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) under conditions of L-arginine depletion. However, there is controversy regarding whether endothelial NOS (eNOS) can also produce O-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 O-2 generation from purified eNOS. With the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), prominent signals of O-2 adduct, DMPO-OOH, were detected from eNOS in the absence of added tetrahydrobiopterin (BH4), and these were quenched by superoxide dismutase. This O-2 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 O-2 generation. BH4 exerted dose-dependent inhibition of the O-2 signals generated by eNOS. Conversely, in the absence of BH4 L-arginine did not decrease this O-2 generation. Thus, eNOS can also catalyze O-2 formation, and this appears to occur primarily at the heme center of its oxygenase domain. O-2 synthesis from eNOS requires Ca2+/calmodulin and is primarily regulated by BH4 rather than L-arginine.
Xia
Y
Y
Molecular and Cellular Biophysics Laboratories, Department of Medicine, Division of Cardiology and the Electron Paramagnetic Resonance Center, The Johns Hopkins University School of Medicine, Baltimore, MD 21224, USA.
Tsai
A L
AL
Berka
V
V
Zweier
J L
JL
D001120:Arginine / Q000494:pharmacology; D001708:Biopterins / Q000031:analogs & derivatives / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000494:pharmacology; D003497:Cyclic N-Oxides / Q000032:analysis / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005609:Free Radicals / Q000032:analysis; D006801:Humans; D009249:NADP / Q000378:metabolism; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012966:Sodium Cyanide / Q000494:pharmacology; D013113:Spin Labels; D013237:Stereoisomerism; D013481:Superoxides / Q000378:metabolism*
D002147:Calmodulin; D003497:Cyclic N-Oxides; D004791:Enzyme Inhibitors; D005609:Free Radicals; D011994:Recombinant Proteins; D013113:Spin Labels; D013481:Superoxides; D001708:Biopterins; D009249:NADP; C017245:5,5-dimethyl-1-pyrroline-1-oxide; C055956:5,5-dimethyl-5-hydroperoxy-1-pyrrolidinyloxy; D001120:Arginine; D019001:Nitric Oxide Synthase; C003402:sapropterin; D012966:Sodium Cyanide; D002118:Calcium; D019331:NG-Nitroarginine Methyl Ester