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, ∼95% decrease, and BH₄ only partially restored this loss of activity. Superoxide (O2-) greatly decreased NO generation, however, BH₄ addition restored this activity. Hydroxyl radical (OH) 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 O₂^.- 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 O₂^.- production.