全 文 ：Relation of vasodilative action of isocorydine to cyclic nucleotides
JIANG Qing-Song1＊ , HUANG Xie-Nan2 , SUN An-Sheng2 , WU Qin2 , XIE Xiao-Long2
(1.Department of Pharmacology , Chongqing University of Medical Sciences , Chongqing　400016 , China;
2.Department of Pharmacology , Zunyi Medical College , Zunyi　563003 , China)
Abstract:The vasodilative mechanism of isocorydine in
isolated rabbit aorta was studied by recording the isotonic
contraction and radioimmunoassaying the levels of cyclic
nucleotides.Isocorydine induced a relaxation with a con-
centration-dependent manner in the isolated rabbit aortic
strips precontracted by norepinephrine (NE , 1 μmol·
L-1)or KCl (30 mmol·L-1), the EC50 was(12.6±
4.4)and(447±38)μmol·L-1 , respectively .The effect
of isocorydine on the precontraction induced by NE was 36
times as strong as that by KCl.The relaxing effect of iso-
corydine on the strip of rabbit aorta precontracted by NE
was partially inhibited by methylene blue (10 μmol·
L-1), but was not affected by glibenclamide , in-
domethacin , propranolol or N-L-nitro-arginine.Further-
more , isocorydine (10 , 100 μmol·L-1)increased the
levels of cGMP , and the effect were inhibited by methy-
lene blue.The results suggest that the vasodilative mech-
anism of isocorydine be related to accumulation of cGMP
by activating guanylate cyclase.
Key words:isocorydine;aorta , thoracic;vasodilation;
cyclic AMP;cyclic GMP
CLC number:R972
Document code:A
Article ID:1000-3002(2001)04-0251-05
Isocorydine , an alkaloid abstracted from the
Chinese traditional herbs Dicranostigma leptopo-
dum Fedde or Doctylicapnos scandens Hutch , pos-
sesses antispasmodic action on isolated vessels and
some visceral smooth muscles[ 1, 2] .Our recent
study[ 3] showed that its vasodilatation is potentially
related with inhibiting the receptor-mediated Ca2+-
influx and Ca
2+-release , and it is a non-cla-
　
　　Received date:2000-09-07　Accepted date:2001-01-09
　　Foundation item:The project supported by Science Founda-
tion of Guizhou Province(943033).
　　Biography:JIANG Qing-Song(1972 -), female , lecturer ,
MM , main research field is cardiovascular pharmacology.
　　＊Corresponding author.Tel:(023)68001318 ,
E-mail:cqjqyang@263.net
ssical Ca
2+
antagonist.In this paper , authors aim
at ascertaining more fully the idiographic tache of
vascular activities affected by isocorydine in isolat-
ed spiral strips of rabbit aorta.
1　MATERIALS AND METHODS
1.1　Chemicals
Isocorydine (white powder , purity >95%)
obtained from Prof.LIAN Xiu-Feng (worked in
the People′s Hospital , Chengcheng County ,
Shanxi)was dissolved in 1%HCl , pH 5.0.Pa-
paverine , methylene blue(MB), N-L-nitro-argi-
nine(L-NNA), indomethacin , glibenclamide and
propranolol were all from Sigma Chemical Co.,
nitroglycerin was from Beijing Yiming Pharmaceu-
tic Factory , and norepinephrine (NE)was from
Guangzhou Mingxing Pharmaceutic Factory , other
chemicals were of AR grade.All solutions were
prepared with deionized distilled water except for
indomethacin and glibenclamide.Indomethacin
was first dissolved in acetone and glibenclamide in
ethanol , then diluted with distilled water casually
when used.Radioimmunoassay kits for cAMP and
cGMP were from Immunotech a Coulter Company ,
France.
1.2　Tissue preparations
Adult New Zealand white rabbits (Experi-
mental Animal Center , Chongqing University of
Medical Sciences)of either sex , weighing (2.3±
0.4)kg( x±s).Rabbits were sacrified by bleed-
ing , and the thoracic aorta was removed and
cleaned of all loosely adherent tissues.The en-
dothelium was intentionally removed.The aorta
was cut into 20 mm×2.5 mm spiral strips , which
were then mounted under a tension of 2 g preload-
ed in a 20mL organ bath containing Krebs physio-
·251·中国药理学与毒理学杂志　　2001 年 8月;15(4):251-255
logical salt solution(mmol·L-1 :NaCl 120 , CaCl2
2.2 , KCl 5.4 , MgCl2 1.0 , NaHCO3 25.0 and
glucose 5.6 , pH 7.4), maintained at 37℃ and
bubbled continuously with a mixture of 95% O2
and 5%CO2.Mechanical activity was recorded
isometrically by a two-pen recorder (L-MS 2B
type , made in Chengdu City Instrument Factory)
with a force transducer.The strips were equili-
brated for 90min and the solution in the baths was
changed every 15 min before stimulated by drugs.
1.3　Contractile and relaxing experiments
Both KCl(final concentration 30 mmol·L-1)
and NE caused the strips to contract rapidly.NE
concentration was adjusted to keep the contractile
responses identically with KCl , usually 1 μmol·
L-1 in our preliminary experiment.The drugs
were then removed from the bath by severalwashes
with Krebs solution and the tension was allowed to
return to baseline.This procedure was repeated
three or more times in each preparation.After
steady state contraction induced by KCl or NE was
obtained(usually in the third time), isocorydine
was added to the bath in a cumulative fashion
(from 0.01 to 300 μmol·L-1), then papaverine
(100μmol·L-1)was added to induce a maximum
vasodilative effect and concentration-relaxation
curves of isocorydine were obtained.The concen-
tration of isocorydine for inducing 50% of maxi-
mum vasodilative effect(EC50)on precontracted
preparations with KCl and NE , was compared.
When KCl was used , quantity of NaCl was
reduced to keep Krebs solution isotonity.
To test the effects of individual inhibitors on
the vasodilatation of isocorydine , the strips pre-
contracted repeatedly with NE (1 μmol·L-1)for
three times were used.After the control concen-
tration-relaxation curve for isocorydine was ob-
tained as above mentioned , the strips were washed
for three or more times and equilibrated for 60
min.Then different inhibitors were added to the
bath 10 min before the addition of NE , respective-
ly , and NE concentration was adjusted to keep
same contractile response as untreatment.Then
another concentration-relaxation curve for isocory-
dine was made to evaluate the effects of inhibitors
on isocorydine vasodilatation.The dissolvant of
isocorydine , indomethacin and glibenclamide had
no effect in our preliminary experiment.
1.4　Measurements of cyclic nucleotides
The aortic strips were divided into six groups
and mounted in 10mL organ baths containing Krebs
solution at 37℃.Then NE (1 μmol·L-1)was
added to bath 8 min before the additions of pa-
paverine(100μmol·L-1), nitroglycerin(10μmol·
L-1), isocorydine (10 μmol·L-1 or 100 μmol·
L-1), or MB (10 μmol·L-1)+isocorydine (100
μmol·L-1).After incubation for 3 min , the strips
were reserved at -70℃.Frozen aortic tissue (20
mg)was homogenized at 4℃ in 1.0 mL of 10%
trichloroacetic acid.The homogenate was cen-
trifuged at 10 000×g (4℃)for 15min and super-
natant was transferred to a 25mL conical glass cen-
trifuge tube to which 5 mL of water-saturated di-
ethyl ether was added.The tubewas agitated with a
vortex mixer at room temperature for 1 min.After
the two phases were separated , the upper ether lay-
er was aspirated off and discarded.The ether ex-
traction was repeated three or more times.After the
last extraction , the tube was placed in a 60℃wa-
ter-bath for 5 min to drive out of all traces of ei-
ther.Aliquots of 0.5 mL , from the remaining
aqueous phase were then blew off with N2
and stored at -70℃.For the measurement of
cyclic nucleotides , the residues were dissolved in
0.05mol·L-1 sodium acetate , and radioimmunoas-
sayed according to the instructions of cAMP and
cGMP kits.
1.5　Relaxation of vessel strips was expressed as
percent of the maximum vasodilative effect induced
by papaverine(100μmol·L-1).The EC50were cal-
culated according to Lineweaver-Burk formula[ 4] .
The data were expressed as x ±s.Significance was
tested using t test or paired comparisons.
2　RESULTS
2.1　Relaxation effects of isocorydine on the
precontractions induced by KCl or norepi-
nephrine
Isocorydine caused a concentration-dependent
·252· Chinese Journal of Pharmacology and Toxicology　2001　Aug;15(4)
relaxation in NE or KCl-precontracted aortic strips ,
but the relaxation effect of isocorydine was more po-
tent in strips precontracted by NE than that by KCl
(Fig 1), the EC50was(12.6±4.4)and(447±38)
μmol·L-1 , respectively.The inhibitive effect of iso-
corydine on the precontraction induced by NEwas 36
times as strong as that by KCl.
Fig 1.　Vasodilatory effects of isocorydine(Isoc)on
rabbit aorta strips precontracted by norepinephrine(1
μmol·L-1 , ★)and KCl(30 mmol·L-1 , ○).Relax-
ation induced by papaverine(100μmol·L-1)was taken as 100%.
x±s , n=8.＊P<0.05 , ＊＊P<0.01 , ＊＊＊P<0.001 , com-
pared with KCl precontracted group.
2.2　Effects of different inhibitors on the
vasodilatation of isocorydine
Pretreatment with MB(10μmol·L-1)inhibit-
ed the dilation of isocorydine (from 0.01 to 10
μmol·L-1)significantly(Fig 2).However , no in-
Fig 2.　Inhibitory effects of methylene blue(MB , 10
μmol·L-1)on the concentration-relaxation curves
for isocorydine on rabbit aorta precontracted by
norepinephrine(NE , 1 μmol·L-1).(★)control;(○)
pretreated with MB 10 min before NE-precontraction.Relaxation
induced by papaverine(100μmol·L-1)was taken as 100%. x±
s , n=7.＊P<0.05 , ＊＊P <0.01 , compared with control.
fluences were observed when pretreatment with
glibenclamide(5 μmol·L-1), indomethacin(1
μmol·L-1), L-NNA(10 μmol·L-1)or propra-
nolol(10μmol·L-1 , P>0.05 , n=5).
2.3　Effects of isocorydine on the production
of cAMP and cGMP
The results(Tab 1)showed that papaverine
increased the cAMP levels beyond the levels with
NE alone , but isocorydine or nitroglycerin did
not.On the other hand , isocorydine and nitro-
glycerin increased the levels of cGMP significant-
ly , and the effect of isocorydine was inhibited by
MB completely.Other drugs did not affect the
levels of cGMP.
Tab 1.　Comparison of effects of papaverine , nitro-
glycerin and isocorydine on the cyclic nucleotide lev-
els and the inhibitory effect of methylene blue on the
action of isocorydine
Drug
/μmol·L-1
cAMP
/ nmol·g-1
tissue
cGMP
/ nmol·g-1
tissue
NE 1 　2.0±0.6 　0.4±0.2
NE 1+papaverine 100 3.3±0.8＊＊ 0.4±0.2
NE 1+nitrogly cerin 10 1.1±0.8 1.1±0.4＊
NE 1+isocorydine 10 2.0±0.8 1.0±0.4＊＊
NE 1+isocorydine 100 1.5±0.6 2.0±0.1＊＊
MB 10+NE 1+isocorydine 100 2.0±0.5 0.4±0.0##
Drugs were added 8 min after NE(1 μmol·L-1)and the levels of
cAMP and cGMP were determined 3 min later. x ± s , n =5
(cAMP), 6(cGMP).＊P <0.05 , ＊＊P<0.01 , compared with
NE alone;##P <0.01 , compared with NE 1+isocorydine 100
μmol·L-1 group.
3　DISCUSSION
Contraction of vascular smooth muscles by
KCl is believed to be dependent on Ca
2+
entry
through the potential-dependent calcium channel
(PDC), but NE-induced vasopressor response is
associated with Ca
2+
influx through the receptor-
operated calcium channel(ROC)and Ca2+ release
from the calcium stores by activating theα1-
adrenoreceptor.In the present study , the in-
hibitory effect of isocorydine on NE-induced con-
traction was 36 times as strong as that on KCl-in-
·253·中国药理学与毒理学杂志　　2001 年 8月;15(4)
duced.The result is in accordance with previous
report[ 3] which showed that the action of isocory-
dine primarily related to the ROC-mediated Ca2+-
influx and Ca2+-release.However , isocorydine
shifted the concentration-response curve for con-
traction induced by NE to right nonparallely , and
the maximum response was decreased , which sug-
gest that isocorydine is not a specificα-adrenore-
ceptor blocker.
The study[ 5] had shown that the inhibitory ef-
fects of some vasodilators , for example , potassium
channel openers , cAMP-producers and cGMP-
producers , on NE-induced contraction were more
potent than that on KCl-induced contraction sig-
nificantly.Therefore , some inhibitors , which act-
ed on different way , were chosen to detect the
concrete link of the vasodilatation of isocorydine.
They were ATP sensitive potassium channel in-
hibitor glibenclamide (blocking ATP sensitive
potassium channel and inhibiting the effects of hy-
perpolarization by K
+-efflux), cyclooxygenase in-
hibitor indomethacin (inhibiting the synthesis of
prostacyclin), β-receptor blocker propranolol(in-
hibiting the vasodilatation of β-receptor coopera-
tion with exciting adenylate cyclase), NO-syn-
thase inhibitor L-NNA (inhibiting the production
of endogenous NO) and guanylate cyclase in-
hibitorMB(reducing the produce of cGMP).
Results showed that glibenclamide , L-NNA ,
indomethacin and propranolol had no effects on
the vasodilatation of isocorydine , but MB at 10
μmol·L-1 partially inhibited the vasodilatation of
isocorydine , which indicate that the effect of iso-
corydine is not related to ATP sensitive potassium
channel , the production of endogenous NO and
prostacyclin , as well as stimulating β-receptor , its
vasodilative effect may be partially by exciting
guanylate cyclase and elevating the levels of
cGMP.
Increases in both cAMP and cGMP can pro-
duce smooth muscle relaxation[ 6] , as the effects
caused by prostaglandin I2 , potassium channel
opener
[ 7]
and papaverine
[ 8] .The results shown in
Tab 1 demonstrated that the cAMP level was not
changed by treatment with isocorydine , but in-
creased by papaverine treatment.It indicates that
the vasodilative effect of isocorydine is not mediat-
ed by cAMP.In other words , isocorydine does not
open potassium channel nor stimulate β-receptor.
Similar results were obtained when propranolol or
glibenclamide was used in strips.Although the
structure of isocorydine is similar to papaver-
ine[ 9] , the characters of the effect of isocorydine
on cAMP level and on the precontractions induced
by NE and KCl , were different from that of pa-
paverine which relaxed the precontractions in-
duced by NE and KCl with a similar potent[ 10] .It
indicates that isocorydine is not a non-specific
phosphodiesterase inhibitor.
On the other hand , isocorydine and nitro-
glycerin were capable of increasing cGMP levels ,
and the effect of isocorydine was inhibited by MB ,
a guanylate cyclase inhibitor.This was consistent
with that obtained from the contractile experiment
of strips.The results suggest that the guanylate
cyclase system is the final pathway for the action
of isocorydine.It means that the vasodilative ef-
fect of isocorydine may be partially mediated by
activating the guanylate cyclase and increasing the
level of cGMP.However , further studies are nec-
essary to ascertain or exclude whether isocorydine
is a NO donor , like nitroglycerin[ 11] .
In conclusion , although the mechanism of
the vasodilatation caused by isocorydine has not
been established , it is suggested that intracellular
accumulation of cGMP by activating guanylate cy-
clase may be an important element of the mecha-
nism by which isocorydine produces the relaxation
in rabbit aortic smooth muscle.
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异紫堇定的扩血管作用与环核苷酸的关系
蒋青松1 , 黄燮南2 , 孙安盛2 , 吴　芹2 , 谢笑龙2
(1.重庆医科大学药理学教研室 , 重庆　400016;2.遵义医学院药理学教研室 , 贵州 遵义　563003)
摘要:应用血管平滑肌等张收缩和放射免疫测定环
核苷酸水平等方法研究了异紫堇定扩血管作用及其
机理.结果表明 ,异紫堇定浓度依赖性地松弛去甲
肾上腺素(NE , 1μmol·L-1)和 KCl(30 mmol·L-1)预
收缩的兔胸主动脉螺旋条 , EC50分别为(12.6±4.4)
和(447±38)μmol·L-1 ,对NE 的作用比对 KCl强 36
倍.亚甲蓝(10μmol·L-1)可部分抑制异紫堇定的扩
血管作用 ,但不受格列本脲 ,吲哚美辛 ,普萘洛尔或
N-L-硝基精氨酸影响.异紫堇定还可促进 cGMP 生
成增加 ,并可被亚甲蓝完全阻断.异紫堇定对 cAMP
的生成无影响.结果提示 ,异紫堇定的扩血管作用
至少部分通过激活鸟苷酸环化酶 ,促进 cGMP 的生
成实现.
关键词:异紫堇定;主动脉 ,胸;血管舒张;环腺苷酸;
环鸟苷酸
　
基金项目:贵州省科委基金资助项目(943033)
(本文编辑　董立春)
·255·中国药理学与毒理学杂志　　2001 年 8月;15(4)