Muscarinic receptors in human SH-SY5Y neuroblastoma cell line: regulation by phorbol ester and retinoic acid-induced differentiation.

The specific binding of the muscarinic ligand [3H](-)quinuclidinyl benzilate [( 3H]QNB) to cell membranes of human SH-SY5Y neuroblastoma cells was studied. Saturation isotherms yielded a Kd = 0.28 +/- 0.06 nM and a Bmax of 337 +/- 47 pmol/g protein. Pirenzepine inhibited [3H]QNB binding; inhibition data showed best fit to a 2-site binding model revealing both a high affinity pirenzepine site (34%, KH = 10 nM) and a low affinity site (66%, KL = 1 microM). These results indicate that muscarinic receptors on SH-SY5Y cells may be subclassified as M1/M2 subtypes. Morphological and biochemical differentiation of these cells after treatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or retinoic acid (RA) resulted in a decrease and an increase in the number of muscarinic binding sites, respectively. Furthermore, TPA- and RA-treated cells showed a significant increase in acetylcholinesterase activity compared with non-treated cells. However, only RA-treated cells showed significant increase in choline acetyltransferase activity compared to non-treated cells. These findings demonstrate that TPA and RA can regulate both the number of muscarinic receptors and the acetylcholinesterase activity in human SH-SY5Y neuroblastoma cells.