Endothelin-induced intracellular Ca2+ mobilization through its specific receptors in murine peritoneal macrophages.

We studied the presence of specific binding sites for endothelin (ET) and the effect of ET on cytosolic free Ca2+ concentration ([Ca2+]i) in murine thioglycolate-activated peritoneal macrophages. Scatchard analysis for binding experiments using [125I]ET-1 or [125I]ET-3 revealed the existence of a single class of binding sites. The binding parameters (Kd and Bmax) for [125I]ET-1 were almost identical to those for [125I]ET-3. In addition, unlabeled 3 ET isopeptides (ET-1, ET-2 and ET-3) inhibited the specific binding of both ET-1 and ET-3 with similar inhibitory potencies. All 3 ET isopeptides caused an increase in [Ca2+]i in the same dose-dependent manner (0.01-100 nM). These results demonstrate the existence of an ET receptor with the same affinity for all isoforms that mediates the ET-induced intracellular Ca2+ mobilization in murine peritoneal macrophages.     

A novel radioligand [125I]BQ-3020 selective for endothelin (ETB) receptors.

A linear endothelin (ET) analog, N-acetyl-LeuMetAspLysGluAlaValTyrPheAlaHisLeu-AspIleIleTrp (BQ-3020), is highly selective for ETB receptors. BQ-3020 displaces [125I]ET-1 binding to ETB receptors (nonselective to ET isopeptides) in porcine cerebellar membranes (IC50: 0.2nM) at a concentration 4,700 times lower than that to ETA receptors (selective to ET-1) on aortic vascular smooth muscle cells (VSMC) (IC50: 940nM). BQ-3020 as well as ET-1 and ET-3 elicits vasoconstriction in the rabbit pulmonary artery. The ETA antagonist BQ-123 failed to inhibit this BQ-3020-induced vasoconstriction. Furthermore, BQ-3020 elicits endothelium-dependent vasodilation. These data indicate that BQ-3020 has ETB agonistic activity. The radioligand [125I]BQ-3020 binds to cerebellar membranes at single high affinity sites (Kd = 34.4pM), whereas it scarcely binds to VSMC. [125I]BQ-3020 binding to the cerebellum was displaced by BQ-3020, ET-1 and ET-3 in a nonselective manner (IC50: 0.07-0.17nM). However, the binding of [125I]BQ-3020 was insensitive to the ETA antagonist BQ-123 and other bioactive peptides. Both [125I]ET-1 and [125I]BQ-3020 show slow onset and offset binding kinetics to ETB receptors. These data indicate that the radioligand [125I]BQ-3020 selectively labels ETB receptors and that the slow binding kinetics of ET-1 are dependent on the peptide sequence from Leu6 to Trp21, but not on the structure formed by its two disulfide bridges.     

A reversible radioligand specific for the ETB receptor: [125I]Tyr13-Suc-[Glu9,Ala11,15]-endothelin-1(8- 21), [125I]IRL 1620.

Suc-[Glu9,Ala11,15]-endothelin(ET)-1(8-21), IRL 1620, is a linear ET-analog specific for the ET-isopeptide-nonselective ETB receptor. The radio-iodinated analog, [125I]IRL 1620, showed a single class of saturable binding to the ETB receptors in porcine lung membranes with a Kd of 18 pM and a Bmax of 930 fmol/mg protein, which are almost comparable to the values obtained with [125I]ET-3 (6 pM and 900 fmol/mg protein). In competitive binding assays with [125I]IRL 1620, unlabeled ET-1, ET-3, IRL 1620 and [monoiodo-Tyr13]-IRL 1620 showed almost identical displacement curves with Ki of 8 to 16 pM. However, [125I]IRL 1620 was dissociated from the binding sites by addition of an excess amount (100 nM) of any of these unlabeled peptides, each with the same t1/2 of 100 min. This was in marked contrast to [125I]ET-3 which was hardly dissociated from the binding sites.     

Endothelin increases [Ca2+]i in rat pancreatic acinar cells by intracellular release but fails to increase amylase secretion.

In individual fura-2 loaded cells of rat pancreatic acini endothelin-1 (ET-1) (10-50 nM) induced sustained oscillations in [Ca2+]i. At higher concentrations a larger, but transient increase in [Ca2+]i was observed, which was largely unaffected by removal of extracellular Ca2+. ET-1 induced the release of Ca2+i from the same store as cholecystokinin (CCK), but with less potency. At concentrations of endothelin which transiently increased Ca2+, ET-1 increased the accumulation of inositol phosphates. Specific binding sites for 125I-endothelin were demonstrated on rat pancreatic acini. A single class of binding sites was identified with an apparent Kd 108 +/- 12 pM and Bmax of 171 +/- 17 fmol/mg for ET-1. The relative potency order for displacing [125I]ET was ET-1 greater than ET-2 greater than ET-3. In contrast to CCK and the non-phorbol ester tumour promoter Thapsigargin (TG) which induce both transient and sustained components of [Ca2+]i elevation, ET-1 failed to increase amylase release over the range 100 pM-1 microM.     

Biological profiles of highly potent novel endothelin antagonists selective for the ETA receptor.

We describe novel potent endothelin (ET) antagonists that are highly potent and selective for the ETA receptor (selective to ET-1). Of the synthetic analogs based on ETA antagonist BE-18257A isolated from Streptomyces misakiensis (IC50 value for ETA receptor on porcine aortic smooth muscle cells (VSMCs); 1.4 microM), the compounds BQ-123 and BQ-153 greatly improved the binding affinity of [125I]ET-1 for ETA receptors on VSMCs (IC50; 7.3 and 8.6 nM, respectively), whereas they barely inhibited [125I]ET-1 binding to ETB receptors (nonselective with respect to isopeptides of ET family) in the cerebellar membranes (IC50; 18 and 54 microM, respectively). Associated with the increased affinity for ETA receptors, these peptides antagonized ET-1-induced constriction of isolated porcine coronary artery. However, there was a small amount of ET-1-induced vasoconstriction resistant to these antagonists, which paralleled the incomplete inhibition of [125I]ET-1 binding in the membrane of the aortic smooth muscle layer. These data suggest that the artery has both ETA and ETB receptors responsible for ET-1-induced vasoconstriction. The antagonists shifted the concentration-response curve to the right for ET-1 in the coronary artery, and increased the apparent dissociation constant in the Scatchard analysis of [125I]ET-1 binding on the VSMCs without affecting the binding capacity, indicative of the competitive antagonism for ETA receptor. In conscious rats, pretreatment with the antagonists markedly antagonized ET-1-induced sustained pressor responses in dose-dependent fashion without affecting ET-1-induced transient depressor action, suggesting that the pressor action is mediated by ETA receptors, while the depressor action is mediated by ETB receptors. In addition, pretreatment with the potent antagonists prevented ET-1-induced sudden death in mice. Thus, these potent ETA antagonists should provide a powerful tool for exploring the therapeutic uses of ETA antagonists in putative ET-1-related disorders.     

Endothelin isopeptides evoke Ca2+ signaling and oscillations of cytosolic free [Ca2+] in human mesangial cells

Isopeptides of the newly discovered peptide family, endothelins (ET), caused a concentration-dependent increase in intracellular free [Ca2+] ([Ca2+]i) in human glomerular mesangial cells. ET isopeptides and sarafotoxin S6b caused transient and sustained [Ca2+]i waveforms which resulted from mobilization of intracellular Ca2+ stores and from Ca2+ influx through a dihydropyridine-insensitive Ca2+ channel. Ca2+ signaling evoked by ET isopeptides underwent a marked adaptive, desensitization response. Although activation of protein kinase C attenuated ET-induced Ca2+ signaling, desensitization by ET isopeptides was independent of protein kinase C. High concentrations of ET-1 and ET-2 also caused oscillations of [Ca2+]i that partially depended on extracellular Ca2+. These results suggest that an increase in [Ca2+]i constitutes a common pathway of signal transduction for the ET peptide family.     

Multiple subtypes of endothelin receptors in porcine tissues: characterization by ligand binding, affinity labeling and regional distribution.

To clarify the existence and the distribution of endothelin (ET) receptor subtypes, we have examined the pharmacological properties and the molecular weight (Mr) of 125I-ET-1 and 125I-ET-3 binding sites in various tissues of pigs. ET-1 and ET-2 showed almost identical potencies in displacing the bound 125I-ET-1 in all the tissues examined. ET-3, sarafotoxin S6b (SRT-b) and sarafotoxin S6c (SRT-c) displaced the 125I-ET-1 with the same sensitivity as ET-1 (IC50 = 0.1-1.4 nM) in brain, kidney, liver and adrenal, whereas the three peptides showed very weak competition (IC50 = 40-500 nM) against 125I-ET-1 binding in cardiac atria, aorta, lung, stomach and uterus. The computer analyses of the binding data suggested the presence of high (Kd1 = 0.04-0.29 nM) and low (Kd2 = 60-190 nM) affinity binding sites for ET-3 and SRT-b in lung and stomach. 125I-ET-3 bound to the high affinity sites in lung and stomach was displaced by ET/SRT isopeptides almost equipotently. Two proteins with Mr of 47,000 and 35,000 were affinity-labeled with 125I-ET-1 in cerebellum, while a protein with Mr of 123,000, in addition to the two proteins, was predominantly labeled in lung. The above findings indicated that two distinct subclasses of ET receptors, namely, ET-1-specific and ET/SRT family-common receptors were distributed in various proportions in mammalian tissues, and suggested that their molecular forms are also different.     

Endothelin receptors in human and guinea-pig gallbladder muscle

We measured contraction of muscle strips caused by endothelin (ET) isopeptides and binding of (125)I-ET-1 to muscle cell membranes prepared from human and guinea-pig gallbladders. Visualization of (125)I-ET-1 binding sites in tissue was performed by autoradiography. Results in human were similar to those in guinea-pig. ET-1 caused tetrodotoxin and atropine-insensitive contraction. The relative potencies for ET isopeptides to cause contraction were ET-1=ET-2>ET-3. ET-1 caused contraction was only slightly inhibited by BQ-123 (potent ET(A) receptor antagonist) and not by BQ-788 (potent ET(B) receptor antagonist). It was inhibited by the combination of both. Autoradiography localized (125)I-ET-1 binding to the smooth muscle layer. Binding of (125)I-ET-1 to muscle cell membranes was saturable and specific. Analysis of dose-inhibition curves demonstrated the presence of two classes of receptors. One class (ET(A) receptor) had a high affinity for ET-1 and ET-2 but a low affinity for ET-3, and the other (ET(B) receptor) a high affinity for ET-1, ET-2 and ET-3. These results demonstrate that similar to guinea-pig, human gallbladder possesses both ET(A) and ET(B) receptors cooperating to mediate muscle contraction.     

Specific receptors for endothelin-3 in cultured bovine endothelial cells and its cellular mechanism of action

Among three endothelin (ET) isopeptides, ET-3 shows the most potent initial depressor response through the endothelium-dependent mechanism. We studied the presence of specific binding sites for ET-3 in cultured bovine endothelial cells (EC) and its cellular mechanism of action. Binding studies revealed the presence of two distinct subclasses of ET-3 receptors with high and low affinities. ET-3 dose-dependently (10(-10)-10(-7) M) increased both intracellular Ca2+ levels ([Ca2+]i) and inositol trisphosphate (IP3) formation. The ET-3-induced increase in [Ca2+]i was not affected by either removal of extracellular Ca2+ or Ca2(+)-channel blockers. These data suggest that ET-3 induces phosphoinositide breakdown and increase in [Ca2+]i in ECs, possibly resulting from intracellular Ca2+ mobilization, thereby leading to vasodilatation.     

Endothelin-stimulated Ca(2+)signaling and endothelin receptor expression are decreased by parathyroid hormone treatment in UMR-106 osteoblastic osteosarcoma cells

Modulation of endothelin (ET-1)-induced [Ca(2+)](i)transients and receptor expression by parathyroid hormone (PTH) was studied in UMR-106 osteoblastic osteosarcoma cells. Ca(2+)signaling was assessed with Fura-2, and ET receptor mRNA expression was determined using ET(A)- and ET(B)-specific primers and RT-PCR amplification. ET-1 binding in UMR-106 cell membranes was also measured. PTH pretreatment for 8 h decreased the [Ca(2+)](i)transients elicited by ET-1 and by the ET(B)-selective agonist sarafotoxin 6c (S6c). When ET(B)receptors were desensitized by pretreatment with S6c or blocked with the ET(B)-selective antagonist BQ-788, the remaining ET(A)component of the signal was also decreased by PTH pretreatment. In contrast, [Ca(2+)](i)transients elicited by PGF(2alpha)and ionomycin were increased following PTH pretreatment, indicating that the effect of PTH to decrease ET-1-stimulated transients was selective. PTH pretreatment also decreased [(125)I]ET-1 binding and ET(A)and ET(B)mRNA, with maximal effects at approximately 8 h. ET-1 was not detectable in medium from either control or PTH treated UMR-106 cultures, suggesting that the decreased expression of ET receptors was not due to enhanced ET production and subsequent homologous desensitization. The downregulation of ET receptors in osteoblasts by PTH pretreatment may serve as a homeostatic mechanism in bone.     

Ontogeny of endothelin and its receptors in rat brain.

The ontogeny of endothelin (ET) system in rats was studied in preterm (18 days of gestation), term (21 days of gestation) and 1 week post term rats. Brains were dissected out and (1) processed for the estimation of endogenous ET-1 by RIA and (2) membranes were prepared for radioreceptor binding. Receptor characteristics, affinity (Kd) and density (Bmax) were determined using [125I] ET-1 and [125I] SRT 6b (which is structurally similar to ET) and cold ET-1 or SRT 6b as displacer. ET levels were found to be 25.66 +/- 3.18 pg/g protein in preterm, 47.37 +/- 5.31 pg/g protein in term and 48.30 +/- 1.90 pg/g protein in post term rats. ET levels were significantly lower in preterm as compared to term and post term rats. Preterm, term and post term rats showed single high affinity binding site for both [125I] ET-1 and [125I] SRT 6b. The Kd values for [125I] ET-1 and [125I] SRT 6b binding were similar in preterm, term and post term rats. The Bmax values of both [125I] ET-1 and [125I] SRT 6b binding were found to be similar in preterm and term rats while they were significantly higher in post term rats. In adult (4 month old) rats the Kd values were similar to neonatal rats while the Bmax values were significantly lower than the post term neonatal rats. It is concluded that ET and its receptors are developmentally regulated and there is a possibility that endogenous ET is involved in the regulation of ET receptor density.     

Ca2+ signaling by distinct endothelin peptides in glomerular mesangial cells.

Ca2+ signaling by peptides of the endothelin (ET) gene family was studied in cultured glomerular mesangial cells. In addition to the increase in cytosolic free [Ca2+] ([Ca2+]i) previously described for ET-1, we also observed that ET-2, ET-3, and sarafotoxin S6b generate similar [Ca2+]i waveforms but with dissimilar potencies and kinetics. The prepro form of ET-1 was inactive, suggesting that mature ET peptides are constrained in an inactive conformation within the preproET species. ET isopeptides caused both release of Ca2+ from intracellular stores and Ca2+ influx via a voltage- and dihydropyridine-insensitive pathway. ET-mediated Ca2+ influx was independent of the increase in [Ca2+]i. Activation of protein kinase C inhibited ET-induced Ca2+ signaling, whereas addition of ET to protein kinase C-depleted cells resulted in enhanced [Ca2+]i waveforms. Mesangial cells also demonstrated a marked adaptive desensitization response to ET. These data demonstrate that Ca2+ signaling is a common response to different ET peptides in glomerular mesangial cells and that activation of protein kinase C down-regulates these Ca2+ signals.     

Effects of endothelins on signal transduction and proliferation in human melanocytes

We demonstrate here that human melanocytes could be regulated by endothelin (ET) derivatives, potent vasoconstrictive peptides synthesized by endothelial cells, to stimulate their proliferation and melanization via a receptor-mediated signal transduction pathway. Receptor-binding assay using [125I]ET indicated that unlabeled ET-1 or ET-2 competitively inhibited each binding of labeled ETs to melanocytes with a concentration for half-maximal inhibition (IC50) of 0.7 or 0.9 nM, respectively. The dissociation constant (Kd) and the number of sites of the specific bindings of ET-1 and those of ET-2 were almost the same (Kd: 1.81 nM, binding sites: 7.0-8.0 x 10(4) per cell). Upon incubation with cultured cells, the mass contents of inositol 1,4,5-trisphosphate and intracellular calcium level were substantially increased by 10 nM ET-1, ET-2, and ET-3, but not by big-ET with maximal response at 80-130-s postincubation. The addition of ET-1 and ET-2 at 1-50 nM concentrations caused human melanocytes to significantly stimulate DNA [( 3H]thymidine incorporation) and melanin synthesis (3H2O release and [14C] thiouracil incorporation). Furthermore, ETs exhibited an additive stimulatory effect on basic fibroblast growth factor-stimulated DNA synthesis. In a long-term serum-free culture system, the strongest stimulation of growth by 10 nM ET-1 or ET-2 was observed in the presence of 10 nM cholera toxin and 0.2% bovine pituitary extract, resulting in a 4.5-fold increase in cell number for 12 culture days. These findings strongly suggest involvement of ET in the mechanism regulating proliferation and melanization of human melanocytes.     

Both endothelin-A and endothelin-B receptors are present on adult rat cardiac ventricular myocytes

Endothelin-A (ET(A)) and endothelin-B (ET(B)) receptors have been demonstrated in intact heart and cardiac membranes. ET(A) receptors have been demonstrated on adult ventricular myocytes. The aim of the present study was to determine the presence of ET(B) and the relative contribution of this receptor subtype to total endothelin-1 (ET-1) binding on adult ventricular myocytes. Saturation binding experiments indicated that ET-1 bound to a single population of receptors (Kd = 0.52 +/- 0.13 nM, n = 4) with an apparent maximum binding (Bmax) of 2.10 +/- 0.25 sites (x 10(5))/cell (n = 4). Competition experiments using 40 pM [125I]ET-1 and nonradioactive ET-1 revealed a Ki of 660 +/- 71 pM (n = 10) and a Hill coefficient (nH) of 0.99 +/- 0.10 (n = 10). A selective ET(A) antagonist, BQ610, displaced 80% of the bound [125I]ET-1. No displacement was observed by concentrations of an ET(B)-selective antagonist, BQ788, up to 1.0 microM. However, in the presence of 1.0 microM BQ610, BQ788 inhibited the remaining [125I]ET-1 binding. Similarly, in the presence of 1.0 microM BQ788, BQ610 inhibited the remaining specific [125I]ET-1 binding. Binding of an ET(B1)-selective agonist, [125I]IRL-1620, confirmed the presence of ET(B). ET(B) bound to ET-1 irreversibly, whereas binding to ET(A) demonstrated both reversible and irreversible components, and BQ610 and BQ788 bound reversibly. Reducing the incubation temperature to 0 degrees C did not alter the irreversible component of ET-1 binding. Hence, both ET(A) and ET(B) receptors are present on intact adult rat ventricular myocytes, and the ratio of ET(A):ET(B) binding sites is 4:1. Both receptor subtypes bind to ET-1 by a two-step association involving the formation of a tight receptor-ligand complex; however, the kinetics of ET-1 binding to ET(A) versus ET(B) differ.     

ET-1 released histamine from guinea pig pulmonary but not peritoneal mast cells.

Endothelin(ET)-1 triggered histamine release of mast cells from pulmonary tissue but not from the peritoneal cavity of guinea pigs. The observed difference in response to ET-1 was attributable to a quantitative difference in ET-1 binding sites between both cells. The concentrations of ET-1 required for half maximal release of histamine and half maximal binding of [125I]ET-1 were approximately 0.05 and 0.08 nM, respectively. The release of histamine by ET-1 was a Ca(2+)-dependent but not a cytotoxic process. These observations, taken together, suggest that ET-1 induces histamine release from mast cells in a receptor-dependent fashion.     

High affinity interaction of endothelin-3 with recombinant ETA receptors

Pharmacological evidence has suggested that endothelin-3 (ET-3) may act via a novel form of ET receptor that is shared by ETA receptor antagonists but not by ETB receptor selective agonists. This study analyses the properties of interaction of ET-3 with recombinant bovine ETA receptor. Apparent Kd(ET-3) values as low as 50 nM were defined from [125I]ET-1 binding experiments performed at low (5 microg/ml) protein concentrations in the assays. Larger (up to 1 microM) values were artefactually obtained in experiments performed at larger protein concentrations. The three monoiodo ET-3 derivatives were synthetized. ([125I]Y14)ET-3 did not recognize ETA receptors. ([125I]Y6)ET-3 labelled 18% of [125I]ET-1 binding sites with a Kd value of 320 pM. ([125I]Y13)ET-3 labelled 44% of [125I]ET-1 binding sites with a Kd value of 130 pM. High affinity ([125I]Y6)ET-3 and ([125I]Y13)ET-3 bindings were prevented by ET-1 (Kd = 5-7 pM), ET-3 (Kd = 70-250 pM), BQ-123 (Kd = 2 nM) and FR139317 (Kd = 2 nM) but not by low concentrations of 4-AlaET-1, sarafotoxin S6c or IRL1620. The three monoiodo ET-3 derivatives bound to recombinant rat ETB receptors with a pM affinity. The results suggest that ET-3, ([125I]Y6)ET-3 and ([125I]Y13)ET-3 should not be considered as ETB receptor specific ligands.     

Autocrine receptors for endothelins in the primary culture of endothelial cells of human umbilical vein.

Human umbilical vein endothelial cells (HUVECs) in primary culture produced and secreted endothelin 1 (ET-1) actively. Specific binding of [125I]ET-1 to these cells was not detectable because of the saturation of ET receptors with endogenously produced ET-1. However, addition of phosphoramidon, an inhibitor of ET-converting enzyme, to the medium reduced the production of ET-1 and thus the receptors on HUVECs were made available for exogenously added [125I]ET-1. Binding studies using phosphoramidon-treated HUVECs indicated the existence of a non-isopeptide-selective type (ETB) of ET receptor with a Kd of 17 pM. This receptor is thought to be involved in ET-induced vasodilation in an autocrine manner in vivo.     

Two classes of endothelin receptors mediating contraction in esophageal muscularis mucosae

Endothelin (ET) causes contraction of the esophageal muscularis mucosae. To characterize the ET receptor subtypes involved in contraction, we measured contraction of isolated muscularis mucosae strips caused by ET-related peptides and binding of (125)I-ET-1 to cell membranes prepared from the guinea pig esophageal muscularis mucosae. Autoradiography demonstrated (125)I-ET-1 binding to the muscularis mucosae and muscularis propria. ET-1 caused tetrodotoxin and atropine-insensitive contraction of esophageal muscularis mucosae strips. The relative potencies for ET isopeptides to cause contraction were ET-1=ET-2>ET-3. FR-139317, an ET(A) receptor antagonist, or BQ-788, an ET(B) receptor antagonist, alone did not alter responses to ET-1. However, the combination of both antagonists almost abolished the ET-1-induced contraction, indicating synergistic inhibition. Desensitization to sarafotoxin S6c, an ET(B) receptor agonist, failed to abolish the response to ET-1, which was completely inhibited by FR-139317. These indicate the involvement of both ET(A) and ET(B) receptors in the contraction. Binding of (125)I-ET-1 to cell membranes of the muscularis mucosae was saturable and specific. Analysis of dose-inhibition curves demonstrated the presence of ET(A) and ET(B) receptors.This study demonstrates that the esophageal muscularis mucosae possesses both ET(A) and ET(B) receptors mediating muscle contraction. There is cooperation between these two subtypes of ET receptors in the esophagus mediating muscle contraction.     

Importance of the phenotypic state of vascular smooth muscle cells on the binding and the mitogenic activity of endothelin

Smooth muscle cells of the rabbit aorta, when grown in vitro, express distinguishable forms of phenotypes (contractile and synthetic). On contractile cells, ET-1 specifically bound to a single class of high affinity (KD = 128 pM) and high capacity (Bmax = 66,000 sites/cell) binding sites. But, whereas affinity of [125I]-ET-1 was not significantly affected by phenotypic modulation, synthetic cells displayed a 10-fold lower [125I]-ET-1 binding capacity than contractile smooth muscle cells. Similarly, the mitogenic effect of ET-1 on smooth muscle cells was considerably lower for synthetic than for contractile cells. The ET-1 receptor on primary cells was recognized by sarafotoxin S6b and the different ET-related peptides with an order of potency [ET-1 greater than S6b greater than ET-3 greater than Big ET-1 much greater than ET(16-21)] identical to that inducing smooth muscle cell growth. Therefore, these data indicate that the binding and the mitogenic effects of ET-1 on smooth muscle cells might be of different magnitudes depending on the phenotypic state of these cells.     

Endothelin ET(A) but not ET(B) receptors mediate contraction of common bile duct

Endothelin (ET) causes contraction of the gallbladder. To investigate effects of ET in the common bile duct, we measured contraction of longitudinal muscle strips from guinea pig common bile ducts induced by ET-related peptides and binding of 125I-ET-1 to cell membranes prepared from the common bile duct. Visualization of 125I-ET-1 binding sites in tissue was performed by autoradiography. ET-1 caused tetrodotoxin and atropine-insensitive contraction. In terms of maximal tension of contraction, ET-1, ET-2 and ET-3 were equal in efficacy. However, sarafotoxin S6c, a selective ET(B) receptor agonist, caused only a negligible contraction. The relative potencies for ET isopeptides to cause contraction were ET-1=ET-2>ET-3. The ET-1-induced contraction was inhibited by BQ-123, an ET(A)-receptor-selective antagonist, but not by BQ-788, an ET(B)-receptor-selective antagonist. In addition, the combination of both antagonists, BQ-123 and BQ-788, inhibited ET-1 induced contraction but did not potentiate the inhibition caused by BQ-123 alone. These indicate that ET(A) but not ET(B) receptors mediate the contraction. Autoradiography localized 125I-ET-1 binding to the smooth muscle layer. Binding of 125I-ET-1 to the smooth muscle cell membranes was saturable and specific. Analysis of dose-inhibition curves indicated the presence of ET(A) and ET(B) receptors. These results demonstrate that ET causes contraction of longitudinal muscle of the common bile duct. Different from the gallbladder, which possesses both ET(A) and ET(B) receptors cooperating to mediate muscle contraction, the common bile duct possesses two classes of ET receptors, but only the ET(A) receptor mediates the contraction.