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 phospha tidylinositol (Ptdlns) and the ability of Li+ to inhibit this after ce llular inositol depletion were studied in 1321N1 astrocytoma cells cul tured in medium +/- inositol (40 mu M). In inositolreplete cells, 1 mM carbachol/10 mM LiCl evoked an initial (0-30 min) similar to greater than or equal to 20-fold activation of phospholipase C, whereas prolon ged (>60 min) stimulation turned over Ptdlns equal to the cellular tot al mass, involving similar to 80% of the cellular Ptdlns pool without reducing Ptdlns concentrations significantly. Ptdlns resynthesis was a chieved by a similar, initial agonist activation of Ptdlns synthase. T he dose dependency for carbachol stimulation of Ptdlns synthase and ph ospholipase C was similar (EC(50) similar to 20 mu M) as was the relat ive intrinsic activity of muscarinic receptor partial agonists. This d emonstrates the tight coupling of phosphoinositide hydrolysis to resyn thesis and suggests this is achieved by a direct mechanism. In inosito l-replete or depleted cells basal concentrations of inositol and CMP-p hosphatidate were respectively similar to 20 mM or less than or equal to 100-500 mu M and similar to 0.1 or similar to greater than or equal to 1-10 pmol/mg of protein. Comparison of the effects of agonist +/- Li+ on the concentrations of these cosubstrates for Ptdlns synthase su ggest that accelerated activity of this enzyme is differentially drive n by stimulated increases in the amounts of CMP-phosphatidate or inosi tol in inositol-replete or depleted cells, respectively. Thus, the pre ferential capacity of Li+ to impair stimulated phosphoinositide turnov er in systems expressing low cellular inositol can be attributed to it s ability to attenuate the stimulated rise in inositol concentrations on which such systems selectively depend to trigger accelerated Ptdlns resynthesis.