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.
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 (approximately 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.
Cioffi
C L
CL
Neuroscience Laboratory, University of Michigan, Ann Arbor 48104-1687.
Fisher
S K
SK
D002118:Calcium / Q000378:metabolism; D003470:Culture Media; D003593:Cytoplasm / Q000378:metabolism; D004224:Diterpenes; D006150:Guanine Nucleotides / Q000494:pharmacology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D006868:Hydrolysis; D007295:Inositol Phosphates / Q000378:metabolism; D009447:Neuroblastoma / Q000378:metabolism* / Q000473:pathology; D009994:Osmolar Concentration; D010716:Phosphatidylinositols / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000378:metabolism*; D012969:Sodium Fluoride / Q000494:pharmacology; D013729:Terpenes / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013873:Thionucleotides / Q000494:pharmacology; D013997:Time Factors; D014407:Tumor Cells, Cultured
D003470:Culture Media; D004224:Diterpenes; D006150:Guanine Nucleotides; D007295:Inositol Phosphates; D010716:Phosphatidylinositols; D011976:Receptors, Muscarinic; D013729:Terpenes; D013873:Thionucleotides; C011309:mezerein; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D012969:Sodium Fluoride; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium