Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Volume 2011, Article ID 130529, 7 pages
EnhancingNO/NOSActivity inSpontaneously HypertensiveRats
HyeSukHwang,Yoo SungKim, Yeon HeeRyu,Ji EunLee,YoungSeop Lee,
EunJinYang,Sun-MiChoi,and Myeong Soo Lee
Department of Medical Research, Korea Institute of Oriental Medicine, 461-24, Jeonmin-dong, Yuseong-gu,
Daejeon 305-811, Republic of Korea
Correspondence should be addressed to Myeong Soo Lee, email@example.com
Received 11 June 2008; Accepted 11 September 2008
Copyright © 2011 Hye Suk Hwang et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
Using spontaneously hypertensive rats (SHR), this study investigated whether electroacupuncture (EA) could reduce early stage
hypertension by examining nitric oxide (NO) levels in plasma and nitric oxide synthase (NOS) levels in the mesenteric resistance
artery. EA was applied to the acupuncture point Governor Vessel 20 (GV20) or to a non-acupuncture point in the tail twice weekly
for 3 weeks under anesthesia. In conscious SHR and normotensive Wistar Kyoto (WKY) rats, blood pressure was determined the
day after EA treatment by the tail-cuff method. We measured plasma NO concentration, and evaluated endothelial NO syntheses
(eNOS) and neuronal NOS (nNOS) protein expression in the mesenteric artery. Systolic blood pressure (SBP) and diastolic blood
pressure (DBP) were lower after 3 weeks of GV20 treatment than EA at non-acupuncture point and no treatment control in SHR.
nNOS expression by EA was significantly different between both WKY and no treatment SHR control, and EA at GV20 in SHR.
eNOS expression was significantly high in EA at GV 20 compared with no treatment control. In conclusion, EA could attenuate
the blood pressure elevation of SHR, along with enhancing NO/NOS activity in the mesenteric artery in SHR.
Electroacupuncture (EA) inhibits sympathetic effects by
regulation of nitric oxide synthase (NOS) expression in the
central nervous system [1–3]. NO, which is produced by
vascular endothelial cells from its precursor, is a potent
vasodilator and plays an important anti-hypertensive role in
blood pressure (BP) homeostasis. The depressor effect of EA
on BP is primarily caused by vasodilation of the mesenteric
vessels due to inhibition of sympathetic vasoconstrictor tone
In spontaneously hypertensive rats (SHR) and other
animals with genetic hypertension, morphological or func-
tional changes within the arterial wall may result in an
increased peripheral vascular resistance, thereby leading to
hypertension [5, 6]. These processes in SHR bear a resem-
blance to that of essential hypertension in humans. Thus,
SHR are widely used as a model to study the mechanism,
pathophysiology and management of essential hypertension.
SHR have irregularities in several vasoregulatory factors,
including an impaired NO/NOS system. Therefore, SHR are
a beneficial model to investigate the mechanism of action
responsible for the effects of acupuncture in the treatment
of essential hypertension.
Underlying mechanism of anti-hypertensive effect of EA
by adjustments of total peripheral resistance which affect
the vascular regulatory system and BP has not been well
understood as much as that by regulation through the
central nervous system . The aim of this study was to
by examining NO levels in plasma in SHR and NOS levels in
the mesenteric resistance artery.
2.1. Animals. A total of 24 4-week-old male SHR/Izm and
age-matched six Wistar Kyoto rats (WKY/Izm) were pur-
chased from Japan Shizuoka Laboratory Center (Shizuoka,
Japan) and used after acclimatization for 1 week. We used
2Evidence-Based Complementary and Alternative Medicine
WKY rats as the normotensive control group because they
are genetically homogeneous. The animals were given food
and water ad libitum. They were housed at a controlled
ambient temperature of 22–25◦C with 55 ± 5% relative
humidity and a 12 h light/12 h dark cycle (lights on at 8:00
AM). Rats were acclimatized into BP measuring conditions
for at least 1 week before undergoing EA treatments.
After measuring BP, SHR were randomly divided into
three groups: untreated group (n = 6), EA at GV20 (n
= 6) and EA at non-acupuncture points group (n = 6).
Animal experiments were carried out in accordance with
the National Institute of Health’s Guide for the Care and
Use of Laboratory Animals, and experimental procedures
were approved by the Institutional Animal Care and Use
Committee at the Korea Institute of Oriental Medicine.
2.2. Measurement of BP. Systolic arterial blood pressure
(SBP) and diastolic blood pressure (DBP) were measured
non-invasively by the tail-cuff method using the Visitech BP-
2000 BP Analysis System (Visitech Systems, Apex, NC, USA)
on the next day of EA treatment. To ensure accuracy and
reproducibility, the rats were trained for 1 week prior to the
experiment, and measurements were taken at the same time
each day. Unanesthetized rats were placed on the specimen
platform, and their tails were placed through tail cuffs and
secured in place with tape. Following a 10min warm-up
period, 10 preliminary cycles were performed to allow the
rats to adjust to the inflating cuff. For each rat of each
group, four cycles were recorded at each time point without
intervention from moving by environmental situations.
2.3. EA Stimulation. EA was applied to the acupuncture
point GV20 or to a non-acupuncture point in the tail on
the first and fourth day of the week for 3 weeks (EA applied
from 6th to 8th week) with a pair of bipolar stimulation
electrodes after placing the rats under isoflurane anesthesia
for reducing stress of electrical stimulations (in the flow of
mixture of oxygen and nitrous oxide; 3% for induction and
1.5% for maintenance). GV20 is located at the vertex, on the
dorsal midline. The methods to determine acupoints in rat
is the transpositional method, which locates the veterinary
acupoints by transforming human acupoints onto animal
anatomy . According to this, the acupoints in tail is
considered as the most suitable control points for avoiding
possible effects of EA.
Two stainless steel acupuncture needles (0.15mm diam-
eter; DB needle CO, LTD, Korea) were mounted in a holder
with 1mm separation between the tips. The needle set
was vertically inserted to a depth of 4mm (cutaneous and
muscle) at the GV20 point on the midsagittal line between
the both of temporalis muscles of rat (equivalent to the
epicranius muscle of human), at the intersection of a line
connecting the right and left ear apices. As a control, a non-
acupuncture point located at the junction between the tail
and buttock was also applied with the same parameters.
Electrical stimulation was performed using a Grass S88
stimulator (Grass Instrument Co., West Warwick, RI, USA)
connected to the pair of needle electrodes. Electrical stimuli
with 10Hz frequencies, 1mA and 1ms duration pulses were
applied to the acupuncture point. The current delivered
was monitored at all times, and the polarity was reversed
every 60s to prevent polarization of the electrodes. The total
of EA, anesthesia was immediately discontinued, and the rats
usually resumed full activity within 5min.
2.4. Tissue Preparation. The animals were anesthetized with
by intraperitoneal injection on the last day of EA treatment,
and perfused intracardially with phosphate-buffered saline
sized artery contribute to the peripheral resistance and
regulate the BP, the mesenteric arteries of four randomly
selected rats in each group were carefully dissected out,
cleared of connective tissue and flushed out with modified
Krebs-Henseleit solution (KHS) at 4◦C; the KHS had the
following composition (millimolar): 119 NaCl, 4.7 KCl, 2.5
CaCl2, 24 NaHCO3, 1.18 KH2PO4, 1.2 MgSO4, 0.01 EDTA,
5.5 glucose. Tissues were immediately snap-frozen in liquid
nitrogen and stored at −70◦C until processed.
2.5. Measurements of Total Nitrate and Nitrite. Blood sam-
ples were collected from cardiac puncture into a polypropy-
lene tube containing sodium-EDTA (3–7 mg ml−1of blood)
as an anti-coagulant, just prior to tissue preparation on
the last day of EA treatment. Samples were centrifuged for
15min at 3000g, and plasma was removed and stored at
≤ −70◦C until use. As blood hemoglobin and other protein
value at 540nm, samples were centrifuged at 14000g at
4◦C for 30min with 10kDa molecular weight cut-off filters
(Millipore Micron YM-10, Bedford, USA). Generation of
NO was determined by measuring nitrite accumulation in
containing 1% sulphanilamide and 0.1% N-(1-naphthyl)-
ethylenediamine dihydrochloride (R&D systems, Minneapo-
lis, MN, USA). Sample and Griess reagent (50μl of each)
were mixed and incubated for 5min, and absorption was
BIO-TEK) at 540nm. Sodium nitrate and nitrite standards
final concentrations ranged from 0 to 200μM.
2.6. Western Immunoblotting Analysis. The mesenteric arter-
ies were ground in a mortar containing liquid nitrogen.
The powdered tissue was suspended in 100μl of lysis buffer
(20mM Tris-HCl, 150mM NaCl, 5mM EDTA, 0.1% SDS,
pH 7.5) containing protease inhibitors (10μgml−1of apro-
tinin, 1mmoll−1PMSF and 10μgml−1of leupeptin) and
agitated at 4◦C for 30min. After centrifugation at 17900g at
4◦C for 30min, the protein concentration in the supernatant
was determined using a Bradford protein assay kit (Bio-
Rad Laboratories Inc., CA, USA). These measurements
were performed to determine the eNOS and nNOS protein
mass. Briefly, thoracic aorta and mesenteric artery tissue
Evidence-Based Complementary and Alternative Medicine3
preparations (30μg of protein for nNOS and eNOS) were
size fractionated on a 10% SDS-PAGE gel at 120V for 2 h.
After electrophoresis, proteins were transferred onto a nitro-
cellulose transfer membrane (Whatman GmbH) at 200mA
for 120min using the Bio-Rad transfer system. Membranes
were blocked with Tris-buffered saline buffer (TBS), pH 7.4,
and 5% skimmed milk, then incubated overnight at 4◦C
with mouse monoclonal anti-nNOS, anti-eNOS (1:2500
dilution, BD Transduction Laboratories, CA, USA), or anti-
β-actin (1:1000, AbCAM, Cambridge, UK) in TBS. β-Actin
was used as an internal control. The membrane was then
washed for 30min in a shaking bath, with the wash buffer
(TBS buffer containing 0.1% Tween-20) changed every
5min. The membranes were subsequently incubated with a
(1:1000, BD Transduction Laboratories) at room tempera-
ture. The washes were repeated before the membrane was
developed with a light-emitting, non-radioactive method
using ECL reagent (Amersham Inc., Buckinghamshire, UK).
The membrane was then subjected to autoluminography for
1–5min. The relative optical density of the respective bands
Solution DT image analysis software (Image and Microscope
Alldata areexpressed asmean ±SEM.Statisticalsignificance
was assessed using one way ANOVA followed by an all
pair-wise multiple comparison procedure (Turkey’s test).
Differences were considered statistically significant at values
of P < .05.
4.1. Relationship between Age and BP. From ages 4 through
12 weeks, the SBP (mean ± SEM) increased progressively
from 152.1 ± 3.02mmHg to 206.8 ± 6.06mmHg in SHR,
but not in WKY (Table 1). Also, the DBP (mean ± SEM)
increased progressively from 119.7 ± 5.52mmHg to 174.3 ±
1.63mmHg in SHR rats. Both SBP and DBP were signifi-
cantly higher than those of age-matched WKY from ages 4
through 12 weeks.
4.2. EA Treatment on GV20 Significantly Delays the Devel-
opment of Hypertension in SHR. The SBP of 8-week-old
SHR was higher than that of the age-matched WKY as
shown in Table 1. And the SBP of 4-week and 12-week-old
SHR were higher than those of the age matched WKY as
listed in Table 1. EA significantly attenuated BP in SHR rats
(Figure 1), but not to levels of age-matched untreated WKY
as shown in Table 1. SBP at 8 weeks increased progressively
to 195.38 ± 2.34 and 193.02 ± 2.97mmHg in SHR and non-
acupuncture point treated group, respectively, while GV20
treatment reduced SBP to 182.2 ± 2.87mmHg in SHR at
the same age. DBP at 8 weeks increased progressively to
169.79 ± 2.5 and 167.21 ± 1.26mmHg in SHR and non-
acupuncture point treated group, respectively, while GV20
Blood Pressure (mmHg)
56789 1011 12
Age (weeks old)
Blood Pressure (mmHg)
Age (weeks old)
Figure 1: (a) SBP in SHR that were anesthetized for the same
period of EA stimulation (SHR+ANES), in Governor Vessel 20
(GV20) treated rats (SHR-GV20), or in non-acupoint treated rats
(SHR-Tail) from 5 to 12 weeks. (b) Diastolic blood pressure in
SHR (SHR+ANES), GV20 (SHR-GV20) or tail acupuncture groups
(SHR-Tail) from 5 to 12 weeks. Results are mean ± SEM for six rats
in each group.∗P < .05, compared with SHR+ANES. The arrows
show the EA treatment for 3 weeks (6–8 weeks).
treatmentreduced DBP to 154.1 ± 4.61mmHg in SHR at the
same age (Figure 1(b)). EA significantly reduced BP in SHR
rats (Figure 1), but not to levels of age-matched untreated
WKY as listed in Table 1.
4.3. NOS Expression in WKY Rats and SHR after EA Treat-
ments. We compared the expression of eNOS and nNOS
in the mesenteric artery of SHR and WKY to delineate
whether they play a differential role in the augmented level
4 Evidence-Based Complementary and Alternative Medicine
Table 1: The systolic and diastolic blood pressure in SHR and age-matched WKY rats at age 4, 8 and 12 weeks.
101.9 ± 4.45
163.9 ± 2.47∗
130.8 ± 3.18
152.1 ± 3.02∗
134.6 ± 1.95
193.1 ± 3.57∗
130.9 ± 6.99
206.8 ± 6.06∗
96.7 ± 5.34
119.7 ± 5.52∗
103.8 ± 5.62
174.3 ± 1.63∗
Results are expressed as mean ± SEM for six rats in each group.∗P < .05 compared with age-matched WKY.
of NO by EA during hypertension. We determined the
expression of eNOS and nNOS protein in the mesenteric
band intensities (Figure 2). The developmental stage of
level. eNOS band intensities relative to the corresponding
β-actin band were 0.31 ± 0.03 (WKY group), 0.33 ±
0.04 (SHR group), 0.57 ± 0.01 (SHR-GV20 group) and
0.35 ± 0.05 (SHR-Tail group). EA treatment increased the
eNOS expression associated with delaying development of
hypertension and showed even higher eNOS expression than
the non-acupuncture point treated SHR and non-treated
(relative to β-actin) was 0.26 ± 0.05 (WKY group), 0.48
± 0.01 (SHR group), 0.34 ± 0.02 (SHR-GV20 group) and
0.46 ± 0.02 (SHR-Tail group). nNOS expression in SHR was
significantly higher than age-matched WKY rats, and EA
treatment significantly decreased it (P < .05) (Figure 2(b)).
4.4. Effect of EA on Plasma Nitrate/Nitrite Levels. The basal
plasma nitrate/nitrite level was significantly lower in SHR
than WKY at 8 weeks (P < .05). EA treatment for 3 weeks
significantly increased plasma nitrate/nitrite levels at 8 weeks
of SHR (P < .05, Figure 3).
The main findings of this study were that long-term
treatment with EA delayed hypertension development, and
this restored NO in the plasma of SHR. In this study,
eNOS expression was also significantly increased by EA in
mesenteric artery of SHR, whereas nNOS expression was
GV20 alone, or in combination with other acupuncture
points, is used for the treatment of hypertension and pre-
hypertension patients [9–11]. Scalp acupuncture decreases
superoxide dismutase activity, reducing the oxidative stress
reaction . Kim et al.  reported that EA treatment
on Tsu-san-li (ST36) controlled the NOS system in the
stomach and cheek pouch tissues, which were on the
stomach meridian, but did not control that in liver tissue,
a non-stomach meridian organ in the two-kidney, one-
clip renal hypertension hamster model. While examining
the distribution of NO in the skin acupuncture points of
rats, Chen et al.  showed that l-arginine-derived NO
synthesis appears to mediate noradrenergic function on skin
sympathetic nerve activation, which contributes to the skin
electrical resistance of acupuncture points and meridians.
Relative Optical Density (NOS/beta-actin)
Figure 2: (a) Representative western blot of eNOS and nNOS
protein in mesenteric artery from WKY and SHR, either control,
EA-treated at GV20 (SHR-GV20) or non-acupoint (SHR-tail)
conditions. (b) Lower panel shows densitometric analysis of the
western blot of eNOS and nNOS protein. Relative abundance of
NOS protein compared with β-actin. Results are expressed as mean
± SEM for four rats in each group. Results are expressed as mean
± SEM for four rats in each group.∗P < .05, compared with WKY
group;†P < .05, compared with no-treated SHR group.
The hypotensive action of NO induced by EA stimulation
remains an unexplained but reproducible observation.
NO is a potent vasodilator that is necessary to maintain
BP homeostasis. Similarly Briones et al.  also reported
than in those of WKY. In contrast, Forte et al.  reported
that basal NO synthesis by endothelial cells is reduced in
patients with untreated essential hypertension. Furthermore,
Hatta et al.  reported that antihypertensive therapy
increases the reduced basal NO levels in SHR and DOCA-
salt rats. Mokuno et al.  have shown that NO production
induced by mechanical stimulation was markedly reduced
in 5-week-old SHR at the pre-hypertensive stage, and
Evidence-Based Complementary and Alternative Medicine5
Nitrate/nitrite levels concentration
in plasma (μM)
Figure 3: Plasma nitrate/nitrite levels in WKY and SHR under
control- or EA-treated at GV20 (SHR-GV20) or non-acupoint
(SHR-tail) conditions. Results are expressed as mean ± SEM for six
rats in each group.∗P < .05, compared with WKY group;†P < .05
compared with no-treated SHR group.
this impairment of nitric oxide production preceded the
onset of hypertension in SHR. These reports support our
findings that basal release of NO is reduced in SHR during
The vascular generation of oxygen species was increased
in development of hypertension  and eNO was rapidly
inactivated in the presence of superoxide anions . Neu-
ronal NO metabolism by superoxide anions in mesenteric
arteries from young SHR is also elevated . Vaziri et
al.  also reported that antioxidant therapy ameliorated
hypertension and mitigated the upregulation of NOS in vas-
cular and renal tissues. Furthermore, the increased oxidative
stress and degradation of NO from eNOS has been described
in SHR [22–24]. Together, these data suggest that anti-
hypertensive treatment restores decreases in NO release by
high BP and enhances NOS bioavailability in the aorta.
In hypertension, where intraluminal pressure, shear
stress and oxidative stress are increased, augmented nNOS-
derived NO may offset decreased eNOS-derived NO, thereby
acting as an adaptive mechanism . eNOS−/−mice
showed elevated BP in conscious states , confirming that
NOderived fromeNOSplaysanimportant roleinregulating
BP as a vasodilator. But nNOS knockout mice, however,
have enlarged stomachs and defects in the inhibitory junc-
tion potential involved in gastrointestinal motility, but not
hypertension [27, 28]. In mice lacking functional eNOS(−/−),
arginine (l-NNA), decreased mean BP , suggesting that
NO derived from isoforms other than eNOS increases BP in
the absence of eNOS activity . These results support that
the eNOS activation could affect to BP˜ a without abnormally
overactivated-nNOS activation in˜ a SHR. Our result shows
EA treatments for 3 weeks prefer enhancing eNOS activity
to nNOS activity compensated eNOS deactivation.
The autonomic nervous system is involved in the
development of hypertension, and chronic imbalance of
the autonomic nervous system is a prevalent, potent risk
factor for adverse cardiovascular events . Both increased
sympathetic nerve firing rates and reduced neuronal nore-
pinephrine re-uptake contribute to sympathetic activation
in hypertension . Acupuncture stimulation seems to
reduce sympathetic nervous system activation via activation
of the cholinergic system or opioid receptors in the rostral
ventrolateral medulla [4, 32]. NO in the central nervous
system plays a very important role in the control of sym-
pathetic outflow and regulation of cardiovascular activities,
 models. Microinjection of nNOS antisense oligos into
the gracile nucleus produces a depressor and inhibitory
cardiovascular response to EA stimulation . Therefore,
along with the cited findings, sympatho-inhibition and
vasodilation may be induced by EA stimulation in various
therapeutic effects of previous clinical trials. According to
previous trials, acupuncture is more effective than sham
only when given in addition to medication [11, 35] and
acupuncture alone is not better than sham [36, 37]. These
results can imply both acupuncture and sham acupuncture
are effective for lowering BP. The result of our study shows
that the suppressive effect of EA on BP seems to be an
immediate one, without any cumulative effects. It would be
useful to investigate the immediate effectsof acupuncture for
lowering BP in the future clinical settings.
In conclusion, our results support the concept that
EA could attenuate the BP elevation of SHR, along with
enhancing NO/NOS activity in the mesenteric artery in
SHR. However, future studies are necessary to investigate the
underlying NOS mechanisms of the peripheral autonomic
nervous system in blood vessels.
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