric oxide release via ET(A) receptor activation

1. 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. 2. 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 ET(A) receptor antagonist, induced a significant leftward shift of the ET-1 concentration-response curve (CRC). This leftward shift was abolished after epithelium removal. 3. L-NAME (3 x 10(-3) M), 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. 4. In intact preparations, the ET(B) 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. 5. IRL 1620, a selective ET(B) 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. 6. 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.     

Difference in the characteristics of ETA-receptor-stimulated response between rat small pulmonary and renal arteries

We investigated the difference in the characteristics of endothelin-1 (ET-1)-induced contraction and the responses of intracellular Ca(2+) concentration ([Ca(2+)](i)) between rat small pulmonary artery and renal artery. ET-1 (30 nM) failed to elicit any contraction in renal arteries pretreated with 3 microM BQ-123, an ETA blocker. However, in the pulmonary artery a combination of BQ-123 and BQ-788, an ETB blocker (5 microM each), only partially inhibited the ET-1-induced contraction (by 25%). To focus on the ETA receptor, in the presence of 5 microM BQ-788, nitric oxide donors (sodium nitroprusside and (+/-)-S-nitroso-N-acetylpenicillamine) and forskolin reduced both the ET-1-induced contraction and increase in [Ca(2+)](i) in both pulmonary and renal arteries. However, the effects were stronger in the renal than in the pulmonary artery. ET-1-induced increase in [Ca(2+)](i) was only partially attenuated by 10 microM verapamil (to 81% of control) in pulmonary arteries but was reduced to 56.1% of control in renal arteries. Our results provide evidence that ET-1 may activate ET receptor(s) insensitive to both BQ-123 and BQ-788 in rat small pulmonary artery, at least under these conditions. Furthermore, the effects of relaxants such as L-type Ca(2+) channel blocker and nitric oxide donors on the ET-1-induced contraction were studied.     

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.     

Comparison of vasodilators in human internal mammary artery: ghrelin is a potent physiological antagonist of endothelin-1

1 The potential vasodilator function of the peptide ghrelin, recently identified as the endogenous ligand of the growth hormone secretagogue orphan receptor (GHS-R), was investigated in human endothelium-denuded internal mammary artery. The peptide endothelin-1 (ET-1) is a potent and long-lasting vasoconstrictor. Comparisons were made with established and putative endogenous vasodilators to determine if any could reverse ET-1-induced vasoconstriction in this vessel. 2 Ghrelin (0.1-300 nM) potently dilated 10 nM ET-1-induced constrictions (pD(2) 8.39+/-0.29; E(MAX) 63+/-5.6%; n=9/14, responders/total). 3 ANP (pD(2) 7.75+/-0.14; E(MAX) 106+/-2.0; n=5/5) and CGRP (pD(2) 8.08+/-0.17; E(MAX) 76+/-15% n=5/6) both produced complete reversal of the constrictor response to ET-1 (E(MAX) not significantly different from 100%, P>0.05 one-sample t-test). 4 The following caused partial reversal of the ET-1 response: Adrenomedullin (n=9/9) and two peptides derived from proadrenomedullin, PAMP-12 (n=6/7) and PAMP-20 (n=9/9) (pD(2) values 7.63+/-0.28, 7.97+/-0.23 and 8.51+/-0.29; E(MAX) 58+/-7.3, 54+/-10 and 51+/-7.8% respectively). Unexpectedly, amylin was only 2 fold less potent than CGRP, although there was less than 50% reversal of the ET-1 constriction (pD(2) 7.86+/-0.30; E(MAX) 41+/-5.4%; n=7/9). CNP (n=6/6) also partially reversed constrictions to ET-1 (E(MAX) 53+/-6.3; pD(2) 8.07+/-0.38). 5 BNP (n=4/5) and PGI(2) (n=6/8) were weak vasodilators, since concentration-response curves failed to reach a maximum within the range tested. PGE(2) caused a small dilatation in some vessels (E(MAX) 17+/-2.1%; pD(2) 8.63+/-0.36; n=4/8). 6 We have demonstrated ghrelin to be an effective, endothelium-independent vasodilator of the long-lasting constrictor ET-1 in human arteries producing responses similar to those of adrenomedullin (P>0.05, ANOVA). British Journal of Pharmacology (2002) 136, 1146-1152     

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.     

Endothelin receptors and calcium translocation pathways in human airways

Tension and phosphatidyl inositol (PI) turnover experiments were conducted to investigate the receptors and signal transduction pathways responsible for contractions elicited by endothelin (ET) ligands in human bronchus. Nicardipine (1 μM), the L-type calcium channel inhibitor, or incubation in Ca²⁺-free medium, produced marked inhibition of contractions to the ET_B receptor-selective agonist, sarafotoxin S6c, and especially those induced by KCl. In contrast, Ca²⁺-free medium was without appreciable effect against contraction produced by endothelin-1 (ET-1), the non-selective ET_A and ET_B receptor agonist. In Ca²⁺-free medium, ryanodine (10 μM), which inhibits intracellular calcium mobilization, reduced sarafotoxin S6c- and ET-1-induced responses, but was without effect on responses to KCl. Similarly, nickel chloride (Ni²⁺; 1 mM) caused marked inhibition of contractions induced by sarafotoxin S6c or ET-1, but had no significant effect on KCl concentration-response curves. The mixed ET_A/ET_B receptor antagonist SB 209670 (3 μM) inhibited responses to sarafotoxin S6c and ET-1 such that concentration-response curves were shifted rightward, at the 30% maximum response level, by 10.0- and 3.8-fold, respectively, whereas BQ-123 (3 μM), the ET_A receptor antagonist, was without effect on responses induced by either agonist. ET-1 (1 nM–0.3 μM) caused a concentration-dependent stimulation of PI turnover, whereas sarafotoxin S6c (0.3 nM–0.1 μM) induced only small and variable increases, except at the highest concentration. The increase in PI turnover evoked by ET-1 was inhibited by SB 209670 (3 μM), and also by BQ-123 (3 μM). This is consistent with linkage of ET_A receptors to activation of inositol phosphate generation in human bronchial smooth muscle cells. Collectively, the data suggest that differences exist in the relative contributions of intracellular and extracellular Ca²⁺ mobilization mechanisms elicited by ET_A and ET_B receptor activation. Thus, sarafotoxin S6c-induced, ET_B receptor-mediated contraction in human bronchial smooth muscle appears to be dependent, in part, upon extracellular Ca²⁺, although a significant component of the response was also mediated by intracellular Ca²⁺ release, including from ryanodine-sensitive stores. ET_A receptor-mediated contraction of human airway smooth muscle was activated largely via the release of intracellular Ca²⁺. Received: 21 July 1998 / Accepted: 26 January 1999     

Relationship between endothelin-1 binding site densities and constrictor activities in human and animal airway smooth muscle.

1. Endothelin-1 (ET-1) binding site densities and constrictor activities were compared in airway smooth muscle preparations of human, guinea-pig, rat and mouse. 2. The mean contractile response to 0.3 microM ET-1 (measured as the % maximum response to 10 microM carbachol, % Cmax +/- s.e.mean) and the mean concentration of ET-1 producing 30% Cmax (95% confidence limits) were respectively; 85.9 +/- 5.4% and 3.4 nM (2.4-5.0) for mouse trachea (n = 11), 88.8 +/- 4.7% and 18.2 nM (11.2-25.2) for rat trachea (n = 6), 71.0 +/- 7.1% and 35.2 nM (5.4-231) for human bronchus (n = 3), and 32.3 +/- 3.0% and 241 nM (125-460) for guinea-pig trachea (n = 6). 3. Light microscopic autoradiography revealed specific [125I]-ET-1 binding sites localized to the smooth muscle band, with very low levels of binding associated with cartilage, submucosal and epithelial cells. 4. Quantitative autoradiographic analyses of the concentration-dependence of specific [125I]-ET-1 binding (0.1-2 nM) to smooth muscle revealed similar dissociation constants but markedly different specific binding site densities for the various animal species. The order of densities of specific [125I]-ET-1 binding sites was rat trachea (69.0 +/- 11.2 amol mm-2) greater than human bronchus (42.7 +/- 17.5 amol mm-2) greater than mouse trachea (28.7 +/- 2.6 amol mm-2) greater than guinea-pig trachea (8.3 +/- 1.8 amol mm-2). 5. A positive relationship between [125I]-ET-1 binding site density and ET-1 constrictor activity was observed in airway smooth muscle preparations from rat, human and guinea-pig.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endothelins-induce cyclicAMP formation in the guinea-pig trachea through an ETA receptor- and cyclooxygenase-dependent mechanism.

1. The non-selective endothelin agonist, endothelin-1 (ET-1), and the selective ETB receptor agonist, sarafotoxin-S6c (SRTX-c), contracted guinea-pig isolated trachea in a concentration-dependent manner. The EC50 value for ET-1 (11 +/- 2.1 nM) was significantly higher than that of SRTX-c (3.2 +/- 0.21 nM) and the maximal developed tension due to SRTX-c was 42.8 +/- 2.3% higher than that produced by ET-1 (P < 0.05). 2. Pretreatment with the ETA antagonist, BQ-610, appreciably enhanced the developed tension due to ET-1 but not SRTX-c. Likewise, the cyclo-oxygenase inhibitor, indomethacin, markedly potentiated the contractile responses to ET-1, but not to SRTX-c. Combining BQ-610 with indomethacin was not more effective than either of them in augmenting ET-1-evoked tension. 3. ET-1 significantly increased cyclic AMP formation in the trachea in concentration- and time-dependent manners. A t1/2 value of 4.3 min, an EC50 value of 20 +/- 3 nM and a maximal cyclic AMP increment of 124% above the basal level, were obtained for ET-1. Similarly but less effectively, ET-3 (0.1 microM) increased cyclic AMP level (35 +/- 3.7% compared to 94 +/- 7.8% for the same concentration of ET-1). By contrast, SRTX-c did not alter the cyclicAMP level when applied in concentrations up to 1 microM. 4. Pre-incubation of the trachea with BQ-610 (1 microM) or indomethacin (1 microM) prevented cyclicAMP formation by either ET-1 or ET-3. 5. The results of the present study indicate a negative regulatory role mediated by the ETA receptor on the ETB-triggered mechanical response. This effect is likely to be mediated by activation of adenylate cyclase through a cyclo-oxygenase-dependent mechanism.     

Vasoconstrictor activity of novel endothelin peptide, ET-1(1 - 31), in human mammary and coronary arteries in vitro.

1. The ability of the putative chymase product of big endothelin-1 (big ET-1), ET-1(1 - 31), to constrict isolated endothelium-denuded preparations of human coronary and internal mammary artery was determined. 2. pD2 values in coronary and mammary artery respectively were 8.21+/-0.12 (n=14) and 8.55+/-0.11 (n=12) for ET-1, 6.74+/-0.11 (n=16) and 7.10+/-0.08 (n=16) for ET-1(1 - 31) and 6.92+/-0.10 (n=15) and 7.23+/-0.11 (n=12) for big ET-1. ET-1(1 - 31) was significantly less potent than ET-1 (P<0.001, Student's t-test) and equipotent with big ET-1. 3. Vasoconstrictor responses to 100 - 700 nM ET-1(1 - 31) were significantly (P<0.05, Student's paired t-test) attenuated by the ET(A) antagonist PD156707 (100 nM). 4. There was no effect of the ECE inhibitor PD159790 (30 microM), the ECE/NEP inhibitor phosphoramidon (100 microM) or the serine protease inhibitor chymostatin (100 microM) on ET-1(1 - 31) responses in either artery. 5. Radioimmunoassay detected significant levels of mature ET in the bathing medium of coronary (1.6+/-0.5 nM, n=14) and mammary (2.1+/-0.6 nM, n=14) arteries, suggesting that conversion of ET-1(1 - 31) to ET-1 contributed to the observed vasoconstriction. 6. ET-1(1 - 31) competed for specific [(125)I]-ET-1 binding to ET(A) and ET(B) receptors in human left ventricle with a pooled K(D) of 71.6+/-7.0 nM (n=3). 7. Therefore, in human arteries the novel peptide ET-1(1 - 31) mediated vasoconstriction via activation of the ET(A) receptor. The conversion of ET-1(1 - 31) to ET-1, by an as yet unidentified protease, must contribute wholly or partly to the observed constrictor response. Chymase generated ET-1(1 - 31) may therefore represent an alternative precursor for ET-1 production in the human vasculature.