On the involvement of multiple muscarinic receptor subtypes in the activation of phosphoinositide metabolism in rat cerebral cortex

We have examined the activation of phosphoinositide metabolism by muscarinic agonists in rat cerebral cortex, in an attempt to delineate the mechanisms by means of which some selective antagonists inhibit this response in a manner that deviates from simple mass action law. The accumulation of [³H]inositol phosphates induced by the full agonist carbamylcholine in cell aggregates preparations was inhibited by muscarinic antagonists with the following order of potency: telenzepine > atropine > 4-diphenylacetoxy-N-methyl-piperidine methbromide > pirenzepine > hexahydro-sila-difenidol > AF-DX 116. The same order of potency was found for the competition of these antagonists with [³H]telenzepine binding to M₁ muscarinic receptors. The inhibition of the formation of [³H]inositol phosphates activated by acetylcholine, carbamylcholine, and oxotremorine-M by pirenzepine and telenzepine showed biphasic curves, with 62-73% of the response being inhibited with high affinity. Atropine, AFDX 116, and pirenzepine shifted the concentration-response curves of oxotremorine-M to the right in a parallel manner. However, pirenzepine at micromolar concentrations showed deviation from linearity of the Schild regression. The blockade by high concentrations of pirenzepine and telenzepine showed less than additive dose ratios when assayed in the presence of atropine, suggesting deviation of their antagonism from simple competition. However, after alkylation with propylbenzilylcholine mustard in the presence of low concentrations of pirenzepine, the response to carbamylcholine and oxotremorine-M showed monophasic inhibition curves by pirenzepine and linear Schild regression for this antagonist. These results support the interpretation that the formation of [³H]inositol phosphates is activated by multiple muscarinic receptor subtypes in rat cerebral cortex. The profile of affinities of muscarinic antagonists indicates that a major component of the response is activated by an M₁ receptor subtype and a minor component is probably mediated by M₃ muscarinic receptors when acetylcholine, carbamylcholine, or oxotremorine-M are used to stimulate the response. Conversely, pirenzepine inhibited the response induced by methacholine and bethanechol in a monophasic manner with high affinity (K, = 13 nM), suggesting that these agonists can selectively stimulate phosphoinositide metabolism through activation of M₁ muscarinic receptors in rat cerebral cortex.