arachidonyltrifluoromethane and Granulomatous-Disease--Chronic

CD40 ligand (CD40L) expression on platelets is mediated by agonists, but the underlying mechanism is still unclear.. CD40L expression was measured in platelets from healthy subjects both with and without the addition of antioxidants or a phospholipase A2 (PLA2) inhibitor and in platelets from 2 patients with an inherited deficiency of gp91phox. Immunoprecipitation analysis was also performed to determine whether normal platelets showed gp91phox expression. Unlike catalase and mannitol, superoxide dismutase inhibited agonist-induced platelet CD40L expression in healthy subjects. Immunoprecipitation analysis also showed that platelets from healthy subjects expressed gp91phox. In 2 male patients with inherited gp91phox deficiency, collagen-, thrombin-, and arachidonic acid-stimulated platelets showed an almost complete absence of superoxide anion (O(2)(-)) and CD40L expression. Incubation of platelets from healthy subjects with a PLA2 inhibitor almost completely prevented agonist-induced O(2)(-) and CD40L expression.. These data provide the first evidence that platelet CD40L expression occurs via arachidonic acid-mediated gp91phox activation.

Topics: Adult; Antioxidants; Arachidonic Acid; Arachidonic Acids; Aspirin; Blood Platelets; Catalase; CD40 Ligand; Collagen; Granulomatous Disease, Chronic; Humans; Male; Mannitol; Membrane Glycoproteins; NADPH Oxidase 2; NADPH Oxidases; Phospholipases A; Phospholipases A2; Platelet Activation; Platelet Aggregation Inhibitors; Reactive Oxygen Species; Superoxide Dismutase; Superoxides; Thrombin

This investigation was undertaken to clarify the mechanisms of superoxide anion (O2-) generation in rat peritoneal mast cells. Compound 48/80, a typical histamine liberator mediated by calcium influx, elicited O2- generation from the mast cells in a dose-dependent fashion. It was demonstrated by immunohistochemical study and Western blot analysis that the mast cells contained the 47-kDa phagocyte oxidase (p47phox) protein, which was one cytosolic component of the NADPH oxidase system. Arachidonic acid stimulated O2- generation in the mast cells, but other unsaturated fatty acids had no effect. On the other hand, 48/80-induced O2- generation was inhibited by phospholipase A2 inhibitors, such as arachidonyl trifluoromethyl ketone and manoalide. Forskolin, isoprenaline, and dibutyryl cyclic AMP inhibited the O2- generation, and KT-5720, a cyclic AMP-dependent protein kinase (A-kinase) inhibitor, markedly enhanced the O2- generation. These findings suggest that O2- is generated by a NADPH oxidase-like enzyme system in mast cells and that this enzyme system is activated by arachidonic acid released by cytosolic phospholipase A2. Thus, it is regulated by the cyclic AMP-A kinase system.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Blotting, Western; Bucladesine; Carbazoles; Colforsin; Cyclic AMP-Dependent Protein Kinases; Granulomatous Disease, Chronic; Immunohistochemistry; Indoles; Isoproterenol; Male; Mast Cells; NADPH Oxidases; p-Methoxy-N-methylphenethylamine; Phosphoproteins; Protein Kinase C; Pyrroles; Rats; Rats, Wistar; Recombinant Proteins; Superoxides; Terpenes