Nitrite (NO2−) 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 μM 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 ∼3.6- and ∼8.3-fold under hypoxia (2 p.p.m. O2) and anoxia (argon) respectively compared with normoxia (∼22 p.p.m. 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 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.
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Research Article|
February 25 2009
Isoform-specific differences in the nitrite reductase activity of nitric oxide synthases under hypoxia
Ivan Mikula;
Ivan Mikula
1
*Laboratory for Optics and Biosciences, INSERM U696, CNRS UMR7645, Ecole Polytechnique, 91128 Palaiseau, France
†Department of Pediatrics and Center for Genomics, First School of Medicine, Charles University of Prague, Prague, Czech Republic
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Suzanne Durocher;
Suzanne Durocher
1
‡Department of Chemistry and Biochemistry, University of Windsor, 401 Sunset Avenue, Windsor, ON, Canada, N9B 3P4
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Pavel Martasek;
Pavel Martasek
†Department of Pediatrics and Center for Genomics, First School of Medicine, Charles University of Prague, Prague, Czech Republic
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Bulent Mutus;
Bulent Mutus
‡Department of Chemistry and Biochemistry, University of Windsor, 401 Sunset Avenue, Windsor, ON, Canada, N9B 3P4
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Anny Slama-Schwok
Anny Slama-Schwok
2
*Laboratory for Optics and Biosciences, INSERM U696, CNRS UMR7645, Ecole Polytechnique, 91128 Palaiseau, France
§Virology et Immunologie Moleculaires, INRA UR892, Domaine de Vilvert, 78350 Jouy en Josas, France
2To whom correspondence should be addressed (email Anny.Schwok@jouy.inra.fr).
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Publisher: Portland Press Ltd
Received:
May 16 2008
Revision Received:
November 03 2008
Accepted:
December 01 2008
Accepted Manuscript online:
December 01 2008
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2009 Biochemical Society
2009
Biochem J (2009) 418 (3): 673–682.
Article history
Received:
May 16 2008
Revision Received:
November 03 2008
Accepted:
December 01 2008
Accepted Manuscript online:
December 01 2008
Citation
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. Biochem J 15 March 2009; 418 (3): 673–682. doi: https://doi.org/10.1042/BJ20080987
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