The Mechanism of Muscarinic Receptor‐Stimulated Phosphatidylinositol Resynthesis in 1321N1 Astrocytoma Cells and Its Inhibition by Li

: The coupling of muscarinic receptor‐stimulated phosphatidylinositol 4,5‐bisphosphate hydrolysis by phospholipase C to resynthesis of phosphatidylinositol (PtdIns) and the ability of Li+ to inhibit this after cellular inositol depletion were studied in 1321N1 astrocytoma cells cultured in medium ± inositol (40 µM). In inositol‐replete cells, 1 mM carbachol/10 mM LiCl evoked an initial (0–30 min) ∼≥20‐fold activation of phospholipase C, whereas prolonged (>60 min) stimulation turned over Ptdlns equal to the cellular total mass, involving ∼80% of the cellular Ptdlns pool without reducing PtdIns concentrations significantly. PtdIns resynthesis was achieved by a similar, initial agonist activation of PtdIns synthase. The dose dependency for carbachol stimulation of PtdIns synthase and phospholipase C was similar (EC50∼ 20 µM) as was the relative intrinsic activity of muscarinic receptor partial agonists. This demonstrates the tight coupling of phosphoinositide hydrolysis to resynthesis and suggests this is achieved by a direct mechanism. In inositol‐replete or depleted cells basal concentrations of inositol and CMP‐phosphatidate were respectively ∼20 mM or ≤100–500 µM and ∼0.1 or ∼≥1–10 pmol/mg of protein. Comparison of the effects of agonist ± Li+ on the concentrations of these cosubstrates for PtdIns synthase suggest that accelerated activity of this enzyme is differentially driven by stimulated increases in the amounts of CMP‐phosphatidate or inositol in inositol‐replete or depleted cells, respectively. Thus, the preferential capacity of Li+ to impair stimulated phosphoinositide turnover in systems expressing low cellular inositol can be attributed to its ability to attenuate the stimulated rise in inositol concentrations on which such systems selectively depend to trigger accelerated PtdIns resynthesis.