Reactive oxygen and nitrogen species regulate inducible nitric oxide synthase function shifting the balance of nitric oxide and superoxide production

Inducible NOS (iNOS) is induced in diseases associated with inflammation and oxidative stress, and questions remain regarding its regulation. We demonstrate that reactive oxygen/nitrogen species (ROS/RNS) dose-dependently regulate iNOS function. Tetrahydrobiopterin (BH4)-replete iNOS was exposed to increasing concentrations of ROS/RNS and activity was measured with and without subsequent BH4 addition. Peroxynitrite (ONOO-) produced the greatest change in NO generation rate, similar to 95% decrease, and BH4 only partially restored this loss of activity. Superoxide (O-2(center dot-)) greatly decreased NO generation, however, BH4 addition restored this activity. Hydroxyl radical ((OH)-O-center dot) mildly decreases NO generation in a BH4-dependent manner. iNOS was resistant to H2O2 With only slightly decreased NO generation with up to millimolar concentrations. In contrast to the inhibition of NO generation, ROS enhanced O-2(center dot-) production from iNOS. while ONOO- had the opposite effect. Thus, ROS promote reversible iNOS uncoupling, while ONOO- induces irreversible enzyme inactivation and decreases both NO and O-2(center dot-) production. (C) 2009 Elsevier Inc. All rights reserved.