Carbachol inhibits insulin-stimulated phosphatidylinositol 3-kinase activity in SH-SY5Y neuroblastoma cells

SH-SY5Y human neuroblastoma cells express muscarinic M(3) receptors as well as insulin receptors, thus offering the opportunity to investigate possible cross-lark following activation of two distinct intracellular signal transduction pathways that convert the precursor phosphatidylinositol (PI) to its 3' phosphate or its 4' phosphate, respectively, In this study, the effect of carbachol on insulin-stimulated PI 3-kinase (PI3K) activity was examined in SH-SY5Y cells, Insulin addition to the cell medium induced a 10-26-fold increase in anti-phosphotyrosine-immunoprecipitable PI3K activity, Preincubation with 1 mM carbachol inhibited the insulin-stimulated PI3K activity in a time-dependent manner, with half-maximal and maximal inhibition times of 4 and 15 min, respectively, Atropine blocked the inhibitory effect of carbachol, Although carbachol did not change the amount of 85-kDa subunit protein regulatory unit associated with tyrosine-phosphorylated proteins, either in control or in insulin-stimulated cells, it appears to decrease the amount of associated 110-kDa catalytic subunit protein in the latter instance, Because PI3K activity from SH-SY5Y cells has been shown to be inhibited in vitro in the presence of cytidine diphosphodiacylglycerol (CDP-DAG) or phosphatidate (PA), we examined the presence of these lipids in SH-SY5Y cells that had been treated with carbachol, Formation of both lipids was increased in a time-dependent manner following carbachol addition, and their increased levels are proposed to account for the observed in vivo inhibition of PI3K. Addition of the cell-permeable homologue didecanoyl-CDP-DAG to intact cells inhibited insulin-stimulated PI3K activity up to 75%, with an IC50 of 0.5 mu M, a result that further supports a proposed lipid-mediated inhibition of PI3K, Exogenously added didecanoyl-PA, however, did not affect PI3K activity. The possibility that stimulation of the PI 4-kinase-mediated signal transduction pathway leads to down-regulation of the PI3K-mediated signal transduction pathway in vivo, via inhibition of PI3K by CDP-DAG or by other consequences of phosphoinositidase C-linked receptor activation, is discussed.