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 (O-2(radical anion)) under conditions of L-arginine depletion. However, there is controversy regarding whether endothelial NOS (eNOS) can also produce O-2(radical anion). 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 O-2(radical anion) generation from purified eNOS. With the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), prominent signals of O-2(radical anion) adduct, DMPO-OOH, were detected from eNOS in the absence of added tetrahydrobiopterin (BH4), and these were quenched by superoxide dismutase. This O-2(radical anion) 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 n-NAME. Pretreatment of the enzyme with the heme blockers cyanide or imidazole also prevented O-2(radical anion) generation. BH4 exerted dose-dependent inhibition of the O-2(radical anion) signals generated by eNOS. Conversely, in the absence of BH, L-arginine did not decrease this OX generation. Thus, eNOS can also catalyze OX formation, and this appears to occur primarily at the heme center of its oxygenase domain. O-2(radical anion) synthesis from eNOS requires Ca2+/calmodulin and is primarily regulated by BH, rather than L-arginine.