Induction of inducible nitric oxide synthase increases the production of reactive oxygen species in RAW264.7 macrophages

Abstract

Macrophages produce a large volume of ROS (reactive oxygen species) through respiratory burst. However, the influence of iNOS [inducible NOS (nitric oxide synthase)] activation on ROS production remains unclear. In the present study, the kinetic generation of ROS in RAW264.7 murine macrophages was monitored by chemiluminescence. PMA induces a robust chemiluminescence in RAW264.7 cells, suggesting PKC (protein kinase C)-related assembly and activation of NOX (NADPH oxidase). The effects of iNOS induction on ROS production were examined. Induction of iNOS expression in RAW264.7 cells with LPS (lipopolysaccharide; 1 microg/ml) causes a significant increase in PMA-induced chemiluminescence, which could be enhanced by the NOS substrate, L-arginine, and could be abolished by the NOS inhibitor, L-NNA (NG-nitro-L-arginine). Further experiments reveal that induction of iNOS expression enhances the PMA-stimulated phosphorylation of the p47phox subunit of NOX, and promotes the relocalization of cytosolic p47phox and p67phox subunits to the membrane. Inhibition of PKCzeta by its myristoylated pseudosubstrate significantly decreased the PMA-stimulated phosphorylation of the p47phox in LPS-pretreated cells, suggesting that PKCzeta is involved in the iNOS-dependent assembly and activation of NOX. Taken together, the present study suggests that the induction of iNOS upregulates the PMA-induced assembly of NOX and leads to the enhanced production of ROS via a PKCzeta-dependent mechanism.