title: Isoform-specific differences in the nitrite reductase activity of nitric oxide synthases under hypoxia.
abstract: Nitrite (NO(2)(-)) recycling to nitric oxide (NO) is catalysed by a number of enzymes and induces a protective vasodilation effect under hypoxia/ischaemia. In the present work, we tested the in vitro ability of the three NOS (nitric oxide synthase) 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 15 muM was specific to the endothelial NOS isoform (eNOS) and did not occur with the neuronal (nNOS) or inducible (iNOS) isoforms. Unlike xanthine oxidase, the eNOS reductase domain did not recycle nitrite to NO, and wild-type eNOS did not reduce nitrate. Our data suggest that structural and, by inference, dynamic differences between nNOS and eNOS in the distal haem side account for eNOS being the only isoform capable of converting nitrite into 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 approximately 3.6- and approximately 8.3-fold under hypoxia (2 p.p.m. O(2)) and anoxia (argon) respectively compared with normoxia ( approximately 22 p.p.m. O(2)) using 10 muM 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 micromolar 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 ischaemia and may explain the observed eNOS-expression-specific protective effect as a short-term response in animal models of acute hypoxia.
authors.affiliation: Laboratory for Optics and Biosciences, INSERM U696, CNRS UMR7645, Ecole Polytechnique, 91128 Palaiseau, France.
authors.forename: Ivan
authors.initials: I
authors.lastname: Mikula
authors.forename: Suzanne
authors.initials: S
authors.lastname: Durocher
authors.forename: Pavel
authors.initials: P
authors.lastname: Martasek
authors.forename: Bulent
authors.initials: B
authors.lastname: Mutus
authors.forename: Anny
authors.initials: A
authors.lastname: Slama-Schwok
mesh_terms: D042783:Endothelial Cells / Q000378:metabolism; D000860:Hypoxia / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D009572:Nitrite Reductases / Q000378:metabolism*; D009573:Nitrites / Q000494:pharmacology
chemical_list: D009573:Nitrites; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D009572:Nitrite Reductases