title: An essential role for phospholipase D in the activation of protein kinase C and degranulation in mast cells.
abstract: Activation of phospholipase D (PLD) and protein kinase C (PKC) as well as calcium mobilization are essential signals for degranulation of mast cells. However, the exact role of PLD in degranulation remains undefined. In this study we have tested the hypothesis that the PLD product, phosphatidic acid, and diacylglycerides generated therefrom might promote activation of PKC. Studies were conducted in two rodent mast cell lines that were stimulated with Ag via FcepsilonRI and a pharmacologic agent, thapsigargin. Diversion of production of phosphatidic acid to phosphatidylbutanol (the transphosphatidylation reaction) by addition of l-butanol suppressed both the translocation of diacylglyceride-dependent isoforms of PKC to the membrane and degranulation. Tertiary-butanol, which is not a substrate for the transphosphatidylation, had a minimal effect on PKC translocation and degranulation, and 1-butanol itself had no effect on PKC translocation when PKC was stimulated directly with phorbol ester, 12-O-tetradecanoylphorbol-13-acetate. Also, in cells transfected with small inhibitory RNAs directed against PLD1 and PLD2, activation of PLD, generation of diacylglycerides, translocation of PKC, and degranulation were all suppressed. Phorbol ester, which did not stimulate degranulation by itself, restored degranulation when used in combination with thapsigargin whether PLD function was disrupted with 1-butanol or the small inhibitory RNAs. However, degranulation was not restored when cells were costimulated with Ag and phorbol ester. These results suggested that the production of phosphatidic acid by PLD facilitates activation of PKC and, in turn, degranulation, although additional PLD-dependent processes appear to be critical for Ag-mediated degranulation.
authors.affiliation: Laboratory of Molecular Immunology, National, Heart, Lung, and Blood Institute, National Institutes of Health, Department of Health and Human Services, Bethesda, MD 20892, USA.
authors.forename: Ze
authors.initials: Z
authors.lastname: Peng
authors.forename: Michael A
authors.initials: MA
authors.lastname: Beaven
mesh_terms: D020001:1-Butanol / Q000037:antagonists & inhibitors / Q000494:pharmacology; D000818:Animals; D015550:Cell Degranulation / Q000187:drug effects / Q000276:immunology*; D002461:Cell Line, Transformed; D045744:Cell Line, Tumor; D004075:Diglycerides / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D004357:Drug Synergism; D004789:Enzyme Activation / Q000187:drug effects / Q000276:immunology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D008407:Mast Cells / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D010712:Phosphatidic Acids / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D010739:Phospholipase D / Q000037:antagonists & inhibitors / Q000502:physiology*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects / Q000276:immunology; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology
chemical_list: D004075:Diglycerides; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D010712:Phosphatidic Acids; D034741:RNA, Small Interfering; D019284:Thapsigargin; D020001:1-Butanol; D011493:Protein Kinase C; D010739:Phospholipase D; D013755:Tetradecanoylphorbol Acetate