Receptor-mediated increases in phosphatidylinositol turnover in neuron-like cell lines

Muscarinic receptors found in the N1E-115 mouse neuroblastoma cell line were tested for their ability to mediate stimulation of phosphatidylinositol (PI) turnover. This study was facilitated by the development of a new solvent system (acetone:butanol:acetic acid:water, 3:5:1:1) for the rapid and consistent separation of PI by one-dimensional thin-layer chromatography. Cholinergic stimulation caused as much as a 680% increase in the incorporation of 32P into PI. Enhanced incorporation of 32P into PI could be measured as early as 4 min after stimulation began. By 20 min, the rate of incorporation by stimulated cells had decreased to that of unstimulated cells, indicating desensitization. The magnitude of the response was dependent on the extent of receptor occupancy, and the response elicited by a saturating dose of carbamylcholine was blocked completely by 10(-7) M atropine, a specific muscarinic antagonist. Chronic stimulation, known to cause a loss of receptor binding sites, led to a 90% decrease in the maximum response even after a 40-min withdrawal period. Replacement of Na+ ions in the medium with choline or K+ severely impaired the ability of the cells to incorporate added 32P into PI (90 and 50%, respectively). Removal of the putative second messenger Ca2+ for short periods of time by the addition of excess EGTA did not alter either basal or muscarinic-stimulated PI turnover.