title: Functional and molecular characterization of beta-adrenoceptors in the internal anal sphincter.
abstract: The purpose of the present study was to characterize different beta-adrenoceptors (beta-ARs) and determine their role in the spontaneously tonic smooth muscle of the internal anal sphincter (IAS). The beta-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-(2-methoxyethyl)phenoxyacetamide], a selective beta(3)-AR agonist, caused a potent and concentration-dependent relaxation of the IAS smooth muscle that was antagonized by the beta(3)-AR antagonist SR 59230A [1-(2-ethylphenoxy)-3-[[(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino]-(2S)-2-propanol hydrochloride]. Conversely, the IAS smooth muscle relaxation caused by beta(1)- and beta(2)-AR agonists (xamoterol and procaterol, respectively) was selectively antagonized by their respective antagonists CGP 20712 [(+/-)-2-hydroxy-5-[2-[[2-hydroxy-3-[4-[1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl]phenoxy]propyl]amino]ethoxy]-benzamide methanesulfonate salt] and ICI 118551. Saturation binding of [(125)I]iodocyanopindolol to beta-AR subtypes revealed the presence of a high-affinity site (K(d1) = 96.4 +/- 8.7 pM; B(max1) = 12.5 +/- 0.6 fmol/mg protein) and a low-affinity site (K(d2) = 1.96 +/- 1.7 nM; B(max2) = 58.7 +/- 4.3 fmol/mg protein). Competition binding with selective beta-AR antagonists revealed that the high-affinity site correspond to beta(1)/beta(2)-AR and the low affinity site to beta(3)-AR. Receptor binding data suggest the predominant presence of beta(3)-AR over beta(1)/beta(2)-AR. Western blot studies identified beta(1)-, beta(2)-, and beta(3)-AR subtypes. The presence of beta(1)-, beta(2)-, and beta(3)-ARs was further demonstrated by mRNA analysis using RT-PCR. The studies demonstrate a comprehensive functional and molecular characterization of beta(1)-, beta(2)-, and beta(3)-ARs in IAS smooth muscle. These studies may have important implications in anorectal and other gastrointestinal motility disorders.
authors.affiliation: Department of Medicine, Division of Gastroenterology and Hepatology, Jefferson Medical College, Thomas Jefferson University, 1025 Walnut Street, Room no. 901 College, Philadelphia, PA 19107, USA.
authors.forename: Sandeep
authors.initials: S
authors.lastname: Rathi
authors.forename: Shiva
authors.initials: S
authors.lastname: Kazerounian
authors.forename: Kuldip
authors.initials: K
authors.lastname: Banwait
authors.forename: Stephanie
authors.initials: S
authors.lastname: Schulz
authors.forename: Scott A
authors.initials: SA
authors.lastname: Waldman
authors.forename: Satish
authors.initials: S
authors.lastname: Rattan
mesh_terms: D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000319:Adrenergic beta-Antagonists; D001003:Anal Canal / Q000737:chemistry* / Q000187:drug effects*; D000818:Animals; D015153:Blotting, Western; D066298:In Vitro Techniques; D020120:Iodocyanopindolol; D007537:Isometric Contraction / Q000187:drug effects; D008297:Male; D008566:Membranes / Q000378:metabolism; D009119:Muscle Contraction / Q000187:drug effects; D009126:Muscle Relaxation / Q000187:drug effects; D009130:Muscle, Smooth / Q000187:drug effects; D009893:Opossums / Q000502:physiology*; D010642:Phenoxyacetates / Q000494:pharmacology; D050198:Phenoxypropanolamines; D017265:Procaterol / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis; D011869:Radioligand Assay; D011943:Receptors, Adrenergic, beta / Q000096:biosynthesis / Q000737:chemistry* / Q000187:drug effects*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017307:Xamoterol / Q000494:pharmacology
chemical_list: D000318:Adrenergic beta-Agonists; D000319:Adrenergic beta-Antagonists; D010642:Phenoxyacetates; D050198:Phenoxypropanolamines; D012333:RNA, Messenger; D011943:Receptors, Adrenergic, beta; C071909:ICI D7114; D017307:Xamoterol; D020120:Iodocyanopindolol; D017265:Procaterol