Diacylglycerol metabolism in SM-3 smooth muscle cells

The metabolism of endogenous and exogenous diacylglycerol (DG) was studied in SM-3 cells derived from rabbit aortic smooth muscle to determine the biochemical step(s) responsible for degrading DG second messengers. Incubation of growth-arrested SM-3 cells with [3H]myristate for 2 h resulted in selective labelling of cellular phosphatidylcholine (PC). Addition of bacterial PC-specific phospholipase C (PC-PLC, 20 U) to [3H]myristate-labelled SM-3 cells resulted in a 7.4-fold increase in 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 microM tetrahydrolipstatin, a DG lipase inhibitor. Similar results were obtained when SM-3 cells were incubated with 1,2-dioctanoyl-[2-3H]glycerol, a cell-permeable DG analog; radioactivity was mainly recovered in the glycerol product of the lipase pathway. Therefore, hydrolysis by a lipase pathway represents the principal metabolic fate of both endogenous and exogenous DG in SM-3 smooth muscle cells.