Peroxynitrite modulates release of inflammatory mediators from guinea pig lung mast cells activated by...

Peroxynitrite (ONOO-), the product of the reaction between the superoxide anion (*O2-) and nitric oxide (NO), is produced during inflammatory disease and may be a major cytotoxic agent. No reports are available as to whether ONOO- generates or modulates inflammatory mediator release from activated guinea pig lung mast cells. In this study, we explored the modulatory role of intracellular ONOO- on inflammatory mediator release (histamine and leukotrienes) from activated mast cells.

Guinea pig lung mast cells were purified by the enzyme digestion, and by using the rough and discontinuous Percoll density gradients. Mast cells were sensitized with IgG1 (anti-ovalbumin) antibody and challenged with ovalbumin (OVA). The intracellular ROS formation was determined by following the oxidative production of 2', 7'-dichlorofluorescein diacetate (DCFH-DA), dihydrorhodamine 123 (DHR), and anti-nitrotyrosine antibody immunofluorescence. Histamine was assayed using a fluorometric analyzer, leukotrienes by radioimmunoassay, intracellular Ca2+ levels by confocal scanning microscopy, and PLA(2) activity using prelabeling of [3H]arachidonic acid.

ROS detected by DCFH-DA weakly increased in mast cells activated with OVA (1.0 g/ml), and the ROS so generated was inhibited by ebselen (50 microM). However, the ROS detected by DHR increased 3-fold under the same conditions. Peroxynitrite scavengers sL-MT, DMTU, and inhibitor FeTPPS inhibited ROS formation but the NADPH oxidase inhibitor diphenyleneiodonium (DPI) only partially inhibited this formation. Dimethyl thiourea (DMTU) and seleno-L-methionine (sL-MT) inhibited the tyrosine nitration of cytosolic proteins, the release of histamine and leukotrienes, Ca2+ influx, and the PLA(2) activity evoked by mast cell activation.

The data obtained suggests that the ROS generated by the antigen/antibody reaction activated mast cells is ONOO-, and that this modulates the release of inflammatory mediators via Ca2+ -dependent PLA(2) activity.