Reduction of Muscarinic Receptor Density and of Guanine Nucleotide‐Stimulated Phosphoinositide Hydrolysis in Human SH‐SY5Y Neuroblastoma Cells Following Long‐Term Treatment with 12‐O‐Tetradecanoylphorbol 13‐Acetate or Mezerein

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Abstract
The actions of tumor promoters on the coupling of muscarinic receptors to the hydrolysis of inositol lipids and the generation of Ca2+ signals were examined in the human neuroblastoma SH-SY5Y cell line. Pretreatment of SH-SY5Y cells with 50 nM 12-O-tetradecanoylphorbol 13-acetate (TPA) for 5 days resulted in neuronal differentiation, a 28% decrease in both N-[3H]methylscopolamine and [3H]-scopolamine binding, and a significantly larger reduction (48%) in agonist-stimulated 3H-inositol phosphate generation. Whereas mezerein could mimic the effects produced by TPA, the biologically inactive 4.alpha.-phorbol 12, 13-didecanoate was without effect on both antagonist binding and agonist-stimulated phosphoinositide (PPI) turnover. A decline (.apprx. 50%) in the agonist-mediated rise in cytoplasmic Ca2+ and a substantial loss of protein kinase C activity also were observed following pretreatment with TPA or mezerein. The ability of fluoride, an agent capable of direct activation of guanine nucleotide binding proteins, to stimulate 3H-inositol phosphate release was significantly reduced in SH-SY5Y cells treated with these agents. Furthermore, pretreatment of SH-SY5Y neuroblastoma cells with TPA or mezerein impaired 3H-inositol phosphate formation induced by the addition of either guanosine 5''-O-(3-thiotriphosphate) or carbamylcholine to digitonin-permeabilized cells, but not that elicited by the addition of 2 mM CaCl2. Although cells cultured in the presence of serum-free media also exhibited neuronal differentiation, no significant alteration in either muscarinic receptor number or agonist-stimulated PPI hydrolysis was observed. The results suggest that TPA and mezerein decrease agonist-stimulated PPI hydrolysis and Ca2+ signaling in SH-SY5Y cells not only by a reduction in muscarinic receptor number but also through an inhibition of guanine nucleotide-stimulated PPI turnover.

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