Journal of zyxwvutsrqponm Neurochemislry Raven Press, Ltd., New York zyxwvutsrqpon 0 1992 International Society for Neurochemistry Lan-1: A Human Neuroblastoma Cell Line With M, and M, Muscarinic Receptor Subtypes Coupled to Intracellular Ca2+ Elevation and Lacking Ca2+ Channels Activated by Membrane Depolarization A. Fatatis, A. Bassi, M. R. Monsurrh, G. Sorrentino, *G. D. Mita, G. F. Di Renzo, and L. Annunziato zyxw Section of Pharmacology, Department of Science of Human Communication, 2nd School of Medicine, University of Naples “Federico II’l and *IIGB (CNR), Naples, Italy zyxw Abstract: The LAN- zyxwvutsr 1 clone, a cell line derived from a hu- man neuroblastoma, possesses muscarinic receptors. The stimulation of these receptors with increasing concentra- tions of carbachol (CCh; 1 zyxwvutsrqponm - 1,000 pM) caused a dose-depen- dent increase of the intracellular free Ca2+concentration ([Ca2+Ii). This increase was characterized by an early peak phase (10 s) and a late plateau phase. The removal of extra- cellular Ca2+ reduced the magnitude of the peak phase to -70% but completely abolished the plateau phase. The muscarinic-activated Ca2+ channel was gadolinium (Gd3+) blockable and nimodipine and w-conotoxin insensitive. In addition, membrane depolarization did not cause any in- crease in [Ca”], . The CCh-induced [Ca2+], elevation was concentration-dependently inhibited by pirenzepine and zyxwvutsr 4-diphenylacetoxy-N-methylpipendine methiodide, two rather selective antagonists of MI and M, muscarinic receptor subtypes, respectively,whereas methoctramine, an M, antagonist, was ineffective. The coupling of MI and M, receptor activation with [Ca2+Ii elevation does not seem to be mediated by a pertussis toxin-sensitive guanine nucleo- tide-binding protein or by the diacylglycerol-protein kinase C system. The mobilization of [Ca”], elicited by MI and M, muscarinic receptor stimulation seems to be dependent on an inositol trisphosphate-sensitiveintracellular store. In ad- dition, ryanodine did not prevent CCh-induced [Ca2+Ii mo- bilization, and, finally, LAN- I cells appear to lack caffeine- sensitive Ca2+ stores, because the methylxanthine was un- able to elicit intracellular Ca2+mobilization, under basal conditions, after a subthreshold concentration of CCh (0.3 pM), or after thapsigargin. Key Words: LAN- 1 cells-Neu- roblastoma cells-MI, M,, and M3 muscarinic receptor subtypes-Intracellular Ca2+ concentration-Ca2+ chan- nels. Fatatis A. et al. LAN- 1 : A human neuroblastoma cell line with MI and M, muscarinic receptor subtypes coupled to intracellular Ca2+elevation and lacking Ca2+ channels activated by membrane depolarization. J. Neurochern. 59, 1-9 ( 1992). LAN-1 is a cell line derived from a human bone marrow metastasis of a neuroblastoma, producing vanillyl mandelic and homovanillic acid. These cells contain large amounts of tyrosine hydroxylase (1 5 times more than that found in the brain) and have multiple cytoplasmic dense cores (Seeger et al., 1977), such as those present in sympathetic neurons, pheochromocytomas, and other neuroblastoma cells. In these cells the existence of muscarinic receptors involved in breakdown of polyphosphoinositides and in elevation of the intracellular free Ca2+concentra- tion ([Ca”],) has been previously demonstrated (Poz- zan et al., 1986; Fisher and Heacock, 1988; Lambert and Nahorski, 1990). The availability nf fluorescent dyes like fura-2 has allowed the measurement of varia- tions in [Ca2+Ii both in cell suspension and in single- cell preparations (Tsien and Poenie, 1986; Malgaroli et al., 1987). Received July 3, 199 1 ; revised manuscript received October 22, I99 I ; accepted December 2, 199 1. Address correspondence and reprint requests to Prof. L. Annun- ziato at Section of Pharmacology, Department of Science of Hu- man Communication, 2nd School of Medicine, University of Na- ples “Federico 11,” Via Pansini 5, 8013 1 Napoli, Italy. Abbreviations zyxwvutsrq used: 8-Br CAMP, 8-bromo cyclic A M P [Ca2’],, intracellular free Ca2+ concentration; CCh, carbachol: w-CgTX, w- conotoxin: 4-DAMP, 4-diphenylacetoxy-N-methylpipendine methi- odide: G protein, guanine nucleotide-binding protein; IP,, inositol 1,4,5-trisphosphate; PK-C, protein kinase C: PTX. pertussis toxin: TMB-8, 8-(N,N-diethylamino)octyl 3.45-trimethoxybenzoate hy- drochloride: TPA. phorbol L 2-myristate 13-acetate: TTX, tetrodo- toxin. 1