Dose dependent effects of reactive oxygen and nitrogen species on the function of neuronal nitric oxide synthase

Reactive nitrogen species (RNS) and oxygen species (ROS) have been reported to modulate the function of nitric oxide synthase (NOS); however, the precise dose-dependent effects of specific RNS and ROS on NOS function are unknown. Questions remain unanswered regarding whether pathophysiological levels of RNS and ROS alter NOS function, and if this alteration is reversible. We measured the effects of peroxynitrite (ONOO−), superoxide (O2·−), hydroxyl radical (.OH), and H2O2 on nNOS activity. The results showed that NO production was inhibited in a dose-dependent manner by all four oxidants, but only O2·− and ONOO− were inhibitory at pathophysiological concentrations (⩽50 μM). Subsequent addition of tetrahydrobiopterin (BH4) fully restored activity after O2·− exposure, while BH4 partially rescued the activity decrease induced by the other three oxidants. Furthermore, treatment with either ONOO− or O2·− stimulated nNOS uncoupling with decreased NO and enhanced O2·− generation. Thus, nNOS is reversibly uncoupled by O2·− (⩽50 μM), but irreversibly uncoupled and inactivated by ONOO−. Additionally, we observed that the mechanism by which oxidative stress alters nNOS activity involves not only BH4 oxidation, but also nNOS monomerization as well as possible degradation of the heme.