Synergism of atenolol and nitrendipine on hemodynamic amelioration and organ protection in hypertensive rats

Abstract

This study was designed to investigate the possible synergism of atenolol and nitrendipine on blood pressure (BP) and blood pressure variability (BPV) reductions, baroreflex sensitivity (BRS) amelioration, and organ protection in hypertensive rats.
The dose was 20 mg/kg for atenolol, 10 mg/kg for nitrendipine and 20 + 10 mg/kg for the combination of these two drugs. In an acute study, a single dose was given via a catheter previously inserted into the stomach in spontaneously hypertensive rats (SHR). In a subacute study, SHR, deoxycorticosterone acetate (DOCA)-salt rats, and two-kidney, one-clip (2K1C) rats were used. They received the same dose by gavage daily for 10 days. BP was measured 24 h after drug administration. In chronic studies, these drugs at the aforementioned dose were mixed into rat chow. SHR were treated for 4 months. BP was then continuously recorded for 24 h. After the determination of BRS, rats were killed for organ-damage evaluation.
In the acute study, it was found that the combination of atenolol and nitrendipine had an obviously greater and longer BP reduction than treatment with each of these two drugs separately. In the subacute study, an effective decrease in BP 24 h after administration was found only in the rats treated with the combination. In chronic studies, it was found that the combination possessed the obvious synergism on BP and BPV reduction, BRS amelioration and organ protection in SHR. Multiple-regression analysis showed that the decrease in left ventricular hypertrophy was most significantly related to the decrease in systolic BPV and BP, the decrease in aortic hypertrophy was most significantly related to the increase in BRS and the decrease in systolic BPV, and amelioration in the renal lesion was most significantly associated with the restoration of BRS.
Treatment with a combination of atenolol and nitrendipine exhibited a rapid and persistent antihypertensive effect and possessed an obvious synergism on BP and BPV reduction, BRS restoration and organ protection in hypertensive rats. The decrease in BPV and the restoration of BRS may importantly contribute to organ protection in SHR with chronic treatment.

Full text

Synergism of atenolol and nitrendipine on hemodynamic
amelioration and organ protection in hypertensive rats
He-Hui Xie, Chao-Yu Miao, Yuan-Ying Jiang and Ding-Feng Su
Objective This study was designed to investigate the
possible synergism of atenolol and nitrendipine on blood
pressure (BP) and blood pressure variability (BPV)
reductions, baroreflex sensitivity (BRS) amelioration, and
organ protection in hypertensive rats.
Method The dose was 20 mg/kg for atenolol, 10 mg/kg
for nitrendipine and 20 10 mg/kg for the combination of
these two drugs. In an acute study, a single dose was
given via a catheter previously inserted into the stomach in
spontaneously hypertensive rats (SHR). In a subacute
study, SHR, deoxycorticosterone acetate (DOCA)-salt rats,
and two-kidney, one-clip (2K1C) rats were used. They
received the same dose by gavage daily for 10 days. BP
was measured 24 h after drug administration. In chronic
studies, these drugs at the aforementioned dose were
mixed into rat chow. SHR were treated for 4 months. BP
was then continuously recorded for 24 h. After the
determination of BRS, rats were killed for organ-damage
evaluation.
Results In the acute study, it was found that the
combination of atenolol and nitrendipine had an obviously
greater and longer BP reduction than treatment with each
of these two drugs separately. In the subacute study, an
effective decrease in BP 24 h after administration was
found only in the rats treated with the combination. In
chronic studies, it was found that the combination
possessed the obvious synergism on BP and BPV
reduction, BRS amelioration and organ protection in SHR.
Multiple-regression analysis showed that the decrease in
left ventricular hypertrophy was most significantly related
to the decrease in systolic BPV and BP, the decrease in
aortic hypertrophy was most significantly related to the
increase in BRS and the decrease in systolic BPV, and
amelioration in the renal lesion was most significantly
associated with the restoration of BRS.
Conclusion Treatment with a combination of atenolol and
nitrendipine exhibited a rapid and persistent
antihypertensive effect and possessed an obvious
synergism on BP and BPV reduction, BRS restoration and
organ protection in hypertensive rats. The decrease in BPV
and the restoration of BRS may importantly contribute to
organ protection in SHR with chronic treatment.
J Hypertens 23:193–201 &2005 Lippincott Williams &
Wilkins.
Journal of Hypertension 2005, 23:193– 201
Keywords: atenolol, nitrendipine, hypertension, end organ damage, blood
pressure variability, baroreflex sensitivity
Department of Pharmacology, Second Military Medical University, Shanghai,
China.
Sponsorship: This project was supported by the High Tech Research and
Development (863) Program of China (2002 AA2Z346C) and the National
Natural Science Foundation of China (39670831 and 30070871).
Correspondence and requests for reprints to Prof. Ding-Feng Su, Department of
Pharmacology, Second Military Medical University, Shanghai 200433, China.
Tel/fax: +86 21 65493951; e-mail: dfsu@citiz.net
Received 7 January 2004 Revised 9 August 2004
Accepted 20 August 2004
Introduction
Recently, the importance of a combination therapy has
been well recognized in the treatment of hypertension
[1,2]. To better control blood pressure (BP) is the main
objective of the combination therapy. Generally speak-
ing, a combination of two drugs belonging to different
classes may possess a synergism in BP reduction. How-
ever, no study could show whether such a combination
possesses a synergism in organ protection.
Preventing or reversing end-organ damage is an impor-
tant objective in the treatment of hypertension. It is
well known that a high BP level induces organ damage
and a decreasing BP level could prevent end-organ
damage. However, a high BP level is not the unique
factor determining hypertensive end-organ damage.
Recently, it has been proposed that blood pressure
variability (BPV) and baroreflex sensitivity (BRS) may
be two important factors determining organ damage in
hypertension [3–6]. Our previous studies demonstrated
that reduction of BPV and restoration of BRS contrib-
uted importantly to the organ protection in spontan-
eously hypertensive rats (SHR) treated with certain
antihypertensive drugs, such as ketanserin [7] and
nitrendipine [8].
Atenolol, a â-adrenoceptor blocker, and nitrendipine, a
calcium antagonist, are two widely used drugs in the
treatment of hypertension. They belong to two differ-
ent classes of antihypertensives and the mechanisms of
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Original article 193
0263-6352 &2005 Lippincott Williams & Wilkins

action are quite different for these two drugs. There-
fore, the present work was designed to investigate the
synergism of atenolol and nitrendipine on BP and BPV
reduction, BRS restoration and organ protection in
hypertensive animals. Considering the sensitivity to a
drug might be different in different animal models, we
used three hypertensive rat models to observe the
synergism of this combination on BP reduction.
Methods
Animals and chemicals
Nitrendipine was purchased from Nanjing Pharma-
ceutical Co. Ltd (Nanjing, China) and atenolol was
purchased from Shanghai Second Pharmaceutical Co.
Ltd (Shanghai, China). Male Sprague–Dawley rats
(used for preparation of hypertensive models) were
purchased from the Sino-British SIPPR/BK Lab Animal
Ltd (Shanghai, China). Male SHR and Wistar – Kyoto
(WKY) rats with an age of 18 weeks were provided by
the animal center of our university. The rats were
housed with controlled temperature (23–258C) and
lighting (0800–2000 h light, 2000–0800 h dark) and
with free access to food and tap water. All the animals
used in this work received humane care in compliance
with institutional animal care guidelines.
Preparation of two-kidney, one-clip (2K1C) hypertensive
rats
Male Sprague–Dawley rats weighing 160 – 180 g were
anesthetized with a combination of ketamine (40 mg/
kg) and diazepam (6 mg/kg). The right renal artery of
each animal was isolated through a flank incision, as
described previously [9], and a silver clip (0.2-mm
internal gap) was placed on the renal artery. All animals
were fed standard rat chow and tap water ad libitum.
The experiments were performed 5 weeks after place-
ment of the clip.
Preparation of deoxycorticosterone acetate-salt
hypertensive rats
Deoxycorticosterone acetate (DOCA)-salt rats were
prepared as previously described [10]. Male Sprague –
Dawley rats weighing 110–130 g were anesthetized
with a combination of ketamine (40 mg/kg) and diaze-
pam (6 mg/kg) and underwent a right nephrectomy via
a flank incision. Rats were given daily subcutaneous
injections of DOCA (50 mg/kg) and 0.9% saline to
drink for 5 weeks.
BP measurement
In subacute studies, the systolic blood pressure (SBP)
of rats was measured by the tail-cuff method (MRS-III;
Shanghai Hypertensive Institute, Shanghai, China).
In acute and chronic studies, the SBP, diastolic blood
pressure (DBP) and heart period (HP) of rats were
continuously recorded using a previously described
technique [11,12]. Briefly, rats were anesthetized with a
combination of ketamine (40 mg/kg) and diazepam
(6 mg/kg). A floating polyethylene catheter was inserted
into the lower abdominal aorta via the left femoral artery
for BP measurement, and another catheter was placed
into the left femoral vein for intravenous injection. The
catheters were exteriorized through the interscapular
skin. After a 2-day recovery period, the animals were
placed for BP recording in individual cylindrical cages
containing food and water. The aortic catheter was
connected to a BP transducer via a rotating swivel that
allowed the animals to move freely in the cage. After
about 14-h habituation, the BP signal was digitized by a
microcomputer. SBP, DBP and HP values from every
heartbeat were determined online. The mean values
and standard deviation of these parameters during a
period of 24 h for each rat were calculated. The standard
deviation of all values obtained during 24 h was denoted
as the quantitative parameter of variability; that is,
systolic blood pressure variability (SBPV), diastolic BPV,
and HP variability for each rat.
BRS measurement
To determine the function of arterial baroreflex in
conscious rats, the methods widely used are derived
from that which Smyth et al. first applied for humans
[13]. The principle of this method is to measure the
prolongation of HP in response to an elevation of BP.
With some modifications, this method was used in
conscious rats [14,15]. A bolus injection of phenylephr-
ine was used to induce an elevation of BP. The dose of
phenylephrine was adjusted to raise the SBP between
20 and 40 mmHg. HP was plotted against SBP for
linear regression analysis and the slope of SBP – HP was
expressed as BRS (ms/mmHg) [15]. As there exists a
delay (about 1 s) between the stimulus and response,
the slopes were calculated by computer with 1 –
10 beats of shift for linear regression analysis and the
slope with the highest correlation coefficient was used
as BRS. A correlation analysis with 5 beats of shift, for
example, means that values of HP6/SBP1,HP
7/SBP2,
HP8/SBP3... were used.
Morphological examination
The animals were weighed and killed by decapitation.
The thoracic and peritoneal cavities were immediately
opened. The right kidney, aorta and heart were excised
and rinsed in cold physiological saline. The right
kidney was blotted, and weighed. The left ventricle
was isolated, blotted, and weighed. At the same time,
the aorta was cleaned of adhering fat and connective
tissue. Just below the branch of the left subclavicular
artery, a 30-mm-long segment of thoracic aorta was
harvested, blotted, and weighed. Ratios of left ventri-
cular weight to body weight (LVW/BW), right ventricu-
lar weight to body weight (RVW/BW), ventricular
weight to body weight (VW/BW), left ventricular
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
194 Journal of Hypertension 2005, Vol 23 No 1

weight to right ventricular weight (LVW/RVW), and
aortic weight to the length of aorta (AW/length) were
calculated [16,17]. Histopathological observation was
also carried out with our conventional method [18].
Briefly, immediately after gross detection, all samples
of left ventricles, in 2–3-mm-thick slices, aortae and
kidney were immersed in formalin solution for more
than 1 week, dehydrated in ethanol, cleared in dimethyl-
benzene and embedded in paraffin. Then 5-ìm-thick
sections were prepared and stained with hematoxylin
and eosin for light microscopic evaluation.
Glomerulosclerosis score
For the semi-quantitative evaluation of glomerulular
damage, the glomerulosclerosis score (GSS) was defined
as previously described [19]. On the light microscopic
specimens, approximately 50 glomeruli from the outer
cortex and the same number of glomeruli from the
inner cortex for each kidney were graded according to
the degree of sclerosis: 0, no mesangial expansion; 1,
mild mesangial expansion (,30% of a glomerular area);
2, moderate mesangial expansion (30 – 60% of a glomer-
ular area); 3, marked mesangial expansion (.60% of a
glomerular area); and 4, sclerosis was global. This was
performed by one observer in a blind fashion using
coded slides. A weighted composite sclerosis score was
then calculated for each kidney according to the follow-
ing formula: glomerulosclerosis score ¼[1 3(number of
grade 1 glomeruli) þ23(number of grade 2 glomeruli)
þ33(number of grade 3 glomeruli) þ43(number of
grade 4 glomeruli)] 3100 / (number of glomeruli ob-
served).
Experimental protocols
Acute studies
SHR were randomly divided into three groups (n¼9
per group) and respectively given atenolol (20 mg/kg),
nitrendipine (10 mg/kg) and the combination of atenolol
and nitrendipine (20 þ10 mg/kg). The drugs were dis-
solved in 0.8% carboxymethylcellulose sodium (CMC)
and given via a catheter inserted into the stomach at
the same time as aortic catheter implantation. The BP
and HP before drug administration and at 0.25, 0.5, 1, 2,
4, 6, 8, 10, 20, 22, and 24 h after drug administration
were determined in conscious freely moving rats. Be-
fore this experiment, six SHR were given a dose of
vehicle (0.8% CMC). No significant changes in BP and
HP were observed in these control rats.
Subacute studies
SHR, 2K1C hypertensive rats, or DOCA-salt hyper-
tensive rats were randomly divided into four groups
(n¼8 per group), respectively, receiving daily vehicle
(0.8% CMC), atenolol (20 mg/kg per day), nitrendipine
(10 mg/kg per day) and the combinations of atenolol
and nitrendipine (20 þ10 mg/kg per day) for 10 days.
The drugs were dissolved in 0.8% CMC and given by
gavage. The SBP was determined by the tail-cuff
method before drug administration and on study days 1,
3, 5, 7, 9 and 10. SBP measurement was performed 24 h
after drug administration each time.
Chronic studies
Studies were performed in four groups of SHR and a
group of WKY rats. Nitrendipine and atenolol were
mixed in the rat chow. The consumption of rat chow
containing drugs was determined previously. The con-
tent of drugs in the rat chow was calculated according
to the chow consumption, and the ingested dose of
atenolol, nitrendipine and combination of atenolol and
nitrendipine were about 20, 10 and 20 þ10 mg/kg per
day, respectively. The control SHR group and WKY
rats received the same diet without the drugs. After
4 months of drug administration, the BP was recorded
during 24 h, then the BPV was calculated and the BRS
was determined in conscious freely moving rats. Histo-
pathological examinations were performed after BP
recording and BRS studies. According to the data
accumulated in our department, the SBP of SHR is
about 170 mmHg at the age of 18 weeks (the start of
the 4-month period) and 190 mmHg at the age of
34 weeks (the end of the 4-month period).
Probability sum test
To determine whether the combination was synergistic,
we tried to use the probability sum test. This came
from classic probability analysis and it was proposed for
evaluating the synergism of the combination of two
drugs (qtest) [20–22]. In the present work, we used
the following criteria. Compared with the mean values
of control rats, treated rats with a decrease in BP (SBP
or DBP) >20 mmHg were defined as responders ac-
cording to clinical experiences. For other parameters,
treated rats with a decrease or increase >20% of the
mean values of the control group were defined arbitra-
rily as responders. The formula is as follows: q¼
PAþB/(PAþPBPA3PB). Here, A and B indicate
drug A and drug B, P(probability) is the percentage of
responders in each group, PAþBis the real percentage
of the responder, (PAþPBPA3PB) is the expected
response rate, (PAþPB) indicates the sum of the
probabilities when drug A and drug B were used alone,
and (PA3PB) is the probability of rats responding to
both drugs when they were used alone (i.e. assuming
the two drugs act independently). When q,0.85, the
combination is antagonistic; when q.1.15, it is syner-
gistic; and when q¼0.85–1.15, it is additive.
Statistical analysis
Data are expressed as the mean standard error of the
mean. Comparisons among values obtained in the same
group were made by repeated-measures analysis of
variance (ANOVA) followed by the Fisher test. Com-
parisons among groups were made by ANOVA followed
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Synergism of atenolol and nitrendipine Xie et al. 195

by the Duncan test. The relationships between hemo-
dynamic parameters and organ damage parameters were
analyzed by classic univariate correlation analysis. Step-
wise multiple-regression analysis was performed to
study which of the hemodynamic parameters is most
significantly a predictor of a measure of organ damage.
Fto enter and Fto remove were set to P,0.05 and
P.0.10, respectively. P,0.05 was considered statisti-
cally significant.
Results
Acute studies
As shown in Figure 1, no obvious change in the SBP
and DBP level was found in atenolol-treated SHR.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
100
140
180
220
Time (h)
SBP (mmHg)
Nit
Ate
Nit 1 Ate
**
**
**
**
**
****
**
**
*
**
(a)
**
40
80
120
160
DBP (mmHg)
*
**
**
** **
**
**
**
**
****
**
**
(b)
*
*
140
170
200
230
HP (ms)
**
*
*
*
*
*
**
**
***
**
****
(c)
**
0246810121416182022242622
Time (h)
0 2 4 6 8 10 12 14 16 18 20 22 24 2622
Time (h)
0246810121416182022242622
Fig. 1
Effects of a single dose of atenolol and nitrendipine alone and in combination on (a) systolic blood pressure (SBP), (b) diastolic blood pressure
(DBP) and (c) heart period (HP) in spontaneously hypertensive rats. Ate, atenolol; Nit, nitrendipine; Ate + Nit, combination of atenolol and
nitrendipine. n¼9. *P,0.05, **P,0.01 versus hour 0 (before drug administration).
196 Journal of Hypertension 2005, Vol 23 No 1

Treatment with nitrendipine significantly decreased
SBP and DBP in SHR, with duration up to 2 and 8 h,
respectively, and with a maximal decrease (29 7 and
41 10 mmHg, respectively) at the 15th minute after
administration. An obvious decrease in SBP and DBP
was found in SHR treated with a combination of
atenolol and nitrendipine, with duration up to 10 and
22 h, respectively, and with a maximal decrease
(65 21 and 60 13 mmHg, respectively) at the 15th
min after administration. It was shown that combination
therapy possessed an obviously greater and longer SBP
and DBP reduction than monotherapy. Nitrendipine
significantly decreased HP in SHR, and a lesser in-
crease in HP was found in rats treated with atenolol or
a combination of atenolol and nitrendipine. Treatment
with vehicle (0.8% CMC) did not influence BP and HP
in SHR (data not shown).
Subacute studies
Subacute treatment (10 days) with atenolol and nitren-
dipine alone did not significantly decrease the SBP
24 h after drug administration in SHR, DOCA-salt
hypertensive rats, and 2K1C hypertensive rats. How-
ever, treatment with a combination of these two drugs
obviously decreased the SBP 24 h after drug adminis-
tration in all the three hypertensive models of rats (Fig.
2). No obvious change in HP was found in all treated
rats and CMC treatment did not influence the BP and
HP (data not shown).
Chronic studies
Effects on BP, BPV, and BRS in SHR
Compared with WKY rats, SHR possessed significantly
lower BRS and higher BP and BPV. Long-term treat-
ment with atenolol and nitrendipine alone or in combi-
nation significantly decreased BP and BPV, and
enhanced BRS in SHR. However, in the rats treated
with a combination of atenolol and nitrendipine, the
extents of BP and BPV reduction and BRS amelioration
were significantly greater than those in atenolol or
nitrendipine-treated rats. Compared with control SHR,
HP was significantly increased in atenolol-treated rats
and slightly decreased in nitrendipine-treated rats. An
increase in HP was also found in rats treated with a
combination of atenolol and nitrendipine, but the in-
crease in HP was less important than that in atenolol-
treated rats (Table 1).
Effects on organ damages in SHR
Compared with WKY rats, SHR possessed significantly
higher LVW/BW, RVW/BW, VW/BW, LVW/RVW,
AW/length and GSS. Long-term treatment with ateno-
lol and nitrendipine alone or in combination signifi-
cantly decreased the LVW/BW, LVW/RVW, AW/
length and GSS in SHR. However, in the rats treated
with combination of atenolol and nitrendipine, the
extents of decrease in LVW/BW, AW/length and GSS
were greater than those in atenolol or nitrendipine-
treated rats (Table 2).
Synergism of atenolol and nitrendipine in SHR
Table 3 presents the result of the probability sum test
in data from SHR treated with atenolol, nitrendipine
and a combination of atenolol and nitrendipine. All q
values were larger than 1.15. It was found that the
combination of atenolol and nitrendipine possesses a
significant synergism on BP and BPV reduction, BRS
amelioration and organ protection in SHR.
Relationships between BP, BPV, BRS and organ damage in
SHR
Relationships between BP, BPV, BRS and organ da-
mage in treated and untreated SHR are presented in
Table 4. It was found that LVW/BW, an index for left
ventricular hypertrophy, AW/length, an index for aortic
hypertrophy, and GSS, an index for renal damage, were
all positively related to BP and BPV, and negatively
related to BRS. Some examples for important correla-
tions are shown in Figure 3.
The relative dependencies of organ damage on hemo-
dynamic parameters were assessed by stepwise multi-
ple-regression analysis. LVW/BW was most significantly
associated with higher SBPV (â¼0.530, P,0.01) and
SBP (â¼0.319, P,0.05). AW/length was most signifi-
cantly associated with lower BRS (â¼0.447, P,
0.01) and higher SBPV (â¼0.369, P,0.01). GSS was
most significantly associated with lower BRS (â¼
0.646, P,0.01).
Discussion
The present work for the first time systematically
studied and clearly demonstrated the synergism of two
antihypertensive drugs on reducing BP and BPV,
restoring BRS and protecting end organs.
In terms of the synergism of the combination of
atenolol and nitrendipine on BP reduction, the main
findings of the present work are as follows. (1) This
synergism was potent. In an acute study in SHR, it was
found that under the tested dose atenolol did not
decrease the BP and nitrendipine slightly decreased
the BP. However, the combination of atenolol and
nitrendipine decreased markedly the BP (both SBP and
DBP): about 40 mmHg 10 h after administration and
about 30 mmHg 20 h after administration. In the
subacute study, atenolol and nitrendipine did not de-
crease the BP when used alone, but significantly de-
creased the SBP (about 20 mmHg) 24 h after drug
administration when used in combination. In chronic
study, qvalues of 1.43 and 1.75 were reported for SBP
and DBP, respectively. These qvalues are markedly
higher than 1.15. This potent synergism may reduce
the doses of each drug required in the treatment of
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Synergism of atenolol and nitrendipine Xie et al. 197

hypertension and then can minimize the clinical and
metabolic side effects of each individual component in
larger dosage when used alone [23]. (2) This synergism
was persistent. As a result, the effect of this combina-
tion lasted for more than 22 h in the acute study and
for more than 24 h in the subacute study. This feature
would decrease the times of drug administration and in
turn decrease the BP fluctuation produced by multiple
drug administrations. The decrease in BP fluctuation
would offer additional benefit in the treatment of
hypertension [24,25]. Furthermore, once a day would
be helpful in enhancing the patient compliance. (3)
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
170
190
210
230
Time (day)
SBP (mmHg)
Nit
Ate
Nit ⫹ Ate
** *
** *
**
(a)
150
170
190
210
SBP (mmHg)
**
*
**
*
(b)
140
160
180
200
SBP (mmHg)
*
*
*
**
****
(c)
01234567891011⫺1
Time (day)
01234567891011⫺1
Time (day)
01234567891011⫺1
Fig. 2
Effects of atenolol and nitrendipine alone and in combination on systolic blood pressure (SBP) in (a) spontaneously hypertensive rats, (b) 2K1C
hypertensive rats, and (c) DOCA-salt hypertensive rats. Ate, atenolol; Nit, nitrendipine; Ate + Nit, combination of atenolol and nitrendipine. n¼8in
each group. *P,0.05, **P,0.01 versus day 0 (before drug administration).
198 Journal of Hypertension 2005, Vol 23 No 1

This synergism was universal. In the present work, a
potent synergism was found existing in all three hyper-
tensive rat models. These different hypertensive mod-
els might possess a different sensitivity to different
classes of antihypertensive drugs. However, no prefer-
ence to a certain model was found for this combination.
This might imply the diminishing requirement of
individualization in the treatment of hypertension when
the combination is used.
Recently, it has been proposed that BPV is an impor-
tant factor determining organ damage in hypertension.
In 1987, Parati et al. found that for nearly any level of
24-h mean BP, patients in whom the BPV was lower
had a lower prevalence and severity of organ damage
than those with higher BPV [24]. Recently, the clinical
prognostic significance of BPV has also been reported
in some clinical studies with ambulatory blood pressure
monitoring [3,4,26]. Our previous study demonstrated
that the severity of organ damage (end-organ damage
score) was positively related to the SBP level (r¼0.32,
n¼50, P,0.05) and to SBPV (r¼0.65, n¼50,
P,0.001) in 60-week-old SHR [27]. A similar result
was obtained in another study [28]. Accordingly, it
seems very important to emphasize the role of BPV in
antihypertensive therapy. However, it is not clear how
to control BPV in the treatment of hypertension. We
have previously proposed two ways to reduce BPV in
antihypertensive therapy [27]: (1) to find antihyperten-
sive drugs with an intrinsic effect on lowering BPV,
such as ketanserin, adenosine analogues, and so on; and
(2) to treat with the long-acting antihypertensive drugs,
such as candesartan, amlodipine, and so on. The
present work might show a third way to control BPV in
the treatment of hypertension: combination therapy. In
the present work, chronic treatment with a combination
of atenolol and nitrendipine markedly decreased BPV
in SHR and an obvious synergism on BPV reduction
was found in combination therapy. These results sug-
gested that combination therapy was more effective in
control of BPV than monotherapy. It is well known that
nitrendipine possesses a rapid and relatively short-
lasting antihypertensive action, and atenolol exhibits a
slow but relatively persistent hypotensive effect. The
hemodynamic features of these two drugs may contri-
bute to the synergism on BPV reduction in combination
therapy. Obviously, this is not the totality of the
possible mechanism underlying the synergism on BPV
reduction.
Arterial baroreflex dysfunction is another feature of
hypertension. It has been well recognized that BRS is
impaired in hypertensive humans and animals [5,6,29].
Our previous study proposed that BRS was one of the
independent variables related to end-organ damage
score, and BRS predicted the end-organ damage in
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Table 1 Effects of long-term treatment with atenolol and nitrendipine alone and in combination on the hemodynamics in spontaneously
hypertensive rats (SHR)
Wistar– Kyoto rats
(n¼10)
SHR
(n¼10)
Atenolol
(n¼10)
Nitrendipine
(n¼10)
Atenolol + nitrendipine
(n¼13)
SBP (mmHg) 138 1.8**#192 3.0 170 4.5**## 173 4.3**## 151 3.4**
DBP (mmHg) 94 1.4**#128 5.4 113 3.8*#113 3.5*#101 2.3**
Heart period (ms) 172 5.1 161 3.0 193 3.2**#156 1.8## 179 4.5**
SBPV (mmHg) 9.0 0.4**## 15.0 0.6 12.5 0.8* 12.8 0.7*#11.1 0.4**
DBPV (mmHg) 7.4 0.3**## 11.6 0.6 10.1 0.8 10.7 1.0 9.4 0.3**
HPV (ms) 25.6 1.6## 29.9 2.5 38.8 1.0** 37.8 0.9* 37.8 2.3*
BRS (ms/mmHg) 1.10 0.08** 0.34 0.05 0.54 0.05*## 0.57 0.05**## 0.98 0.07**
Data presented as mean standard error of the mean. SBP, systolic blood pressure; DBP, diastolic blood pressure; SBPV, systolic blood pressure variability; DBPV,
diastolic blood pressure variability; HPV, heart period variability; BRS, baroreflex sensitivity. *P,0.05 and **P,0.01 versus SHR; #P,0.05 and ## P,0.01 versus
atenolol and nitrendipine in combination.
Table 2 Effects of long-term treatment with atenolol and nitrendipine alone and in combination on pathological changes in ventricles,
kidneys and aortae in spontaneously hypertensive rats (SHR)
Wistar– Kyoto rats
(n¼10)
SHR
(n¼10)
Atenolol
(n¼10)
Nitrendipine
(n¼10)
Atenolol + nitrendipine
(n¼13)
LVW/BW (mg/g) 2.35 0.06**## 3.62 0.09 3.35 0.08*## 3.25 0.08**#2.95 0.08**
RVW/BW (mg/g) 0.666 0.015* 0.739 0.023 0.782 0.022 0.808 0.043 0.736 0.032
VW/BW (mg/g) 3.02 0.07**## 4.36 0.10 4.08 0.08#4.06 0.13#3.69 0.10**
LVW/RVW 3.53 0.07**#4.93 0.16 4.24 0.12** 4.08 0.14** 4.07 0.14**
AW/length (mg/mm) 0.98 0.06** 1.50 0.03 1.14 0.05**#1.23 0.03**## 1.02 0.03**
GSS 28.0 1.7**## 59.2 2.6 49.32.0**## 47.6 1.9**## 41.1 1.3**
Data presented as mean standard error of the mean. LVW, left ventricular weight; BW, body weight; RVW, right ventricular weight; VW, ventricular weight; AW, aortic
weight; GSS, glomerulosclerosis score. *P,0.05 and **P,0.01 versus SHR; #P,0.05 and ## P,0.01 versus atenolol and nitrendipine in combination.
Synergism of atenolol and nitrendipine Xie et al. 199

hypertension [28]. In the present work, chronic treat-
ment with a combination of atenolol and nitrendipine
markedly enhanced BRS in SHR, and an obvious
synergism on BRS restoration was found in combination
therapy.
The present work for the first time clearly demon-
strated that chronic treatment with atenolol, nitrendi-
pine, or a combination of these two drugs possessed
obvious effects on organ protection in SHR, and an
obvious synergism on organ protection was found in
combination therapy. It was found that LVW/BW, AW/
length, and GSS were all positively related to BP and
BPV, and negatively related to BRS. In multiple-
regression analysis, a decrease in left ventricular hyper-
trophy was most significantly related to the decrease in
SBPV and SBP. The decrease in aortic hypertrophy
was most significantly related to the increase in BRS
and decrease in SBPV, and amelioration in the renal
lesion was most significantly associated with the in-
crease in BRS. These results suggest that the decrease
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Table 4 Linear regression coefficient (r) between blood pressure, blood pressure
variability and baroreflex sensitivity values and organ damage in treated and untreated
spontaneously hypertensive rats (n43).
Left ventricular
weight/body
weight
Aortic
weight/length
Glomerulosclerosis
score
Systolic blood pressure 0.555** 0.603** 0.588**
Diastolic blood pressure 0.397** 0.399** 0.450**
Systolic blood pressure variability 0.636** 0.619** 0.505**
Diastolic blood pressure variability 0.421** 0.355* 0.271
Baroreflex sensitivity 0.561** 0.648** 0.646**
*P,0.05, **P,0.01.
Table 3 Results of the probability sum test in spontaneously
hypertensive rats treated with long-term nitrendipine and atenolol
Parameter Pvalue qvalue
Systolic blood pressure
Atenolol PAte ¼50%
Nitrendipine PNit ¼40% 1.43
Atenolol + nitrendipine PAte þNit ¼100%
Systolic blood pressure variability
Atenolol PAte ¼30%
Nitrendipine PNit ¼30% 1.66
Atenolol + nitrendipine PAte þNit ¼85%
Diastolic blood pressure
Atenolol PAte ¼30%
Nitrendipine PNit ¼20% 1.75
Atenolol + nitrendipine PAte þNit ¼77%
Diastolic blood pressure variability
Atenolol PAte ¼30%
Nitrendipine PNit ¼20% 1.40
Atenolol + nitrendipine PAte þNit ¼62%
Baroreflex sensitivity
Atenolol PAte ¼50%
Nitrendipine PNit ¼60% 1.25
Atenolol + nitrendipine PAte þNit ¼100%
Left ventricular weight/body weight
Atenolol PAte ¼0
Nitrendipine PNit ¼20% 1.92
Atenolol + nitrendipine PAte þNit ¼38%
Aortic weight/body weight
Atenolol PAte ¼60%
Nitrendipine PNit ¼20% 1.36
Atenolol + nitrendipine PAte þNit ¼92%
Glomerulosclerosis score
Atenolol PAte ¼30%
Nitrendipine PNit ¼40% 1.59
Atenolol + nitrendipine PAte þNit ¼92%
PAte,PNit,andPAte þNit were the percentages of animals possessing an effective
decrease in systolic blood pressure, diastolic blood pressure, systolic blood
pressure variability, baroreflex sensitivity, left ventricular weight/body weight,
aortic weight/body weight, or glomerulosclerosis score produced by atenolol,
nitrendipine, and a combination of atenolol and nitrendipine. q>1.15 means
synergism.
r ⫽ 0.636
P ⬍ 0.01
2
3
4
5
SBPV (mmHg)
LVW/BW (mg/g)
0.5
1
1.5
2
AW/length (mg/mm)
30
35
40
45
50
55
60
65
70
75
80
BRS (ms/mmHg)
GSS
0.5
1
1.5
2
AW/length(mg/mm)
81318
SBPV (mmHg)
81318
r ⫽ 0.619
P ⬍ 0.01
00.511.5
r ⫽ ⫺0.646
P ⬍ 0.01
BRS (ms/mmHg)
00.511.5
r ⫽ ⫺0.648
P ⬍ 0.01
Fig. 3
Examples of correlation between hemodynamic parameters and organ-
damage parameters in treated and untreated spontaneously
hypertensive rats in chronic studies. n¼43. LVW, left ventricular
weight; BW, body weight; SBPV, systolic blood pressure variability;
AW, aortic weight; GSS, glomerulosclerosis score; BRS, baroreflex
sensitivity.
200 Journal of Hypertension 2005, Vol 23 No 1

in BP and BPV and the restoration of baroreflex func-
tion may co-contribute to the organ protective action of
drugs in SHR.
In conclusion, treatment with the combination of
atenolol and nitrendipine exhibited a rapid and persis-
tent antihypertensive effect and possessed an obvious
synergism on BP and BPV reduction, BRS restoration
and organ protection in hypertensive animals. Chronic
treatment with atenolol, nitrendipine, or a combination
of these two drugs possessed obvious organ protection
in SHR. Besides the BP reduction, the decrease in
BPV and the restoration of BRS may importantly
contribute to this organ protection.
References
1 Materson BJ, Reda DJ, Cushman WC, Massie BM, Freis ED, Kochar MS,
et al. Single-drug therapy for hypertension in men. A comparison of six
antihypertensive agents with placebo. The Department of Veterans Affairs
Cooperative Study Group on Antihypertensive Agents. N Engl J Med
1993; 328:914– 921.
2 Chobanian AV, Bakris GL, Black HR, Cushman WC, Green LA, Izzo JL Jr,
et al. The Seventh Report of the Joint National Committee on prevention,
detection, evaluation, and treatment of high blood pressure: the JNC 7
report. JAMA 2003; 289:2560– 2572.
3 Kikuya M, Hozawa A, Ohokubo T, Tsuji I, Michimata M, Matsubara M,
et al. Prognostic significance of blood pressure and heart rate variabil-
ities: the Ohasama study. Hypertension 2000; 36:901–906.
4 Parati G, Mancia G. Blood pressure variability as a risk factor. Blood
Press Monit 2001; 6:341– 347.
5 Su DF, Miao CY. Functional studies of arterial baroreflex in conscious
rats. Acta Pharmacol Sin 2002; 23:673– 679.
6 Sleight P. The importance of autonomic nervous system in health and
disease. Aust NZ J Med 1997; 27:467– 473.
7 Du WM, Miao CY, Liu JG, Shen FM, Yang XQ, Su DF. Effects of long-
term treatment with ketanserin on blood pressure variability and end-
organ damage in spontaneously hypertensive rats. J Cardiovasc Pharma-
col 2003; 41:233– 239.
8 Liu JG, Xu LP, Chu ZX, Miao CY, Su DF. Contribution of blood pressure
variability to the effect of nitrendipine on end-organ damage in sponta-
neously hypertensive rats. J Hypertens 2003; 21:1961–1967.
9 Guan S, Fox J, Mitchell KD, Navar LG. Angiotensin and angiotensin-
converting enzyme tissue levels in two-kidney, one clip hypertensive rats.
Hypertension 1992; 20:763– 767.
10 Hirata Y, Matsuoka H, Suzuki E, Hayakawa H, Sugimoto T, Matsuda Y,
et al. Role of endogenous ANP in DOCA-salt hypertensive rats: effects of
a novel non-peptide antagonist for ANP receptor. Circulation 1993;
87:554– 561.
11 Shan ZZ, Dai SM, Su DF. Arterial baroreflex deficit induced organ
damage in sinoaortic denervated rats. J Cardiovasc Pharmacol 2001;
38:427– 437.
12 Norman RA Jr, Coleman TG, Dent AC. Continuous monitoring of arterial
pressure indicates sinoaortic denervated rats are not hypertensive. Hyper-
tension 1981; 3:119– 125.
13 Smyth HS, Sleight P, Pickering GW. Reflex regulation of arterial pressure
during sleep in man: a quantitative method of assessing baroreflex
sensitivity. Circ Res 1969; 24:109– 121.
14 Su DF, Chen L, Kong XB, Cheng Y. Determination of arterial baroreflex-
blood pressure control in conscious rats. Acta Pharmacol Sin 2002;
23:103– 109.
15 Su DF, Cerutti C, Barres C, Julien C, Vincent M, Paultre C, et al. Arterial
baroreflex control of heart period is not related to blood pressure
variability in conscious hypertensive and normotensive rats. Clin Exp
Pharmacol Physiol 1992; 19:767– 775.
16 Hayakawa H, Raij L. The link among nitric oxide synthase activity,
endothelial function, and aortic and ventricular hypertrophy in hyper-
tension. Hypertension 1997; 29:235– 241.
17 Miao CY, Xie HH, Wang JJ, Su DF. Candesartan inhibits sinoaortic
denervation-induced cardiovascular hypertrophy in rats. Acta Pharmacol
Sin 2002; 23:713– 720.
18 Miao CY, Tao X, Gong K, Zhang SH, Chu ZX, Su DF. Aterial remodeling
in chronic sinoaortic-denervated rats. J Cardiovasc Pharmacol 2001;
37:6– 15.
19 Kimula K, Tojo A, Matsuoka H, Sugimoto T. Renal arteriolar diameters in
spontaneously hypertensive rats: vascular cast study. Hypertension
1991; 18:101– 110.
20 Jin ZJ. About the evaluation of drug combination. Acta Pharmacol Sin
2004; 25:146– 147.
21 Su DF, Xu LP, Miao CY, Xie HH, Shen FM, Jiang YY. Two useful methods
for evaluating antihypertensive drugs in conscious freely moving rats. Acta
Pharmacol Sin 2004; 25:148– 151.
22 Xu LP, Miao CY, Shen FM, Jiang YY, Su DF. Synergism of atenolol and
amlodipine on lowering and stabilizing blood pressure in spontaneously
hypertensive rats. Fundam Clin Pharmacol 2004; 18:33–38.
23 Sica DA. Fixed-dose combination antihypertensive drugs. Do they have a
role in rational therapy? Drugs 1994; 48:16 –24.
24 Parati, G, Pomidossi, G, Albini, F, Malaspina D, Mancia G. Relationship of
24-hour blood pressure mean and variability to severity of target-organ
damage in hypertension. J Hypertens 1987; 5:93 –98.
25 Mancia G, Frattola A, Parati G, Santucciu C, Ulian L. Blood pressure
variability and organ damage. J Cardiovasc Pharmacol 1994; 24(suppl
A):S6– S11.
26 Sander D, Kukla C, Klingelhofer J, Winbeck K, Conrad B. Relationship
between circadian blood pressure patterns and progression of early
carotid atherosclerosis: a 3-year follow-up study. Circulation 2000;
102:1536– 1541.
27 Su DF, Miao CY. Blood pressure variability and organ damage. Clin Exp
Pharmacol Physiol 2001; 28:709– 715.
28 Shan ZZ, Dai SM, Su DF. Relationship between baroreceptor reflex
function and end-organ damage in spontaneously hypertensive rats. Am J
Physiol 1999; 277:H1200– H1206.
29 Bristow JD, Honour AJ, Pickering GW, Sleight P, Smyth HS. Diminished
baroreflex sensitivity in high blood pressure. Circulation 1969; 39:
48– 54.
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Synergism of atenolol and nitrendipine Xie et al. 201