A Putative M 3 Muscarinic Cholinergic Receptor of High Molecular Weight Couples to Phosphoinositide Hydrolysis in Human SK-N-SH Neuroblastoma Cells

Abstract

The M 1 -selective (high affinity for pirenzepine) muscarinic acetylcholine receptor (mAChR) antagonist pirenzepine displaced both N -[ 3 H]methylscopolamine ([ 3 H]NMS) and [ 3 H]qui-nuclidinylbenzilate from intact human SK-N-SH neuroblastoma cells with a low affinity ( K i = 869–1,066 nM), a result indicating the predominance of the M 2 or M 3 (low affinity for pirenzepine) receptor subtype in these cells. Whereas a selective M 2 agent, AF-DX 116 {11–2[[2-[(diethylamino)methyl]-1-piperidinyl]-acetyl]-5,11-dihydro-6 H -pyrido[2,3- b ][1,4]benzodiazepin-6-one} bound to the mAChRs with a very low affinity ( K i = 6.0 Μ M ), 4-diphenylacetoxy- N -methylpiperidine methiodide (4-DAMP), an agent that binds with high affinity to the M 3 subtype, potently inhibited [ 3 H]NMS binding ( K i = 7.2 n M ). 4-DAMP was also 1,000-fold more effective than AF-DX 1 16 at blocking stimulated phosphoinositide (PPI) hydrolysis in these cells. Covalent labeling studies (with [ 3 H]propylbenzilylcholine mustard) suggest that the size of the SK-N-SH mAChR (M r = 81.000–98,000) distinguishes it from the predominant mAChR species in rat cerebral cortex (M r =66,000), an M 1 -enriched tissue. These results provide the first demonstration of a neural M 3 mAChR subtype that couples to PPI turnover.