The Binding of [³H]af-dx 384 to Rat Ileal Smooth Muscle Muscarinic Receptors

Abstract

The tritiated cardioselective muscarinic antagonist AF-DX 384 (5, 11-dihydro-11-[[2–(-[8–dipropylamino)methyl]-1-piperidinyl]-ethyl]amino]-carbonyl]-6H-pyrido[2,3–b] [1,4]benzodiazepin-6–one) was used to label muscarinic receptors in the rat ileum. Saturation binding to membrane suspensions revealed a high affinity binding site with K_d of 9.2 nM. The maximal number of binding sites labeled in this tissue (B_max) is 237 fmol/mg protein. The association and dissociation kinetics were well represented by single exponential reactions, and the dissociation constant obtained from the ratio of rate constants was in agreement with that derived from saturation experiments. Specific binding was inhibited by muscarinic antagonists with a rank order of potencies of atropine (pK_i: 8.80) > 4–DAMP (pK_i: 8.23) = AF-DX 384 (pK_i: 8.20) > AF-DX 116 (pK_i: 7.09) = hexahydro-sila-difenidol (pK_i: 6.97) > pirenzepine (pK_i: 6.49) and is consistent with the interaction of [³H]AF-DX 384 with muscarinic receptors of the M₂ subtype. It can be concluded that [³H]AF-DX 384 can be used to selectively label M₂ muscarinic receptors in heterogeneous receptor populations.