A Putative M3 Muscarinic Cholinergic Receptor of High Molecular Weight Couples to Phosphoinositide Hydrolysis in Human SK‐N‐SH Neuroblastoma Cells

Abstract

The M1-selective (high affinity for pirenzepine) muscarinic acetylcholine receptor (mAChR) antagonist pirenzepine displaced both N-[3H]methylscopolamine ([3H]NMS) and [3H]quinuclidinylbenzilate from intact human SK-N-SH neuroblastoma cells with a low affinity (Ki = 869-1,066 nM), a result indicating the predominance of the M2 or M3 (low affinity for pirenzepine) receptor subtype in these cells. Whereas a selective M2 agent, AF-DX 116 {11-2[[2-[(diethylamino)methyl]-1-piperidinyl]-acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one} bound to the mAChRs with a very low affinity (Ki = 6.0 .mu.M), 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), and agent that binds with high affinity to the M3 subtype, potently inhibited [3H]NMS binding (Ki = 7.2 nM), 4-DAMP was also 1,000-fold more effective than AF-DX 116 at blocking stimulated phosphoinsositide (PPI) hydrolysis in these cells. Covalent labeling studies (with [3H]propylbenzilylcholine mustard) suggest that the size of the SK-N-SH mAChR (Mr = 81,000-98,000) distinguishes it from the predominant mAChR species in rat cerebral cortex (Mr = 66,000), an Mr-enriched tissue. These results provide the first demonstration of a neural M3 mAChR subtype that couples to PPI turnover.