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.
Footnotes
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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.
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DOI: 10.1124/jpet.102.048462
- Abbreviations:
- β-AR
- β–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
- [125I]CYP
- [125I]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
- Received December 20, 2002.
- Accepted February 4, 2003.
- The American Society for Pharmacology and Experimental Therapeutics
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