[3H]Pirenzepine and (-)-[3H]quinuclidinyl benzilate binding to rat cerebral cortical and cardiac muscarinic cholinergic sites. II. Characterization and regulation of antagonist binding to putative muscarinic subtypes

M. Watson, W. R. Roeske, H. I. Yamamura

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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 μM) showed little effect on K(d) (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 K(d) 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°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 (n(H) 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 n(H) < 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.

Original languageEnglish (US)
Pages (from-to)419-427
Number of pages9
JournalJournal of Pharmacology and Experimental Therapeutics
Issue number2
Publication statusPublished - 1986
Externally publishedYes


ASJC Scopus subject areas

  • Pharmacology

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