ORIGINAL ARTICLE

Pharmacological Efficacy of CPU 86017 on Hypoxic

Pulmonary Hypertension in Rats
Mediated by Direct Inhibition of Calcium Channels and Antioxidant
Action, but Indirect Effects on the ET-1 Pathway
Tian-Tai Zhang, PhD, Bing Cui, MS, De-Zai Dai, MD, and Xiao-Yun Tang, MS

investigation have been conducted to reveal more understand-

Abstract: Endothelin-1 (ET-1) plays a key role in the pathogenesis ing of the mechanisms underlying this disease in recent years.
of pulmonary hypertension. The present study was conducted to The first orally effective drug to declare that involvement of an
examine the effects of a novel compound p-chlorobenzyltetrahy- activated endothelin system plays a crucial role in the
droberberine (CPU 86017) on endothelin-1 system of hypoxia- pathologic progression of pulmonary hypertension was
induced pulmonary hypertension in rats. SD male rats were divided Bosentan, launched in 2001.2 Suppression on an overactive
into control, untreated pulmonary hypertension, nifedipine (10 mg/kg endothelin system is likely to provide a reduction in the raised
p.o.), and CPU 86017 (80, 40, and 20 mg/kg p.o.) groups. The pressure in the pulmonary artery. Pulmonary hypertension can
pulmonary hypertension was established by housing the rats in a be created experimentally by sustained hypoxia in which an
hypoxic (10 6 0.5% oxygen) chamber 8 hours per day for 4 weeks. exaggerated endothelin-1 (ET-1) release and oxidative stress
Hemodynamic and morphologic assessment exhibited a significant participation are involved.3 The increase in pulmonary
increase in the central vein pressure (CVP), right ventricular systolic resistance is attributed to an imbalance between the
pressure (RVSP), and pulmonary arteriole remodeling in the pul- compromised vasodilating substances (ie, NO) against an
monary hypertensive rats, which were improved by CPU 86017 80 enhanced vasoconstricting ET-1.4 A damage by hypoxia to
and 40 mg/kg administration (P , 0.01). The elevated pulmonary pulmonary endothelium and myocardium results in an
endothelin-1 level and the over-active preproET-1 and iNOS mRNA overproduction of ET-1 during the process of formation of
expression were also decreased significantly (P , 0.01) in CPU pulmonary hypertension,5 and presumably an exaggerated
86017 groups. The maladjustment of redox enzyme system in pulmo- release of ET-1 from the damaged myocardium and vascular
nary hypertension rats was corrected after treatment. We concluded endothelium play a crucial role in deteriorating the progression
that CPU 86017 improves pulmonary hypertension mainly to sup- of PH. A rise in intracellular calcium is harmful to impair
press the endothelin-1 pathway at the upstream and downstream via endothelium and vascular smooth muscle in pulmonary
calcium antagonism and antioxidative action, then, resulting in a arterioles, contributing to further excessive release of ET-1
relief in pathogenesis of the disease. in response to sustained hypoxia exposure.
Key Words: CPU 86017, endothelin 1, inducible nitric oxide A new compound, p-benzyl-tetra-hydro-berberine, re-
synthase, pulmonary hypertension, reactive oxygen species ferred to as CPU 86017 (Fig. 1) possesses calcium channels
blockade and anti-oxidation effects6 and is effective to treat
(J Cardiovasc Pharmacol TM 2005;46:727–734) chronic congestive heart failure with an associated suppression
on the ET-1 system.7 It is hypothesized that an activated ET
pathway plays an important role in the development of hypoxia
PH and an interruption at the sites of either the upstream or the
P ulmonary hypertension (PH) is characterized with an
abnormally sustained elevation of the blood pressure in the
pulmonary circulation, leading to the right ventricle hyper-
downstream of the ET pathway is likely to relieve the PH. We
intended to test if CPU 86017 possessing calcium antagonism
trophy and dysfunction, with high incidence of morbidity and and antioxidative action could be beneficial to chronic PH
mortality.1 At present, the definite mechanisms of this disease developed by hypoxic exposure via its suppression on the ET-1
still remain unknown; fortunately, more exploration and pathway.

Received for publication June 6, 2005; accepted August 3, 2005. METHODS

From the Research Division of Pharmacology, China Pharmaceutical
University, Nanjing, China. Animals
This work was supported by a key project from National Natural Science Sprague–Dawley (SD) male rats (weighing 200–220 g)
Foundation of China. No: 30230170. were divided randomly into 6 groups (n = 13): control,
Reprints: De-Zai Dai, Professor, Research Division of Pharmacology, China
Pharmaceutical University, Nanjing 210009, China (e-mail: dezaidai@ untreated PH model, CPU 86017 80, 40, and 20 mg/kg and
vip.sina.com). nifedipine 10 mg/kg (Nif), each was given 15 g forage every
Copyright Ó 2005 by Lippincott Williams & Wilkins day and free access to water. The room temperature was

J Cardiovasc Pharmacol ä  Volume 46, Number 6, December 2005 727

Zhang et al J Cardiovasc Pharmacol ä  Volume 46, Number 6, December 2005

Weight Index of the Right Ventricle and Lungs

After hemodynamic measurement, animals were killed
to remove hearts and lungs. Hearts were washed by 0.9% NaCl
solution and carefully divided into the right ventricle and left
ventricle (including septum). The weight index of right
ventricle and lungs were computed against body weight as:
RV/BW, RV/LV, and Lung/BW, separately.
Biochemistry Assays
Pulmonary tissue samples were weighted and homog-
enized in 9 volumes of ice-cold 0.9% NaCl solution. The
homogenates were centrifuged (4000 rpm, 4°C, 10 min) and
aliquots of supernatants were collected for superoxide dis-
mutase (SOD), malondialdehyde (MDA), NO, and cNOS
assays. Protein contents of the samples were detected by the
Coomassie Brilliant Blue method. Blood samples were centri-
fuged (4000 rpm, 4°C, 10 min) and the aliquots of serum were
FIGURE 1. The chemical structure of CPU 86017, MW: 465.5. collected for the assay of NO.
The measurement of total SOD activity was based on the
controlled between 20°C and 25°C. All procedures performed generation of nitrite that derives from the reaction between
on the animals in the present study were conducted in hydroxylamine and the superoxide radicals produced by xan-
accordance with the Animal Regulations of Jiangsu Province. thine oxidase-xanthine system. MDA content was evaluated by
thiobarbituric acid (TBA) reaction.
The cNOS activity in pulmonary tissue was measured by
Chemicals and Drug Administration NADPH oxidation, using L-arginine as a substrate. The NO
p-Chlor-benzyl-tetrahydroberberine chloride (CPU 86017) concentration was assessed by the measurement of NO meta-
was synthesized by the New Drug Center of China Phar- bolites, nitrite and nitrate, using the Griess reaction.
maceutical University and provided by Shandong Xinhua All the procedures performed in the SOD, MDA, NOS,
Pharmaceutical Industry, Zibo, Shandong province. CPU and NO assays were in accordance with the instructions of kits
86017 was suspended freshly in 0.5% CMC-Na and admin- (Nanjing Jiancheng Bioengineering Institute, China).
istrated in 3 dosages of 80, 40, and 20 mg/kg by p.o. Nifed-
ipine (10 mg/kg p.o.) was administrated as a positive control, Measurement of Endothelin-1
and vehicle was given in control and PH model groups. Drug The blood samples were treated with aprotinin and 10%
administration was performed once a day and lasted for EDTA; after centrifugation (3000 rpm, 15 min, 4°C) the plas-
4 weeks (28 d) from the first day of hypoxia exposure. ma were removed and stored under 220°C before use. The
pulmonary tissues were homogenized in 1 mol/L acetic acid
Hypoxic Pulmonary Hypertension and centrifuged (3000 rpm, 15 min, 4°C), the supernatants were
For the establishment of pulmonary hypertension, rats collected and stored under 220°C before use. The ET-1 con-
were exposed to hypoxia according to the reference with some centration of pulmonary tissue and plasma were determined by
modification.8 The control group was exposed to air in the the RIA method,9 using the [125I]-ET-1 radioimmunoassay kit
same room. Briefly, hypoxic rats were housed in an organic (Beijing Northern Bioengineering Institute, China).
glass chamber 8 hours per day and the O2 concentration was Evaluation of Remodeling of
well kept at 10 6 0.5% by an instant monitoring system and
Pulmonary Arterioles
blowing of nitrogen. The hypoxia exposure lasted for 4 weeks
(28 days) to induce pulmonary hypertension. Soda lime 20 g The pulmonary tissues were dipped in 10% formalin,
and anhydrous calcium chloride 30 g were placed inside the embedded by paraffin, and sliced into 4-mm-thick pieces. The
chamber to absorb CO2 and moisture. H&E staining was performed according to the previous
practice.7 A color graph analysis system (Quantment 500,
Leica, German) was used for the morphologic assessment.
Hemodynamic Measurement Pulmonary arterioles wall thickness (WT) was measured and
After 28 days of hypoxia exposure, rats were anesthetized the relative values of WT% was calculated as follows: WT% =
with urethane (1.5 g/kg, i.p.) and the cannulation operation was (external diameter 2 internal diameter)/external diameter 3 100.
conducted. Briefly, the jugular vein and carotid artery were ex- We took 10 visual fields for each slide and got the mean value.
posed and PE-90 polyethylene catheters were cannulated into
the right carotid and the right ventricle to measure the central mRNA Expression of preproET-1, cNOS, and
vein pressure (CVP) and right ventricular systolic pressure iNOS in Pulmonary Tissue
(RVSP), respectively. The hemodynamic analysis was per- The extraction of RNA followed the chloroform-
formed with a real-time hemodynamic analyzer (MPA2000, hydroxybenzene method. Briefly, the pulmonary tissue
Shanghai Secondary Military Medical University). was homogenized in TRIZOL as fully as possible. After

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 Pharmacological Efficacy
J Cardiovasc Pharmacol ä  Volume 46, Number 6, December 2005 of CPU 86017 on Hypoxic PH in Rats

centrifugation (5000 rpm, 4°C, 10 min), the supernatants were

collected and treated with chloroform and hydroxybenzene to
remove protein. The RNA was precipitated in isopropanol and
washed by ethanol. Following the reverse transcription, the
cDNA was obtained and stored at 270°C before use.
The PCR experiment was performed in 25 mL final
volume with the following profile: pre-denaturation 5 minutes
at 94°C, denaturation 40 seconds at 94°C, annealing 40
seconds at 64°C for preproET-1(pp-ET-1), at 58°C for cNOS,
at 58°C for iNOS, and at 60°C for b-actin, extension 1 minute
at 72°C. The last cycle was followed by a final extension step
of 10 minutes at 72°C. The PCR products were separated by
gelose electrophoresis and stained by ethidium bromide before
being detected by a semi-quantitative analyzer (GDS8000,
Syngene, England).

Statistical Analysis
All data were expressed as mean 6 SE and the one-way
ANOVA assay was performed. The differences between the
two groups were compared by Student-Newman-Keuls (SNK)
test. P , 0.05 was considered statistically significant.

RESULTS
Relief in Pulmonary Hypertension
The RVSP and CVP of hypoxic PH rats were elevated
significantly by + 72% (P , 0.01) and +712% (P , 0.01)
respectively versus the control group after exposure to hypoxia
for 28 days, suggesting the successful establishment of hyp-
oxic pulmonary hypertension. Significant decreases in RVSP
and CVP in CPU 86017 80, 40, and 20 mg/kg and Nif groups
(P , 0.01) were achieved (Fig. 2).
FIGURE 2. A, Central vein pressure of PH, CPU 86017 80, 40,
Regression of Right Ventricular Hypertrophy and 20 mg/kg and nifedipine groups after 28 days of exposure
After 28 days of hypoxic exposure, the untreated rats to hypoxia. CPV elevated significantly in PH model group and
exhibited elevated RV/BW, by +50% (P , 0.01) and recovered by CPU 86017 80, 40, and 20 mg/kg and nifedipine
RV/(LV+S) by +53% (P , 0.01), showing the right ventricle 10 mg/kg administration p.o. cP , 0.01 versus Control; fP ,
hypertrophy induced by hypoxia. Regressions of the cardiac 0.01 versus PH. B, Right ventricular systolic pressure of PH, CPU
weight index RV/BW and RV/(LV+S) were significant (P , 86017 80, 40, and 20 mg/kg and nifedipine groups after
0.01) in CPU 86017 80, 40, and 20 mg/kg and Nif groups 28 days of exposure to hypoxia. RVSP increased in PH model
against PH model. There were no significant differences in group by hypoxia and recovered to a different extent by CPU
Lung/BW among each group (Fig. 3). 86017 80, 40, and 20 mg/kg and nifedipine 10 mg/kg
administration p.o. cP , 0.01 versus Control; fP , 0.01
versus PH.
Attenuation of an Exaggerated
Endothelin System
Endothelin-1 is a potent endogenous substance for ves-
sel constriction secreted by endothelial and myocardial cells. Suppression on Oxidative Stress
After 28 days of hypoxia exposure, an elevation of the ET-1 After 28 days of hypoxia exposure, the SOD activity in
concentration in plasma (+67%, P , 0.01) and pulmonary plasma and pulmonary tissue decreased by 231% (P , 0.01)
tissue (+93%, P , 0.01) was significant. In CPU 86017 and 221% (P , 0.01), respectively. In CPU 86017 80, 40, and
80 mg/kg and Nif groups, the plasma ET-1 concentration 20 mg/kg groups, significant increment in the plasma SOD
showed a significant decline (P , 0.01), and in CPU 86017 80 activity was found, by 29% (P , 0.01), +15% (P , 0.05), and
and 40 mg/kg groups the pulmonary ET-1 concentration was +11% (P , 0.05) versus PH model, respectively. The SOD
reduced significantly against PH model (P , 0.01). activity in pulmonary tissue was also increased by +21%
The amount of mRNA expression of preproET-1 mRNA (P , 0.01), +4% (P , 0.05), +5% (P , 0.05) respectively. An
in PH model group was nearly 2-fold greater than the control increment of the plasma and pulmonary SOD activity in Nif
group (P , 0.01). In the CPU 86017 80 mg/kg group it de- group was significant, by +33% and +17% (P , 0.01) against
creased dramatically against the PH model (P , 0.01) (Fig. 4). PH model, respectively.

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Zhang et al J Cardiovasc Pharmacol ä  Volume 46, Number 6, December 2005

Effects on Nitric Oxide and Nitric

Oxide Synthase
The NO concentration in plasma and pulmonary tissue
of the PH model group decreased by 41% (P , 0.01) and
233% (P , 0.01) versus control, respectively. In CPU 86017
80, 40, and 20 mg/kg and Nif groups it increased by +65%
(P , 0.01), +54% (P , 0.01), +24% (P , 0.05), and +48%
(P , 0.01) in plasma and by +50% (P , 0.01), +31% (P ,
0.01), +19% (P . 0.05), and +32% (P , 0.01) in pulmonary
tissue against the PH model, respectively (Fig. 6).
The activity of cNOS in pulmonary tissue declined signifi-
cantly after chronic exposure to hypoxia (246%, P , 0.01).
In CPU 86107 80 mg/kg and Nif groups, it increased by +67%
(P , 0.05), and +70% (P , 0.01) compared with the PH
model, respectively.
FIGURE 3. RV/(LV+S) of PH, CPU 80, 40, and 20 mg/kg and The abundance of the cNOS and iNOS mRNA expres-
nifedipine groups after 28 days of exposure to hypoxia. The sion in the PH model group was increased significantly against
ratio of RV/(LV+S) increased significantly in the PH model control (P , 0.01). Declined mRNA expression of cNOS (P ,
group, and decreased obviously in the CPU 86017 80, 40, and 0.01), iNOS (P , 0.01) in CPU 86017 and Nif groups were
20 mg/kg and nifedipine 10 mg/kg groups. cP , 0.01 versus found, compared with the PH model group (Fig. 7).
Control; fP , 0.01 versus PH.
Regression on Remodeling of Small
Pulmonary Arterioles
In the PH model group, the WT% was increased by
The plasma and pulmonary tissue MDA level in the PH +58.8% (P , 0.01) against the control, and was decreased by
model increased significantly by +43% (P , 0.01) and +38% 244.4% (P , 0.01), 223.1% (P , 0.05), and 29.5% respec-
(P , 0.01) versus control. A decrease in plasma MDA in CPU tively in CPU 86017 80, 40, and 20 mg/kg groups against
86017 80 mg/kg group was significant against the PH model the PH model (Fig. 8).
(P , 0.01), and no reduction was found in the other two CPU
86017 groups. The MDA level in pulmonary tissue was
decreased by 224% (P , 0.01) and 221% (P , 0.05) in CPU DISCUSSION
86017 80 and 40 mg/kg groups versus PH model, respectively. CPU 86017 is derived from chemical modification of the
In the Nif group, a decline in the pulmonary MDA level by moiety of berberine, which possesses a blockade on the Ca2+
221% (P , 0.05), but no effects on the plasma MDA level and K+ channels;10,11 however, berberine is water insoluble
were found (Fig. 5). and very poor in oral absorption. Therefore the chemical

FIGURE 4. A, The plasma ET-1

content of PH, CPU 86017 80, 40,
and 20 mg/kg and nifedipine groups
after 28 days of exposure to hypoxia.
c
P , 0.01 versus Control; eP , 0.05
versus PH; fP , 0.01 versus PH.
B, Content of ET-1 of pulmonary
tissue in PH, CPU 86017 80, 40, and
20 mg/kg and nifedipine groups
after 28 days of exposure to hypoxia,
c
P , 0.01 versus Control; eP , 0.05
versus PH; fP , 0.01 versus PH. C,
RT-PCR analysis of preproET-1 mRNA
expression in pulmonary tissue.
Lanes 1, 2, 3, 4, 5, and 6 represent
Control, PH, CPU 86017 80 mg/kg,
CPU 86017 40 mg/kg, CPU 86017
20 mg/kg, and nifedipine groups.
c
P , 0.01 versus Control; eP , 0.05
versus PH; fP , 0.01 versus PH.

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 Pharmacological Efficacy
J Cardiovasc Pharmacol ä  Volume 46, Number 6, December 2005 of CPU 86017 on Hypoxic PH in Rats

FIGURE 5. A, Pulmonary tissue SOD activity of PH, CPU 86017 FIGURE 6. A, Content of NO in serum in PH after 28 days of
80, 40, and 20 mg/kg and nifedipine groups. In PH model exposure to hypoxia. Serum NO was decreased in the hypoxia
group, SOD activity sharply decreased and it was significantly PH model, and recovered in CPU 86017 80, 40, and 20 mg/kg
higher in the CPU 86017 80, 40 mg/kg and nifedipine 10 mg/kg p.o. and nifedipine 10 mg/kg p.o. groups. N = 13. cP , 0.01
groups. cP , 0.01 versus Control; eP , 0.05 versus PH; fP , versus Control; eP , 0.05 versus PH; fP , 0.01 versus PH.
0.01 versus PH. B, Pulmonary tissue MDA content of PH, CPU B, Content of NO in pulmonary tissue in PH. The pulmonary
86017 80, 40, and 20 mg/kg and nifedipine groups after NO was reduced in hypoxia model and recovered by treatment
28 days exposure to hypoxia. In the PH model group, pulmonary with CPU 86017 80, 40, and 20 mg/kg p.o. and nifedipine 10
tissue MDA increased significantly, in the CPU 86017 80, mg/kg p.o. after 28 days of exposure to hypoxia. N = 13. cP ,
40 mg/kg and nifedipine 10 mg/kg groups; it was significantly 0.01 versus Control; eP , 0.05 versus PH; fP , 0.01 versus PH.
lower against that of PH model group. cP , 0.01 versus
Control; eP , 0.05 versus PH; fP , 0.01 versus PH.

increase an influx of calcium and an increment in the intercellular

reformation is needed to improve bioavailability by producing free calcium concentration.17 The recent studies have
a new compound CPU 86017, which retains the profile of the indicated that increased intracellular calcium is also a deter-
blocking action on multi-channels with a significant enhance- minant to promote cell proliferation as a signal transform
ment of the calcium antagonism.12 It has been shown that CPU factor.18 The MAPK and PKC are involved in the events to
86017 is effective by oral medication to remove abnormalities follow an activation of the ETA receptors, and the processes of
of biochemical measurements, mRNA expression of the the transcript in the nuclear are also accelerated and
components involved in the ET-1- iNOS- ROS pathway, and reinforced.19,20
the hemodynamics and the pulmonary arterioles remodeling in CPU 86017 blocks the L-type calcium channel to reduce
hypoxic pulmonary hypertension. the influx of extracellular calcium; therefore, a decrease of
Hypoxia impairs pulmonary artery endothelium and intracellular calcium release is anticipated, leading to the dila-
myocardium to secrete an excess of ET-1,13,14 which initiates tation of pulmonary arterioles, regression of vascular and right
and exacerbates the development of pulmonary hypertension ventricular remodeling, and relief in the insult of the endo-
through its vasoconstrictive and pro-mitogenic properties.15,16 thelium and myocardium. Therefore, calcium channel block-
The binding of an excessive ET-1 to ETA receptors stimulates ade reveals an improvement to the endothelial and myocardial
both the L-type calcium channels, which are coupled to the function, which is compromised in hypoxia exposure, by
ETA receptors and phospholipase C through G protein to correcting an elevation of ET-1 level in pulmonary tissue and

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Zhang et al J Cardiovasc Pharmacol ä  Volume 46, Number 6, December 2005

FIGURE 7. A, The pulmonary

tissue cNOS activity of PH. CPU
86017 80, 40, and 20 mg/kg and
nifedipine groups after 28 days of
exposure to hypoxia. cP , 0.01
versus Control; eP , 0.05 versus
PH; fP , 0.01 versus PH. B, RT-PCR
analysis of cNOS mRNA expres-
sion in pulmonary tissue. Lanes 1,
2, 3, 4, 5, and 6 represent Control,
PH, CPU 86017 80 mg/kg, CPU
86017 40 mg/kg, CPU 86017
20 mg/kg, and nifedipine groups.
c
P , 0.01 versus Control; eP ,
0.05 versus PH; fP , 0.01 versus
PH. C, RT-PCR analysis of the
mRNA expression of iNOS in
pulmonary tissue. Lanes 1, 2, 3,
4, 5, and 6 representative Control,
PH, CPU 86017 80 mg/kg, CPU
86017 40 mg/kg, CPU 86017
20 mg/kg, and nifedipine groups.
c
P , 0.01 versus Control; eP ,
0.05 versus PH; fP , 0.01 versus PH.

plasma and the preproET-1 mRNA expression in the hypoxic pulmonary vasculature and, thereafter, to the right ventricular
pulmonary hypertension. myocardium can be produced by an activation of the ET-1
The ET-1 pathway comprises the iNOS and ROS in its pathway and in turn, the damaged vascular and myocardium
downstream and is involved in the pathologic process of some could release more ET-1 in the response to the insult. An
diseases.21 An increase in ET-1 deteriorates the downstream interruption at the cycle between the activation of the ETA, the
events to tissue such as an activation of iNOS and ROS in the signal pathway in the cytosol (an elevated intracellular calcium
pathway.22 The oxidative stress in the hypoxic pulmonary and ROS) and an enhancement of the transcription in the
hypertension is predominant and CPU 86017 decreases the nuclear provides a relief of the insult; therefore, an improve-
MDA level and increases SOD activity substantially by offer- ment of the pulmonary hypertension is expected.
ing an effect to suppress oxidative stress in accordance with The decreased level of NO and cNOS and increased
the previous data of CPU 86017.6 An increment of ROS, iNOS in pulmonary tissue are revealed as the consequences to
which diffuses into the nuclear to promote the transcription an impairment of pulmonary arterioles endothelium induced
process provides an increase in the abundance of mRNA of by hypoxia. The vascular NO is synthesized and released by
prepro-ET-1 and iNOS.23,24 The ET-1 is a sort of cytokine endothelial cell in a constant manner to mediate the vascular
and exerts an inflammatory effect on the endothelium and tone by combating the vasoconstrictive response of ET-1.
myocardium;25 the iNOS and ROS are the important elements However, the cNOS activity and NO content in the PH group
in an inflammatory process involved in the pathologic changes was significantly decreased but the cNOS mRNA expression
by chronic hypoxia exposure.23 The profound damage to the was increased. The same result was reported by Resta et al.26
pulmonary vasculature is associated with an excess of ET-1, Such a contradiction may be attributed to the endothelium
iNOS, and ROS activity in the lung. In fact an insult to the impairment with which the cNOS cannot be converted into

732 q 2005 Lippincott Williams & Wilkins

 Pharmacological Efficacy
J Cardiovasc Pharmacol ä  Volume 46, Number 6, December 2005 of CPU 86017 on Hypoxic PH in Rats

FIGURE 8. A, The wall thickness is increased in pulmonary small artery of PH in rats. CPU 86017 80, 40, and 20 mg/kg and
nifedipine groups after 28 days of exposure to hypoxia. cP , 0.01 versus Control; eP , 0.05 versus PH; fP , 0.01 versus PH.
B, Changes of WT% of pulmonary small artery in PH, CPU 86017 80, 40, and 20 mg/kg and nifedipine groups after 28 days of
exposure to hypoxia, cP , 0.01 versus Control; eP , 0.05 versus PH; fP , 0.01 versus PH. C, Micrographs of pulmonary arterioles
wall (diameter , 150 mm) of Control, PH, CPU 86017 80, 40, and 20 mg/kg and nifedipine groups.

a normally active form due to phosphorylation of Thr,27 which ACKNOWLEDGMENTS

causes a defective activity of the cNOS contributing to PAH We are grateful to Prof. Peng Si-Xun, Huang Wen-Long,
progression.28 On the other hand, in many cases, the amount and Zhang Can who synthesized the novel compound orig-
of mRNA transcription is not in parallel with the protein inally and many thanks for the kind supply of the tested com-
production. CPU 86017 increases NO and cNOS activity and pound CPU 86017 by Shandong Xinhua Pharmaceutical Co.
decreases iNOS activity in pulmonary tissue, suggesting an
improvement in pulmonary vascular endothelium function.
In conclusion, the ET-1–ROS pathway is involved in REFERENCES
the pathogenesis of hypoxic pulmonary hypertension and 1. Benisty JI. Pulmonary hypertension. Circulation. 2002;106:192–194.
2. O’Callaghan D, Gaine SP. Bosentan: a novel agent for the treatment of
the beneficial effects of CPU 86017 on the pulmonary hyper- pulmonary arterial hypertension. Int J Clin Pract. 2004;58:69–73.
tension are mediated by an indirect suppression on the ET-1 3. Presberg KW, Dincer HE. Pathophysiology of pulmonary hypertension
pathway by a calcium antagonist and antioxidative action. due to lung disease. Curr Opin Pulm Med. 2003;9:131–138.

q 2005 Lippincott Williams & Wilkins 733

Zhang et al J Cardiovasc Pharmacol ä  Volume 46, Number 6, December 2005

4. Humbert M, Morrell NW, Archer SL, et al. Cellular and molecular pathobi- 17. Chew DK, Orshal JM, Khalil RA. Elastase induced suppression of
ology of pulmonary arterial hypertension. J Am Coll Cardiol. 2004;43:13–24. endothelin-mediated Ca2+ entry mechanisms of vascular contraction.
5. Touyz RM, Schiffrin EL. Role of endothelin in human hypertension. Can Hypertension. 2003;42:818–824.
J Physiol Pharmacol. 2003;81:533–541. 18. Li SJ, Sun NL. Regulation of intracellular Ca2+ and calcineurin by NO/PKG
6. Tang YQ, Dai DZ, Zhao J, et al. Protective effect of pretreatment with in proliferation of vascular smooth muscle cells. Acta Pharmacol Sin.
tetrahydroberberine chloride in hypoxic damage in mouse cerebral 2005;26:323–328.
mitochondria. Chin J Pharmacol Toxicol. 1996;10:31–32. 19. Mazzocchi G, Rossi GP, Malendowicz LK, et al. Endothelin-1 [1-31],
7. Wang YQ, Shi YP, Dai DZ. The therapeutic effects of CPU 86017 on acting as an ETA-receptor selective agonist, stimulates proliferation
acute and chronic congestive cardiac failure mediated by reducing ET-1, of cultured rat zona glomerulosa cells. FEBS Lett. 2000;29:194–
NOS and oxidative stress in rats. Drug Dev Res. 2004;63:22–32. 198.
8. Pauvert O, Bonnet S, Rousseau E, et al. Sildenafil alters calcium signaling 20. Piacentini L, Gray M, Honbo NY, et al. Endothelin-1 stimulates cardiac
and vascular tone in pulmonary arteries from chronically hypoxic rats. Am fibroblast proliferation through activation of protein kinase C. J Mol Cell
J Physiol Lung Cell Mol Physiol. 2004;287:577–583. Cardiol. 2000;32:565–576.
9. Venuti A, Salani D, Manni V, et al. Expression of endothelin 1 and 21. Xu FP, Chen MS, Wang YZ, et al. Leptin induces hypertrophy via
endothelin A receptor in HPV-associated cervical carcinoma: new endothelin-1–reactive oxygen species pathway in cultured neonatal rat
potential targets for anticancer therapy. FASEB J. 2000;14:2277–2283. cardiomyocytes. Circulation. 2004;110:1269–1275.
10. Dai DZ, Feng Y, Li HT, et al. Blockade on sodium, potassium, and 22. Kalinowski L, Malinski T. Endothelial NADH/NADPH-dependent
calcium channels by a new antiarrhythmic agent CPU 86017. Drug Dev enzymatic sources of superoxide production: relationship to endothelial
Res. 1996;39:138–146. dysfunction. Acta Biochim Pol. 2004;51:459–469.
11. Wang HL, Li SB, Dai DZ. Effects of CPU 86017 on L-type calcium 23. Sham JS. Hypoxic pulmonary vasoconstriction: ups and downs of reactive
current in single guinea pig hypertrophic ventricular myocytes. J China oxygen species. Circ Res. 2002;18:649–651.
Pharmaceut Univ. 2004;35:259–262. 24. Wedgwood S, Black SM. Role of reactive oxygen species in vascular
12. Dai DZ, He Y, Huang FH, et al. Comparison of inhibitory activity of remodeling associated with pulmonary hypertension. Antioxid Redox
berberine derivate CPU 86017 and berberine on vascular contraction. Signal. 2003;5:759–769.
J China Pharmaceut Univ. 2000;31:447–450. 25. Goraca A. New views on the role of endothelin. Endocr Regul. 2002;36:
13. Pellicelli AM. Cytokines in portopulmonary hypertension. Gut. 2004;53: 161–167.
1721. 26. Resta TC, Chicoine LG, Omdahl JL, et al. Maintained upregulation of
14. Giaid A, Michel RP, Stewart DJ, et al. Expression of endothelin-1 in the pulmonary eNOS gene and protein expression during recovery from
lungs of patients with pulmonary hypertension. N Engl J Med. 1993;328: chronic hypoxia. Am J Physiol. 1999;276:699–708.
1732–1739. 27. Fleming I, Fisslthaler B, Dimmeler S, et al. phosphorylation of Thr495
15. Freitas CF, Faro R, Dragosavac D, et al. Role of endothelin-1 and thromboxane regulates Ca2+/calmodulin-dependent endothelial nitric oxide synthase
A2 in the pulmonary hypertension induced by heparin-protamine interaction activity. Circ Res. 2001;88:68–75.
in anesthetized dogs. J Cardiovasc Pharmacol. 2004;43:106–112. 28. Murata T, Sato K, Hori M, et al. Decreased endothelial nitric-oxide
16. Kim H, Yung GL, Marsh JJ, et al. Endothelin mediates pulmonary synthase (eNOS) activity resulting from abnormal interaction between
vascular remodeling in a canine PH model of chronic embolic pulmonary eNOS and its regulatory proteins in hypoxia-induced pulmonary
hypertension. Eur Respir J. 2000;15:640–648. hypertension. J Biol Chem. 2002;277:44085–44092.

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