title: Role of phospholipase D-derived diradylglycerol in the activation of the human neutrophil respiratory burst oxidase. Inhibition by phosphatidic acid phosphohydrolase inhibitors.
abstract: An agonist-activated phospholipase D/phosphatidic acid phosphohydrolase (PAH) pathway was recently demonstrated in human neutrophils, and evidence suggests that phosphatidic acid (PA) and/or diradylglycerol (DG) generated from this pathway participates in activation of the O2(-)-generating respiratory burst. We have used a series of cationic amphiphilic compounds (sphingosine, propranolol, chlorpromazine, and desipramine) and antibiotics (clindamycin, trimethoprim, and roxithromycin) all of which inhibit the respiratory burst, to investigate the role of the phospholipase D/PAH pathway in neutrophil activation. The phosphatidylcholine (PC) pool in intact cells was first labeled using [3H]-1-O-alkyl-lysoPC; released [3H]-PA and [3H]-DG were then quantified after the addition of either chemo-attractant or PMA. Using either agonist, all compounds showed a dose-dependent inhibition of [3H]-DG generation which correlated with inhibition of O2- generation, but compounds failed to inhibit directly the NADPH oxidase in a cell-free system. For either activator, a plot of the ID50 values for O2- generation vs those for DG generation was linear over four orders of magnitude. In many cases, inhibition of [3H]-DG generation corresponded to an increase in [3H]-PA, implicating PAH as the locus of inhibition. Superoxide generation was inhibited under conditions where PA was either elevated or minimally affected. Neither O2- release nor DG generation showed any selectivity for stereoisomers of propranolol, suggesting that this inhibition does not act via a specific binding site on PAH. No evidence was obtained for an effect of the inhibitors on PA mobility as monitored by electron spin resonance studies of spin-labeled PA in a model membrane system. Data are consistent with an effect of the inhibitors at the level of the interaction of PAH with the membrane and/or its substrate. These data imply that DG produced via the phospholipase D/PAH pathway functions in the activation or maintenance of the respiratory burst.
authors.affiliation: Department of Biochemistry, Emory University Medical School, Atlanta, GA 30322.
authors.forename: D K
authors.initials: DK
authors.lastname: Perry
authors.forename: W L
authors.initials: WL
authors.lastname: Hand
authors.forename: D E
authors.initials: DE
authors.lastname: Edmondson
authors.forename: J D
authors.initials: JD
authors.lastname: Lambeth
mesh_terms: D004075:Diglycerides / Q000502:physiology*; D004789:Enzyme Activation; D006801:Humans; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000502:physiology*; D010711:Phosphatidate Phosphatase / Q000037:antagonists & inhibitors / Q000502:physiology*; D010712:Phosphatidic Acids / Q000378:metabolism; D010739:Phospholipase D / Q000502:physiology*; D010766:Phosphorylation; D011433:Propranolol / Q000494:pharmacology; D016897:Respiratory Burst; D013481:Superoxides / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D004075:Diglycerides; D010712:Phosphatidic Acids; D013481:Superoxides; D009240:N-Formylmethionine Leucyl-Phenylalanine; C035561:diarachidonyl diglyceride; D011433:Propranolol; D010711:Phosphatidate Phosphatase; D010739:Phospholipase D; D013755:Tetradecanoylphorbol Acetate