METABOLISM OF A LONG-CHAIN DIACYLGLYCEROL BY PERMEABILIZED A10 SMOOTH-MUSCLE CELLS

The regulatory effects of diacylglycerol (DAG) second messengers will be terminated by metabolism. A long-chain DAG, 1-palmitoyl-2-[1-C-14]o leoyl-sn-glycerol (2-[C-14]POG), was metabolized by cultured A10 smoot h muscle cells after permeabilization by preincubation with 340 U/ml a lpha-toxin from Staphylococcus aureus. In contrast to results with the cell-permeable DAG analogue. dioctanoylglycerol ([H-3]diC8), no appre ciabie 2-[C-14]POG degradation could be detected in control A10 cells not treated with alpha-toxin. With permeabilized A10 cells, 2-[C-14]PO G was mainly converted into lipolytic products of a lipase pathway, mo noacylglycerol (MG) and fatty acid (FA); very little radioactivity was incorporated into triacylglycerol (TG) or phospholipid (PL) via react ions catalyzed by either DAG acyltransferase, cholinephosphotransferas e, or DAG kinase. Similar results were obtained in experiments with 1- stearoyl-2-[1-C-14]arachidonoyl-sn-glycerol. The conversion of 2-[C-14 ]POG into PL and TG was not enhanced by the addition of 1 mM ATP-MgCl2 , 1 mM CDP-choline, or 1 mM oleoyl-CoA to the alpha-toxin-treated A10 cells. The formation of FA and MG by permeabilized A10 cells was inhib ited by DAG lipase inhibitors, U-57,908 (50 muM) and tetrahydrolipstat in (1-25 nM). The predominant contribution of the lipase pathway to th e metabolism of a long-chain DAG, 2-[C-14]POG, by alpha-toxin-treated A10 cells is similar to results for the degradation of [H-3]diC8 by in tact A10 cells.