Studies on the regulation of agonist-sensitive second messenger responses mediated by recombinant m1-muscarinic receptors.

The m1-muscarinic receptor is a G protein-coupled receptor that preferentially couples to the Gq/11 and the phosphoinositidase C (PIC) pathway. The acute regulation of agonist-stimulated PIC activation by recombinant m1-muscarinic receptors expressed in CHO cells was investigated. The time courses for methacholine- and arecoline stimulated inositol (l,4,5)-trisphosphate (Ins(l,4,5)P3) generation were both biphasic-consisting of a peak phase that attenuated within seconds of agonist addition and a sustained phase that persisted for at least 10 minutes. Arecoline was also shown to be a partial agonist for the 10 second Ins(l,4,5)P3 response. Prestimulation with methacholine for 5 minutes followed by a 3 minute agonist wash-out period resulted in desensitisation of agonist-dependent Ins(l,4,5)P3 production. Desensitisation of the methacholine response was apparent as an approximately 4 fold increase in the EC50 with no reduction in maximal responsiveness. In contrast, desensitisation of the arecoline response was manifest as a decrease in the maximal Ins(l,4,5)P3 response. Desensitisation was rapid being approximately halt maximal within 10 seconds of agonist addition and the dose response relationship for desensitisation indicated that it was unlikely to be a highly amplified response. Studies on [3H]inositol labelled CHO-m1 cells indicated that methacholine-stimulated depletion of [3H]phosphatidylinositol-(4,5)-bisphosphate, but not of [3H]phosphatidylinositol-4-phosphate or [3H]phosphatidylinositol, was also desensitised by agonist prestimulation. Moreover, these results indicated that desensitisation resulted from a de facto decrease in the efficacy of receptor coupling to PIC rather than a sustained depletion in phosphoinositides or enhanced metabolism of Ins(l,4,5)P3. An antiserum was raised against the carboxy terminal domain of the m1-muscarinic receptor and was shown to specifically recognise and quantitatively immunoprecipitate m1-muscarinic receptors. Immunoprecipitation of m1-muscarinic receptors from [32P]orthophosphate labelled CHO-m1 cells indicated that the receptors underwent rapid agonist-induced phosphorylation by a receptor kinase distinct from PKA, PKC or Ca2+/calmodulin dependent protein kinase.