Abstract
The purpose of the present study was to ascertain the role of adenylate (AC) versus guanylate cyclase (GC) signaling pathways in the internal anal sphincter (IAS) smooth muscle relaxation by β1-, β2-, and β3-adrenoceptor (AR) activation by xamoterol, procaterol, and disodium 5-[(2R)-2-(3-chlorophenyl)-2-hydroxy-ethyl]amino)propyl]-1,3-benzodioxole-2,2-dicarboxylate (CL 316243), respectively. The above-mentioned agonists produced concentration-dependent relaxation of the smooth muscle strips. Both the selective Gi/oα and Gsα antagonists 8,8′-(carbonylbis(imino-3,1-phenylene))bis-(1,3,5-naphthalene trisulfonic acid) (NF 023) and 4,4′,4″,4‴-(carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid (NF 449), respectively, inhibited the relaxation induced by procaterol. However, only NF 023 inhibited the relaxation induced by xamoterol and CL 316243. 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one, a soluble GC inhibitor, significantly inhibited the relaxation induced by different agonists. In contrast, the selective AC inhibitor [9-(tetrahydro-2′-furyl)adenine] (SQ 22536) inhibited only the relaxation induced by procaterol. (9R,10S,12S)-2,3,9,10,11,12-Hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg: 3′,2′,1′-kl]pyrrolo[3,4-l][1,6]benzodiazocine-10-carboxylic acid, hexyl ester (KT 5720), a cAMP-dependent protein kinase inhibitor, attenuated the relaxation by procaterol, whereas (9S,10R,12R)-2,3,9,10,11,12, hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9.12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-I][1,6]benzodiazocine-10-carboxylic acid methyl ester (KT 5823), a selective cGMP-dependent protein kinase (PKG) inhibitor, attenuated the relaxation induced by xamoterol and CL 316243. Xamoterol produced significant increase in cGMP levels, whereas only procaterol enhanced the cAMP levels. Western blot analysis confirmed the presence of β1, β2, and β3-AR subtypes in the IAS. In summary, β2-AR activates both Gsα and Gi/oα-protein subunits and induces relaxation in the rat IAS via both cAMP/cGMP pathways. In contrast, the β1/β3-ARs activation causes the smooth muscle relaxation via Gi/oα-protein subunit/GC/GMP/PKG pathway. These studies are important for the understanding of intracellular mechanisms underlying IAS smooth muscle relaxation and in turn the pathophysiology of certain anorectal motility disorders.
Footnotes
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The studies were supported by National Institutes of Diabetes and Digestive and Kidney Diseases Grant DK-35385 and an institutional grant from Thomas Jefferson University (Philadelphia, PA).
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DOI: 10.1124/jpet.103.060145.
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ABBREVIATIONS: IAS, internal anal sphincter; β-AR, β-adrenergic receptor; CL 316243, disodium 5-[(2R)-2-(3-chlorophenyl)-2-hydroxy-ethyl] amino)propyl]-1,3-benzodioxole-2,2-dicarboxylate; AC, adenylate cyclase; GC, guanylate cyclase; CRC, concentration response curve; NF 023, 8,8′-(carbonylbis(imino-3,1-phenylene))bis-(1,3,5-naphthalene trisulfonic acid); NF 449, 4,4′,4″,4-‴(carbonylbis(imino-5,1,3-benzenetriylbis-(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; SQ22536, 9-(tetrahydro-2′-furyl)adenine; KT 5720, (9R,10S,12S)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-l] [1,6]benzodiazocine-10-carboxylic acid, hexyl ester; KT 5823, (9S,10R,12R)-2,3,9,10,11,12, hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9.12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-I][1,6]benzodiazocine-10-carboxylic acid methyl ester; SNP, sodium nitroprusside; IBMX, 3-isobutyl-1-methylxanthine; BSA, bovine serum albumin; NCM, nitrocellulose membrane; IOD, integrated optical density; DMSO, dimethyl sulfoxide; ANOVA, analysis of variance; PKA, cAMP-dependent protein kinase; PKG, cGMP-dependent protein kinase; xamoterol, xamoterol hemifumarate; procaterol, (±)-erythro-8-hydroxy-5-[1-hydroxy-2-(isopropylamino) butyl]carbostyril.
- Received September 16, 2003.
- Accepted November 20, 2003.
- The American Society for Pharmacology and Experimental Therapeutics
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