Superoxide generation from endothelial nitric-oxide synthase: A Ca²⁺/calmodulin-dependent and tetrahydrobiopterin regulatory process

It has been previously shown that besides synthesizing nitric oxide (NO), neuronal and inducible NO synthase (NOS) generates superoxide (O₂/^- ·) under conditions of L-arginine depletion. However, there is controversy regarding whether endothelial NOS (eNOS) can also produce O₂/^-·. Moreover, the mechanism and control of this process are not fully understood. Therefore, we performed electron paramagnetic resonance spin-trapping experiments to directly measure and characterize the O₂/^-· generation from purified eNOS. With the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), prominent signals of O₂/^-· adduct, DMPO-OOH, were detected from eNOS in the absence of added tetrahydrobiopterin (BH₄), and these were quenched by superoxide dismutase. This O₂/^-· formation required Ca²⁺/calmodulin and was blocked by the specific NOS inhibitor N-nitro-L-arginine methyl ester (L- NAME) but not its non-inhibitory enantiomer D-NAME. A parallel process of Ca²⁺/calmodulin-dependent NADPH oxidation was observed which was also inhibited by L-NAME but not D-NAME. Pretreatment of the enzyme with the heme blockers cyanide or imidazole also prevented O₂/^-· generation. BH₄ exerted dose-dependent inhibition of the O₂/^-· signals generated by eNOS. Conversely, in the absence of BH₄ L-arginine did not decrease this O₂/^-· generation. Thus, eNOS can also catalyze O₂/^-· formation, and this appears to occur primarily at the heme center of its oxygenase domain. O₂/^-· synthesis from eNOS requires Ca²⁺/calmodulin and is primarily regulated by BH₄ rather than L-arginine.