Direct binding and functional studies on muscarinic cholinoceptors in porcine coronary artery

Abstract

The muscarinic cholinoceptors in porcine coronary artery were identified and characterized by a binding assay using (-)-[3H]quinuclidinyl benzilate (QNB) and also by pharmacological method. Specific (-)-[3H]QNB binding in the coronary artery was saturable and of high affinity (K(d) = 0.08 nM), and it showed a pharmacological specificity as well as stereoselectivity which characterized muscarinic receptors. Muscarinic antagonists completed with the (-)-[3H]QNB binding in order: nonlabeled QNB > dexetimide > atropine > pirenzepine > AF-DX 116 > levetimide > gallamine. Dexetimide was approximately 2000 times as potent as levetimide. The potencies (pK(i)) of these muscarinic antagonists in competing for (-)-[3H]QNB binding sites in porcine coronary artery correlated well with their pharmacological potencies (pA2 for antagonistic effect of acetylcholine-induced contraction of coronary artery). The decrease in the (-)-[3H]QNB binding by atropine and pirenzepine was due to a reduction in the apparent affinity with little change in the number of maximal binding sites, suggesting a competitive antagonism. Specific (-)-[3H]QNB binding (K(d) and maximal number of binding sites) in porcine coronary artery was not changed by the removal of endothelium. We conclude: 1) (-)-[3H]QNBselectively labels the physiologically relevant muscarinic receptors in porcine coronary artery and 2) the majority of these receptors is localized on vascular smooth muscles and the receptors mediate the acetylcholine-induced contractile response of coronary artery.