Functional and Molecular Characterization of -Adrenoceptors in the Internal Anal Sphincter

The purpose of the present study was to characterize different -adrenoceptors ( -ARs) and determine their role in the spontaneously tonic smooth muscle of the internal anal sphincter (IAS). The -AR subtypes in the opossum IAS were investigated by functional in vitro, radioligand binding, Western blot, and reverse transcription-polymerase chain reaction (RT-PCR) studies. ZD 7114 [(S)-4-[2-hydroxy-3-phenoxypropylaminoethoxy]-N-(2methoxyethyl)phenoxyacetamide], a selective 3-AR agonist, caused a potent and concentration-dependent relaxation of the IAS smooth muscle that was antagonized by the 3-AR antagonist SR 59230A [1-(2-ethylphenoxy)-3-[[(1S)-1,2,3,4-tetrahydro-1naphthalenyl]amino]-(2S)-2-propanol hydrochloride]. Conversely, the IAS smooth muscle relaxation caused by 1and 2-AR agonists (xamoterol and procaterol, respectively) was selectively antagonized by their respective antagonists CGP 20712 [( )-2hydroxy-5-[2-[[2-hydroxy-3-[4-[1-methyl-4-(trifluoromethyl)-1Himidazol-2-yl]phenoxy]propyl]amino]ethoxy]-benzamide methanesulfonate salt] and ICI 118551. Saturation binding of [I]iodocyanopindolol to -AR subtypes revealed the presence of a high-affinity site (Kd1 96.4 8.7 pM; Bmax1 12.5 0.6 fmol/mg protein) and a low-affinity site (Kd2 1.96 1.7 nM; Bmax2 58.7 4.3 fmol/mg protein). Competition binding with selective -AR antagonists revealed that the high-affinity site correspond to 1/ 2-AR and the low affinity site to 3-AR. Receptor binding data suggest the predominant presence of 3-AR over 1/ 2-AR. Western blot studies identified 1-, 2-, and 3-AR subtypes. The presence of 1-, 2-, and 3-ARs was further demonstrated by mRNA analysis using RT-PCR. The studies demonstrate a comprehensive functional and molecular characterization of 1-, 2-, and 3-ARs in IAS smooth muscle. These studies may have important implications in anorectal and other gastrointestinal motility disorders. It is well known that postjunctional -adrenoceptors ( ARs) mediate the inhibitory effects of sympathetic nerve stimulation in different smooth muscles including those of the gastrointestinal tract (Manara et al., 1995b; Gauthier et al., 2000). The intestinal -AR was originally described as a 1and 2-AR (Lands et al., 1967). Further studies with gastrointestinal preparations from several species established the relaxant effect of classical -AR ( 1 and 2) agonists (Bennett, 1965; Hedges and Turner, 1969; De Ponti et al., 1996a). Subsequently, studies investigating -ARs in gastrointestinal smooth muscle from several species demonstrated relaxation responses that were resistant to propranolol and displayed lower affinity to other conventional -AR antagonists (Arch and Kaumann, 1993; Goldberg and Frishman, 1995; Strosberg, 1997; Manara et al., 2000). This finding, along with the emergence of a new class of -AR agonists described first in adipocytes (Feve et al., 1991), suggested the presence of an “atypical” class of -ARs. In 1989, Emorine et al. (1989) cloned and sequenced 3-AR and found that it shared the pharmacological characteristics of the atypical -AR. The 3-AR has been found in a variety of mammalian tissues (Berkowitz et al., 1995) including white and brown adipocytes (Muzzin et al., 1991), trachea (Webber and Stock, The studies were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-35385 and an institutional grant from Thomas Jefferson University, Philadelphia, Pennsylvania. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. DOI: 10.1124/jpet.102.048462. ABBREVIATIONS: -AR, –adrenergic receptor; GI, gastrointestinal; IAS, internal anal sphincter; RT-PCR, reverse transcription-polymerase chain reaction; CRC, concentration-response curve; ECmax, concentration causing maximal relaxation; EC50, concentration causing 50% of maximal relaxation; ZD 7114 hydrochloride, (S)-4-[2-hydroxy-3-phenoxypropylaminoethoxy]-N-(2-methoxyethyl)phenoxyacetamide); CGP 20712A methanesulfonate salt, ( )-2-hydroxy-5-[2-[[2-hydroxy-3-[4-[1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl]phenoxy]propyl]amino]ethoxy]-benzamide methanesulfonate salt; ICI 118,551 hydrochloride, ( )-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol; SR 59230A hydrochloride, 1-(2-ethylphenoxy)-3-[[(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino]-(2S)-2-propanol hydrochloride; NCM, nitrocellulose membrane; [I]CYP, [I]iodocyanopindolol; DMSO, dimethyl sulfoxide; bp, base pair; CHO, Chinese hamster ovary; CL 316,243, 5-[2-(R)-2([(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]amino)popyl]-1,3-benzodioxole-2,2-dicarboxylate. 0022-3565/03/3052-615–624$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 305, No. 2 Copyright © 2003 by The American Society for Pharmacology and Experimental Therapeutics 48462/1061453 JPET 305:615–624, 2003 Printed in U.S.A. 615 at A PE T Jornals on A ril 9, 2017 jpet.asjournals.org D ow nladed from 1992), heart (Kaumann and Molenaar, 1996; Gauthier et al., 2000), gastrointestinal tract (De Ponti et al., 1995; Bardou et al., 1998), and urinary tract (Tomiyama et al., 1998). In the GI tract, recent studies have focused on the ability of 3-AR specific agonists to cause relaxation in a number of different smooth muscle tissues including rat ileum, jejunum, colon, guinea pig ileum, and duodenum (Manara et al., 1995b). One of the problems in delineating the pharmacology of -ARs in the gastrointestinal tract has been the lack of subtype-selective agonists and antagonists, especially those for 3-AR. Recent in vivo studies have demonstrated the selective, potent, and prolonged relaxant effect of CL 316,243, a selective 3-AR agonist, on the sphincteric smooth muscles of the opossum lower esophageal sphincter (DiMarino et al., 2002), without the significant systemic cardiovascular side effects that are associated with 1and 2-AR agonists. In the past few years, 3-agonists have emerged as potential therapeutic agents for several gastrointestinal motility disorders including irritable bowel syndrome (Scarpignato and Pelosini, 1999). Anorectal dysfunctions such as Hirschsprung’s disease, constipation, anal fissures, and hemorrhoids may also be associated with either hypertensive IAS or failure of sphincteric relaxation in response to the rectoanal inhibitory reflex (Azpiroz and Whitehead, 2002). Characterization of neurohumoral receptors that mediate selective, potent, and prolonged relaxation of IAS and other GI smooth muscles without untoward systemic effects will be of considerable interest in the treatment of anorectal and other GI motility disorders. The present investigation was carried out to characterize -AR in the gastrointestinal tonic smooth muscle of the IAS by comprehensive studies using a combination of classical pharmacology, receptor binding, and molecular biology approaches. The aim of the present study is to determine the presence of and characterize the -AR subtypes involved in mediating relaxation of the IAS smooth muscle. We used selective agonists and antagonists to determine the receptor binding profiles of each -AR subtype. The presence of membrane bound -AR and mRNA encoding for the three -AR subtypes was determined through Western blot studies and reverse transcription-polymerase chain reaction (RT-PCR) analysis, respectively. Materials and Methods Preparation of Smooth Muscle Strips. Adult male opossums (Didelphis virginiana) weighing 2.5 to 3.5 kg were anesthetized with sodium pentobarbital (50 mg/kg i.p.). Laparotomy was performed, and a part of the rectum along with the anal canal was removed using sharp dissection. The IAS was identified by manometry as high-pressure zone and marked by means of sutures in situ. The animals were sacrificed by exsanguinations; the anorectal region was then dissected out and transferred immediately to oxygenated (95% O2 5% CO2) Krebs’ physiological solution of the following composition: 118.07 mM NaCl, 4.69 mM KCl, 2.52 mM CaCl2, 1.16 mM MgSO4, 1.01 mM NaH2PO4, 25 mM NaHCO3, and 11.10 mM glucose. A longitudinal incision along the length of isolated anorectal region was made, and the tissue was pinned flat in a Sylgard (Dow Corning Corp., Midland, MI)-coated Petri dish. Once the lumen was fully exposed, the mucosa and submucosa were removed carefully by sharp dissection. The tissue was then turned on the serosal side, and all extraneous tissue including the outer longitudinal muscle was removed. Circular smooth muscle strips of the IAS (approximately 1 10 mm) were prepared and tied on either end using 3-0 silk suture in preparation for measurement of isometric tension. The experimental protocol of the study was approved by the Institutional Animal Care and Use Committee of Thomas Jefferson University in accordance with the recommendations of the American Association for the Accreditation of Laboratory Animal Care. Measurement of Isometric Tension. The smooth muscle strips were transferred to 2-ml muscle baths (Radnoti Glass Technology, Inc., Monrovia, CA) containing oxygenated Krebs’ solution at 35°C. One end of the muscle strip was anchored at the bottom of the muscle bath while the other end was connected to a force transducer (model FT03; Grass Instruments, Quincy, MA). Isometric tension was recorded by the PowerLab/8SP data acquisition system using Chart 4.1.2 (ADInstruments, Grand Junction, CO). Each smooth muscle strip was initially stretched to a tension of 0.7 g. The muscle strips were then given at least an hour to equilibrate, during which time they were washed with Krebs’ solution every 15 min. Only smooth muscle strips that developed spontaneous tone and responded to electrical field stimulation were used in this study. The changes in tension from various drugs were expressed as the percent maximal relaxation achieved by 50 mM EGTA, at the end of each experiment. Each smooth muscle served as its own control. Drug Responses. To determine the concentration-response curves (CRCs) with 1-, 2-, and 3-AR agonists on the basal tone of the IAS smooth muscles, xamoterol, procaterol, and ZD 7114 [(S)-4-[2-hydroxy3-phenoxypropylaminoethoxy]-N-(2-methoxyethyl)phenoxyacetamide], respectively, were added to the muscle bath in cumulative concentrations (Rattan and Moummi, 1989). Successive concentrations of the agonists were not added until the response of the previous concentration stabilized. When no effect was observed, 10 minutes were allowed between additions of different concentrations. In preliminary studies, when a single concentration was used, we noted that this was an appropriate time to gauge the maximal effect of a given concentration of the agonist. No difference in the results occurred with longer exposures. To determine the effects of 1-, 2-, and 3-AR antagonists, CGP 20712A [( )-2-hydroxy-5-[2-[[2-hydroxy-3-[4-[1-methyl-4-(trifluoromethyl)-1Himidazol-2-yl]phenoxy]propyl]amino]ethoxy]-benzamide methanesulfonate salt], ICI 118551, and SR 59230A [1-(2-ethylphenoxy)-3-[[(1S)1,2,3,4-tetrahydro-1-naphthalenyl]amino]-(2S)-2-propanol hydrochloride], respectively (in concentrations ranging from 1 10 8 to 1 10 6 M), were added 30 min before obtaining the CRC of the test agonist. -Adrenoceptor ( -AR) Analysis by Western Blot. Western blot analysis of 1-, 2-, and 3-AR in the IAS and rectum of the opossum was performed according to the protocol of Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Circular smooth muscles tissues of the IAS and rectum were cut into small pieces (2 2 mm cubes) and rapidly homogenized in 3 ml of boiling lysis buffer (1% SDS, 1.0 mM sodium orthovanadate, 10 mM Tris, pH 7.4) and then put into the microwave for 10 s. The homogenates were centrifuged (16,000g, 4°C) for 15 min. The pellet obtained was dissolved in Krebs’ buffer (composition already described) containing 1 mM EDTA, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride (combined pH of 7.6). The protein contents were determined by the method described by Lowry et al. (1951) using bovine serum albumin