Characterization of the muscarinic receptor subtype that mediates the contractile response of acetylcholine in the swine myometrium

The aim of the present study was to characterize the subtype of muscarinic receptor that mediates acetylcholine-induced contractions in the nonpregnant proestrus swine myometrium by means of mechanical, radioligand ([H-3]quinuclidinyl benzilate) binding and biochemical (measurement of cyclic AMP) approaches. Acetylcholine (- logEC(50), 6.12), oxotremorine-methiodide (6.47), methacholine (6.35), carbachol (6.18) and muscarine (6.33) caused contractile responses of the uterine circular muscle, with a similar maximum amplitude, but pilocarpine and McN-A-343 (4-(m-chlorophenyl-carbamoyloxy)-2-butynyltrimethylammonium) were ineffective in causing contraction. The contractile response to acetylcholine was antagonized by the following muscarinic receptor antagonists in a competitive manner (with pA(2) values in parentheses): atropine (8.95), 4-diphenylacetoxy-N-methylpiperidine (4-DAMP, 8.83), tropicamide (7.07), himbacine (7.01), pirenzepine (6.42) and 11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5, 11-dihydro-6H-pyrido[2,3b][1,4]benzodiazepin-6-one (AF-DX116, 5.96). Electrical field stimulation (10 Hz) caused tetrodotoxin- and atropine-sensitive contractions in the circular muscle. All muscarinic receptor antagonists decreased the electrical field stimulation-induced contraction in a concentration-dependent manner. The order of inhibition (-logIC(50)) was 4-DAMP (8.35)> tropicamide (6.72)> himbacine (6.54)> pirenzepine (6.31)> AF-DX116 (6.13). Acetylcholine did not affect the cytoplasmic cyclic AMP level, regardless of the presence or absence of forskolin, suggesting the absence of functional muscarinic M-2, and/or Mg receptors in the swine myometrium. The receptor binding study indicated that circular muscle layers of the swine myometrium contained a single class of [3H]quinuclidinyl benzilate binding site (K-d = 0.92 nM; B-max = 126.6 fmol/mg protein). Specific binding was displaced by muscarinic receptor antagonists in the following order (with pK(i) value and Hill coefficient in parentheses): atropine (8.22 and 0.93)> 4-DAMP (8.18 and 0.94)> tropicamide (6.78 and 0.93)> pirenzepine (5.46 and 0.92)> AF-DX116 (5.12 and 0.94). The present results suggest that in circular muscle layers of the swine myometrium, exogenous and endogenous acetylcholine cause contraction through activation of muscarinic M-3, receptors present on smooth muscle cells. (C) 1999 Elsevier Science B.V. All rights reserved.