Muscarinic receptor activation of phosphatidylcholine hydrolysis. Relationship to phosphoinositide hydrolysis and diacylglycerol metabolism

• Published in: The Journal of biological chemistry 1989-09, Vol.264 (25), p.14748-14754
• Main Authors: MARTINSON, E. A, GOLDSTEIN, D, HELLER BROWN, J
• Format: Article
• Language: English
• Subjects: 550501 - Metabolism- Tracer Techniques; 560300 - Chemicals Metabolism & Toxicology; ACETYLCHOLINE; ALCOHOLS; ALKALINE EARTH METALS; AMINES; AMMONIUM COMPOUNDS; Analytical, structural and metabolic biochemistry; ANIMAL CELLS; ANIMALS; ARACHIDONIC ACID; Arachidonic Acids - metabolism; Astrocytoma; AUTONOMIC NERVOUS SYSTEM AGENTS; BASIC BIOLOGICAL SCIENCES; Biological and medical sciences; BIOLOGICAL EFFECTS; BIOSYNTHESIS; CALCIUM; Calcium - metabolism; Carbachol - pharmacology; CARBON 14 COMPOUNDS; CARBOXYLIC ACIDS; CARCINOGENS; Cell Line; CHOLINE; Choline - metabolism; Diglycerides - metabolism; DRUGS; ELEMENTS; ENZYME ACTIVITY; ENZYMES; ESTERS; Fundamental and applied biological sciences. Psychology; Glycerides - metabolism; GLYCEROL; Humans; HYDROGEN COMPOUNDS; Hydrolysis; HYDROXY COMPOUNDS; Intracellular Fluid - metabolism; ISOTOPE APPLICATIONS; LABELLED COMPOUNDS; LIPIDS; LIPOTROPIC FACTORS; MAMMALS; MAN; MEMBRANE PROTEINS; METABOLISM; METALS; Miscellaneous; MONOCARBOXYLIC ACIDS; Myristic Acid; Myristic Acids - metabolism; NEUROREGULATORS; ORGANIC ACIDS; ORGANIC COMPOUNDS; ORGANIC PHOSPHORUS COMPOUNDS; Other biological molecules; PARASYMPATHOMIMETICS; PHORBOL ESTERS; Phosphatidic Acids - metabolism; phosphatidylcholine; Phosphatidylcholines - metabolism; Phosphatidylinositols - metabolism; PHOSPHOLIPIDS; PHOSPHORUS-GROUP TRANSFERASES; PHOSPHOTRANSFERASES; PRIMATES; Protein Kinase C - metabolism; PROTEINS; QUATERNARY COMPOUNDS; RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT; RECEPTORS; Receptors, Muscarinic - drug effects; Receptors, Muscarinic - physiology; SYNTHESIS; Tetradecanoylphorbol Acetate - pharmacology; TRACER TECHNIQUES; TRANSFERASES; Tritium; TRITIUM COMPOUNDS; TUMOR CELLS; VERTEBRATES
• ISSN: 0021-9258; 1083-351X

We examined the relationship between phosphatidylcholine (PC) hydrolysis, phosphoinositide hydrolysis, and diacylglycerol (DAG) formation in response to muscarinic acetylcholine receptor (mAChR) stimulation in 1321N1 astrocytoma cells. Carbachol increases the release of [3H]choline and [3H]phosphorylcholine ([3H]Pchol) from cells containing [3H]choline-labeled PC. The production of Pchol is rapid and transient, while choline production continues for at least 30 min. mAChR-stimulated release of Pchol is reduced in cells that have been depleted of intracellular Ca2+ stores by ionomycin pretreatment, whereas choline release is unaffected by this pretreatment. Phorbol 12-myristate 13-acetate (PMA) increases the release of choline, but not Pchol, from 1321N1 cells, and down-regulation of protein kinase C blocks the ability of carbachol to stimulate choline production. Taken together, these results suggest that Ca2+ mobilization is involved in mAChR-mediated hydrolysis of PC by a phospholipase C, whereas protein kinase C activation is required for mAChR-stimulated hydrolysis of PC by a phospholipase D. Both carbachol and PMA rapidly increase the formation of [3H]phosphatidic acid ([3H]PA) in cells containing [3H]myristate-labeled PC. [3H]Diacylglycerol ([3H]DAG) levels increase more slowly, suggesting that the predominant pathway for PC hydrolysis is via phospholipase D. When cells are labeled with [3H]myristate and [14C]arachidonate such that there is a much greater 3H/14C ratio in PC compared with the phosphoinositides, the 3H/14C ratio in DAG and PA increases with PMA treatment but decreases in response to carbachol. By analyzing the increase in 3H versus 14C in DAG, we estimate that the DAG that is formed in response to PMA arises largely from PC. Muscarinic receptor activation also causes formation of DAG from PC, but approximately 20% of carbachol-stimulated DAG appears to arise from hydrolysis of the phosphoinositides.