Characterization and quantitative autoradiographic distribution of [ 3H]acetylcholine muscarinic receptors in mammalian brain. Apparent labelling of an M 2-like receptor sub-type

Abstract

[ 3H]Acetylcholine receptor binding characteristics (under muscarinic conditions) have been investigated using membrane binding assays and in vitro receptor autoradiography. In rat, guinea-pig and monkey brain membrane preparations, [ 3H]acetylcholine binds with high affinity (25–50 nM) to an apparently single class of sites which is differentially distributed across brain regions. The ligand selectivity pattern reveals that the potency of (−)quinuclidinyl benzylate is greater than (>) atropine > scopolamine > oxotremorine > carbamylcholine > pirenzepine > methylcarbamyl-choline = nicotine in competing for [ 3H]acetylcholine binding sites, indicating that [ 3H]acetylcholine selectively binds to muscarinic sites under these incubation conditions. Moreover, the low potency of pirenzepine suggests that [ 3H]acetylcholine does not label a significant proportion of the M 1 receptor sub-type but most likely binds to putative M 2-like receptor sites. This hypothesis is also supported by the autoradiographic distribution of [ 3H]acetylcholine binding sites in all species studied here. High densities of [ 3H]acetylcholine binding sites are seen in various nuclei of the medulla and pons, certain thalamic nuclei, medial septum, laminae III, V and VI of the cortex and just above the pyramidal cell layer of the hippocampus. Such localization is much different from that seen with the non-selective antagonist [ 3H]quinuclidinyl benzylate and the selective M 1 receptor ligand [ 3H]pirenzepine, although it resembles that of the selective M 2 receptor antagonist [ 3H]AF-DX 116. Thus, [ 3H]acetylcholine apparently mostly binds with high affinity mainly to non-M 1 muscarinic receptor types in mammalian brain tissues. Moreover, the ligand selectivity pattern and in vitro receptor autoradiographic data suggest that at low concentrations (10–20 nM) most of [ 3H]acetylcholine labelled sites are of the M 2-like receptor class.