Muscarinic binding sites in a catecholaminergic human neuroblastoma cell line

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 B_max 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 M) thus suggesting the presence of an M₃ receptor. Acetylcholine (100 M) plus eserine (50 M) and BW284c55 (1 M), 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 M), 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 -bungarotoxin (-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.