Metabolic fate of exogenous diacylglycerols in A10 smooth muscle cells

The metabolic fate of exogenous diacylglycerols, l-palmitoyl-2-[l -C-14]oleoyl-sn-glycerol (2-[C-14]POG) and 1-stearoyl-2-[1-C-14]arachidonoyl-sn-glycerol (2-[C-14]SAG), was determined after incubation of A10 smooth muscle cells with liposomal suspensions. Hydrolysis through a diacylglycerol (DG) lipase pathway was the predominant metabolic fate; more than 80% of cell-associated radioactivity from 2-[C-14]POG and 2-[C-14]SAG was recovered in lipolytic products, monoacylglycerol (MG) and fatty acids (FA), which were present in the incubation medium. Hydrolysis of 2-[C-14]POG was reduced completely by tetrahydrolipstatin, a lipase inhibitor. Very little radioactivity from either 2-[C-14]POG or 2-[C-14]SAG was incorporated into triacylglycerol or phospholipids. DG lipase and kinase activities were measured by in vitro enzyme assays. l-[l-C-14]Palmitoyl-2-oleoyl-sn-glycerol (1-[C-14]POG) was phosphorylated (kinase activity) to a greater extent than 2[C-14]SAG in assays with both soluble and particulate subcellular fractions from A10 cells. DG lipase activity (hydrolysis of l-[C-14]POG and 2-[C-14]SAG) was markedly stimulated by the addition of 20mM MgCl2 and 20mM ATP to the assay. Under optimal assay conditions, DG lipase activity exhibited little substrate specificity. Our findings indicate that exogenous DG are mainly hydrolyzed by DG and MG lipases in A10 smooth muscle cells; as a result, signalling mechanisms responding to DG second messengers will be attenuated. (C) 1998 Elsevier Science B.V.