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1 Molecular and Cellular Biophysics Laboratories and the Electron Paramagnetic Resonance Center, Cardiology Division, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21224; 2 Disciplina de Emergências Clínicas, Faculdade de Medicina da Universidade de São Paulo; and 3 Instituto do Coração, Faculdade de Medicina da Universidade de São Paulo, São Paulo 05403-000, Brazil
An NAD(P)H oxidase has
been hypothesized to be the main source of reactive oxygen species
(ROS) in vessels; however, questions remain about its function and
similarity with the neutrophil oxidase. Therefore, vascular superoxide
generation was measured by electron paramagnetic resonance spectroscopy
using the spin-trap 5,5'-dimethly-pyrroline-N-oxide in
aortas from wild-type (WT) and gp91phox-deficient mice
(gp91phox
/), which do not have a functioning neutrophil
NADPH oxidase. There was no significant difference between radical
adduct formation by WT or gp91phox/ mouse aortas either
at baseline or after stimulation with NADPH or NADH. Also, spin-adduct
formation was identical in the 100,000-g pellets obtained
from WT and gp91phox/ mouse aortas. SOD mimetics and
the flavoenzyme inhibitor diphenyleneiodonium blocked spin-adduct
formation from both intact vessels and particulate fractions. Other
pharmacological inhibitors of metabolic pathways involved in ROS
generation had no effect on this phenomenon. To examine the role of
this enzyme in vascular tone control, aortic rings were suspended in
organ chambers and preconstricted with phenylephrine to reach
half-maximal contraction. Exposure to NADPH elicited a 20% increase in
vascular tone, which was decreased by SOD mimetics in a
concentration-dependent manner, suggesting that superoxide was
responsible for this phenomenon. NADH had no effect on vascular tone.
Thus superoxide is generated in the vessel wall by an NAD(P)H-dependent
oxidase, which modulates vascular contractile tone. This enzyme is
structurally and genetically distinct from the neutrophil NADPH oxidase.
superoxide; electron paramagnetic resonance; reactive oxygen species; superoxide dismutase; NADH; NADPH; diphenyleneiodonium; gp91phox knockout mice
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