[H-3] PIRENZEPINE AND (-)-[H-3]QUINUCLIDINYL BENZILATE BINDING TO RAT CEREBRAL CORTICAL AND CARDIAC MUSCARINIC CHOLINERGIC SITES .2. CHARACTERIZATION AND REGULATION OF ANTAGONIST BINDING TO PUTATIVE MUSCARINIC SUBTYPES

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  • 1 May 1986
    • journal article
    • research article
    • Vol. 237  (2) , 419-427
Abstract
Studies show [3H]PZ identified selectively a subpopulation of muscarinic binding sites compared to classical antagonists like (-)-[3H]QNB in many central and peripheral tissues. We characterized the binding and regulation of selected antagonists to high-affinity [3H]PZ (putative M1) and low-affinity PZ(putative M2) sites in rat cerebral cortex (predominantly M1) and heart (predominantly M2). Saturation isotherms of [3H]PZ and (-)-[3H]QNB were performed under various conditions. Guanyl-5''-yl-imidodiphosphate (30 .mu.M) showed little effect on Kd (dissociation constant) or total binding capacity (total receptor density) values. Higher ionic strength buffers yielded lower affinity values for [3H]PZ and (-)-[3H]QNB. Kinetic studies confirmed high affinity Kd values seen in steady-state assays. We conducted inhibition studies of selected muscarinic antagonists including the reportedly cardioselective (putative M2) drug, AF-DX 116 {11-[(2-(diethylamino)methyl-1-piperidinyl)-acetyl]-5,11-dihydro-6H-pyrido(2,3-b)(1,4)-benzodiazepine-6-one}, the reportedly M1 selective compound, PZ, and the classical antagonist (-)QNB, using [3H]PZ and (-)-[3H]QNB-labeled cerebral cortical and cardiac homogenates. Assays were done with and without guanyl-5''-yl imidophosphate at 25.degree. C in 10 mM Na-K-phosphate, 50 mM Na-K-phosphate and modified Krebs-phosphate buffer. Studies showed antagonists generally had higher affinity in 10 mM Na-K-phosphate buffer, were insensitive to guanyl-5''-yl imidodiphosphate and had Hill values (nH) nearly equal to one. Cardiac PZ/[3H]QNB curves were steep. Whereas cortical PZ/[3H]PZ inhibition curves were also steep, PZ/(-)-[3H]QNB and AF-DX 116/(-)-[3H]QNB curves had nH < 1 and were better fit to a 2-site model. AF-DX 116 had a selectivity profile inverse to PZs. Thus, direct and indirect studies show PZ, unlike (-)-QNB, discriminates high- and low-affinity (putative M1 and M2) sites in each of our assay conditions in these membranes. Many antagonists were M1 selective. Only AF-DX 116 was putative M2 selective. Orders of affinity and selectivity for muscarinic antagonists support the concept of pharmacologically distinct (putative M1 and M2) muscarinic receptor subtypes.