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Muscarinic binding sites in a catecholaminergic human neuroblastoma cell line

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Abstract

Tyrosine hydroxylase (TH) a characteristic enzyme activity for the catecholaminergic clonal cell line LA-N-1 and choline acetyltransferase (ChAT) a characteristic enzyme activity for the cholinergic clonal cell line LA-N-2 were previously shown to be increased in these cells exposed to 10−5 M retinoic acid (RA) as differentiating agent. An investigation of the receptor characteristics suggests a complementarity between the two cell lines. The binding of QNB, a muscarinic ligand, was undetectable with the LA-N-2 cells but was present in the LA-N-1 cells and possessed a kD of 1.8 nM and 2.2 nM and a Bmax of 0.56 and 0.68 for control and RA grown cells respectively. There was a gradual increase in QNB binding to LA-N-1 cells from 2 days in vitro (DIV) until 6 DIV in both control and RA grown cells. An IC50 of 2.5×10−8 M and 0.9×10−8 M for atropine inhibition was obtained for the control and RA grown cells respectively. The corresponding values for carbachol inhibition were 7×10−2 M and 3×10−2 M respectively. The inhibition by the agonist oxotremorine is comparable to that of carbachol and 1 mM pilocarpine inhibited the binding by 21%. QNB binding showed a low affinity for pirenzepine and for AF-DX-116 but was inhibited with a rather high affinity by 4-DAMP (IC50:110 μM) thus suggesting the presence of an M3 receptor. Acetylcholine (100 μM) plus eserine (50 μM) and BW284c55 (1 μM), an acetylcholinesterase inhibitor, reduced the binding of QNB by approximately 25%. Nicotine (1 mM) caused a 36% reduction of binding and hemicholinium-3 (HC-3) (1 μM), an inhibitor of choline uptake, inhibited the binding by 53%. There was a down regulation of QNB binding observed with cells grown for 24 hours with either the antagonist atropine or the agonists carbachol or oxotremorine. Low amounts of α-bungarotoxin (α-BgTx) binding sites were barely detectable in both LA-N-1 and LA-N-2 cells. The LA-N-1 muscarinic receptor is coupled to polyphosphoinositide hydrolysis without increased cyclic AMP formation further suggesting its being an M3 receptor.

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Author notes

  1. Giuseppe Sorrentino

    Present address: 1a Facoltà di Medicina, Istituto di Scienze Neurologiche, Via Pansini 5, 80131, Napoli, Italy

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, The University of Manitoba, Faculty of Medicine, 770 Bannatyne Avenue, R3E 0W3, Winnipeg, Manitoba, Canada

    Giuseppe Sorrentino, Indrapal N. Singh & Julian N. Kanfer

  2. Centre de Neurochimie-Cronenbourg, BP20 CR, 67037, Strasbourg Cedex, France

    Alphonse Hubsch, Serge Mykita & Raphael Massarelli

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  1. Giuseppe Sorrentino
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  4. Julian N. Kanfer
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Sorrentino, G., Singh, I.N., Hubsch, A. et al. Muscarinic binding sites in a catecholaminergic human neuroblastoma cell line. Neurochem Res 17, 215–222 (1992). https://doi.org/10.1007/BF00966662

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