Affinities of muscarinic drugs for [3H]N-methylscopolamine (NMS) and [3H]oxotremorine (OXO) binding to a mixture of M1−M4 muscarinic receptors: Use of NMS/OXO-M ratios to group compounds into potential agonist, partial agonist, and antagonist classes

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Abstract
The relative affinities of various muscarinic drugs in the antagonist ([3H]N-methyl scopolamine ([3H]NMS)) and agonist ([3H]Oxotremorine-m ([3H]OXO-M)) binding assays using a mixture of tissues containing M1−M4 receptor subtypes have been determined. [3H]NMS bound with high affinity (Kd=25±5.9 pM; n=3) and to a high density (Bmax=11.8±0.025 nmol/g wet weight) of muscarinic receptors. [3H]OXO-M appeared to bind to two binding sites with differing affinities (Kd1=2.5±0.1 nM; Kd2=9.0±4.9 μM; n=4) and to a different population of binding sites (Bmax1=5.0±0.26 nmol/g wet weight; Bmax2=130±60 nmol/g wet weight). Well known antagonists exhibited high affinity for [3H]NMS binding but a lower affinity for [3H]OXO-M binding. The opposite was true for acetylcholine and other known agonists. However, pilocarpine and McN-A-343 had similar affinities for sites labeled by both radioligands. Using the ratios of antagonist-to-agonist binding affinities, it was possible to group compounds into apparently distinct full agonist (ratios of 180–665; e.g. carbachol, muscarine, OXO-M, OXO-S and arecoline), partial agonist (ratios of 14–132; e.g. McN-A-343, pilocarpine, aceclidine, bethanechol, OXA-22 and acetylcholine) and antagonist (ratios of 0.22–1.9; e.g. atropine, NMS, pirenzepine, methoctramine, 4-DAMP and p-fluorohexahydrosialo-difenidol) classes. These data suggest that the NMS/OXO-M affinity ratios using a mixture of M1−M4 muscarinic receptors may be a useful way to screen and group a large number of compounds into apparent agonist, partial agonist, and antagonist classes of cholinergic agents.

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