European Journal of Pharmacology - Molecular Pharmacology Section, 206 (1991) 181-189 © 1991 Elsevier Science Publishers B.V. 0922-4106/91/$03.50 A DONIS 092241069100076A 181 EJPMOL 90148 M 3 muscarinic cholinoceptors are linked to phosphoinositide metabolism in rat cerebellar granule cells Emma M. Whitham, R.A. John Challiss and Stefan R. Nahorski Department of Pharmacolog), and Therapeutics, Universi O, of Leicester, Leicester, U.K. Received 18 July 1990. revised MS received 30 October 1990; accepted 20 November 1990 Primary cultures of rat cerebellar granule cells are shown to possess a high density (283 _+48 fmol/mg of protein) of muscarinic receptor sites, defined using N-[3H]methylscopolamine ([3H]NMS), with a K o of 0.18 +_0.01 nM measured after culture in vitro for 7 days. Displacement of specific [3H]NMS binding demonstrated a muscarinic receptor with low affinity for pirenzepine (Ki: 240 nM); further investigation using antagonists, AF-DX 116 and 4-DAMP to discriminate between M 2 and M 3 receptors respectively, revealed low M 2 affinity (Ki: 600 nM) and high M 3 affinity (Ki: 2.4 nM), indicative of the M 3 receptor subtype. The robust muscarinic receptor stimulation of [3H]inositol phosphate formation, previously observed in these cells, was confirmed. Inhibition of this response followed a similar profile to the binding data, exhibiting weak inhibitory effects for pirenzepine ( Ki: 710 nM) and AF-DX 116 (Ki: 5000 nM), but a potent action for 4-DAMP (Ki: 2.4 nM). The opposite profile seen for AF-DX 116 and 4-DAMP is indicative of a M 3 receptor subtype expressed on these cells and linked to phosphoinositide hydrolysis. Further studies demonstrated that M 3 receptor stimulation caused a rapid, transient increase in the second messenger inositol 1,4,5-trisphosphate, suggesting that potential Ca2+-homeostatic and neuromodulatory effects may be mediated by this response. Cerebellar granule cells; Muscarinic M 3 receptors; Phosphoinositide metabolism; Inositol 1.4,5-trisphosphate 1. Introduction The muscarinic acetylcholine group of receptors has been established to exhibit considerable heterogeneity, based on pharmacological and molecular cloning ap- proaches (Birdsall and Hulme, 1983; Hammer et al., 1986; Kubo et al., 1986; Bonnet et al., 1987; 1988; Peralta et al., 1987). There is now general agreement that M~, M 2 and M 3 receptors characterised pharmaco- logically, correspond to appropriate gene products, but the significance of two further types remain to be established (Hulme et al., 1990). In the CNS, expression of Mt-M 4 subtypes can be demonstrated (Buckley, 1990), though M~ sites prob- Correspondence to: Stefan R. Nahorski. Department of Pharmacol- ogy and Therapeutics, University of Leicester, P.O. Box 138, Medical Sciences Building, University Road, Leicester LE1 9HN, U.K. Abbreviations: AF-DX 116. 11[[2](diethylamino)methyl][-1-piper- idinyl]-acetyl-5,1 l-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6- one; 4-DAMP, 4-diphenylacetoxy-N-methylpiperidine methoiodide; DIV, days in vitro; InsP t. InsPz, InsP 3 and InsP4; myo-inositol mono-, bis-, tris-, and tetrakis- phosphate derivatives with assignment of phosphate Iocants in parentheses where appropriate. ably predominate and have been established to be lin- ked to phosphoinositide metabolism (Gill and Wolfe, 1984; Lazareno et al., 1985). Recent work however, in human neuroblastoma cell lines, has identified the pres- ence of the M 3 muscarinic receptor subtype coupled functionally to phosphoinositide hydrolysis (in SK-N- SH (Fisher and Heacock, 1988) and SH-SY5Y (Lam- bert et al., 1989) neuroblastoma cells). Although there are indications of a non-M t site linked to phos- phoinositide metabolism in guinea-pig striatum (Fisher and Snyder, 1987), a functional M 3 site has yet to be established in central neurones. Primary cultures of rat cerebellar granule cells, used in this study, comprise a highly homogenous neuronal preparation and have already been shown to express many receptor types that are coupled to a number of biochemical effector systems (Xu and Wojcik, 1986; Xu and Chuang, 1987a, 1987b) and ion channels (Wroblew- ski et al., 1985). These include a muscarinic linkage to phosphoinositidase C that shows a low affinity for pirenzepine (Xu and Chuang, 1987a). The following experiments provide further evidence to suggest that the classification of the muscarinic receptor subtype ex- pressed in this cell system is M 3 and is positively coupled to PI hydrolysis. Furthermore, we establish that this neuronal preparation has the capacity to produce