D. Yang et al
Oxygen-derived free radicals and EDCF
109
or deferoxamine could not gain access to the site of
production of oxygen-derived free radicals. Superoxide anion
is a primary oxygen-derived free radical, it can be dismutated
to form hydrogen peroxide by superoxide dismutase. Metal-
catalyzed interaction between hydrogen peroxide and super-
oxide anion produces hydroxyl radical (Fridovich, 1986). The
vascular level of superoxide anion is determined by the
superoxide dismutase activity and the amount of nitric oxide
released by endothelial cells (Huie & Padmaja, 1993; Oury et
al., 1996). Since the present experiments were performed in
the presence of an inhibitor of nitric oxide synthase,
inactivation of superoxide anion by nitric oxide can be ruled
out. An increased production of superoxide anions should
result in an increase in hydrogen peroxide and hydroxyl
radicals and favour the occurrence of endothelium-dependent
contractions as observed in present study and earlier work
(Katusic & Vanhoutte, 1989). Inhibition of the endogenous
Cu/Zn superoxide dismutase activity with diethyldithiocarba-
mic acid should reduce the formation of hydrogen peroxides
and hydroxyl radicals. Tiron should eectively scavenge
intracellular superoxide anions resulting in decreased genera-
tion of hydrogen peroxides and hydroxyl radicals in the
vascular wall. In the presence of diethyldithiocarbamic acid,
the increased level of intracellular superoxide anion, resulting
from the inhibition of endogenous Cu/Zn superoxide
Furthermore, the chronic treatment with dimethylthiourea
unmasked an inhibitory eect of superoxide dismutase plus
catalase or deferoxamine on the reduced endothelium-
dependent contractions to acetylcholine. These ®ndings
strongly support the interpretation that oxygen-derived free
radicals contribute to endothelium-dependent contractions in
the SHR aorta and that hydroxyl radicals are involved. Since
the in vitro administration of dimethylthiourea does not
decrease endothelium-dependent contractions to acetylcholine
in aortas from untreated control SHR, the attenuation of the
response in blood vessels from treated animals can be
attributed to a decreased generation of hydroxyl radicals in
vivo. Likewise, the unmasking of an inhibitory eect of
superoxide dismutase plus catalase or deferoxamine on
endothelium-dependent contractions in dimethylthiourea-
treated SHR indicates that the chronic limitation of the
production of hydroxyl radicals allows an eective scaven-
ging by superoxide dismutase plus catalase or deferoxamine.
Like that to acetylcholine, the responses to oxygen-derived
free radicals generated by xanthine plus xanthine oxidase in
aortas without endothelium from dimethylthiourea-treated
SHR were reduced by the chronic treatment. This probably
re¯ects a diminished hypersensitivity of the hypertensive
smooth muscle exposed to exaggerated oxidative stress. This
decrease in sensitivity of the smooth muscle to oxygen-
derived free radicals following the chronic treatment with
dimethylthiourea, parallels a similar eect on the response to
acetylcholine, and this supports the role of oxygen-derived
free radicals in endothelium-dependent contractions in the
hypertensive aorta. The chronic inhibition of the production
of oxygen-derived free radicals in vivo could result in
inhibition of endothelium-dependent contractions because of
either a decreased synthesis of EDCF and/or a reduced
sensitivity to endothelial mediator.
dismutase activity (Heikkila
& Cohen, 1977), was not
associated with an increase in endothelium-dependent con-
tractions. This indicates that superoxide anions per se are not
sucient to evoke contractions, but that they must be
dismutated ®rst into hydroxyl radicals. This then indirectly
con®rms the role of hydroxyl radicals in endothelium-
dependent contractions. The lack of full inhibition of
endothelium-dependent contractions with either diethyldithio-
carbamic acid or Tiron is explained best by their incomplete
intracellular action. Indeed, for example, in canine basilar
arteries, even higher concentration of diethyldithiocarbamic
acid only inhibits half of the total vascular superoxide
dismutase activity (Wambi-Kiesse & Katusic, 1999).
To curtail the presence of oxygen-derived free radicals in
the aorta from SHR, the animals were treated chronically
with dimethylthiourea at a dose known to scavenge hydroxyl
radicals in vivo and to limit their production (Mayhan &
Patel, 1998; Pieper et al., 1996). The endothelium-dependent
contractions to acetylcholine in the treated SHR were
decreased signi®cantly compared with untreated animals.
In summary, the present study suggests that reactive
oxygen species, and in particular hydroxyl radicals and/or
hydrogen peroxide, play a key role in endothelium-dependent
contractions to acetylcholine in the aorta of the sponta-
neously hypertensive rat.
This study was supported by an Educational grant from the
Institut de Recherches Servier, France and by a grant from the
Education Administration of Jiangsu Province, China.
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