Stimulation of Hydrolysis of Phosphatidic Acid by Cholinergic Agents in Guinea Pig Synaptosomes

Light nerve endings from guinea pig cerebral cortex were incubated with [32P]orthophosphate, myo[3H]inositol, [3H]glycerol, or [3H]glucose in the presence or absence of cholinergic agents. Acetylcholine and carbamylcholine increased incorporation of 32Pi into phosphatidic acid 100% and into various classes of phosphatidylinositol 20 to 70%. These agents also stimulated the labeling of phosphatidylinositol from myo[3H]inositol but did not affect the labeling of either phosphatidylinositol or phosphatidic acid from [3H]glycerol or from [3H]glucose. The specific activity of synaptosomal [γ-32P]ATP was unchanged. In a series of two-step incubations, lipids were first labeled with 32Pi, then excess unlabeled Pi or 2,4-dinitrophenol was added to lower the specific activity or the amount of [γ32P]-ATP. The addition of acetylcholine stimulated the rate of disappearance of [32P]phosphatidic acid by 80%. This effect of acetylcholine was blocked by atropine. We conclude that cholinergic agents increase phospholipid labeling by increasing the hydrolysis of phosphatidate.