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

Abstract

The actions of tumor promoters on the coupling of muscarinic receptors to the hydrolysis of inositol lipids and the generation of Ca 2+ signals were examined in the human neuroblastoma SH-SY5Y cell line. Pretreatment of SH-SY5Y cells with 50 n M 12- O -tetradecanoylphorbol 13-acetate (TPA) for 5 days resulted in neuronal differentiation, a 28% decrease in both N -[ 3 H]methylscopolamine and [ 3 H]-scopolamine binding, and a significantly larger reduction (48%) in agonist-stimulated 3 H-inositol phosphate generation. Whereas mezerein could mimic the effects produced by TPA, the biologically inactive 4 Α -phorbol 12,13-didecanoate was without effect on both antagonist binding and agonist-stimulated phosphoinositide (PPI) turnover. A decline (∼ 50%) in the agonist-mediated rise in cytoplasmic Ca 2+ 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 3 H-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 3 H-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 m M CaCl 2 . 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 Ca 2+ 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.