DIACYLGLYCEROL METABOLISM IN SM-3 SMOOTH-MUSCLE CELLS

The metabolism of endogenous and exogenous diacylglycerol (DG) was stu died in SM-3 cells derived from rabbit aortic smooth muscle to determi ne the biochemical step(s) responsible for degrading DG second messeng ers. Incubation of growth-arrested SM-3 cells with [H-3]myristate for 2 h resulted in selective labelling of cellular phosphatidylcholine (P C). Addition of bacterial PC-specific phospholipase C (PC-PLC, 20 U) t o [H-3]myristate-labelled SM-3 cells resulted in a 7.4-fold increase i n endogenous radiolabelled DG and activation of the epsilon isoform of protein kinase C. Subsequent incubation of PC-PLC-treated SM-3 cells resulted in the incorporation of radioactivity primarily into products of a lipase pathway, monoacylglycerol and fatty acid. The hydrolysis of endogenous PC-derived DG was inhibited by 0.25 mu M tetrahydrolipst atin, a DG lipase inhibitor. Similar results were obtained when SM-3 c ells were incubated with 1,2-dioctanoyl-[2-H-3]glycerol, a cell-permea ble DG analog; radioactivity was mainly recovered in the glycerol prod uct of the lipase pathway. Therefore, hydrolysis by a lipase pathway r epresents the principal metabolic fate of both endogenous and exogenou s DG in SM-3 smooth muscle cells.