Endothelial nitric oxide synthase catalyzes superoxide generation

We have previous showed that besides synthesizing NO. neuronal and inducible NO synthase (NOS) generate Superoxide in L-arginine-depleted cells. However, il is not known if endothelial NOS (eNOS) also generates superoxide. It is also not clear how this process is regulated. Therefore, we directly measured and characterized superoxide generation from purified eNOS using electron paramagnetic resonance spin-trapping techniques. Recombinant human eNOS was purified from a barulovirus expression system by affinity chromatography. With the spin trap 5.5-dirnethyI-1-pyrroline-N-oxide, prominent superoxide signal-4 were detected from eNOS and these we.re parallel to marked NADPH oxidation. Superoxide, dismutase quenched these signals. This superoxide formation required Ca-4+/calmodulin and was more than 90% blocked by the specific NOS inhibitor N-nitro-L-arginine methyl ester (L-NAMK. 1 mM). but not by its non-inhibitory enantiome.r D-NAMK. Pre-treatmcnt of the enzyme with the homo blocker cyanide also prevented superoxide generation. L-arginine did not decrease the superoxide formation from eNOS. Conversely, tetrahydrobiopterin dose-dependently inhibited this superoxide generation. Thus. eNOS can also catalyze superoxide formation and this appears to primarily occur at the home center of its oxygenase domain. Superoxide synthesis from eNOS requires ("a2+/calmodulin and is triggered and controlled by depletion of ti'tralivdrobiopterin rather than I.-arg'inine.