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 (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 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.