Antagonistic affinity profile of imperialine on different subtypes of muscarinic receptors

Abstract

In order to identify the receptor subtype responsible for acetylcholine (ACh)-induced relaxation of bovine coronary artery, we determined the affinity of six subtype-selective muscarinic antagonists and compared them with affinity estimates obtained for bovine left atria. At low concentrations, ACh potently relaxed circular strips of coronary artery with endothelium (EC50 0.15 μM), but contracted them at higher agonist concentrations with potencies that depended on the presence or absence of endothelium: EC50 1.8 μM (without endothelium); 4.6 JμM (with endothelium). The pA2 values obtained for antagonism of relaxant responses to ACh were: pirenzepine (M1-selective) 7.38 ± 0.12; AF-DX 116 (11-[2-(diethylamino-methyl)-1-piperidinylacetyl]-5, 11-dihydro-6H-pyrido(2,3-b)l,4-benzodiazepine-6-one; M2-selective) 5.79 ± 0.09; and 4-diphenylacetoxy-N-methylpiperidine-methobromide (4-DAMP; M3/M1-selective) 9.07 ± 0.12. The corresponding Schild slopes were 0.98 ± 0.07 for pirenzepine, 1.17 ± 0.09 for AF-DX 116 and 1.01 ± 0.04 for 4-DAMP. For the following three antagonists, pKg values were determined at two different antagonist concentrations: dicyclomine (M1-selective) 7.49 ± 0.10, cyclohexylphenyl-(2-piperidinoethyl)-silanol (CPPS; M3-selective) 8.0 ± 0.10, and parafluoro-hexahydrosila-difenidol (pFHHSiD; M3-selective) 7.87 ± 0.10. For comparison, the antagonism of methacholine-induced negative inotropy in left atria was determined for three antagonists, yielding the following pA2 values: pirenzepine 5.98 ± 0.14; AF-DX 116 6.81 ± 0.14 and 4-DAMP 7.99 ± 0.14. The slopes of the corresponding Schild plots were 1.05 ± 0.10,1.14 ± 0.12 and 1.08 ± 0.08, respectively. The results indicate that ACh relaxes precontracted circular strips of bovine coronary arteries with intact endothelium by activation of muscarinic receptors most closely corresponding to the M3 subtype.