Differential inhibition of [³H]-oxotremorine-M and [³H]-quinuclinidyl benzilate binding to muscarinic receptors in rat brain membranes with acetylcholinesterase inhibitors

Abstract.

The potential interaction of acetylcholinesterase inhibitors with cholinergic receptors may play a significant role in the therapeutic and/or side-effects associated with this class of compound. In the present study, the capacity of acetylcholinesterase inhibitors to interact with muscarinic receptors was assessed by their ability to displace both [³H]-oxotremorine-M and [³H]-quinuclinidyl benzilate binding in rat brain membranes. The [³H]-quinuclinidyl benzilate/[³H]-oxotremorine-M affinity ratios permitted predictions to be made of either the antagonist or agonist properties of the different compounds. A series of compounds, representative of the principal classes of acetylcholinesterase inhibitors, displaced [³H]-oxotremorine-M binding with high-to-moderate potency (ambenonium>neostigmine=pyridostigmine=tacrine>physostigmine>edrophonium=galanthamine>desoxypeganine) whereas only ambenonium and tacrine displaced [³H]-quinuclinidyl benzilate binding. Inhibitors such as desoxypeganine, parathion and gramine demonstrated negligible inhibition of the binding of both radioligands. Scatchard plots constructed from the inhibition of [³H]-oxotremorine-M binding in the absence and presence of different inhibitors showed an unaltered B _max and a reduced affinity constant, indicative of potential competitive or allosteric mechanisms. The capacity of acetylcholinesterase inhibitors, with the exception of tacrine and ambenonium, to displace bound [³H]-oxotremorine-M in preference to [³H]-quinuclinidyl benzilate predicts that the former compounds could act as potential agonists at muscarinic receptors. Moreover, the rank order for potency in inhibiting acetylcholinesterase (ambenonium>neostigmine=physostigmine=tacrine>pyridostigmine=edrophonium=galanthamine>desoxypeganine>parathion>gramine) indicated that the most effective inhibitors of acetylcholinesterase also displaced [³H]-oxotremorine-M to the greatest extent. The capacity of these inhibitors to displace [³H]-oxotremorine-M binding preclude their utilisation for the prevention of acetylcholine catabolism in rat brain membranes, the latter being required to estimate the binding of acetylcholine to [³H]-oxotremorine-M-labelled muscarinic receptors. However, fasciculin-2, a potent peptide inhibitor of acetylcholinesterase (IC₅₀ 24 nM), did prevent catabolism of acetylcholine in rat brain membranes with an atypical inhibition isotherm of [³H]-oxotremorine-M binding, thus permitting an estimation of the "global affinity" of acetylcholine (K _i 85 nM) for [³H]-oxotremorine-M-labelled muscarinic receptors in rat brain.