Endothelium-independent vasorelaxation effects of 16-O-acetyldihydroisosteviol on isolated rat thoracic aorta

Article abstract of DOI:10.1016/j.lfs.2014.08.010

Aims: The aim of this study is to investigate the vasorelaxant effect of 16-O-acetyldihydroisosteviol (ADIS) and its underlying mechanisms in isolated rat aorta. Main methods: Rat aortic rings were isolated, suspended in organ baths containing Kreb's solution, maintained at 37°C, and mounted on tungsten wire and continuously bubbled with a mixture of 95% O₂ and 5% CO₂ under a resting tension of 1 g. The vasorelaxant effects of ADIS were investigated by means of isometric tension recording experiment. Key findings: ADIS (0.1 μM-3 mM) induced relaxation of aortic rings pre-contracted by phenylephrine (PE, 10 μM) and KCl (80 mM) with intact-endothelium (E_max = 79.26 ± 3.74 and 79.88 ± 3.79, respectively) or denuded-endothelium (E_max = 88.05 ± 3.69 and 78.22 ± 6.86, respectively). In depolarization Ca²⁺-free solution, ADIS inhibits calcium chloride (CaCl₂)-induced contraction in endothelium-denuded rings in a concentration-dependent manner. In addition, ADIS attenuates transient contractions in Ca²⁺-free medium containing EGTA (1 mM) induced by PE (10 μM) and caffeine (20 mM). By contrast, relaxation was not affected by tetraethylammonium (TEA, 5 mM), 4-aminopyridine (4-AP, 1 mM), glibenclamide (10 μM), barium chloride (BaCl₂, 1 mM), and 1H-[1,2,3]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ, 1 μM). Significance: These findings reveal the vasorelaxant effect of ADIS, through endothelium-independent pathway. It acts as a Ca^{2 +} channel blocker through both intracellular and extracellular Ca^{2 +} release.

Full text of DOI:10.1016/j.lfs.2014.08.010

Life Sciences 116 (2014) 31–36
Contents lists available at ScienceDirect
Life Sciences
journal homepage: www.elsevier.com/locate/lifescie
Endothelium-independent vasorelaxation effects of
6-O-acetyldihydroisosteviol on isolated rat thoracic aorta
1
a
b
c
d
Rungusa Pantan , Amnart Onsa-ard , Jiraporn Tocharus , Orawan Wonganan ,
d
a,
Apichart Suksamrarn , Chainarong Tocharus ⁎
Department of Anatomy, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand
Department of Biochemistry, Faculty of Medical Science, Phayao University, Phayao 56000, Thailand
Department of Physiology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand
a
b
c
d
Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Ramkhamhaeng University, Bangkok 10240, Thailand
a r t i c l e i n f o
a b s t r a c t
Article history:
Aims: The aim of this study is to investigate the vasorelaxant effect of 16-O-acetyldihydroisosteviol (ADIS) and its
underlying mechanisms in isolated rat aorta.
Main methods: Rat aortic rings were isolated, suspended in organ baths containing Kreb's solution, maintained at
Received 24 March 2014
Accepted 11 August 2014
Available online 21 August 2014
2 2
37 °C, and mounted on tungsten wire and continuously bubbled with a mixture of 95% O and 5% CO under a
resting tension of 1 g. The vasorelaxant effects of ADIS were investigated by means of isometric tension recording
experiment.
Key findings: ADIS (0.1 μM–3 mM) induced relaxation of aortic rings pre-contracted by phenylephrine (PE,
Keywords:
16-O-Acetyldihydroisosteviol
Vasorelaxation
Endothelium-independent
Aorta
1
0 μM) and KCl (80 mM) with intact-endothelium (Emax = 79.26 ± 3.74 and 79.88 ± 3.79, respectively)
2
+
or denuded-endothelium (Emax = 88.05 ± 3.69 and 78.22 ± 6.86, respectively). In depolarization Ca
-
2
free solution, ADIS inhibits calcium chloride (CaCl )-induced contraction in endothelium-denuded rings
2
+
in a concentration-dependent manner. In addition, ADIS attenuates transient contractions in Ca -free
medium containing EGTA (1 mM) induced by PE (10 μM) and caffeine (20 mM). By contrast, relaxation
was not affected by tetraethylammonium (TEA, 5 mM), 4-aminopyridine (4-AP, 1 mM), glibenclamide
(
10 μM), barium chloride (BaCl
2
, 1 mM), and 1H-[1,2,3]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ, 1 μM).
Significance: These findings reveal the vasorelaxant effect of ADIS, through endothelium-independent pathway.
2
+
2+
It acts as a Ca channel blocker through both intracellular and extracellular Ca release.
©
2014 Elsevier Inc. All rights reserved.
Introduction
and hypotension (Humboldt and Boech, 1977) and continuous oral ad-
ministration tea prepared from S. rebaudiana daily for 30 days also
showed a slight hypotensive effect (Boeckh and Humboldt, 1981). Fur-
thermore, isosteviol (ent-16-oxobeyeran-19-oic acid), a tetracyclic
diterpenoid obtained by acid hydrolysis of stevioside, has been reported
as exhibiting pharmacological activity. Isosteviol attenuates myocardial
damage induced by ischemia–reperfusion in isolated perfused guinea
pig heart (Xu et al., 2007a), rat heart in vivo (Xu et al., 2007b), and rat
brain in vivo (Xu et al., 2008). Moreover, isosteviol also shows the
Stevioside is a diterpene glycoside isolated from the leaves
of the plant Stevia rebaudiana (Bertoni) Bertoni, and it has been
popularly used as an artificial sweetener worldwide (Geuns,
2
003; Chatsudthipong and Muanprasat, 2009; Goyal et al., 2010).
Stevioside is the main component of S. rebaudiana extract and
has shown the effects similar to calcium antagonists were shown
on arterial pressure and renal function (Melis, 1992) and it lowered
arterial pressure in the form of an intravenous injection in anesthetized
spontaneously hypertensive rats (SHRs) (Chan et al., 1998). Additional-
ly, studies in humans demonstrated that stevioside cause bradycardia
+
vasorelaxant effect via opening the K channels in rat aorta (Wong
et al., 2004). Recently, the chemical modification of isosteviol was per-
formed by sodium borohydride reduction of isosteviol, followed by
acetylation to give 16-O-acetyldihydroisosteviol (ADIS) (Fig. 1). The
vasorelaxant effect in rat aortic rings was found to be greater in the
case of ADIS than in the case of isosteviol about 12-fold (Wonganan
⁎
Corresponding author. Tel.: +66 53 945312; fax: +66 53 945304.
E-mail address: chainarongt@hotmail.com (C. Tocharus).
http://dx.doi.org/10.1016/j.lfs.2014.08.010
024-3205/© 2014 Elsevier Inc. All rights reserved.
0

32
R. Pantan et al. / Life Sciences 116 (2014) 31–36
rings were immersed in a 2 ml chamber bath which contained Krebs so-
lution (composition, mM: NaCl, 122; KCl, 4.9; HEPES, 10; KH PO , 0.5;
NaH PO , 0.5; MgCl , 1.0; glucose, 11.0; CaCl , 1.8, pH 7.3), maintained
2
4
2
4
2
2
at a 37 °C temperature, mounted on tungsten wire and continuously
bubbled with oxygen. A resting tension of 1 g was applied to each tissue
and equilibrated at least 1 h. During the equilibrium period, the Krebs
solution was changed every 15 min. After equilibration, the endothelial
integrity was verified with a sub-maximal pre-contraction of PE
(
10 μM). After the tension was stabilized, ACh (10 μM) was directly
added into the chamber bath in order to detect and evaluate the pres-
ence or absence of the endothelial cell layer; more than 90% relaxation
of the rings was considered to be an endothelium-intact ring, whereas
less than 10% relaxation was considered to be endothelium-denuded
ring. Relaxation was calculated as a percentage of the maximal contrac-
tion induced by PE. Before each experimental protocol was performed,
the presence or absence of the endothelial cell layer was tested and
washed out with the Krebs solution for at least 30 min. Changes in ten-
sion were detected using FT-104 isometric force transducers (Iworx
System, Inc., NH, USA) coupled with Power Lab data acquisition system
Fig. 1. The chemical structure of 16-O-acetyldihydroisosteviol (ADIS).
et al., 2013). This is of special interest as far as evaluating the mecha-
nism of ADIS on the vasorelaxant effect is concerned. Thus, the purpose
of this study is to investigate the mechanism of ADIS as regarding
vasorelaxant activity, as it would be beneficial in evaluating natural
products, which may be considered as an alternative modality for
hypertensive patients.
(
ADIntruments, Sydney, Australia), and then connected to a computer
Materials and methods
equipped with Lab Chart 7 Software program (ADIntruments, Sydney,
Australia).
Drugs and chemicals
Aorta ring contraction assay
Phenylephrine (PE), acetylcholine (ACh), ethylene glycol-bis(β-
aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), glibenclamide,
tetraethylammonium, 4-aminopyridine (4-AP), 1H-[1,2,4]oxadiazolo
The vasorelaxant effects of ADIS were investigated in both
endothelium-intact and endothelium-denuded aortic rings. After
the rings were pre-equilibrated, they were pre-contracted with PE
[
4,3-a]quinoxalin-1-one (ODQ), and caffeine were purchased from
Sigma (St. Louis, MO, USA).
(
10 μM) until the stability of tension was established, which was follow-
ed by cumulative exposure to ADIS (0.1 μM–3 mM).
Preparation of ADIS
+
Role of K channels on ADIS-induced relaxation
The pulverized, dry S. rebaudina leaves were successively extracted
with n-hexane, ethyl acetate, and methanol. The methanol extract was
subjected to silica column chromatography to yield stevioside. ADIS
was obtained from stevioside as described previously (Wonganan
et al., 2013). Briefly, stevioside was dissolved in 20% sulfuric acid solu-
tion and then heated at 60–70 °C for 6 h with stirring. The mixture
was extracted with ethyl acetate. The solvent was evaporated and the
product was purified by column chromatography to give isosteviol.
Sodium borohydride reduction of isosteviol in tetrahydrofuran pro-
duced dihydroisosteviol, which upon acetylation with acetic anhydride
+
The role of the K channels was elucidated by the vasorelaxation re-
sponse upon pre-incubating the endothelium-denuded aortic rings
with one of the following specific K channel blockers: TEA (5 mM),
2
-AP (1 mM), glibenclamide (10 μM), and BaCl (1 mM) for 30 min be-
fore PE (10 μM) pre-contraction. Then, ADIS (0.1 μM–3 mM) was added
cumulatively.
+
4
Role of soluble guanylyl cyclase (sGC) in ADIS-induced relaxation
The mechanism of vasorelaxation induced by ADIS (0.1 μM–3 mM)
was further tested by using ODQ, a selective inhibitor of sGC, to focus
on the role of soluble guanylyl cyclase (sGC) in the relaxant activity.
The procedure was conducted by pre-incubating the endothelium-
denuded aortic ring with ODQ (1 μM) for 30 min before PE (10 μM)
induced contraction was performed. After the pre-contraction stabi-
lized, ADIS (0.1 μM–3 mM) was added. The vasorelaxation abilities of
ADIS in the presence and absence of ODQ were compared.
1
in pyridine yielded ADIS. The spectroscopic data ( H-NMR and mass
spectra) of the synthesized ADIS were identical to those reported previ-
ously (Wonganan et al., 2013).
Animals
Male Wistar rats (200–250 g) were obtained from the National Lab-
oratory Animal Center, Mahidol University, Salaya, Nakornpathom,
Thailand. All the animals were housed under a 12:12 h light–dark
cycle condition, with a steady temperature maintained (24 ± 1 °C).
The animals were allowed free access to rodent diet and tap water.
The experimental protocol was approved by the Animal Ethics Commit-
tee in accordance with the guidelines for the care and use of laboratory
animals, as prepared by Chiang Mai University.
Endothelium denudation effect of ADIS contraction induced by PE and KCl
After the equilibration period, the aortic rings were pre-contracted
with PE (10 μM) or KCl (80 mM). Then, the tension was allowed to
stabilize, which was followed by exposure to ADIS (0.1 μM–3 mM) cu-
mulatively. The extent of relaxation was expressed as the percentage
of PE-induced or KCl-induced contraction.
Preparation of isolated rat thoracic aortic rings
1
Role of α -receptor in inhibitory vasoconstriction effect of ADIS.
Rats were anesthetized by intraperitoneal injection using sodium
pentobarbital (60 mg/kg BW). After they attained complete uncon-
sciousness, the rats were sacrificed and the thoracic aorta was immedi-
ately removed to be cleaned of the adhering connective tissue and fat.
The vessels were cut into rings approximately 3 mm in length. For the
endothelium-denuded rings, the endothelial layer was removed by
gently rubbing the internal surface of the vascular lumen. The aortic
Endothelium-denuded rings were tested in normal Krebs solution.
ADIS was added in concentrations of 1 μM, 10 μM, and 100 μM to
the aortic rings for 20 min before vasoconstriction was performed.
The contractions of the aortic rings were induced by PE (0.1 nM–
10 μM) which was added cumulatively. The obtained results were
shown as percentages of contraction compared with the absence
of ADIS (control).

R. Pantan et al. / Life Sciences 116 (2014) 31–36
33
2+
Effect of ADIS on extracellular Ca influx to SMC. Further to examine the
mechanism of ADIS-induced vasorelaxation on Ca2 channel, the
+
endothelium-denuded rings were used to investigate the CaCl
2
concen-
tration–response curve. Briefly, after the aortic rings stabilized under
2
+
the Ca free Krebs solution containing EGTA (1 mM) for 20 min, the
2+
+
rings were allowed in Ca free solution containing 80 mM K to pro-
duce depolarization in the smooth muscle cell for the opening up of
the voltage-operated calcium channels (VOCCs). Then, CaCl
0.01–10 mM) was added cumulatively. After the maximal response
2
(
2
+
was obtained, the rings were washed out with Ca free Krebs solution
2
+
for 20 min. Next, Ca
incubating with ADIS (1 μM, 10 μM, and 100 μM) and nifedipine
1 μM). The percentages of contraction induced by CaCl in the absence
and presence of ADIS were compared.
free KCl (80 mM) was re-exposed after pre-
(
2
2
+
Role of ADIS on intracellular Ca release. To determine whether ADIS
could attenuate the release of Ca2 from sarcoplasmic reticulum (SR),
the endothelium-denuded rings were used in this experiment. The
+
2
+
rings were pre-contracted with KCl (80 mM) to provide Ca loading
into SR. Then, the aortic rings were washed and exposed to Ca2 free
Krebs solution containing EGTA (1 mM), followed by activating tran-
Fig. 2. The cumulative concentration–response curve of ADIS (0.1 μM–3 mM) on
endothelium-intact (+EC) and endothelium-denuded (−EC) aortic rings pre-
contracted with PE (10 μM). Data are analyzed by one way ANOVA followed by Dunnett's
multiple comparison test and expressed as mean ± SEM of six rats. **p b 0.01 vs. control
group (0.01% DMSO).
+
2
+
sient contraction by PE (10 μM) or caffeine (20 mM) in Ca free solu-
tion before and after the pre-incubation with ADIS (1 μM, 10 μM, and
1
00 μM). The percentages of contraction in the absence of ADIS and in
relaxation induced by ADIS, which had an Emax value of 78.25 ± 7.22%
the presence of ADIS, both activated by PE and caffeine, were compared.
(
vs. control group Emax value of 88.05 ± 3.31%).
Statistical analysis
ADIS relaxation in PE and KCl contracted endothelium denuded rings
All the data were expressed as mean ± SEM. The statistical analysis
was performed using unpaired Student's t-test between two groups and
one-way-analysis of variance (ANOVA) by following Dunnett's post hoc
test for the comparison between multiple groups by SPSS. A value of
p b 0.05 was considered to be having significance. Concentration–
response curves were plotted and experimental data obtained by
using a nonlinear curve-fit program (GraphPad Prism 5).
In endothelium-denuded aortic rings, ADIS (0.1 μM–3 mM) sig-
nificantly relaxed PE (10 μM) and KCl (80 mM) pre-contracted aortic
rings in a concentration-dependent manner (Fig. 4). The EC50 values
amounted to 41.99 ± 7.14 μM and 51.89 ± 1.42 μM for PE and KCl in-
duced contractions, respectively. The Emax value of PE was at
7
8
9.26 ± 3.74% of the control and the Emax value of KCl was at
2.12 ± 2.61% of the control.
Results
1
Role of α -receptor in inhibitory vasoconstriction effect of ADIS
Role of endothelium in ADIS-induced relaxation in rat aortic rings
To verify the ability of ADIS to prevent contractile effect conveyed
through the α -receptor, the aortic rings were pre-incubated with
ADIS (1 μM, 10 μM, and 100 μM) before exposing them to PE (0.1 nM–
0 μM), which was added cumulatively. The results revealed that all
the three concentrations of ADIS attenuated the maximal contraction
of PE to 83.70 ± 3.20%, 70.01 ± 5.67%, and 64.20 ± 3.53%, respectively
1
In the rat aortic rings with or without endothelium, PE (10 μM) in-
duced a steady contraction. ADIS caused concentration-dependent
vasorelaxant effect on PE-induced contraction in both endothelium-
intact and endothelium-denuded arteries, with Emax values of 79.26 ±
1
3
.74 and 88.05 ± 3.31%, respectively (vs. control group Emax value of
(
vs. control group 89.86 ± 10.02%), as demonstrated in Fig. 5.
1
1.98 ± 4.19%, p b 0.01). The EC50 values of the relaxing effect of ADIS
for the endothelium-intact arteries and the endothelium-denuded arter-
ies were 41.99 ± 7.14 μM and 54.5 ± 1.76 μM, respectively. These two
values were not significantly different (Fig. 2).
2+
Effect of ADIS on extracellular Ca -induced contraction
To investigate the effect of ADIS on the voltage-dependent calcium
channels (VOCCs) pathway, Ca2 -free high KCl (80 mM) solution was
+
+
Role of K channels on ADIS-induced relaxation
2
applied to induce stable contraction. CaCl (0.01–10 mM) was then
added cumulatively to induce a progressive increase in the contraction
of the aortic rings. ADIS (1 μM, 10 μM, and 100 μM) pre-incubation
To define the role of K+ channels on vasorelaxation induced by ADIS,
the endothelium-denuded rings were pre-incubated with four different
+
K
(
(
channel blockers such as TEA (5 mM), 4-AP (1 mM), glibenclamide
(1 mM). The vasorelaxant effect of ADIS on PE
10 μM) pre-contracted aortic rings was not altered by the pre-
Table 1
10 mM), and BaCl
2
Effect of various K+ channel inhibitors on the negative logarithm of EC50 (pD
2
) and max-
imal vasorelaxation (Emax) induced by ADIS on endothelium-denuded, pre-contracted
with PE (10 μM).
+
incubation of the rings with four types of K channel blockers
Table 1 and Fig. 3A).
(
pD2 (M)
Emax (%)
Control
-AP
TEA
4.07 ± 0.12
4.16 ± 0.16
4.14 ± 0.08
4.45 ± 0.20
4.37 ± 0.11
87.53 ± 3.10
62.60 ± 7.23
78.84 ± 5.48
75.69 ± 12.09
89.65 ± 6.99
Role of soluble guanylyl cyclase (sGC) in ADIS-induced relaxation
4
To investigate the effect of ADIS on the NO-cyclic guanosine
monophosphate (cGMP) pathway, the aortic rings were pre-incubated
with ODQ (1 μM). As is evident from Fig. 3B, ODQ did not alter the
Glibenclamide
BaCl
2
The data are expressed as mean ± SEM (n = 6).

34
R. Pantan et al. / Life Sciences 116 (2014) 31–36
Fig. 3. The effects of K+ channel blockers: 4-aminopyridine (4-AP, 1 mM), tetraethylammonium (TEA, 5 mM), glibenclamide (10 μM), and barium chloride (BaCl
, 1 mM) (A) and soluble
2
guanylyl cyclase; ODQ (1 μM) (B) on ADIS (0.1 μM–3 mM) induced relaxation in endothelium-denuded aortic rings pre-contracted with PE (10 μM). Data are analyzed by one way ANOVA
followed by Dunnett's multiple comparison test and expressed as mean ± SEM, where n = 6 rats.
significantly inhibited the contraction induced by extracellular Ca2+ to
2.87 ± 8.52%, 87.28 ± 8.12%, and 70.63 ± 6.16%, respectively (vs.
control group 103.80 ± 5.80%). In addition, pretreatment with nifedi-
contraction to 79.58 ± 3.59, 79.18 ± 3.12, and 64.61 ± 3.20, respec-
tively (vs. control group 91.19 ± 3.06).
9
2+
pine (1 μM), the Ca channel blocker, also inhibited the contraction in-
duced by the extracellular Ca2 (Emax = 30.69 ± 4.04%), as presented
in Fig. 6A.
Discussion
+
ADIS has been studied for its efficiency in vasorelaxation and has
been found to have more potential than isosteviol, which is the parent
compound, by approximately 12-fold (Wonganan et al., 2013). This
study is intended to verify the underlying mechanism of ADIS on the va-
sorelaxation response. Vasorelaxation is the response of the vascular
smooth muscle cell which is regulated via the endothelium-dependent
and the endothelium-independent signaling pathways. In the present
study, ADIS showed relaxant effects on PE pre-contracted endothelium-
intact as well as endothelium-denuded aortic rings. These findings sug-
gest that the vasorelaxant effect caused by ADIS was endothelium-
independent. To reduce experimental complexity we investigated the
Role of ADIS in sarcoplasmic reticulum calcium release induced by PE or
caffeine
2
+
The inhibitory effect of ADIS on a transient contraction due to Ca
release from intracellular stores was carried out in a Ca2 free solution
+
by using endothelium-denuded rings which were activated by PE
(
10 μM) and caffeine (20 mM). As shown in Fig. 6B, pretreatment
with ADIS (1 μM, 10 μM, and 100 μM) reduced the percentage of PE-
induced transient contraction to 66.44 ± 3.42, 64.86 ± 3.36, and
relaxing effect of ADIS on endothelium denuded rings. The opening of
+
4
1.09 ± 3.45, respectively (vs. control group 93.26 ± 2.61). In addition,
K
channels in the vascular smooth muscle cell is one of the mechanisms
ADIS also decreased the percentage of caffeine-induced transient
involved in the regulation of muscle contractility (Nelson and Quayle,
Fig. 5. The inhibitory effects of ADIS (1 μM, 10 μM, and 100 μM) on the contraction induced
by PE (0.1 nM–10 μM) in endothelium-intact rat aortic rings. Data are analyzed by one
way ANOVA followed by Dunnett's multiple comparison test and expressed as mean ±
SEM of six rats. *p b 0.05 vs. control group (0.01% DMSO).
Fig. 4. The effect of ADIS (0.1 μM–3 mM) on PE (10 μM) and KCl (80 mM) pre-contracted
endothelium-denuded aortic rings. Data are analyzed with Student's t-test and expressed
as mean ± SEM of six rats.

R. Pantan et al. / Life Sciences 116 (2014) 31–36
35
Fig. 6. The effect of ADIS (1 μM, 10 μM, and 100 μM) and nifedipine (1 μM) on the cumulative-concentration curve induced by extracellular Ca2+ in endothelium-denuded rings
pre-challenged with KCl (80 mM), in Ca2+ free solution (A) and inhibitory effect of ADIS on PE (10 μM) and caffeine (20 mM) induced transient contractions in Ca free Krebs
solution in endothelium-denuded aortic rings (B). Data are analyzed by one way ANOVA followed by Dunnett's multiple comparison test and expressed as mean ± SEM of six
2+
⁎
⁎⁎
rats. p b 0.05 and p b 0.01 vs. control group (0.01% DMSO).
1
995; Quayle et al., 1997; Jackson, 2000; Brayden, 2002). TEA (5 mM),
was expected, the ADIS' relaxation effects could be observed in contrac-
tion induced by the alpha adrenergic agonist. To determine the role of
the VOCCs, we further evaluated the concentration–response curve of
+
2
glibenclamide (10 μM), 4-AP (1 mM), and BaCl (1 mM) are the K
channel blockers used to investigate potential K channel-related
vasorelaxant effects of various agents. The vasorelaxant effects of ADIS
were not affected by pre-treatment with these K blockers. Our study
indicated that the relaxant effect of ADIS is not related to the opening
of the K channels. Moreover, we have tested the role of soluble guanylyl
cyclase (sGC) which effects an increase in the amount of cGMP in the
smooth muscle cell and causes vasorelaxation (Waldman and Murad,
+
2+
2
CaCl in a Ca free medium containing 80 mM KCl which affects the in-
+
2+
flux of Ca into cytoplasm. The results show that ADIS significantly re-
duced the KCl-induced vascular smooth muscle contraction in the
endothelium-denuded rings, in a concentration-dependent manner,
and clearly reduced the Ca2 -induced contraction in the aortic rings ex-
posed to KCl. In addition, the contraction was reduced by nifedipine, a
typical L-type voltage-operated calcium channel blocker, confirming
the involvement of L-type voltage-operated calcium channels in the
contraction response (Hockerman et al., 1997). These results demon-
+
+
1987; Warner et al., 1994; Lucas et al., 2000). The result revealed that in-
hibition of the sGC activity by using ODQ did not have any effect on the
relaxation of the aortic ring induced by ADIS. Therefore, this is a clear in-
dication that the action of ADIS on the K channels and the sGC activities
+
2+
strate that ADIS acts by inhibiting the Ca influx through the mem-
in the aortic ring can be ruled out.
brane of the smooth muscle, through interference with the VOCCs.
Next, we investigated whether ADIS could exert its vasorelaxant ef-
fects by interfering with the Ca released from intracellular stores by
The vascular smooth muscle contraction in also regulated by the
inhibition of extracellular Ca2 influx via voltage and/or receptor-
operated calcium channels (VOCCs and/or ROCCs) and the release of
+
2+
phosphoinositide production, following the receptor activation. PE and
caffeine were used as the contractile agents; PE induces mobility of
2+
Ca
from intracellular stores (Hurwitz, 1986; Van Breemen and
2+
2+
Saida, 1989; Hori et al., 1993; Nelson and Quayle, 1995; Horowitz
et al., 1996). In addition, the binding of alpha adrenergic agonist mole-
cule such as phenylephrine (PE) to its receptor activates IP and induces
3
release of calcium from intracellular stores. However, elevated intracel-
lular calcium might activate voltage dependent calcium channels
through the PKC pathway (Chen et al., 2009; Lee et al., 2013). Moreover,
it is widely known that KCl induces smooth muscle contraction through
the activation of VOCCs and the subsequent release of calcium from SR,
whereas PE-induced vasoconstriction is mediated by extracellular Ca2
influx through ROCCs and by internal calcium release from the specific
3
Ca from SR by IP R activity, whereas caffeine induces Ca release
from the SR by ryanodine receptor activity (Ferris and Snyder, 1992;
Meissner, 1994). ADIS significantly inhibited the transient contraction
induced by both PE and caffeine. Thus, it seems likely that the vascular
effects of ADIS involved the reduction of IP
from SR sensitive to PE.
-dependent Ca2+ releases
3
Conclusion
+
Taken together, our results suggest that ADIS induces relaxation in
aortic rings through an endothelium-independent pathway, involving
both blockage of voltage dependent cytoplasma membrane calcium
channels and calcium release from intracellular stores in vascular
smooth muscle cells.
3 3
IP receptor (IP R) channels in the SR membrane (Ruzycky and
Morgan, 1989; Van Breemen and Saida, 1989; Meissner, 1994). Thus,
both the contractile agents produce a significant increase in the intracel-
lular calcium concentration through calcium influx. It should be noted
that ADIS possesses vasorelaxation via both VOCCs and ROCCs which re-
spond to KCl and PE, respectively. The blocking activity of the VOCC and
the ROCC effect are result in a decrease of intracellular calcium, which is
followed by the relaxation of the vascular smooth muscle (Ehrlich and
Watras, 1988). In addition, this study clearly demonstrated that pre-
incubation with ADIS reduces the aortic tension induced by PE cumula-
tively in a concentration-dependent manner. It is well known that PE
Conflict of interest
The authors have declared no conflict of interest.
Acknowledgments
regulates vasoconstriction through α
1
-adrenergic receptors and also
This work was supported by the Higher Education Research Promo-
affects the ROCCs (Ferris and Snyder, 1992; Hori et al., 1993). As it
tion (HERP) Program, the Office of the Higher Education Commission,

36
R. Pantan et al. / Life Sciences 116 (2014) 31–36
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