The mechanisms of compound 48/80-induced superoxide generation mediated by A-kinase in rat peritoneal mast cells

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 dosedependent 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 0₂/^- 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.