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 microM), 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), an 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 phosphoinositide (PPI) hydrolysis in these cells. Covalent labeling studies (with [3H]propylbenzilycholine 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 M1-enriched tissue. These results provide the first demonstration of a neural M3 mAChR subtype that couples to PPI turnover.

Muscarinic receptors coupled to phosphoinositide hydrolysis and elevated cytosolic calcium in a human neuroblastoma cell line SK‐N‐SH

“…The EC50 value for carbachol-stimulated increase in intracellular calcium was 10.7 + 3.2upM (n = 4). The actions of carbachol were shown to be atropine- tion of the various [3H]-inositol phosphates, which confirms and extends the previous studies of Yu & Sadee (1986), Fisher & Snider (1987) and Fisher & Heacock (1988). These cells clearly display a coupling of muscarinic receptors to phospholipase C, although in contrast to results obtained in cerebral cortical tissue (Batty & Nahorski, 1985; Berridge, 1984).…”

“…However, preliminary investigations revealed that these cells displayed little or no production of inositol phosphates in response to muscarinic agonists (Baird & Nahorski, unpublished observations). In contrast the human cell line SK-N-SH, which was originally derived from bone marrow metasteses of a four year old girl (Biedler et al, 1973), expresses a relatively high density of muscarinic receptors linked to phosphoinositide metabolism (Fisher & Snider, 1987;Fisher & Heacock, 1988). We have recently confirmed the relatively high density of M3-muscarinic receptors on SK-N-SH cells by use of the radioligand [3H]-Nmethylscopolamine ([3H]-NMS) and here examine the linkage to phosphoinositide metabolism and calcium metabolism in this cell line.…”

Quantitative comparisons of muscarinic and bradykinin receptor‐mediated Ins (1,4,5)P3 accumulation and Ca2+ signalling in human neuroblastoma cells

“…Indeed, phosphorylation of a number of PLC-linked receptors has been reported (Klueppelberg et al, 1992;Kwatra et al, 1993) including that of the m3 receptor by PKC and an additional kinase distinct from either protein kinase A (PKA), Ca2+-calmodulin-dependent kinase or P-ARK . (Yu & Sadee, 1986) or SH-SY5Y cell lines (Ogino & Costa, 1993 (Yu & Sadee, 1986;Fisher & Heacock, 1988;Fisher et al, 1989;Baird et al, 1989) and SH-SY5Y cells (Lambert & Nahorski, 1990b;Lambert et al, 1991 (Kenakin, 1987 [Ca2J]i is an 'all-or-none' response (Bootman et al, 1992;Shao & McCarthy, 1993 release whereby the release of a small amount of Ca2l initiates the release of further Ca2" from ryanodine-sensitive stores (see Putney & Bird, 1993 receptor activation (Bezprozvanny et al, 1991 all-or-none phenomenon. Whatever the mechanism, the potency difference defines a step which features as an important amplification stage within the signal transduction pathway.…”

Phosphatidylinositol 4,5-Bisphosphate Modifies Tubulin Participation in Phospholipase Cβ1Signaling

“…The net absorbance of microtubule polymerization reactions without added phosphoinositides was 1.09 Ϯ 0.12 (control). *p Ͻ 0.05; **p Ͻ 0.01. contain m3 muscarinic receptors, G␣q, and PLC␤ 1 (Fisher and Heacock, 1988).…”