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

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Abstract
The purpose of the present study was to characterize different β-adrenoceptors (β-ARs) and determine their role in the spontaneously tonic smooth muscle of the internal anal sphincter (IAS). The β-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 β3-AR agonist, caused a potent and concentration-dependent relaxation of the IAS smooth muscle that was antagonized by the β3-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 β1- and β2-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 [125I]iodocyanopindolol to β-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 β-AR antagonists revealed that the high-affinity site correspond to β1/β2-AR and the low affinity site to β3-AR. Receptor binding data suggest the predominant presence of β3-AR over β1/β2-AR. Western blot studies identified β1-, β2-, and β3-AR subtypes. The presence of β1-, β2-, and β3-ARs was further demonstrated by mRNA analysis using RT-PCR. The studies demonstrate a comprehensive functional and molecular characterization of β1-, β2-, and β3-ARs in IAS smooth muscle. These studies may have important implications in anorectal and other gastrointestinal motility disorders.