Isoform-specific differences in the nitrite reductase activity of nitric oxide synthases under hypoxia

Ivan Mikula , Suzanne Durocher , Pavel Martasek , Bulent Mutus , Anny Slama-Schwok (1)

1 VIM (UR 0892) - Unité de recherche Virologie et Immunologie Moléculaires

Nitrite (NO2-) recycling to nitric oxide (NO) is catalyzed by a number of enzymes and induces a protective vasodilatory effect under hypoxia/ ischemia. In the present work, we tested the in vitro ability of the three NOS isoforms to release NO from nitrite under anoxia using electrochemical detection, chemiluminescence and absorption spectroscopy. The release of free NO from anoxic nitrite solutions at 15µM was specific to the endothelial isoform eNOS and did not occur with neuronal nNOS and inducible iNOS. Unlike xanthine oxidase, eNOS reductase domain did not recycle nitrite to NO and WT eNOS did not reduce nitrate. Our data suggest that structural—and by inference—dynamic differences between nNOS and eNOS in the distal heme domains account for eNOS being the only isoform capable of converting nitrite to NO at pH=7.6. In human dermal microvascular endothelial cells under careful control of oxygen tension, the rates of NO formation determined by chemiluminescence were enhanced ~3.6-fold and ~8.3-fold under hypoxia (2ppm O2) and anoxia (argon) respectively compared to normoxia (~22ppm O2) using 10 µM extracellular nitrite. NOS inhibitors inhibited this hypoxic NO release. Our data show that eNOS is unique in that it releases NO under all oxygen levels from normoxia to complete anoxia at physiological µM nitrite concentrations. The magnitude of the hypoxic NO release by the endothelial cells suggest that the endothelium could provide an appropriate response to acute episodic ischemia and may explain the observed eNOS-expression specific protective effect as a short-term response in animal models of acute hypoxia.

Mots clés: Life Sciences

Ivan Mikula, Suzanne Durocher, Pavel Martasek, Bulent Mutus, Anny Slama-Schwok. Isoform-specific differences in the nitrite reductase activity of nitric oxide synthases under hypoxia. Biochemical Journal, 2009, 418 (3), pp.673-682. ⟨10.1042/BJ20080987⟩. ⟨hal-00479026⟩