TY - JOUR
T1 - [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
AU - Watson, M.
AU - Roeske, W. R.
AU - Yamamura, H. I.
PY - 1986
Y1 - 1986
N2 - 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.
AB - 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.
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M3 - Article
C2 - 3754581
AN - SCOPUS:0022560526
SN - 0022-3565
VL - 237
SP - 419
EP - 427
JO - Journal of Pharmacology and Experimental Therapeutics
JF - Journal of Pharmacology and Experimental Therapeutics
IS - 2
ER -