1-palmitoyl-2-oleoyl-sn-glycerol and 1-stearoyl-2-arachidonoylglycerol

Though most of the studies have focused on the effects of free fatty acids on T-cell activation, fatty acids incorporated into plasma membrane phospholipids may also affect cell signaling via diacylglycerol (DAG), generally produced by phospholipid hydrolysis. In the present study, we have synthesized a DAG-containing oleic acid and studied its implication in the modulation of calcium signaling in human Jurkat T-cells. 1-palmitoyl-2-oleoyl-sn-glycerol (POG) induced a dose-dependent increase in [Ca(2+)](i). This effect was due to the presence of oleic acid at the sn-2 position as no differences were observed between POG and 1-stearoly-2-oleoyl-sn-glycerol (SOG). However, the substitution of oleic acid with arachidonic acid at the sn-2 position of the DAG moiety exerted a different response on the increases in [Ca(2+)](i) in these cells. POG-evoked increases in [Ca(2+)](i) were not due to its metabolites. Furthermore, POG-induced increases in [Ca(2+)](i) were due to the opening of TRPC3/TRPC6 channels as silencing of TRPC3 and TRPC6 genes by shRNA abolished calcium entry. Moreover, disruption of lipid rafts with methyl-β-cyclodextrin completely abolished POG-evoked increases in [Ca(2+)](i). In conclusion, our results demonstrate that oleic acid can influence T-lymphocyte functions, in the conjugated form of DAG, via opening TRPC3/6 channels.

Topics: beta-Cyclodextrins; Calcium; Calcium Signaling; Caveolae; Diglycerides; Dose-Response Relationship, Drug; Gene Expression; Humans; Ion Transport; Jurkat Cells; Membrane Microdomains; Microscopy, Fluorescence; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; T-Lymphocytes; TRPC Cation Channels; TRPC6 Cation Channel

The regulatory effects of diacylglycerol (DAG) second messengers will be terminated by metabolism. A long-chain DAG, 1-palmitoyl-2-[1-14C]oleoyl-sn-glycerol (2-[14C]POG), was metabolized by cultured A10 smooth muscle cells after permeabilization by preincubation with 340 U/ml alpha-toxin from Staphylococcus aureus. In contrast to results with the cell-permeable DAG analogue, dioctanoyl-glycerol ([3H]diC8), no appreciable 2-[14C]POG degradation could be detected in control A10 cells not treated with alpha-toxin. With permeabilized A10 cells, 2-[14C]POG was mainly converted into lipolytic products of a lipase pathway, monoacylglycerol (MG) and fatty acid (FA); very little radioactivity was incorporated into triacylglycerol (TG) or phospholipid (PL) via reactions catalyzed by either DAG acyltransferase, cholinephosphotransferase, or DAG kinase. Similar results were obtained in experiments with 1-stearoyl-2-[1-14C]arachidonoyl-sn-glycerol. The conversion of 2-[14C]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 inhibited by DAG lipase inhibitors, U-57,908 (50 microM) and tetrahydrolipstatin (1-25 nM). The predominant contribution of the lipase pathway to the metabolism of a long-chain DAG, 2-[14C]POG, by alpha-toxin-treated A10 cells is similar to results for the degradation of [3H]diC8 by intact A10 cells.

Topics: Animals; Cell Line; Cell Membrane Permeability; Cyclohexanones; Diglycerides; Lactones; Lipoprotein Lipase; Muscle, Smooth, Vascular; Orlistat