Original article 741
Enhanced superoxide anion formation in vascular tissues
from spontaneously hypertensive and desoxycorticosterone
acetate-salt hypertensive rats
Rong Wua , Esther Milletteb , Lingyun Wua and Jacques de Champlaina
Objectives To investigate the basal and NADH-stimulated
superoxide ( . O2 2 ) production and inactivation by Cu/Zn
superoxide dismutase (SOD) in aorta from spontaneously
hypertensive rats (SHR) and from desoxycorticosterone
acetate (DOCA)-salt hypertensive (DOCA-HT) rats.
Methods Tissue . O2 2 levels were estimated with the
lucigenin-enhanced chemiluminescence method in aorta
and cultured smooth muscle cells (SMCs) from SHR and in
aorta from DOCA-HT rats treated for 4 weeks.
Results The basal aortic . O2 2 generation was increased
by 135 and 100%, and the NADH stimulated . O2 2
production was also increased 37 and 22% in SHR and in
DOCA-HT rats compared to their normotensive controls,
respectively. Although no difference existed in blood
pressure as well as in basal and in NADH stimulated . O2 2
production between Wistar±Kyoto (WKY) rats and SHR
rats at age of 6 weeks, O2 2 production and blood pressure
increased concomitantly in SHR aged 9 and 12 weeks.
Basal and NADH-stimulated . O2 2 production, in cultured
SMCs, was also 80 and 64% higher, respectively, in SHR
compared to WKY rats. The NADH oxidase activity was
found to be increased in aorta from both SHR and DOCA-
HT rats but SOD activity was reduced only in aorta from
DOCA-HT rats.
Conclusions An enhanced . O2 2 formation resulting from
Introduction
Increasing evidences suggest that oxidative stress may
contribute to vascular diseases, such as hypertension
and atherosclerosis, by decreasing the availability of
NO, by promoting vascular smooth muscle prolifera-
tion, and by inducing neutrophils in®ltration [1]. Oxida-
tive stress may result from either excessive production
of reactive oxygen species, especially the superoxide
anion ( . O2 ÿ ), or from reduced antioxidant levels.
Several studies have demonstrated that in normotensive
animals, the membrane-bound NADH/NADPH oxi-
dase pathway accounts for the majority of vascular
. O2 ÿ generation [2], while the Cu/Zn superoxide
dismutase (Cu/Zn SOD) contributes the most in
scavenging the vascular generated non-mitochondrial
. O2 ÿ [3,4]. In hypertensive patients, decreased con-
centrations of antioxidants and SOD activity have been
0263-6352 & 2001 Lippincott Williams & Wilkins
an increased NADH oxidase activity was found in aorta
from SHR and DOCA-HT rats. Cultured arterial SMCs from
SHR also generated excessive . O2 2 formation under
basal and stimulated conditions. The age-related increase
in vascular . O2 2 formation in association with the rise in
blood pressure in SHR suggests that the oxidative stress
might contribute to the development of hypertension.
NADH oxidase activity was greater in aorta of both
hypertension models, but a decrease of Cu/Zn SOD
activity could also contribute to the high level of aortic
. O2 2 in DOCA-HT rats. J Hypertens 19:741±748 & 2001
Lippincott Williams & Wilkins.
Journal of Hypertension 2001, 19:741±748
Keywords: superoxide anion, NADH oxidase, superoxide dismutase,
spontaneously hypertensive rats, DOCA-salt hypertension
a
Research Group on Autonomic Nervous System, Department of Physiology,
Faculty of Medicine and b Faculty of Pharmacy, University of Montreal, Montreal,
Quebec, Canada.
Sponsorship: This research was supported by a grant from the Medical Research
Council of Canada. J.de C is the holder of an associateship from the J.C. Edward
Foundation, E.M is the holder of FCAR-FRSQ studentship, L.W is the holder of a
studentship of the Heart and Stroke Foundation of Canada.
Correspondence and requests for reprints to Jacques de Champlain, Department
of Physiology, Faculty of Medicine, University of Montreal, CP 6128, Succursale
Centre-ville, MontreÂal, QueÂbec H3C 3J7, Canada.
Tel: ‡1514 3437562; fax: ‡1514 3432257;
e-mail: grsna@ere.umontreal.ca
Received 21 July 2000 Revised 11 December 2000
Accepted 13 December 2000
documented [5,6]. Increased . O2 ÿ generation and
reduced NO production were also reported in neutro-
phils and platelet from essential hypertensive patients
[7]. Moreover, Hamilton et al. [8] have reported that an
in-vitro treatment with SOD potentiated the NO-
dependent relaxation in human thoracic artery and
saphenous vein.
In animal studies, arterial tissue . O2 ÿ levels were
reported to be increased in several hypertensive mod-
els, including spontaneously hypertensive rats (SHR)
and spontaneously hypertensive stroke-prone rats
(SHRSP) [9,10], angiotensin II-induced hypertension
[11,12,13], aortic banding-induced hypertension [14]
and Dahl salt-sensitive hypertensive rats [15]. Various
kinds of vessel cells, such as the endothelium [16],
smooth muscles cells [17], adventitial ®broblasts [18] or
742 Journal of Hypertension 2001, Vol 19 No 4
the migratory in¯ammatory cells [19] were reported to
contribute to the vascular . O2 ÿ production in the
diseased vessel, but the relative role of each of those
speci®c cells in the production of vessel . O2 ÿ in
hypertension remains unclear. Although the membrane-
bound NADH oxidase pathway has been demonstrated
to be the major source of vascular . O2 ÿ production [2],
the only direct evidence supporting the critical role of
this enzyme in . O2 ÿ generation in hypertension comes
from the angiotensin II infusion-induced hypertensive
model [12,13], where the chronic angiotensin II infu-
sion was found to increase the NAD(P)H oxidase
activity and the biosynthesis of some components of
this enzyme in smooth muscle cells (SMCs) and in
vessel ®broblasts. Several studies have also suggested
that the decrease of endogenous Cu/Zn SOD activity
might be implicated in the high level of vascular . O2 ÿ
[4,20]. Ito et al. [21] have reported that, compared to
Wistar±Kyoto (WKY) rats, the myocardium Cu/Zn
SOD activity was decreased and the tissue . O2 ÿ level
was elevated in SHR hypertrophied heart.
The present study was designed to measure . O2 ÿ
production in whole aorta and cultured aortic SMCs
and to investigate the relative role of . O2 ÿ production
and/or inactivation in controlling the aortic . O2 ÿ level
in the genetic (SHR) and the acquired desoxycorticos-
terone acetate (DOCA)-salt (DOCA-HT) models of
hypertension in rat.
Materials and methods
Animals
Studies were performed in male rats for the two models
of experimental hypertension: (i) spontaneously hyper-
tensive rats (SHR) and their age-matched control WKY
rats; (ii) DOCA-salt hypertensive rats and their intact-
control (intact) or uninephrectomized control (Sham)
normotensive Sprague±Dawley rats. The SHR and
WKY rats (Charles River Laboratories, St Constant,
Quebec, Canada) were studied at the age of 6, 9 and
12 weeks. Systolic blood pressure was measured by an
indirect tail cuff plethysmography, 1 day before the
study. DOCA-salt hypertension was induced in male
Sprague±Dawley rats weighing 110±125 g (aged 7
weeks) as described previously [22]. Brie¯y, the rats
were ®rst uninephrectomized under pentobarbital
anaesthesia (80 mg/kg intraperitoneally). After 3 days of
recovery, randomly selected uninephrectomized rats
were distributed to DOCA-salt-treated group or Sham-
treated group. DOCA-salt hypertensive rats received
weekly subcutaneous injection of deoxycorticosterone
acetate (10 mg/rat per week) for 4 weeks and were
given free access of 0.9% saline in drinking water
(DOCA-HT group), while the Sham-treated rats were
free of drug treatment and were given tap water to
drink (Sham group). The intact-control rats did not
undergo surgical ablation of one kidney and were given
tap water to drink (intact group). The systolic blood
pressure was measured with tail cuff method at least
three times before and at the end of the treatment. At
the end of the treatment, the rats were sacri®ced by
decapitation after light anaesthesia with CO2 and the
thoracic aorta was quickly excised and immersed in ice-
cold Krebs±Hepes buffer solution containing (mmol=l):
NaCl 99.01, KCl 4.69, CaCl2 1.87, MgSO4 1.20,
K2 HPO4 1.03, NaHCO3 25.0, Na-Hepes 20.0, glucose
11.1 (saturated with 95% O2 and 5% CO2 , pH 7.4). The
aortic periadventitial tissue was carefully removed and
the aorta was cut into 2 mm ring segments. The ring
segments were washed at least three times with Krebs-
Hepes buffer before any . O2 ÿ measurement.
Aortic SMC culture
Aortic SMCs from 12-week-old SHR and WKY rats
were isolated and cultured as described previously [23].
Brie¯y, rat aortas were isolated and connective tissues
as well as endothelium were removed. The vessel was
cut and enzymatically digested with collagenase-dis-
pase, elastase and collagenase in a stepwise manner.
The dispersed cells were plated in the tissue culture
¯asks and cultured in Dulbecco's modi®ed Eagle's
medium containing 10% fetal calf serum in a CO2
incubator at 378C. Cultured SMCs were passed once a
week by harvesting with trypsin treatment and splitting
at a ratio of 1 : 4. The medium was changed twice
weekly. Cultured cells between passages 10 and 12
were used in the present study. SMCs were identi®ed
by the fact that their á-actin was positively stained with
mouse anti-á-actin antibody and anti-mouse immuno-
globulin G ¯uorescent isothiocyanate conjugate. More
than 90% of cultured cells expressed á-actin in the
present study. The SMCs scraped mechanically were
washed twice with centrifugation and the concentration
of the cells was estimated by a hemacytometer. Our
preliminary experiments did not show any signi®cant
differences in the . O2 ÿ production among passages
10±12 in those cultured SMCs.
Superoxide anion measurement
The superoxide anion production was measured using
the lucigenin-enhanced chemiluminescence method as
described previously [24,25]. Brie¯y, after vessel pre-
paration, a 2 mm ring segment (approximately 2±4 mg)
was placed into the Krebs-Hepes buffer (saturated with
95% O2 and 5% CO2 , at room temperature). For
enzyme inhibiting experiment, a speci®c inhibitor was
added to this buffer. After equilibration for 10 min, the
aortic ring segment was transferred to a scintillation vial
containing 250 ìmol=l lucigenin in a total volume of
2 ml of Krebs-Hepes buffer for determining the basal
. O2 ÿ level. NADH (100 ìmol=l) was added into the
vial to evaluate the NADH-stimulated . O2 ÿ genera-
tion. Various enzymatic inhibitors were also added to
the scintillation vial to investigate the corresponding
 Enhanced ´O2ÿ formation in HT rats Wu et al. 743

enzyme's activity. For cultured SMCs, approximately Fig. 1

106 cells were added into the counting vials. The
chemiluminescence was recorded every minute for (a) Basal
15 min by a liquid scintillation counter (Wallac 1409, 12
* **
Turku, Finland) switched to the out-of-coincidence **
mode. The respective background was subtracted from 10

c.p.m. 3 103 per mg tissue

total count. At the end of the chemiluminescence
measurement, the fresh aortic ring was weighed and 8
total SMC protein was determined with the Lowry's
method [26]. The superoxide generation was expressed 6
as counts 3 103 per min/mg fresh tissue (c.p.m. 3 103
per mg tissue) for aortic ring or c.p.m. per ìg protein 4
for cultured SMCs.
2
Drugs
Lucigenin, NADH, diphenylene iodonium (DPI), 0
diethyldithiocarbamic acid (DDC), were purchased WKY SHR Intact Sham DOCA
from Sigma (St Louis, Missouri, USA). All drug solu-
tions were prepared fresh before each experiment. The (b) Stimulated
*
total drug volume added into the test tube was less 350 *
than 1% to avoid the possible buffer dilution. DPI was *
initially prepared in pure dimethyl sulfoxide and then
c.p.m. 3 103 per mg tissue
300
diluted to the needed concentration in water. All other
drugs were prepared in distilled water. 250

200
Statistical analysis
Data are expressed as mean  SEM. Statistical com- 150
parisons were made by Student's t-test or by one-way
ANOVA followed by Tukey HSD analysis. P , 0.05 100
was considered statistically signi®cant.
50

Results
Systolic blood pressure in 12-week-old SHR and in DOCA-
salt-treated rats
The systolic blood pressure was 136  2 mmHg and
206  5 mmHg in 12-week-old WKY rats and SHR,
respectively (P , 0.01). In Sprague±Dawley rats, the
average blood pressure before surgery or treatment was
103  1 mmHg for all three groups. At the end of
4 weeks treatment, the systolic blood pressure was
118  1 mmHg for intact, 119  2 for Sham and
194  2 mmHg for DOCA-HT rats (P , 0.01 for
DOCA-HT versus intact and Sham groups).
Basal and NADH-activated superoxide anion production in
aorta of control and hypertensive animals
In 12-week-old WKY rats, the aortic basal . O2 ÿ level
was 4.0  0.7 counts 3 103 per min/mg fresh tissue
(c.p.m. 3 103 per mg tissue, n ˆ 10), but was increased
by 135% to 9.4  1.5 c.p.m. 3 103 per mg tissue in 12-
week-old SHR rats (n ˆ 10, P , 0.05 versus WKY rats)
(Fig. 1a). In the DOCA-salt hypertensive model, the
basal . O2 ÿ levels were 4.0  0.3 (n ˆ 10) and 3.6  0.3
(n ˆ 11) c.p.m. 3 103 per mg tissue for the intact and
Sham normotensive rats, respectively, but was in-
creased to 7.2  0.6 c.p.m. 3 103 per mg tissue in
0
WKY SHR Intact Sham DOCA
(a) Basal superoxide production in aortic rings from normotensive and
hypertensive rats. Superoxide production measured with the lucigenin
(250 ìmol=l)-enhanced chemiluminescence method was signi®cantly
increased in both hypertensive rats [spontaneously hypertensive rats
(SHR) and desoxycorticosterone acetate-salt hypertensive (DOCA-HT)
rats]. (b) NADH (100 ìmol=l)-stimulated superoxide production in
aortic rings. The stimulated superoxide production was signi®cantly
increased in SHR, DOCA-HT and Sham normotensive rats suggesting
an enhanced NADH oxidase activity in the aorta from those animals.
WKY, Wistar±Kyoto rats. *P , 0.05, **P , 0.01.
hypertensive rats (n ˆ 16, P , 0.01 versus both intact
and Sham) (Fig. 1a).
To determine the membrane-bound NADH oxidase
activity, the NADH (100 ìmol=l)-stimulated . O2 ÿ
production was evaluated in aortic rings from the
normotensive and hypertensive rats. In SHR rats, the
NADH-stimulated . O2 ÿ production was increased by
36.6% to 309.1  27.6 from the control value of 226.3 
19.1 c.p.m. 3 103 per mg tissue in WKY rats (P , 0.05)
(Fig. 1b). In intact rats, the stimulated . O2 ÿ produc-
tion was 254.6  8.4 c.p.m. 3 103 per mg tissue, but
was increased to 297.0  21.6 in Sham (increased
744 Journal of Hypertension 2001, Vol 19 No 4

16.6%, P , 0.05 versus intact) and to 310.7  Fig. 2

19.6 c.p.m. 3 103 per mg tissue in DOCA-HT rats
(increased 22.0%, P , 0.05 versus intact) (Fig. 1b). 220 (a) BP **

Age-related changes in superoxide anion production and 200

**
blood pressure in SHR
180
The average systolic blood pressures were 102  1
(n ˆ 6) and 103  1 (n ˆ 6) mmHg in WKY rats and

BP (mmHg)
160
SHR at 6 weeks, respectively. Thereafter, SHR blood
pressure increased in an age-dependent manner to 140
reach 185  1 mmHg (n ˆ 6) at 9 weeks and 206 
5 mmHg (n ˆ 10) at 12 weeks while age-matched WKY 120
rat blood pressure increased only to 134  2 (n ˆ 6) and
WKY
136  2 (n ˆ 10) mmHg, respectively, at 9 and 100
SHR
12 weeks (P , 0.01 versus SHR) (Fig. 2a). Correspond-
80
ingly, at the age of 6 weeks, the aortic basal and 6 9 12
NADH-stimulated . O2 ÿ levels were similar in WKY Age of animals (weeks)
rats (n ˆ 6) and SHR (n ˆ 6) (basal: 2.5  0.2 and
2.6  0.2 (Fig. 2b); NADH-stimulated: 234.0  7.2 and 12 (b) Basal *
234.2  10.9 (Fig. 2c) c.p.m. 3 103 per mg tissue for
WKY rats and SHR, respectively). Compared to age- 10 WKY
matched WKY rats, SHR basal and NADH-stimulated c.p.m. 3 103 per mg tissue
SHR
. O2 ÿ levels were increased by 67.9 and 43.4%, respec- 8
tively, at 9 weeks (n ˆ 6, P , 0.05), and by 135.0 and *
36.6% at 12 weeks (n ˆ 10, P , 0.05 SHR versus WKY 6
rats) (Fig. 2b,c). Regression analysis of the blood pres-
sure and basal . O2 ÿ level in all aged WKY rats and
4

SHR showed that it exist a signi®cant linear relation- 2

ship between these two parameters with r ˆ 0.68
(P , 0.001). 0
6-week 9-week 12-week
Basal and stimulated superoxide formation in cultured
aortic smooth muscle cells from WKY rats and SHR 350 (c) Stimulated
* *
The cultured aortic SMCs from 12-week-old WKY rats
300
and SHR were used in this study. The basal and
c.p.m. 3 103 per mg tissue

NADH-stimulated . O2 ÿ productions which were 250

39.1  6.8 and 15235  3119 c.p.m. per ìg protein,
200
respectively, for SMCs from WKY rats (n ˆ 5), were
increased by 80% to 70.2  4.0 and by 64% to 150
24935  4421 c.p.m. per ìg protein in SMCs from SHR
(n ˆ 5, P , 0.001 SHR versus WKY rats) (Fig. 3). 100

Effects of NADH oxidase or superoxide dismutase
inhibitors on aortic superoxide level
NADH-oxidase inhibitor
The relative contribution of the NADH oxidase path-
way to the basal . O2 ÿ production was evaluated with
its selective inhibitor, DPI (100 ìmol=l) (Fig. 4a).
Previous studies have reported that the IC50 or Ki for
DPI to inhibit NADH oxidase are between 0.9 and
5.6 ìmol=l [27]; therefore, the concentration of DPI
used in this experiment was approximately 100 times
higher than its IC50 . The incubation with DPI lowered
the basal tissue . O2 ÿ level by ±89.1  2.3% in SHR
(n ˆ 5) which was signi®cantly greater than the de-
crease of ±74.6  4.2% observed in WKY rats (n ˆ 5,
P , 0.05). Similarly, the DPI-induced tissue . O2 ÿ
50
0
6-week 9-week 12-week
Age-dependent superoxide production and blood pressure (BP)
changes in Wistar±Kyoto (WKY) rats and spontaneously hypertensive
rats (SHR) rats. (a) Blood pressure levels at different ages in WKY rats
and SHR. Blood pressure which was identical for WKY rats and SHR
at age 6 weeks was increased in an age-dependent manner in SHR,
which was signi®cantly higher than their age-matched WKY rats at age
of 9 and 12 weeks. (b) Basal aortic superoxide production in different
age of rats. Basal aortic superoxide levels, which were similar in 6-
week-old WKY rats and SHR, also increased in an age-dependent
manner, and were signi®cantly higher in SHR than in their age-matched
WKY rats at 9 and 12 weeks. (c) NADH-stimulated superoxide
production which was similar at 6 weeks between the two groups of
rats became signi®cantly higher in 9- and 12-week-old SHR than in
their age-matched WKY rats. *P , 0.05, **P , 0.01.
 Enhanced ´O2ÿ formation in HT rats Wu et al. 745

Fig. 3 Fig. 4

** ** 30000 (a) NADH-oxidase inhibition

70 WKY SHR Intact Sham DOCA
0
25000
60

Basal O22 change (%)

c.p.m. per µg protein

c.p.m. per µg protein

220
50 20000

40 240
15000
30 260
10000
20
280
5000
10
**
0 0 * **
WKY SHR WKY SHR
Basal NADH-stimulated **
(b) SOD inhibition
* *
Basal and NADH-stimulated superoxide production in cultured aortic
smooth muscle cells. The basal and the stimulated superoxide 120
production were both signi®cantly increased in cells from

Basal O22 change (%)

spontaneously hypertensive rats (SHR) when compared with the cells 100
from Wistar±Kyoto (WKY) rats. **P , 0.01.
80

60

40
lowering was also signi®cantly greater in DOCA-HT
20
rats (±86.2  2.3%, n ˆ 7) than in intact (±70.1  0.7%,
n ˆ 6, P , 0.01) and Sham rats (±59.9  5.6%, n ˆ 5, 0
P , 0.001). WKY SHR Intact Sham DOCA

Effect of diphenylene iodonium (DPI) (100 ìmol=l), an NADH-oxidase

Superoxide dismutase inhibitor
The contribution of the activity of Cu/Zn SOD to the
basal aortic tissue . O2 ÿ level in normotensive and
hypertensive rats was also evaluated by using the Cu/
Zn SOD speci®c inhibitor, DDC (100 ìmol=l) (Fig. 4b).
In the presence of the DDC, the basal . O2 ÿ produc-
tions were similarly increased by 35.2 and 28.7% from
their control values of 4.3  0.1 and 8.0  0.3
c.p.m. 3 103 per mg tissue in aortic ring from WKY rats
(n ˆ 5) and SHR (n ˆ 5), respectively. In the DOCA-
HT experimental model, an increase of 99.4  28.8%
was observed in aortic rings from normotensive Sham
rats (n ˆ 5), and an increase of 44.7  5.1% was ob-
served in intact rats (n ˆ 6) following DDC, whereas
this treatment induced only an increase of 12.3  2.7%
in the basal . O2 ÿ production in aortic rings from
DOCA-HT rats (n ˆ 6) (P , 0.05, Sham versus intact
and DOCA-HT, P , 0.01 DOCA-HT versus intact).
Discussion
The major ®ndings of this study are as follows: (i) aortic
basal levels of . O2 ÿ as well as NADH-stimulated
. O2 ÿ formation were increased in tissues from both
SHR and DOCA-HT rats; (ii) similar increases in basal
and NADH-stimulated . O2 ÿ generation were observed
in cultured aortic SMCs from SHR; (iii) aortic basal
. O2 ÿ level, NADH-stimulated . O2 ÿ generation and
the systolic blood pressure increased concomitantly in
inhibitor and diethyldithiocarbamic acid (DDC) (100 ìmol=l), an
superoxide dismutase (SOD) inhibitor, on the aortic basal superoxide
production. (a) Effect of DPI on the basal superoxide production. DPI
induced signi®cantly more important decrease in basal superoxide
production in both spontaneously hypertensive rats (SHR) and
desoxycorticosterone acetate-salt hypertensive (DOCA-HT) rats than in
their normotensive control animals. (b) Effect of DDC on basal
superoxide level. DDC induced a similar increased in aortic basal
superoxide level in Wistar±Kyoto (WKY) rats and SHR, but a
signi®cantly higher increase was observed in Sham and a signi®cantly
lower increase was observed in DOCA-HT rats compared to intact-
control rats. *P , 0.05, **P , 0.01.
an age-dependent manner in 6-, 9- and 12-week-old
SHR; and (iv) an enhanced NADH oxidase activity
appears to contribute to the increased . O2 ÿ generation
in the aorta in both experimental models of hyper-
tension, while a decrease in the Cu/Zn SOD activity
appears to contribute to the enhanced . O2 ÿ formation
only in DOCA-HT rats.
The present study showed that the aortic tissue basal
. O2 ÿ levels were increased approximately two-fold in
SHR and DOCA-HT rats, and that an increased
NADH oxidase activity was a major contributor for this
excess . O2 ÿ formation. In previous studies, a similar
enhancement of basal . O2 ÿ generation was also re-
ported in arterioles from SHR [10] and in the aorta
from SHRSP rats [9]. However, at variance from our
746 Journal of Hypertension 2001, Vol 19 No 4
study, the glucocorticoid-dependent xanthine oxidase
and the endothelial NO synthase were suggested to be
the main mechanisms contributing to the excess . O2 ÿ
generation in SHR and in SHRSP, respectively.
It is well known that, in mammalian cells, the . O2 ÿ
generating NADH oxidase is located in plasma mem-
brane and NADH can not penetrate through cell
membrane. In the present study, the basal . O2 ÿ was
measured in absence of added exogenous NADH. The
DPI inhibition results (see below) demonstrated that
this basal . O2 ÿ was mainly generated by NADH
oxidase. Differently, the NADH stimulated . O2 ÿ
generation was determined by adding a high concentra-
tion of exogenous NADH (100 ìmol=l), which in-
creased the . O2 ÿ production by aortic rings and by
intact cultured SMCs approximately 40-fold and 400-
fold, respectively. Similar results were also reported
previously in intact cultured ®broblasts [28,29] and
mesangial cells [30] as well as in intact vessel ring [31].
An explanation for these observations could be the
presence of an outer membrane active site for NADH
oxidase or the presence of a transport of reducing
equivalents into cells, resulting in an indirect increase
of intracellular NADH concentration. The present data
indicate that the activity of this external NADH
dependent . O2 ÿ generating system is high and is
signi®cantly enhanced in the two hypertensive models.
This might suggest that this system plays a role in
vascular regulation and might be implicated in the
physiopathology of hypertension. However, the real
physiological function of this . O2 ÿ generating system
is still unknown.
The mechanisms modulating the NADH oxidase activ-
ity in hypertensive rats remain unclear. Previous studies
have demonstrated that angiotensin II exerts a stimu-
lating effect on NADH oxidase by increasing its
biosynthesis in aorta [32], in cultured vascular SMCs
[17] and in vascular ®broblasts [18]. Several studies
have demonstrated that, although the circulating renin
and angiotensin concentrations in SHR and DOCA-salt
hypertensive rats are normal or subnormal, the vascular
local renin±angiotensin system (RAS) was reported to
be activated [33±35], and the renal angiotensin II AT1
receptors were found to be upregulated in SHR and in
DOCA-salt hypertensive rats [36]. Therefore, the local
activated RAS in SHR and DOCA-salt hypertensive
rats might be involved in the higher levels of . O2 ÿ
production observed in aortic rings from SHR or
DOCA-salt hypertensive rats, as well as in arterial
smooth muscle cells from SHR [37], but further studies
with an angiotensin II antagonist are needed to con®rm
this hypothesis.
An interesting ®nding in the present study is that the
NADH oxidase activity was equally increased in Sham
normotensive rats. This result suggests that the in-
crease of the aortic NADH oxidase activity is indepen-
dent of the rise of the blood pressure. This ®nding is in
keeping with previous reports that, although infusion of
angiotensin II and noradrenaline (NA) induced a simi-
lar degree of hypertension, the aortic superoxide pro-
duction was only increased in angiotensin II infused
but not in noradrenaline infused rats [12]. The en-
hanced NADH oxidase activity observed in normoten-
sive Sham but uninephrectomized rats might be a
consequence of the nephrectomy. In fact, nephrectomy
has been reported to increase vascular angiotensin II
release despite the reduction in circulating renin and
angiotensin II [38,39].
Another interesting ®nding in the present study was
that the basal and NADH-stimulated vascular . O2 ÿ
production in young SHR (6 weeks) was identical to
that of age-matched WKY rats. Thereafter, the vascular
. O2 ÿ level and the NADH oxidase activity in SHR
increased in an age-dependent manner in concordance
with the development of an elevated blood pressure.
Regression analysis also showed a linear relationship
between blood pressure and basal aortic . O2 ÿ level.
Our present results from Sham animal and cultured
SMCs, as well as the previous data from angiotensin II
and NA induced hypertension in rats [12] and from
cultured endothelial cells [40], indicated that the vascu-
lar . O2 ÿ production is independent of the blood
pressure. These observations thus support the potential
role of enhanced vascular . O2 ÿ level in the develop-
ment of hypertension in the SHR model. However, the
possibilities that an increase in . O2 ÿ may be secondary
to an increase in blood pressure is not excluded and
requires further study.
The cellular and enzymatic sources of the excess
generated . O2 ÿ and the importance of those sources
for vascular oxidative stress in hypertension are still
debated. Several studies have demonstrated that angio-
tensin II stimulated the vascular . O2 ÿ formation by
SMCs through the activation of the NADH oxidase
pathway [17,12], while others have reported that the
aortic adventitial ®broblasts and their NADH oxidase
were the main source of vascular . O2 ÿ production in
the SHR hypertension model [18,13]. Kerr et al. [9]
have reported that, in spontaneously hypertensive
stroke-prone rats (SHRSP), the endothelium and its
NO synthase pathway were the main sources of the
excess vascular . O2 ÿ generation. The present study
showed that the . O2 ÿ level in vascular SMCs from
SHR was increased by a similar magnitude to that
observed in whole aortic ring from the same animals,
and that the increase in basal . O2 ÿ level in SMCs was
also accompanied by an enhancement of the NADH
oxidase activity. These results thus suggest that vascu-
lar SMCs might contribute, at least in part, to the high

level of vascular . O2 ÿ formation in SHR, possibly
through an increase in NADH oxidase activity. This
postulate needs to be further proved in whole vessels
with microhistological and immunological techniques,
since cultured SMCs often undergo dedifferentiation
and change their phenotype. Although the exact con-
sequence of this dedifferentiation on . O2 ÿ generation
is not clear, extrapolation of our results from cultured
SMCs to whole animals should be made with caution.
In our previous study, it was shown that . O2 ÿ stimu-
lated inositol 1,4,5-triphosphate formation and de-
creased the cGMP level in cultured aortic SMCs, and
that those effects were potentiated in cells from SHR
[41]. Taken together, these results thus suggest that
the higher levels of . O2 ÿ could modify the function of
cultured SMCs and their responsiveness to vasomotory
factors.
The relative contribution of the NADH oxidase path-
way to the aortic basal . O2 ÿ tissue level was evaluated
by studying the effect of a selective inhibitor of this
enzyme, DPI, which at a concentration of 100 ìmol=l
should completely inhibit the NADH oxidase activity
[27]. The degree of decrease in the tissue . O2 ÿ
formation induced by DPI re¯ected the relative con-
tribution of this enzyme to the basal formation of
. O2 ÿ . Our results showed that the NADH oxidase-
produced . O2 ÿ was the major aortic source of . O2 ÿ in
aortic tissues from hypertensive and normotensive rats,
but that some other minor pathways could also be
involved in the aortic basal . O2 ÿ generation. This
result is in agreement with previous reports showing
that the NADH oxidase pathway is a major source of
. O2 ÿ in arterial SMC and endothelium [2]. In addi-
tion, our results showed that, in both SHR and DOCA-
HT rats, the aortic . O2 ÿ generation was decreased by
more than 85% by DPI treatment, which was signi®-
cantly higher than the decrease observed in their
control normotensive animals (WKY rats, 75%; intact or
Sham control, 60±70%) suggesting that the contribution
of NADH oxidase activity in the aorta of hypertensive
rats in both models was relatively increased. These
®ndings indicate that the increased spontaneous . O2 ÿ
production in the two hypertensive models is mainly
generated by the enhanced activity of the NADH
oxidase pathway.
Superoxide dismutase, one of the major cellular antiox-
idant enzymes, catalyses the reaction of . O2 ÿ with an
electron and two protons to form H2 O2 . Three mamma-
lian SODs have been identi®ed: cytosolic Cu/Zn SOD,
extracellular SOD and mitochondrial Mn SOD. The
cytosolic and extracellular SODs are the major non-
mitochondrial regulator for vascular . O2 ÿ level [3,4],
and they both contain one copper and one zinc atom in
their molecule, so that they are sensitive to the copper
chelator DDC [42]. When the Cu/Zn SOD is comple-
Enhanced ´O2ÿ formation in HT rats Wu et al. 747
tely inhibited, such as in the present study using a
treatment with DDC, the degree of enhancement in
tissue . O2 ÿ levels re¯ects the tissue initial Cu/Zn
SOD activity. In the present study, DDC produced a
similar increase of basal aortic . O2 ÿ levels in WKY rats
and SHR suggesting no difference in Cu/Zn SOD
activity between these two strains. On the other hand,
DDC induced a much higher increase (99%) in the
basal . O2 ÿ level of Sham normotensive rats than in
intact-control (45%) suggesting that the SOD enzy-
matic activity is elevated in Sham rats. This elevated
SOD activity might represent a compensatory mechan-
ism counteracting the increase in . O2 ÿ production
associated with the enhanced activity of NADH oxi-
dase, thus maintaining the basal aortic tissue . O2 ÿ
levels within the normal range in those animals. In
contrast, in DOCA-HT rats, DDC induced a signi®-
cant, but smaller increase in . O2 ÿ level (only 12%)
than that in intact or Sham animals indicating that the
aortic Cu/Zn SOD activity in DOCA-HT rats was
markedly reduced. Therefore, in DOCA-HT rats, both
an enhanced . O2 ÿ production by NADH oxidase and
a reduced . O2 ÿ inactivation by Cu/Zn SOD, may
contribute to the higher basal aortic . O2 ÿ levels. The
explanation for the variation in Cu/Zn SOD activity
changes in the present study remains unknown.
In conclusion, our results indicate that an enhanced
formation of aortic . O2 ÿ occurs in both SHR and
DOCA-HT models of hypertension in rats. Arterial
SMC generated excessive . O2 ÿ formation might con-
tribute to the oxidative stress in SHR, and it is
suggested that a gradual increase in vascular . O2 ÿ
formation could contribute to the development of
hypertension in that model. Although increased NADH
oxidase activity is implicated in both hypertensive
models, a decrease in aortic tissue Cu/Zn SOD activity
appears to play an important role in the high level of
aortic . O2 ÿ formation only in DOCA-HT rats.
Acknowledgements
The authors would like to express their gratitude to Jo-
Anne Le Guerrier and Diane Papin for their technical
expertise and Carole Champagne for her editorial
assistance.
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