Abstract
[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-3β-ol and granatan-3α-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.
- Copyright © 1978 by Academic Press, Inc.
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