Involvement of cytosolic phospholipase A(2), and the subsequent release ofarachidonic acid, in signalling by Rac for the generation of intracellularreactive oxygen species in Rat-2 fibroblasts

Although there have been a number of recent studies on the role of Rac in t he generation of reactive oxygen species (ROS), details of the signalling p athway remain unclear. In the present study we analysed the extent to which the activation of cytosolic phospholipase A(2) and the resultant release o f arachidonic acid (AA) are involved in the Rac-mediated generation of ROS. Transfection of Rat-2 cells with RacV12, a constitutively active form of R ad, induced elevated levels of ROS, as reflected by increased H2O2- sensiti ve fluorescence of 2',7'-dichlorofluorescein. These effects could be blocke d by inhibiting phospholipase A(2) or 5-lipoxygenase but not by inhibiting cyclo-oxygenase. The application of exogenous AA increased levels of ROS bu t the effect was dependent on the further metabolism of AA to leukotrienes C-4/D-4/E-4 by 5-lipoxygenase. Indeed, the exogenous application of a mixtu re of leukotrienes C-4/D-4/E-4 elicited transient elevations in the levels of ROS that were blocked by catalase. These findings indicate that phosphol ipase A(2) and subsequent AA metabolism by 5-lipoxygenase act as downstream mediators in a Rac signalling pathway leading to the generation of ROS.