POSITIVE COOPERATIVITY OF ACETYLCHOLINE AND OTHER AGONISTS WITH ALLOSTERIC LIGANDS ON MUSCARINIC ACETYLCHOLINE-RECEPTORS

It is well known that allosteric modulators of muscarinic acetylcholin e receptors can both diminish and increase the affinity of receptors f or their antagonists. We investigated whether the allosteric modulator s can also increase the affinity of receptors for their agonists. Twel ve agonists and five allosteric medulators were tested in experiments on membranes of CHO cells that had been stably transfected with genes for the M-1-M-4 receptor subtypes. Allosterically induced changes in t he affinities for agonists were computed from changes in the ability o f a fixed concentration of each agonist to compete with [H-3]N-methyls copolamine for the binding to the receptors in the absence and the pre sence of varying concentrations of allosteric modulators. The effects of allosteric modulators varied greatly depending on the agonists and the subtypes of receptors. The affinity for acetylcholine was augmente d by (-)-eburnamonine on the M-2 and M-4 receptors and by brucine on t he M-1 and M-3 receptors. Brucine also enhanced the affinities for car bachol, bethanechol, furmethide, methylfurmethide, pilocarpine, 3-(3-p entylthio-1 ,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1 -methylpyridine (pentylthio-TZTP), oxotremorine-M, and McN-A-343 on the M-1, M-3, and M-4 receptors, for pentylthio-TZTP on the M-2 receptors, and for arec oline on the M-3 receptors. (-)-Eburnamonine enhanced the affinities f or carbachol, bethanechol, furmethide, methylfurmethide, pentylthio-TZ TP, pilocarpine, oxotremorine and oxotremorine-M on the M-2 receptors and for pilocarpine on the M-4 receptors. Vincamine, strychnine, and a lcuronium displayed fewer positive allosteric interactions with the ag onists, but each allosteric modulator displayed positive cooperativity with at least one agonist on at least one muscarinic receptor subtype . The highest degrees of positive cooperativity were observed between (-)-eburnamonine and pilocarpine and (-)-eburnamonine and oxotremorine -M on the M-2 receptors (25- and 7-fold increases in affinity, respect ively) and between brucine and pentylthio-TZTP on the M-2 and brucine and carbachol on the M-1 receptors (8-fold increases in affinity). The discovery that it is possible to increase the affinity of muscarinic receptors for their agonists by allosteric modulators offers a new way to subtype-specific pharmacological enhancement of transmission at ch olinergic (muscarinic) synapses.