Evidence for muscarinic cholinergic receptors in dog portal vein: Binding of [ 3H] quinuclidinyl benzilate

Abstract

Muscarinic cholinergic receptor sites in dog portal veins were analyzed directly using [ 3H] quinuclidinyl benzilate (QNB) as a ligand. Specific [ 3H] QNB binding to crude membrane preparations from the isolated veins was saturable, reversible and of high affinity (K D = 15.5 ± 2.8 pM) with a Bmax of 110 ± 14.7 fmol/mg protein. Scatchard and Hill plot analyses of the data indicated one class of binding sites. From kinetic analysis of the data, association and dissociation rate constants of 1.91 × 10 9 M −1 min −1 and 0.016 min −1, respectively, were calculated. The dissociation constant calculated from the equation K D = K −1/K +1 was 8.3 pM, such being in good agreement with the Scatchard estimate of K D (15.5 pM). Specific binding of [ 3H] QNB was displaced by muscarinic agents. Nicotinic cholinergic agents, α-bungarotoxin, nicotine and hexamethonium, were ineffective in displacing [ 3H] QNB binding at 10 μM. Our findings provide direct evidence for the existence of muscarinic cholinergic receptors in dog portal veins.