Molecular Pharmacology, Vol 14, 751-767, Copyright © 1978 by the American Society for Pharmacology and Experimental Therapeutics

Comparative Binding Studies with Cholinergic Ligands and Histrionicotoxin at Muscarinic Receptors of Neural Cell Lines

¹ Laboratory of Chemistry, National Institute of Arthritis, Metabolism and Digestive Diseases, and Laboratory of Biochemical Genetics, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20014

[³H]Scopolamine and [³H]quinuclidinyl-benzilate (QNB) have been used to study inhibitory acetylcholine receptors of neuroblastoma clone N1E-115 and excitatory acetylcholine receptors of NG108-15 neuroblastoma x glioma hybrid cells. Both [³H]ligands bind with high affinity to muscarinic acetylcholine receptors of the cells. The apparent dissociation constants are 0.4 nM (N1E-115) and 0.5 nM (NG108-15) for [³H]scopolamine, 0.06 nM (N1E-115) and 0.1 nM (NG108-15) for [³H]QNB. The receptor concentration is 25 fmol/mg in N1E-115 and 40 fmol/mg in NG108-15. Binding and release of [³H]-scopolamine are kinetically biphasic processes. [³H]QNB has a similar rate of binding but a much slower rate of release from the receptor. Binding of both [³H]ligands is competitively inhibited by compounds known to interact with muscarinic acetylcholine receptors. With 1 nM [³H]ligand a 50% inhibition is caused by nanomolar concentrations of muscarinic antagonists, by 1 to 100 micromolar concentrations of muscarinic agonists, and by >100 micromolar concentrations of nicotinic cholinergic compounds. Slopes of approximately 1 were found for receptor antagonists and approximately 0.5 for receptor agonists in logit-log plots of competition data. Antagonist binding can therefore be described as interaction with a noncooperative class of receptors, whereas agonist binding exhibits negative cooperativity or heterogeneity in binding sites. The interaction of dihydroiso-histrionicotoxin (H₂-HTX) with muscarinic acetylcholine receptors of N1E-115 cells has also been studied by inhibition experiments. H₂-HTX inhibits [³H]scopolamine binding in a noncompetitive manner causing a 50% inhibition at an applied concentration of 70 µM. The local anesthetic, tetracaine, shows almost identical characteristics. Dihydro-adaline, granatan-3-ol and granatan-3-ol are less strong inhibitors. Structural comparison of these compounds with H₂-HTX suggests that the two aliphatic side chains and the proximity of the hydroxyl and amino groups may be important features for the interaction of H₂-HTX with the muscarinic acetylcholine receptor.

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