Characterization of muscarinic cholinergic receptors in the lower esophageal sphincter of the cat: Binding of [3H]quinuclidinyl benzilate

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We have studied the binding of the potent muscarinic antagonist [3H]quinuclidinyl benzilate to characterize muscarinic receptors in the lower esophageal sphincter of the cat. Specific binding of [3H]quinuclidinyl benzilate reached equilibrium in 25–30 min at 37 °C, was linear with tissue protein concentration, and was saturable. Estimates of the apparent equilibrium dissociation constant (KD) of 1.1 ± 0.1 nM and maximum receptor density (Bmax) of 147 ± 5.5 fmol/mg protein were determined from Scatchard plots of the binding data. The Hill coefficient for binding was 1.06 ± 0.07, indicating the absence of cooperative interactions. From kinetic analysis of the data association (k1) and dissociation (k2) rate constants of 6.3 × 107 M−1 min−1 and 5.0 × 10−3 min−1, respectively, were calculated. Inhibition of binding was stereoselective, with the (-)-isomer of quinuclidinyl benzilate being 37-fold more potent in inhibiting specific binding than the (+)-isomer. Various muscarinic agents inhibited binding in accordance with their estimated affinities for the muscarinic receptor; nicotinic and noncholinergic drugs had virtually no affinity for [3H]quinuclidinyl benzilate binding sites.In functional studies, atropine and (±)-quinuclidinyl benzilate antagonized bethanechol-induced contraction of the lower esophageal sphincter in accordance with their abilities to inhibit specific binding of [3H]quinuclidinyl benzilate.The regional distribution of specific binding along the length of the esophagus paralleled the distribution of smooth muscle in the tunica muscularis. Binding to duodenal, jejunal, ileal, and nonsphincter esophageal muscle was similar to that in the sphincter, but was fourfold greater to fundic muscle.