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 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 4α‐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 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. Copyright © 1990, Wiley Blackwell. All rights reserved