Endothelin ETA but not ETB receptors mediate contraction of common bile duct

Regulatory Peptides 113 (2003) 131 – 138 www.elsevier.com/locate/regpep

Endothelin ETA but not ETB receptors mediate contraction of common bile duct Shih-Che Huang * Department of Internal Medicine, Tzu Chi General Hospital and Tzu Chi University, 707 Section 3, Chung-Yang Road, Hualien 970, Taiwan Received 20 September 2002; received in revised form 23 December 2002; accepted 23 December 2002

Abstract 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 ETB 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 ETA-receptor-selective antagonist, but not by BQ-788, an ETB-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 ETA but not ETB 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 ETA and ETB receptors. These results demonstrate that ET causes contraction of longitudinal muscle of the common bile duct. Different from the gallbladder, which possesses both ETA and ETB receptors cooperating to mediate muscle contraction, the common bile duct possesses two classes of ET receptors, but only the ETA receptor mediates the contraction. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Smooth muscle; Binding; Autoradiography; Motility

1. Introduction Endothelins (ETs) are a family of peptides with 21amino-acid residues. ET-1 was identified as a potent vasoconstrictor produced by vascular endothelial cells. Following the discovery of ET-1, three distinct isoforms of ET, i.e. ET-1, ET-2 and ET-3, have been shown to exist in a variety of tissues. In addition to these endogenous ligands, sarafotoxins, which are snake venom peptides with similar structure and biological effects to ET, have been included as members of the ET superfamily. ET was first identified as a potent vasoconstrictor and later found to cause contraction of cardiovascular, tracheal, gastrointestinal, urinary and uterine smooth muscle [1– 3]. In the gastrointestinal system, ET causes contraction of strips of the esophageal muscularis mucosae and stomach, as well as dispersed gastric and * Tel.: +886-3-8561825x2139; fax: +886-3-8577161. E-mail address: [email protected] (S.-C. Huang).

intestinal smooth muscle cells [4 –8]. In strips of the ileum and lower esophageal sphincter, ET causes relaxation followed by contraction [9,10]. In the hepatobiliary system, ET causes contraction of hepatic stellate (Ito) cells, the gallbladder and circular muscle of the common bile duct [11– 15]. In mammalian species, two classes of ET receptors, ETA and ETB, have been cloned. ETA receptors have higher affinities for ET-1 and ET-2 than ET-3, while ETB receptors have the same affinities for ET-1, ET-2 and ET-3 [2,16,17]. In the cardiovascular system, ET-1 interacts with ETA receptors that predominate on the smooth muscle to cause vasoconstriction. In some vessels, a small population of ETB receptors also mediates constriction [18]. In the gastrointestinal system, ET causes smooth muscle contraction through interaction with ETA receptors, ETB receptors or both ETA and ETB receptors, depending on the tissue type [2,4 –9,13 – 15]. In the esophagus and gallbladder, ET interacts with both ETA and ETB receptors to induce muscle

0167-0115/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0167-0115(03)00004-1

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contraction [4,5,13 – 15]. In the stomach and ileum, either ETA or ETB receptors alone are involved in muscle contraction [6– 9]. Gallbladder epithelial cells produce ET-1, which can act in a paracrine manner on muscle and control bile flow [19]. In animal studies, ligation of the common bile duct causes increase of ET-1 levels in the bile and blood, which may contribute to the renal and pulmonary damage after the ligation. Thus, ET may be involved in the pathogenesis of hepatorenal and hepatopulmonary syndrome in cirrhosis [20,21]. At the present time, not much information is available about the effect of ET in the common bile duct. A recent report described ET-1-induced contraction of isolated rings of the common bile duct after pretreatment with indomethacin [12]. Little is known about the mechanism of ET-induced contraction on common bile duct muscle. No data are available about whether ET causes contraction of longitudinal muscle of the common bile duct and the types of ET receptors mediate this effect. The aim of this study is to investigate the effects of ET in longitudinal muscle of the common bile duct and to characterize the receptors mediating the effect.

7.40 F 0.05. The common bile duct was cut longitudinally to make a 0.4  1-cm strip. Common bile duct strips were attached along the longitudinal axis to an organ bath using surgical silk sutures and incubated at 37 jC in the standard incubation solution continuously gassed with 95% O2 – 5% CO2. The strips were connected to an isometric transducer (Grass FT.03), which was connected to an integrated amplifier and recorder (Gould). The basal tension of the muscle strips was adjusted to 0.5 g. Experiments were started after a 45-min equilibration period. KCl (80 mM)induced contraction was used as a reference to express contractile response to agonist-related peptides. All experiments with ET-related agonists were performed in a cumulative manner because of lack of desensitization of the common bile duct muscle to the cumulative administration of these agents [4,5,12 – 15]. Only one cumulative concentration – response curve with or without a receptor antagonist or tetrodotoxin was constructed with each preparation. For studies using receptor antagonists or tetrodotoxin, muscle strips were exposed to the indicated concentrations of these agents for 6 min and then to the various concentrations of the peptides.

2. Materials and methods

2.2. Measurement of contraction of gallbladder muscle strips

Male Hartley guinea pigs (300 – 350 g) were obtained from the Animal Center, National Science Council of Taiwan. N-[2-hydroxyethyl]piperazine-NV-[2-ethanesulfonic acid] (HEPES), piperazine-N,NV-bis(2-ethanesulfonic acid) (PIPES), Tris, bovine serum albumin (BSA), soybean trypsin inhibitor, leupeptin, phosphoramidon, bestatin, bacitracin, indomethacin and atropine were from Sigma, St. Louis, MO; ET-related reagents, ET-1, ET-2, ET-3, BQ-123, BQ788, sarafotoxin S6c as well as other peptides were from Peninsula (Bachem), Belmont, CA or RBI (Sigma), Natick, MA; 125I-ET-1 was from NEN (Perkin-Elmer), Boston, MA; protein assay kit was from Bio-Rad, Hercules, CA, USA. Tetrodotoxin was from Tocris Cookson, Avonmouth Bristol, UK. The protocol for this work has been reviewed and approved by the Institutional Animal Care and Use Committee of the Tzu Chi University. 2.1. Measurement of contraction of common bile duct muscle strips Measurements of the contraction of muscle strips from the common bile duct were performed according to the procedure published previously with minor modifications [5,15,22]. In brief, the guinea pig was sacrificed with CO2 and the common bile duct, from the outer surface of the duodenum to the junction with the hepatic duct, was removed and placed in standard incubation solution, containing 118 mM NaCl, 25 mM NaHCO3, 4.7 mM KCl, 14 mM glucose, 1.2 mM NaH2PO4, 1.8 mM CaCl2, gassed with 95% O2 – 5% CO2. The final pH at 37 jC was

Measurements of the contraction of muscle strips from the guinea pig gallbladder were performed according to the procedure published previously [15]. 2.3. Autoradiographic studies for common bile ducts Guinea pig common bile ducts, when removed, were rapidly frozen at 20 jC. Tissue sections (30 Am) of common bile ducts were cut longitudinally with a cryostat microtome at 20 jC and mounted onto gelatin-coated glass slides. They were dried overnight at 70 jC. Binding of radiolabeled peptide (125I-ET-1) to the tissue sections was performed using the method described previously for the gallbladder [15]. In brief, tissue sections were incubated with 40 pM radiolabeled peptide with or without 1 AM ET-1 for 60 min and washed three times after incubation. After washing, tissue sections were partially dried with a stream of cold air and completely dried in a desiccator under partial pressure for 24 h. The dried tissue sections were exposed to Biomax-MS films (Kodak) for 24 h in an X-ray cassette. Autoradiographic localization of the binding sites was obtained by superimposing the developed film with the stained histological section (hematoxylin and eosin). 2.4. Cell membrane-binding experiments Cell membranes from the common bile duct were prepared according to the method described previously for gallbladder [15,23]. Briefly the common bile duct

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was cut longitudinally, washed in ice-cold homogenization buffer, 20 mM HEPES (pH7.4) containing 0.1% bacitracin, 0.01% trypsin inhibitor, 100 AM bestatin and 3 AM phosphoramidon. The strip was then cut into small pieces and homogenized with a polytron homogenizer. The homogenate was centrifuged at 200  g for 10 min. Then the supernatant was centrifuged at 48,000  g for 20 min at 4 jC. The pellet, containing the membranes, was resuspended in membrane solution containing 10 mM PIPES (pH6.5), 5 mM MgCl2, 0.1% bacitracin, 0.01% trypsin inhibitor, 100 mM bestatin and 3 mM phosphoramidon. For binding to membranes, incubations were performed with 0.1 mg/ml membranes in membrane solution, 20 pM 125 I-ET-1 and the indicated concentration of cold ligands at 22 jC. Nonspecific binding was obtained by adding 1 AM ET-1 to the incubation tubes. After the incubation, 300 Al of samples was filtered through Whatman GF/C glass filters over a vacuum-filtering manifold (Millipore). Membranes retained on the filters were washed three times with 5 ml of ice-cold membrane solution. Radioactivity associated with membranes was determined using a gamma counter. All binding data in this paper, unless otherwise specified, represent specific binding, i.e. binding measured with 125I-ET-1 alone (total binding) minus binding in the presence of 1 AM ET-1 (nonspecific binding). In all experiments, nonspecific binding was < 20% of total binding. Membrane protein was measured according to the method published previously [24] using a commercially available protein assay kit. 2.5. Analysis of data Results are expressed as means F S.E.M. Statistical evaluation is performed using Student t test; p < 0.05 is considered statistically significant. Dose – inhibition curves are analyzed with the use of a nonlinear least-squares program, LIGAND [5,7,15,23,25], to determine the binding parameters (Kd, dissociation constant and Bmax, sites per milligram protein). The number of classes of binding sites is determined by repetitive fits with increasing classes of sites and is established by determining the fewest number of classes that statistically best fit the data using F test and p < 0.05 as the level of significance.

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3. Results 3.1. Effects of ET-related peptides on the common bile duct muscle contraction To test the ability of ET related peptides to interact with ET receptors and cause muscle contraction, muscle strips of guinea pig common bile ducts were prepared and responses to ET isopeptides, ET-1, ET-2, ET-3, and sarafotoxin S6c (SX6c), an ETB receptor selective agonist [2 – 5], were studied. In the muscle strips isolated from guinea pig common bile ducts, ET-1 induced a slow contraction (Fig. 1), which was not affected by tetrodotoxin or atropine, 1 AM each (data not shown). ET-1 did not cause any relaxation in resting muscle strips. In terms of the maximal tension of contraction, ET-1, ET-2 and ET-3 were equal in efficacy (Fig. 2). ET-1 caused detectable contraction of common bile duct muscle strips at 1 nM, half-maximal at 3.5 F 0.7 nM and maximal contraction at 30 nM. The maximal tension caused by 30 nM ET-1 was 51 F 5% of the tension caused by 80 mM KCl. ET-2 was as potent as ET-1 and caused half-maximal contraction at 2.8 F 1.0 nM. ET-3 was 17fold less potent than ET-1 and caused half-maximal contraction of common bile duct muscle strips at 59 F 3 nM ( p < 0.05; Fig. 2 and Table 1). SX6c, the ETB receptor agonist, caused only a negligible contraction (maximal tension = 3 F 3% KCl, 80 mM) at concentrations from 3 nM to 1 AM (Fig. 2). To test whether SX6c caused relaxation of the common bile duct muscle, SX6c was added after muscle had been precontracted with carbachol, which caused a sustained contraction of the common bile duct muscle strip. The maximal contractile response to carbachol, 1 AM, was 94 F 3% KCl. SX6c, 0.1 AM, did not affect the contraction caused by 1 AM carbachol. 3.2. Effect of SX6c on the gallbladder muscle contraction To compare the ability of SX6c to cause contraction in the guinea pig gallbladder with that in the common bile duct, muscle strips of the guinea pig gallbladder were prepared and responses to SX6c were studied. In contrast

Fig. 1. A typical tracing showing the contraction of guinea pig common bile duct muscle on KCl (80 mM) and cumulative addition of ET-1.

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Fig. 2. Ability of ET-1, ET-2, ET-3 and SX6c to cause contraction of muscle strips from guinea pig common bile ducts. Values are expressed as percent of KCl (80 mM)-induced contraction. Results given are from at least four experiments. Vertical bars represent F S.E.M.

to a negligible contraction in the common bile duct, SX6c caused a marked contraction in the strips from the gallbladder. Detectable contraction occurred with 1 nM SX6c, halfmaximal with 4.9 F 0.9 nM and maximal contraction with 100 nM. The maximal tension caused by 100 nM SX6c in the gallbladder strips was 91 F 10% of the tension caused by 80 mM KCl. 3.3. Effects of ET receptor antagonists on the ET-1-induced common bile duct muscle contraction To further characterize the ET receptors mediating the common bile duct muscle contraction, the abilities of two

potent ET receptor antagonists, BQ-123 (ETA receptor selective antagonist) and BQ-788 (ETB receptor selective antagonist), to inhibit ET-1 induced contraction were determined. BQ-123 (10 AM) or BQ-788 (1 AM) alone did not cause contraction. BQ-788 (1 AM) did not significantly affect the ET-1 induced contraction (EC50 of ET-1 alone = 5.6 F 1.1 nM; EC50 of ET-1 plus 1 AM BQ-788 = 7.3 F 1.2 nM; p>0.05; Fig. 3). On the other hand, BQ-123 (10 AM) dramatically inhibited ET-1 induced contraction. As shown in Fig. 3, BQ-123 (10 AM) shifted the ET-1 concentration– response curve to the right, with >50-fold change at the EC50 level for the guinea pig common bile duct (EC50 of ET-1 alone = 5.6 F 1.1 nM; EC50 of ET-1 plus 10 AM BQ-123>300 nM). The combination of both antagonists, BQ-123 (10 AM) and BQ-788 (1 AM), inhibited ET-1-induced contraction but did not potentiate the inhibition caused by BQ-123 (10 AM) alone (EC50 of ET-1 plus 10 AM BQ-123 and 1 AM BQ788>300 nM; Fig. 3). 3.4. Autoradiographic studies Autoradiographic studies were performed to localize binding sites for 125I-ET-1 in the common bile duct tissue sections. The autoradiographs of the sections after incubation with 125I-ET-1 alone or 125I-ET-1 plus ET-1, 1 AM, were compared with the histology of adjacent sections after staining with hematoxylin and eosin. Fig. 4A shows the stained histological section of the common bile duct containing, from left to right, the mucosa, submucosa, smooth muscle layer and serosa. Comparing the autoradiograph of the section after incubation with 125I-ET-1 alone (Fig. 4B) with the histological section (Fig. 4A) demonstrates that binding of 125I-ET-1 is localized to the smooth muscle layer, including the longitudinal and circular muscle. Adding 1 AM ET-1 caused a complete reduction

Table 1 Abilities of ET receptor agonists to cause contraction of longitudinal muscle strips and inhibit binding of 125I-ET-1 to muscle cell membranes prepared from guinea pig common bile ducts Peptide Contraction Binding of EC50 (nM) IC50 (pM) ET-1 ET-2 ET-3 SX6c

3.5 F 0.7 2.8 F 1.0 59 F 3* –a

125

I-ET-1

Site 2 (ETB Site 1 (ETA receptor) Kd (pM) receptor) Kd (pM)

45 F 7 5.4 F 2.2 56 F 6 5.2 F 1.3 240 F 80* 2200 F 1100* 3100 F 800* 120,000 F 50,000*

5.4 F 2.2 5.2 F 1.3 8.7 F 2.5 140 F 30*

Values of EC50 and IC50 (means F S.E.M.) are derived from original data of at least four experiments. Dose – inhibition curves in Fig. 5 were analyzed using a nonlinear least-squares curve-fitting program (LIGAND) [25]. 125IET-1 binding was best fit by a model with two binding sites, Site 1 and Site 2. One class of binding sites, Site 1 (ETA receptor), had a high affinity for ET-1 and ET-2 but a lower affinity for ET-3. In contrast, the other binding site, Site 2 (ETB receptor), had similar high affinities for ET-1, ET-2 and ET-3. Site 2 (ETB receptor), with a Bmax of 22 F 2 fmol/mg protein, was present four times in numbers to Site 1 (ETA receptor) with a Bmax of 5.2 F 0.9 fmol/mg protein. * P < 0.05 compared with ET-1. a SX6c, 3 nM to 1 AM, did not cause significant contraction.

Fig. 3. Ability of ET receptor antagonists BQ-123 and BQ-788 to inhibit ET-1 induced contraction of muscle strips from guinea pig common bile ducts. Values are expressed as percent of KCl (80 mM) induced contraction. Results given are from at least four experiments. Vertical bars represent F S.E.M.

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3.5. 125I-ET-1 binding to common bile duct muscle cell membranes To determine affinities of ET related peptides for ET receptors in the guinea pig common bile duct, we studied the ability of ET-1, ET-2, ET-3 and SX6c to inhibit binding of 125I-ET-1 to common bile duct cell membranes. In common bile duct muscle cell membranes, binding of 125 I-ET-1 reached maximal at 60 min and remained constant for additional 60 min. To examine the specificity of binding of 125I-ET-1 to muscle cell membranes, various peptides were tested for their abilities to inhibit binding of 125I-ET-1. Cholecystokinin-8 (CCK-8), gastrin-I, substance P, neurokinin A, bombesin and peptide YY, 1 AM each, did not alter binding of 125I-ET-1 (data not shown). 3.6. Saturation binding studies The specific binding of 125I-ET-1 (5 to 320 pM) was saturable in the cell membranes of the common bile duct (data not shown). Scatchard analysis using the nonlinear, least-squares curve fitting program (LIGAND) demonstrated a single class of binding sites with high affinity (Kd = 7.9 F 3.2 pM) and a Bmax of 20 F 1 fmol/mg protein. 3.7. Competition binding studies ET-1, ET-2, ET-3 and SX6c dose-dependently inhibited the 125I-ET-1 binding to cell membranes of the common bile duct (Fig. 5). ET-1 caused detectable inhibition at 3 pM, half-maximal inhibition at 45 F 7 pM and maximal inhibition at 100 nM. The relative potencies for inhibiting 125IET-1 binding were ET-1 = ET-2 (IC50 = 56 F 6 pM)>ET-3 (IC50 = 240 F 80 pM; p < 0.05, compared with ET-1)>SX6c

Fig. 4. Photomicrograph of a representative tissue section of the guinea pig common bile duct stained with hematoxylin-eosin and corresponding autoradiographs for binding of 125I-ET-1 with or without 1 AM ET-1. (A) Tissue section stained with hematoxylin-eosin. (B) Autoradiograph of section adjacent to that in A showing binding of 125I-ET-1 (silver grains) after incubation with 40 pM 125I-ET-1 for 1 h. (C) Autoradiograph of section adjacent to that in (B), showing binding of 125I-ET-1 with 1 AM ET-1. Arrows indicate smooth muscle layer in each panel.

of silver grains, demonstrating a high degree of specific binding to ET receptors (Fig. 4C). No specific binding of 125 I-ET-1 was observed in the mucosa, submucosa or serosa.

Fig. 5. Ability of ET-1, ET-2 and ET-3 to inhibit binding of 125I-ET-1 to cell membranes of guinea pig common bile duct muscle. Cell membranes were incubated for 60 min at 22 jC with 20 pM 125I-ET-1 plus indicated concentrations of peptides. Specific binding of 125I-ET-1 is expressed as the percentage of radioactivity bound in the absence of added peptides. Results given are from at least four experiments. Vertical bars represent F S.E.M.

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(IC50 = 3.1 F 0.8 nM; p < 0.05, compared with ET-1). These dose – inhibition data were analyzed using the nonlinear least-squares curve-fitting program (LIGAND). The dose – inhibition data with ET-1 and ET-2 fit best with a one-site model. However, when the dose – inhibition data with ET-1 and ET-3 were analyzed together by the least-squares curvefitting program, the data were best fit by a model with two binding sites, Site 1 and Site 2 ( p < 0.05, compared with one-site). These two binding sites had same affinities for ET-1 and ET-2 but different affinities for ET-3 (Table 1). Specifically, one class of binding sites, Site 1, had high affinity for ET-1 (dissociation constant, Kd = 5.4 F 2.2 pM) and ET-2 (Kd = 5.2 F 1.3 pM) but a 410 times lower affinity for ET-3 (Kd = 2.2 F 1.1 nM), similar to that described in other tissues for the ETA receptor [3,5,7,15]. In contrast, the other binding site, Site 2, had similar high affinities for ET-1 (Kd = 5.4 F 2.2 pM), ET-2 (Kd = 5.2 F 1.3 pM) and ET-3 (Kd = 8.7 F 2.5 pM) and thus resembled the ETB receptor described in other tissues [3,5,7,15]. Site 2 was present four times in numbers to Site 1 (22 F 2 vs. 5.2 F 0.9 fmol/mg protein). The dose– inhibition data with SX6c were best fit by a model with two binding sites, too. For SX6c, Site 2 had a 860-fold higher affinity (Kd = 0.14 F 0.03 nM) than Site 1 (Kd = 120 F 50 nM). To further characterize binding to ETA and ETB receptors, dose – inhibition curves for ET receptor antagonists, BQ-123 and BQ-788, were performed for binding of 125IET-1 to the cell membranes. Each antagonist showed a broad dose –inhibition curve. BQ-123 caused half-maximal inhibition at >10 AM and BQ-788, 3.3 F 1.4 nM (Fig. 6). Computer analysis of the BQ-788 dose – inhibition

Fig. 6. Ability of ET receptor antagonists BQ-123 and BQ-788 to inhibit binding of 125I-ET-1 to cell membranes of guinea pig common bile duct muscle. Cell membranes were incubated for 60 min at 22 jC with 20 pM 125 I-ET-1 plus indicated concentrations of peptides. Specific binding of 125 I-ET-1 is expressed as the percentage of radioactivity bound in the absence of added peptides. Results given are from at least four experiments. Vertical bars represent F S.E.M.

curve demonstrated two binding sites, Site 1 and Site 2. Site 1 had a Kd = 34 F 23 nM, whereas Site 2 had a Kd = 0.13 F 0.04 nM for BQ-788. Computer analysis of the dose – inhibition curve of BQ-123 was not performed because BQ-123, at the highest concentration tested, inhibited only 50% of the binding of 125I-ET-1 to the cell membranes.

4. Discussion Previous studies have shown that functional ETA and ETB receptors mediate contraction in the guinea pig and human gallbladder and ET induces contraction of circular muscle of the guinea pig common bile duct [12 – 15]. The present study demonstrates that ET causes contraction of longitudinal muscle of the guinea pig common bile duct and that the common bile duct muscle possesses two classes of ET receptors, ETA and ETB, but only the ETA receptor mediates contraction. In the present study, the ability of ET to cause contraction of guinea pig common bile duct strips was not influenced by tetrodotoxin or atropine. These indicate that ET interacts with receptors on the smooth muscle. 125I-ET-1 binding was localized by autoradiography to the smooth muscle layer of the guinea pig common bile ducts. These results demonstrate that most of the ET receptors are on the smooth muscle cells of the common bile duct. ET-1 was equipotent to ET-2 but more potent than ET-3 in causing contraction of the common bile duct muscle strips, demonstrating the presence of functional ETA receptors, which have high affinity for ET-1 and ET-2 but low affinity for ET-3. In addition, SX6c, a selective ETB receptor agonist, caused only a negligible contraction. Furthermore, BQ-123, an ETA-receptor-selective antagonist, inhibited the ET-1-induced contraction, but BQ-788, an ETB-receptorselective antagonist, did not. The combination of both antagonists, BQ-123 and BQ-788, did not show synergistic inhibition. These observations indicate that ET-1 interacts only with ETA receptors in the guinea pig common bile duct to cause the contraction. The present study demonstrates that 125I-ET-1 binds to ET receptors on guinea pig common bile duct muscle cell membranes. Binding of 125I-ET-1 was time-dependent, saturable and specific. The fact that two different classes of ET receptors are present on guinea pig common bile duct muscle is confirmed by several findings. Binding of 125I-ET1 is inhibited by ET-1 in a narrow dose – inhibition curve but by ET-3 in a broad curve, spanning more than 3 log units, with biphasic configuration. When the abilities of ET-1 and ET-3 to inhibit binding of 125I-ET-1 are analyzed together by a least-squares curve-fitting program, the data are best fitted by a two-binding site model. One class (ETA receptor) has a high affinity for ET-1 and a 410 times lower affinity for ET3. The other class (ETB receptor) has equally high affinity for ET-1 and ET-3. To further confirm this conclusion, we

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analyzed the broad and biphasic dose – inhibition curve of 125 I-ET-1 by SX6c and BQ-788. Again, the data are best fitted with a two-binding-site model. Both ETA and ETB receptors are present in the smooth muscle of the common bile duct, gallbladder, esophagus and stomach. In the gallbladder and esophagus, both ETA and ETB receptors cooperate to mediate contraction, but in the common bile duct and stomach, only ETA receptors mediate contraction [4,5,7,13 –15]. In the common bile duct, SX6c did not alter carbachol-induced contractions, indicating that ETB receptors do not mediate relaxation; in the gastric smooth muscle, ETB receptors do not mediate relaxation either [7]. At the present time, the function of ETB receptors in the smooth muscle of the stomach or common bile duct is unclear. In some other cell systems, ETB receptors are involved in stimulation of cell growth or removing ET-1 from the circulation [2,18]. In the common bile duct, CCK receptors, muscarinic M3 receptors and tachykinin NK1, NK2 as well as NK3 receptors mediating contraction have been described [22,26,27]. This study demonstrates ETA receptors in the common bile duct mediating contraction. Previous reports show that gallbladder epithelial cells produce ET-1 and ligation of the common bile duct induces increase of ET-1 levels in the bile and blood [19,21]. It is likely that during obstruction of the biliary tract, much ET is produced to contract the bile duct muscle and overcome the obstruction. In conclusion, these results demonstrate that different from the gallbladder, which possesses both ETA and ETB receptors cooperating to mediate muscle contraction, the common bile duct possesses two classes of ET receptors but only the ETA receptor mediates contraction.

Acknowledgements This study was supported by National Science Council of Taiwan (NSC grant 89-2315-B-303-001) and Tzu Chi General Hospital. The author thanks Keng-Hsien Kuo, Chia-Yi Wang, Yu-Jen Chen, Yu-Jen Yeh, Ling-Jung Chiu and Cheng-Tsiu Yang for their excellent technical assistance.

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