Vol. 305, Issue 2, 615-624, May 2003

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

Sandeep Rathi, Shiva Kazerounian, Kuldip Banwait, Stephanie Schulz, Scott A. Waldman and Satish Rattan

Department of Medicine, Division of Gastroenterology and Hepatology (San.R., K.B., Sat.R.); and Clinical Pharmacology (S.K., S.S., S.A.W.), Jefferson Medical College of Thomas Jefferson University, Philadelphia, Pennsylvania

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-(2-methoxyethyl)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-1-naphthalenyl]amino]-(2S)-2-propanol hydrochloride]. Conversely, the IAS smooth muscle relaxation caused by ₁- and 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 [¹²⁵I]iodocyanopindolol to -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 -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.