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Research ArticleGASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

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

Sandeep Rathi, Shiva Kazerounian, Kuldip Banwait, Stephanie Schulz, Scott A. Waldman and Satish Rattan
Journal of Pharmacology and Experimental Therapeutics May 2003, 305 (2) 615-624; DOI: https://doi.org/10.1124/jpet.102.048462
Sandeep Rathi
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Shiva Kazerounian
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Kuldip Banwait
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Stephanie Schulz
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Scott A. Waldman
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Satish Rattan
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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.

Footnotes

  • 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.

  • 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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Journal of Pharmacology and Experimental Therapeutics: 305 (2)
Journal of Pharmacology and Experimental Therapeutics
Vol. 305, Issue 2
1 May 2003
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Research ArticleGASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

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

Sandeep Rathi, Shiva Kazerounian, Kuldip Banwait, Stephanie Schulz, Scott A. Waldman and Satish Rattan
Journal of Pharmacology and Experimental Therapeutics May 1, 2003, 305 (2) 615-624; DOI: https://doi.org/10.1124/jpet.102.048462

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Research ArticleGASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

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

Sandeep Rathi, Shiva Kazerounian, Kuldip Banwait, Stephanie Schulz, Scott A. Waldman and Satish Rattan
Journal of Pharmacology and Experimental Therapeutics May 1, 2003, 305 (2) 615-624; DOI: https://doi.org/10.1124/jpet.102.048462
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