PHARMACOLOGICAL CHARACTERIZATION OF THE NOVEL CHOLINOMIMETIC L-689,660 AT CLONED AND NATIVE BRAIN MUSCARINIC RECEPTORS

I-Azabicyclo[2,2,2]octane,3-(6-chloropyrazinyl)maleate (L-689, 660) reportedly is an agonist with selectivity for Ml and M3 muscarinic receptors. We confirmed this in functional assays of brain muscarinic receptors and of cloned human muscarinic receptors transfected into Chinese hamster ovary (CHO-Kl) cells.For stimulation of phosphoinositide turnover in rat cortical and hippocampal dissociated tissue, L-689,660 was a partial agonist (24% and 26% intrinsic activity, respectively, relative to oxotremorine-M) with EC50 values of 71 muM and 118 muM, respectively. At putative M4 receptors coupled to cyclic AMP inhibition in rat striatum, however, L-689,660 acted as a competitive antagonist (K(B) = 0.4 muM). Furthermore, at putative M2/M4 autoreceptors that regulate acetylcholine release in the hippocampus, the drug also behaved as an antagonist (K(B) = 2.1 muM). These data indicated that L-689,660 behaves as a postsynaptic agonist/presynaptic antagonist at central cholinergic synapses. Further aspects of the selectivity of the drug for specific muscarinic receptor subtypes were revealed with phosphoinositide turnover assays of cloned muscarinic receptors expressed in CHO-K1 cells. L-689,660 was a partial agonist at transfected hm1 and hm3 receptors and was more potent than oxotremorine-M; however, the drug was inactive at transfected hm5 receptors. Partial agonist activity at hm1 and hm3 muscarinic receptors was present even after using alkylation to reduce receptor numbers to levels comparable to that level found in the hm5 cell line. Thus, with functional assays either with brain tissue or with transfected cell lines, L-689,660 was shown to be an agonist for the M1 and M3 receptors but not for M5 or M4 receptors. Because previous studies have shown that L-689,660 also does not activate M2 receptors, these data taken as a whole reveal a unique pharmacological profile for this agonist, i.e., it is a drug that activates only Ml and M3 receptors. This in vitro profile suggests that L-689,660 might be a useful cholinomimetic for restoring cholinergic tone at central synapses in the diseased brain.