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1 Molecular and Cellular Biophysics Laboratories, Cardiology Division, Department of Medicine, and the Electron Paramagnetic Resonance Center, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21224; 2 Disciplina de Emergências Clínicas and 3 Instituto do Coração, Faculdade de Medicina da Universidade de São Paulo, São Paulo 01246-903, Brazil
In
vascular tissues, an NAD(P)H oxidase is the main source of superoxide;
however, there has been much uncertainty regarding its activity and the
levels of superoxide it generates. This problem has limited overall
progress in this field. Therefore, studies were performed and
techniques developed to quantitatively assess the function of the
vascular NAD(P)H oxidase, measuring its rate of superoxide production
and substrate consumption in rat aortic homogenates and intact
segments. NADPH/NADH oxidation was measured spectrophotometrically, and
oxygen consumption was measured by electrochemical probe. Superoxide
was detected and quantitated by electron paramagnetic resonance spin
trapping. Under basal conditions, superoxide generation and oxygen
consumption were negligible. After addition of NADPH or NADH (0.1 mM),
superoxide was generated at rates of 0.41 ± 0.03 or 0.36 ± 0.04 nmol · mg protein
1 · min1, respectively.
Oxygen was consumed with a similar time course at rates of 1.5 ± 0.2 or 1.3 ± 0.3 nmol · mg
protein1 · min1, and NADPH or NADH
were oxidized at rates of 1.8 ± 0.4 and 1.5 ± 0.3 nmol · mg protein1 · min1,
respectively. In intact aortic rings, superoxide was generated with
rates of 4.0 ± 0.7 or 3.7 ± 0.7 pmol · mg
tissue1 · min1, whereas oxygen was
consumed at rates of 22.1 ± 5.0 or 14.5 ± 3.3 pmol · mg tissue1 · min1,
for NADPH or NADH, respectively. These values are lower than those
previously measured using lucigenin, which uncouples flavoenzymes, triggering additional superoxide generation. This quantitative approach
for characterization of the vascular NAD(P)H oxidase activity should
facilitate the further identification and cellular characterization of
this enzyme(s) and its functional and signaling roles.
vascular oxidase; free radicals; electron paramagnetic resonance; spin trap; 5,5'-dimethyl-pyrroline-N-oxide; lucigenin
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