Superoxide Generation from Endothelial Nitric-oxide Synthase

Abstract

It has been previously shown that besides synthesizing nitric oxide (NO), neuronal and inducible NO synthase (NOS) generates superoxide (OG¿¬2) under conditions ofl-arginine depletion. However, there is controversy regarding whether endothelial NOS (eNOS) can also produce OG¿¬2. 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 OG¿¬2 generation from purified eNOS. With the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), prominent signals of OG¿¬2 adduct, DMPO-OOH, were detected from eNOS in the absence of added tetrahydrobiopterin (BH4), and these were quenched by superoxide dismutase. This OG¿¬2formation required Ca2+/calmodulin and was blocked by the specific NOS inhibitor N-nitro-l-arginine methyl ester (l-NAME) but not its non-inhibitory enantiomerd-NAME. A parallel process of Ca2+/calmodulin-dependent NADPH oxidation was observed which was also inhibited by l-NAME but notd-NAME. Pretreatment of the enzyme with the heme blockers cyanide or imidazole also prevented OG¿¬2 generation. BH4 exerted dose-dependent inhibition of the OG¿¬2 signals generated by eNOS. Conversely, in the absence of BH4 l-arginine did not decrease this OG¿¬2 generation. Thus, eNOS can also catalyze OG¿¬2formation, and this appears to occur primarily at the heme center of its oxygenase domain. OG¿¬2 synthesis from eNOS requires Ca2+/calmodulin and is primarily regulated by BH4 rather than l-arginine