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Calcium Channel Blockade in Vascular Smooth Muscle Cells:
Major Hypotensive Mechanism of S-Petasin, a Hypotensive
Sesquiterpene from Petasites formosanus
GUEI-JANE WANG, ANDREW YAU-CHIK SHUM,
1
YUN-LIAN LIN, JYH-FEI LIAO,
1
XI-CHEN WU,
2
JUN REN,
3
and
CHIEH-FU CHEN
National Research Institute of Chinese Medicine, Taipei, Taiwan, Republic of China
Received November 6, 2000; accepted January 3, 2001 This paper is available online at http://jpet.aspetjournals.org
ABSTRACT
In vivo and in vitro studies were carried out to examine the
putative hypotensive actions of S-petasin, a sesquiterpene ex-
tracted from the medicinal plant Petasites formosanus. Intrave-
nous S-petasin (0.1–1.5 mg/kg) in anesthetized rats produced a
dose-dependent hypotensive effect. In isolated aortic ring, iso-
metric contraction elicited by KCl or the L-type Ca
2⫹
channel
agonist Bay K 8644 was reduced by S-petasin (0.1–100
M), an
action not affected by the cyclooxygenase inhibitor indometh-
acin, nitric-oxide synthase inhibitor N
-nitro-L-arginine, guany-
lyl cyclase inhibitor methylene blue, or removal of vascular
endothelium. Pretreatment with S-petasin for 10 min shifted the
concentration-response curve for KCl (15–90 mM)-induced
contraction to the right and reduced the maximal response. In
Ca
2⫹
-depleted and high K
⫹
-depolarized aortic rings preincu-
bation with S-petasin attenuated the Ca
2⫹
-induced contraction
in a concentration-dependent manner, suggesting that S-peta-
sin reduced Ca
2⫹
influx into vascular smooth muscle cells
(VSMCs). Moreover, in cultured VSMCs, whole-cell patch-
clamp recording indicated that S-petasin (1–50
M) inhibited
the L-type voltage-dependent Ca
2⫹
channel (VDCC) activities.
Intracellular Ca
2⫹
concentration ([Ca
2⫹
]
i
) estimation using the
fluorescent probe 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzo-
furan-5-oxy]-2-(2⬘-amino-5⬘-methylphenoxy)-ethane-N,N,N,N-
tetraacetic acid pentaacetoxymethyl ester indicated that S-
petasin (10, 100
M) suppressed the KCl-stimulated increase in
[Ca
2⫹
]
i
. Taken together, the results suggested that a direct
Ca
2⫹
antagonism of L-type VDCC in vascular smooth muscle
may account, at least in part, for the hypotensive action of
S-petasin.
Extracts from Petasites plants (Compositae) have been
used for thousands of years for therapeutic purposes in folk
medicine. They have been claimed to improve conditions in
respiratory diseases such as asthma and cough (Ziolo and
Samochowiec, 1998), gastrointestinal pain, as well as spasms
of the urogenital tract (Brune et al., 1993). P. formosanus is
an indigenous species of Petasites in Taiwan and has been
used as a folk medicine to treat hypertension. However, its
active ingredients are unknown and the mechanisms of ac-
tion obscure. In attempting to assess its potential as an
antihypertensive agent, a series of in vivo and in vitro exper-
iments were conducted to systematically verify its hypoten-
sive properties, identify the active ingredients, and define its
hypotensive mechanisms. A series of compounds, mainly ses-
quiterpenes, were isolated from the aerial part of P. formo-
sanus (Lin et al., 1998). Of these the major one was S-petasin
(Fig. 1). Intravenous administration of S-petasin in anesthes-
tized rat caused a dose-dependent fall in blood pressure. In
defining its mechanism of action, first of all the isolated
aortic ring was used. It was found that S-petasin concentra-
tion dependently relaxed the contraction induced by various
vasoconstricting agents, thus providing a basis for hypoten-
sive action. Endothelium dependence was then examined
using endothelium-intact and -denuded preparations as well
as inhibitors of endothelium-related mediators such as pros-
tacyclin, NO, and guanylyl cyclase (Marczin et al., 1992;
Ribeiro et al., 1992; Schro¨r, 1993). Further mechanistic stud-
ies were carried out in cultured VSMCs with the focus on the
role of Ca
2⫹
because it plays a central role in the regulation
This work was supported by Grant NSC89-2320-B077-009 from the Na-
tional Science Council of the Republic of China to G.J.W. Some information
contained in this article was presented in preliminary form at the Experimen-
tal Biology 98 in San Francisco, CA (Wang et al., 1998).
1
Current address: Department and Institute of Pharmacology, National
Yang-Ming University, Taipei, Taiwan, Republic of China.
2
Current address: Heritage Medical Research Center, Cardiology of Medi-
cine, University of Alberta, Edmonton, Alberta, Canada.
3
Current address: Department of Pharmacology, Physiology, and Thera-
peutics, University of North Dakota School of Medicine, Grand Forks, ND
58203.
ABBREVIATIONS: NO, nitric oxide; VSMC, vascular smooth muscle cell; [Ca
2⫹
]
i
, intracellular Ca
2⫹
concentration; Fura-2/AM, 1-[2-(5-carboxyox-
azol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2⬘-amino-5⬘-methylphenoxy)-ethane-N,N,N,N-tetraacetic acid pentaacetoxymethyl ester; VDCC, volt-
age-dependent Ca
2⫹
channel; MAP, mean arterial blood pressure; L-NNA, N
-nitro-L-arginine; I-V, current-voltage.
0022-3565/01/2971-240–246$3.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 297, No. 1
Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics 3523/893779
JPET 297:240–246, 2001 Printed in U.S.A.
240
of vascular tension. Subtle alterations in the activity of Ca
2⫹
regulatory mechanism can have profound effects on [Ca
2⫹
]
i
,
which in turn can affect muscle tone and vascular resistance.
Possible changes in [Ca
2⫹
]
i
associated with S-petasin-in-
duced vasorelaxation were monitored using the fluorescent
dye Fura-2/AM. Finally, the L-type VCDD, which is electri-
cally activated by membrane depolarization, represents the
principal route by which Ca
2⫹
enters vascular smooth muscle
(Bolton, 1979) and plays an essential role in excitation-con-
traction coupling (van Breemen and Saida, 1989). Whole-cell
patch-clamp technique was applied to single cultured rat
VSMCs to study the Ca
2⫹
fluxes.
The results indicated that S-petasin relaxed precontracted
isolated rat aortic rings. Such vasorelaxation was indepen-
dent of the endothelium or the associated mediators such as
prostacyclin, NO, and guanylyl cyclase. On the other hand,
reduction in [Ca
2⫹
]
i
and Ca
2⫹
influx in VSMCs suggested
that prevention of Ca
2⫹
entry from the extracellular fluid
through L-type VDCC, leading to the reduction in [Ca
2⫹
]
i
in
VSMCs, could account, at least in part, for the hypotensive
action of S-petasin.
Materials and Methods
Rats
Male Sprague-Dawley rats, weighing 250 to 350 g (Laboratory
Animal Science Center of the National Yang-Ming University, Tai-
pei, Taiwan), were used. The rats were allowed to acclimate in
environmentally controlled quarters with temperature maintained
at 20–22°C and lighting at 12-h light/dark cycles for at least a week
before being used in experiments. Standard laboratory chow (Labo-
ratory rodent diet no. 5P14; PMI Feeds Inc., Richmond, IN) and
drinking water were provided ad libitum.
Assessment of Effects on Blood Pressure
The rats were anesthetized by i.p. administration of sodium pen-
tobarbital (50 mg/kg) and kept on a heating pad for the maintenance
of body temperature at 37 ⫾1°C. The right femoral artery and vein
were cannulated using PE-50 tubing (Clay Adams, Parsippany, NJ)
for the monitoring of pulse pressure and mean arterial pressure
(MAP), and for i.v. bolus administration of S-petasin, respectively.
To record changes in blood pressure, the arterial cannula was con-
nected to a Gould 2400 polygraph (Valley View, OH) via a P23XL
pressure transducer (Viggo-Spectramed, Oxnard, CA). Changes in
MAP after S-petasin administration were compared with those after
the injection of the same volume of vehicle.
In Vitro Vascular Tension Study
The details of the experimental procedures have been described
previously (Wang et al., 1999). Briefly, isolated aortic rings 3 to 4 mm
in length were fixed isometrically in organ chambers under passive
tension of 1 g for 60 min. After equilibration, near maximal contrac-
tion was induced by phenylephrine (0.3
M). When the rings
achieved a stable contractile tension, acetylcholine (1
M) was added
to the bath to assess endothelial integrity. In some preparations, the
intima was gently frayed with a cotton swab to disrupt the endothe-
lium. The absence of acetylcholine-induced relaxation indicated suc-
cessful endothelial denudation.
Relaxation of Agonist-Induced Contraction. For the evalua-
tion of relaxation, S-petasin (0.01–100
M) or vehicle was added in a
cumulative manner during the tonic phase of contraction induced by
KCl (60 mM) in both endothelium-intact and -denuded aortic rings.
A 10-min time interval was required to obtain the maximal effect
with each concentration of S-petasin. The construction of concentra-
tion-response curves for S-petasin was based on the percentage of
relaxation of the agonist-induced contraction. A 100% relaxation was
considered attained when the precontracted rings returned to the
baseline position. A second assessment of the vasorelaxing effect of
S-petasin was also carried out in endothelium-denuded aortic rings
preconstricted by the L-type VDCC activator Bay K 8644 (50 nM).
Because a partial depolarization of the cell membranes is required to
obtain responses to Bay K 8644 (Schramm et al., 1983), contractions
to this Ca
2⫹
agonist were obtained in a medium that contained 15
mM KCl.
Effects of Endothelial Mediators on the Vasorelaxation of
S-Petasin. To investigate the possible involvements of prostacyclin,
NO, and guanylyl cyclase in the vasorelaxing effects of S-petasin,
endothelium-intact aortic rings were preincubated separately with
the cyclooxygenase inhibitor indomethacin (10
M), the NO synthase
inhibitor L-NNA (100
M), and the guanylyl cyclase inhibitor meth-
ylene blue (10
M) for appropriate periods. Cumulative concentra-
tions of S-petasin (0.01–100
M) were then applied during the sus-
tained phase of phenylephrine (0.3
M)-induced contraction. The
effects of the various inhibitors were studied by comparing the de-
grees of vasorelaxation induced by S-petasin in the absence or pres-
ence of these inhibitors. The concentrations of the inhibitors used
had been reported to be adequate to produce the necessary prosta-
cyclin (Garcia-Cohen et al., 2000), NO (Pieper and Siebeneich, 1997),
and guanylyl cyclase (Terluk et al., 2000) inhibition.
Inhibition of KCl-Induced Contraction. The contraction gen-
erated by cumulative concentration of KCl (15–90 mM) was first
recorded in endothelium-denuded preparations. Following washing
and recovery for 60 min, the tissue was then treated with S-petasin
(1–100
M) or vehicle for 10 min and finally application of the same
concentration of KCl again. Only one S-petasin concentration was
tested per tissue. The treatment time of 10 min was chosen based on
the maximal relaxation relative to each concentration. Concentra-
tion-response curves of S-petasin were constructed and compared.
Effects of Extracellular Ca
2ⴙ
on S-Petasin’s Modulation of
KCl-Induced Contraction. An aortic ring depolarized and con-
tracted by Ca
2⫹
was chosen as the model to investigate the effects of
S-petasin on the contraction dependent on Ca
2⫹
influx from VDCC.
Experiments were carried out under Ca
2⫹
-free conditions after equil-
ibration. Subsequent to the addition of K
⫹
(60 mM) to depolarize the
membrane potential, cumulative concentrations of Ca
2⫹
(0.1–3 mM)
were applied. The stepwise increments in tension represented the
vasoconstriction dependent on extracellular Ca
2⫹
influx induced by
K
⫹
. The aortic rings were then washed and equilibrated for 60 min,
followed by repetition of the experiment in the presence of S-petasin
(1–100
M) or vehicle for 10 min. Only one S-petasin concentration
was tested per tissue. Concentration-response curves to the added
Ca
2⫹
were constructed and compared.
In Vitro Whole-Cell Patch-Clamp Recording
Cell Culture. VSMCs were isolated by collagenase-elastase dis-
sociation from the rat thoracic aorta using previously published
procedures developed in our laboratory (Wang et al., 1996). Before
being used in studies, the cells were incubated in trypsin solution for
1 to 2 min, washed with Hanks’ balanced salt solution, and divided
into small groups ranging in number from 1 to 30 cells for each dish.
After dispersion, cells were allowed to reattach to the culture dish
with only one experiment conducted per dish. VSMCs were used
within 10 to 24 h after they were plated and were of passage between
Fig. 1. Chemical structure of S-petasin.
S-Petasin Blocks Voltage-Dependent Ca
2ⴙ
Channels 241
3 and 6. The limited time after isolation helped to maximize Ca
2⫹
current amplitudes of the cells.
Electrophysiology. Ca
2⫹
channel activity was determined in
single VSMCs by the whole-cell version of the patch-clamp technique
as described previously (Wang et al., 1999). In all experiments, Ba
2⫹
was used as the charge carrier. Because the inward Ba
2⫹
currents
were small and the series resistance was less than 0.1 ohms, the
series resistance compensation was not usually used. The currents
were monitored through digital oscilloscope (Nicolet Instrument Co.,
Madison, WI) and filtered with a low pass filter (Axon Instruments,
Inc., Foster City, CA) at 1 kHz. The software pCLAMP and a lab-
master interface were used for the generation of test pulses and
storage and analysis of data. Leakage and capacitive currents were
subtracted during analysis while simultaneously slow records were
taken on an SC 284 chart recorder (Gould). All recording was done at
room temperature (20–22°C).
Effects of S-Petasin on Ca
2ⴙ
Channel Activity in VSMCs. To
generate current-voltage (I-V) curves, the Ba
2⫹
current through the
Ca
2⫹
channels was elicited by depolarizing the VSMCs from a test
pulse of ⫺30 mV to more positive test potentials at a frequency of 0.1
Hz. The duration of the depolarizing test pulses was 250 ms at
intervals of 5 s. Peak currents were attained for constructing the I-V
relationships. Only cells showing stable channel activity for at least
5 min were used to test the effects of S-petasin. The I-V relationships
were measured repeatedly for 5 min after the addition of S-petasin
(1–50
M) or vehicle in the medium.
[Ca
2ⴙ
]
i
Measurement in Individual VSMCs
[Ca
2⫹
]
i
was measured with the ratiometric fluorescent dye Fura-
2/AM by a method modified from that of Wang et al. (1996). Briefly,
VSMCs were seeded on a sterile glass coverslip at an appropriate
density to allow imaging of 10 to 20 single cells. After 24 h, these
attached cells were loaded with Fura-2/AM (2
M) for 40 min in a
dark place at room temperature. The dye-loaded cells were gently
washed three times with a medium containing 145 mM NaCl, 5 mM
KCl, 1 mM MgCl
2
, 10 mM glucose, 1 mM CaCl
2
, 0.5 mM NaH
2
PO
4
,
and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH
7.4). The cells were kept in medium for a further 20 min to allow the
hydrolysis of Fura-2/AM into Ca
2⫹
-sensitive free acid form (Fura-2)
by cell esterases. The coverslip with attached cells was then trans-
ferred to a 1-ml thermoregulated chamber (22°C) on the stage of a
Olympus IX-70 inverted microscope (Tokyo, Japan) and viewed un-
der bright and UV illumination via a 40⫻(1.35 numerical aperture)
oil immersion fluorescence objective. The Merlin imaging system
(PerkinElmer Life Science, Cambridge, UK) connected to a cooled
charge-coupled device camera (Kodak KAF 1600, Rochester, NY) was
used for digital imaging of the changes of [Ca
2⫹
]
i
in individual cells.
The advantages of this system are that multiple cells can be exam-
ined simultaneously and that the cells under investigation can be
imaged throughout the experiment. Data were analyzed for [Ca
2⫹
]
i
changes by measurement of the 340- and 380-nm excitation signals
and emission signal at 510 nm. Maximal and minimal fluorescences
were obtained by adding ionomycin (10
M) and EGTA (5 mM)
sequentially at the end of the experiment. Ratio values were con-
verted to an estimate of [Ca
2⫹
]
i
as described previously (Grynkiewicz
et al., 1985) assuming a K
d
of 155 nM. All procedures and experi-
ments were performed at room temperature to minimize compart-
mentalization and cell extrusion of the dye.
Effects of S-Petasin on [Ca
2ⴙ
]
i
in VSMCs. To study the effect
of S-petasin on Ca
2⫹
influx from VDCC, the VSMCs were challenged
with KCl (60 mM) in the presence of S-petasin (10, 100
M) or
vehicle for 10 min, and the changes in [Ca
2⫹
]
i
were recorded.
Drugs
The following drugs were used: S-petasin (mol. wt. ⫽334) was
isolated and purified by the National Research Institute of Chinese
Medicine (Taipei, Taiwan, Republic of China) (Lin et al., 1998);
acetylcholine, indomethacin, methylene blue, L-NNA, and phenyl-
ephrine were purchased from Sigma Chemical Co. (St. Louis, MO);
and Fura-2/AM was obtained from Molecular Probes (Eugene, OR).
Indomethacin was dissolved in absolute ethanol. Fura-2/AM was
dissolved in dimethyl sulfoxide, whereas the rest of the drugs were
dissolved in distilled water and kept at ⫺20°C with the exception of
S-petasin. S-Petasin was dissolved in dimethyl sulfoxide, ethanol,
and medium mixture (0.05:0.51:0.44) to make 0.1 to 100 mM stock
solutions. The final concentration of the vehicle in the solution did
not exceed 0.1%, and it had no effects on vascular tension, magni-
tude/kinetics of the inward current and fluorescence imaging of
VSMCs.
Statistical Analyses
The data are presented as mean ⫾S.E. and nrepresents the
number of experiments. In line graphs, S.E. values are indicated by
error bars (in some cases the error bars were so small they were
obliterated by the line symbols). For representation of the Ca
2⫹
current data, the peak inward currents were used in most cases.
Statistical analyses were carried out by Student’s paired or unpaired
ttests when applicable. Pvalues of less than 0.05 were considered to
be significant.
Results
Effect of S-Petasin on MAP. The mean MAP before
S-petasin treatment in 10 anesthetized rats was 105 ⫾3mm
Hg. Mean body weight for this experiment was 288 ⫾6g.
Figure 2 demonstrates the dose dependence of the effect of
S-petasin (0.1–1.5 mg/kg) on MAP. Although the vehicle
alone slightly decreased MAP, the changes were significantly
higher in the presence of S-petasin at doses of 0.1 mg/kg and
higher. Within seconds of injection of S-petasin the MAP fell
(⫺34 ⫾2 mm Hg) and remained lower than the preinjection
value for the next 2 to 3 min at the maximal dose of 1.5
mg/kg.
Relaxation of Agonist-Induced Contraction. S-Peta-
sin given alone did not alter the baseline tension of the aortic
rings (data not shown). In KCl (60 mM)-precontracted aortic
rings, S-petasin (0.01–100
M) produced concentration-de-
pendent vasorelaxation compared with the vehicle-treated
group (data not shown). The vasorelaxing effect of S-petasin
on precontracted aortic rings showed no significant differ-
ence in the presence or absence of endothelium, implying
that S-petasin acted directly on the arterial smooth muscle
Fig. 2. The dose-dependent decreases in MAP after i.v. bolus injection of
S-petasin in anesthetized Sprague-Dawley rats. Values are mean ⫾S.E.
and, when no S.E. is shown, it was smaller than the symbol for the mean.
Eand F, vehicle and S-petasin treatment, respectively; n⫽10 for each
group. *
,
***Statistically significant difference (P⬍0.05 and P⬍0.001,
respectively) between vehicle and S-petasin-treated groups.
242 Wang et al.
(Fig. 3). The IC
50
and the maximal relaxation obtained by
100
MS-petasin were 6.6 ⫾1.4
M and 100%, respectively.
S-Petasin also induced vasorelaxation in Bay K 8644-precon-
tracted endothelium-denuded aortic rings (Fig. 4). The IC
50
and the maximal relaxation obtained by 100
MS-petasin
were 4.2 ⫾0.8
M and 96%, respectively.
Effect of Endothelial Mediators in S-Petasin-In-
duced Vasorelaxation. In the rat thoracic aorta, phenyl-
ephrine (0.3
M) caused an initial phasic and then a tonic
contraction, which lasted for at least 30 min. During the tonic
contraction induced by phenylephrine, endothelium-intact
aortic rings showed a significant relaxation in response to
acetylcholine (95 ⫾4%) (data not shown). The concentration-
response curves for cumulative S-petasin (0.01–100
M)
treatment in endothelium-intact aortic rings, before and af-
ter treatment with indomethacin (10
M), L-NNA (100
M),
or methylene blue (10
M) are illustrated in Fig. 5. The
results indicated that treatment with these inhibitors did not
significantly affect either the basal vascular tone or the re-
laxing effect of S-petasin in endothelium-intact aortic rings.
Inhibition of KCl-Induced Contraction. In endotheli-
um-denuded aortic preparations, the cumulative concentra-
tion-effect curves for KCl (15–90 mM) in the absence and
presence of five concentrations of S-petasin (1–100
M) are
shown in Fig. 6. Pretreatment with S-petasin (3–100
M) for
10 min suppressed the cumulative concentration contrac-
tions induced by KCl. The maximal inhibition obtained with
100
MS-petasin was approximately 89%. Vehicle treatment
had no significant effects.
Effects of Extracellular Ca
2ⴙ
on S-Petasin’s Modula-
tion of KCl-Induced Contraction. In Ca
2⫹
-free, high K
⫹
(60 mM) solution, the cell membrane of aortic smooth muscle
was depolarized and VDCCs were activated. The lack of Ca
2⫹
entry was verified by the failure of KCl to produce vasocon-
striction in the aortic rings in the absence of extracellular
Ca
2⫹
(data not shown). Figure 7 shows that cumulative ad-
dition of Ca
2⫹
(0.1–3 mM) caused a stepwise increase of
contraction of the rat aorta, apparently caused by Ca
2⫹
en-
tering the depolarized cell through VDCC. The maximal ten-
sion attained at 3 mM Ca
2⫹
was 1.64 ⫾0.12 g in the presence
of vehicle and was taken to be 100%. When the aortic ring
was treated with S-petasin at 1 to 100
M, 10 min before KCl, the KCl-induced contraction was attenuated in a con-
centration-dependent manner, suggesting that Ca
2⫹
influx
through VDCC was probably inhibited by S-petasin. The IC
50
value was calculated to be 8.2 ⫾0.6
MataCa
2⫹
concen-
tration of 3 mM.
Effects of S-Petasin on Ca
2ⴙ
Channel Activity in
VSMCs. VSMCs were depolarized from ⫺30 to 60 mV with
the ramp protocol to investigate the channel openings. Ba
2⫹
currents through L-type VDCC were observed in VSMCs.
During a 5-min application of the vehicle alone, no significant
changes (⫺0.5 ⫾1.2%) in the kinetics and I-V relationship of
L-type VDCC current occurred (data not shown). Figure 8
shows that a 5-min application of S-petasin (1–50
M) re-
duced the L-type VDCC current to below the immediately
preceding current measured in vehicle-treated specimen. The
decrease in the magnitude of L-channel currents induced by
S-petasin was evident within 2 to 3 min and reached a
steady-state level within 5 min. Figure 9 summarizes the
results from several experiments. The maximal reduction
caused by S-petasin was 75.45 ⫾13.08%.
Fig. 3. Vasorelaxing effect of S-petasin on endothelium-intact (F) and
endothelium-denuded (E) Sprague-Dawley rat thoracic aortic rings con-
tracted with KCl (60 mM). The tensions developed in the absence of
vehicle and S-petasin in intact and denuded rings were 1.50 ⫾0.17 and
1.75 ⫾0.15 g, respectively (considered as 100%). Values are mean ⫾S.E.;
n⫽7 to 8 for each group.
Fig. 4. Comparison the vasorelaxing effects of S-petasin in endothelium-
denuded aortic rings between KCl (60 mM, E)- and Bay K 8644 (50 nM,
䡺)-induced contractions. The tensions developed by KCl and Bay K 8644
in the absence of S-petasin in denuded rings were 1.75 ⫾0.15 and 1.81 ⫾
0.13 g, respectively (considered as 100%). Values are mean ⫾S.E.; n⫽8
to 10 for each group.
Fig. 5. Effects of indomethacin (10
M, ƒ), L-NNA (100
M, 䡺), and
methylene blue (10
M, ‚) treatments on S-petasin (0.01–100
M)-
induced relaxation in endothelium-intact (F) thoracic aorta isolated from
Sprague-Dawley rats. Aortic rings were precontracted with phenyleph-
rine, and the change in tension is expressed as a percentage of the active
tension originally generated by phenylephrine. The tension developed in
the absence of S-petasin and agents in endothelium-intact rings was
1.92 ⫾0.03 g. Values are mean ⫾S.E.; n⫽8 to 10 for each group.
S-Petasin Blocks Voltage-Dependent Ca
2ⴙ
Channels 243
Effects of S-Petasin on [Ca
2ⴙ
]
i
in Individual VSMCs.
The average basal [Ca
2⫹
]
i
in single VSMCs was 112.06 ⫾
2.52 nM. [Ca
2⫹
]
i
increased to 149.68 ⫾2.66 nM when the
VSMCs were stimulated by KCl (60 mM). S-Petasin (10, 100
M) suppressed the KCl-induced increase of [Ca
2⫹
]
i
by
39.6 ⫾2.43 and 66.8 ⫾1.97%, respectively (Fig. 10). S-
Petasin itself did not alter the basal [Ca
2⫹
]
i
in VSMCs (data
not shown).
Discussion
Although folk medicines are popular in many parts of the
world and in certain cases can ably complement or supple-
ment mainstream medicine where ineffective, inadequate, or
low therapeutic indices exist (Marshall, 1994). Unfortunately
in many cases the claims are shaky or inadequately substan-
tiated, the mechanisms of action undefined, and the pharma-
cology mystified, impeding their acceptance and develop-
ment. Complicating further their therapeutic application is
the fact that many folk medicines are empirical with multiple
components and lack of batch-to-batch consistency, although
the claim is that the interactions of these components may be
beneficial. The first goal of the present study was to identify
the active ingredients. Following chemical isolation and iden-
tification, the major component of P. formosanus turned out
to be the sesquiterpene of S-petasin. Intravenous adminis-
tration of S-petasin in anesthetized rats produced a dose-
dependent hypotensive effect. However, no accompanying
Fig. 6. Inhibitory effect of S-petasin (1–100
M) on the contraction
induced by KCl (15–90 mM) in Sprague-Dawley rat thoracic aortic rings
with denuded endothelium. Aorta was preincubated with vehicle (E)or
S-petasin (1
M, F;3
M, Œ;10
M, ;30
M, ⽧; 100
M, f) at 37°C for
10 min, and then cumulative concentrations of KCl (15–90 mM) were
used to trigger the contraction. The mean maximal contractile responses
induced by KCl (90 mM) in the absence of S-petasin in endothelium-
denuded rings was 1.46 ⫾0.07 g. Values are mean ⫾S.E.; n⫽10 to 12
for each group. *
,
**
,
***Statistically significant difference (P⬍0.05, P⬍
0.01, and P⬍0.001, respectively) between the vehicle and the S-petasin-
treated group.
Fig. 7. Inhibitory effect of S-petasin (1–100
M) on the contraction,
dependent on extracellular Ca
2⫹
influx, induced by KCl (60 mM) in
Ca
2⫹
-free Krebs’ solution of endothelium-denuded thoracic aortic rings
from Sprague-Dawley rats. Aorta was preincubated with vehicle (E)or
S-petasin (1
M, F;3
M, Œ;10
M, ;30
M, ⽧; 100
M, f) at 37°C for
10 min, and then cumulative concentrations of Ca
2⫹
(0.1–3.0 mM) were
used to trigger the contraction. The mean maximal contractile responses
induced by Ca
2⫹
(3.0 mM) in the absence of S-petasin in endothelium-
denuded rings was 1.64 ⫾0.12 g. Values are mean ⫾S.E.; n⫽10 to 12
for each group. *
,
***Statistically significant difference (P⬍0.05 and P⬍
0.01, respectively) between the vehicle and the S-petasin-treated group.
Fig. 8. Representative I-V plots and recordings in single cultured VSMCs
isolated from Sprague-Dawley rat thoracic aorta. The peak amplitude of
L-type VDCC currents, carried by Ba
2⫹
, was inhibited by S-petasin (1–50
M). The Ba
2⫹
current through the Ca
2⫹
channels was elicited by depo-
larizing the VSMC from a test pulse of ⫺30 to 60 mV at a frequency of 0.1
Hz. The duration of the depolarizing test pulses was 250 ms at intervals
of 5 s. The I-V relationships were measured repeatedly for 5 min after the
addition of S-petasin or vehicle (0.1%) in the medium.
Fig. 9. Inward Ca
2⫹
currents carried by Ba
2⫹
were inhibited by S-petasin
(1–50
M) in cultured VSMCs isolated from Sprague-Dawley rat thoracic
aorta. Values are mean ⫾S.E.; n⫽5 in each group. *
,
**Statistically
significant difference (P⬍0.05 and P⬍0.01, respectively) between the
vehicle and the S-petasin-treated group.
244 Wang et al.
reflex tachycardia was observed. Related studies involving
isolated atria and cell cultures are in progress and will be
reported in due course. Preliminary indications are that S-
petasin may exert direct cardiac depressant effects, which
may even be beneficial if S-petasin were to be developed to be
an antihypertensive agent. For the present purpose, S-peta-
sin appeared to be the or one of the hypotensive principles in
P. formosanus. Mechanistic studies focusing on S-petasin
thus followed.
In the vascular tension studies, the S-petasin induced re-
laxation in endothelium-denuded or -intact aorta precon-
tracted by KCl or Bay K 8644, suggesting that vasorelaxation
may be a basis for its hypotensive action. The role of the
endothelium, being known to be involved in the regulation of
cardiovascular functions, was then being examined. Endo-
thelium-intact and -denuded preparations as well as inhibi-
tors of the known vasorelaxing mediators prostacyclin, NO,
and guanylyl cyclase were used. The results indicated that
the vasorelaxing actions of S-petasin were not affected even
in the presence of indomethacin, L-NNA, or methylene blue.
Indomethacin is known to block the generation of prostacy-
clin, whereas L-NNA and methylene blue have been reported
to inactivate the NO system or inhibit the activation of gua-
nylyl cyclase, respectively (Thorin et al., 1998). It thus ap-
peared that the vasorelaxation caused by S-petasin was en-
dothelium-independent and not mediated by prostacyclin or
the NO-guanylyl cyclase pathway but rather acted directly
on the arterial smooth muscle. The rest of the experiments
were therefore conducted in endothelium-denuded aortic
preparations or cultured VSMCs.
An increase in free cytoplasmic Ca
2⫹
levels is required for
excitation-contraction coupling of vascular smooth muscle.
Vasoconstrictors can increase the [Ca
2⫹
]
i
by activating sev-
eral different pathways. VDCC represents the principal
route by which Ca
2⫹
enters vascular smooth muscle cells
(Bolton, 1979) and plays an essential role in the sustained
phase of contraction (Cauvin and Malik, 1984). Drugs that
block the Ca
2⫹
channel have proven clinically effective for
the treatment of a multitude of cardiovascular disorders. It
has been reported that increased KCl depolarizes smooth
muscle cells, leading to the opening of VDCC, with subse-
quent Ca
2⫹
influx and contraction (Karaki and Weiss, 1979).
In the present study, pretreatment with S-petasin sup-
pressed, in a concentration-dependent manner, the aortic
contractile response to high K
⫹
. The maximum inhibition
produced by S-petasin at 100
M was about 89%. When
S-petasin was cumulatively added during the tonic contrac-
tion induced by high K
⫹
, it exerted 100% vasorelaxation.
Similar results were also obtained as the aortic preparations
were challenged with the VDCC activator Bay K 8644. These
observations suggested that S-petasin might interfere with
these Ca
2⫹
channels in the aortic smooth muscle, possibly
resulting in the decrease of Ca
2⫹
influx and contraction.
Furthermore, in Ca
2⫹
-depleted and high K
⫹
medium, the cell
membrane of aortic smooth muscle was depolarized and the
VDCCs were activated but without contraction due to the
lack of extracellular Ca
2⫹
. Addition of Ca
2⫹
produced sus-
tained contraction that was produced by the Ca
2⫹
influx
through VDCC. Preincubation with S-petasin could effec-
tively antagonize, in a concentration-dependent manner,
Ca
2⫹
-induced contraction, implying that S-petasin probably
blocked Ca
2⫹
influx through VDCC in isolated aortic smooth
muscle cells. However, the antagonism was noncompetitive
in nature because there was a nonparallel shift to the right
and suppression of the maximal response. A plausible reason
is probably that much higher concentrations of Ca
2⫹
would
be needed to achieve the maximal contraction because Ca
2⫹
in high concentrations can be autoinhibitory and decrease
the permeability of the cell membrane for Ca
2⫹
.
With K
⫹
and Na
⫹
channels blocked, whole-cell patch-
clamp was studied in isolated cultured VSMCs. These data
provide strong evidence that S-petasin inhibited Ca
2⫹
-gen-
erated currents in the L-type VDCC, the predominant Ca
2⫹
channels in VSMCs. The fact that S-petasin can inhibit
VDCC activity suggests that S-petasin may reduce the in-
crease in [Ca
2⫹
]
i
elicited by KCl, resulting in decreased Ca
2⫹
entry and [Ca
2⫹
]
i
. In our Fura-2 studies, the measurement of
[Ca
2⫹
]
i
in cultured VSMCs confirmed this interpretation.
S-Petasin indeed produced a significant reduction in [Ca
2⫹
]
i
induced by KCl, which indicated that the direct effect of
S-petasin on blood vessels was probably related to interfer-
ence with Ca
2⫹
transport and consequently the contraction.
Taken together, the vasorelaxant action induced by S-peta-
sin in KCl-contracted aortic rings appeared to be mediated
via direct inhibition of VDCC activity, leading to decreased
Ca
2⫹
entry and [Ca
2⫹
]
i
. The attenuation of KCl-induced
Ca
2⫹
transients by S-petasin may explain its observed hypo-
tensive effects in vivo. It thus seems that S-petasin exerts its
hypotensive action by decreasing vascular reactivity to en-
dogenous pressor agents, at least in part, through inhibition
of the VDCC activity and the net inward flow of Ca
2⫹
.
In conclusion, the present studies identified S-petasin as
the principal active ingredient in P. formosanus and verified
its hypotensive effect. Mechanistic studies suggested vasore-
laxation via inhibition of Ca
2⫹
being the likely underlying
mechanism. Vascular endothelium and related vasorelax-
ation mediators play small roles. These findings may be
helpful in the establishment of S-petasin as a potential an-
tihypertensive agent, elucidation of its pharmacological ac-
tions and its further development as a therapeutic agent.
Acknowledgments
We thank Professor Peter Pang of the University of Hong Kong,
Faculty of Medicine, for proofreading the manuscript and Shu-Jen
Fig. 10. Inhibitory effect of S-petasin (10, 100
M) on the peak increment
of [Ca
2⫹
]
i
induced by KCl (60 mM) in cultured VSMCs isolated from
Sprague-Dawley rat thoracic aorta. The cells were challenged by KCl (60
mM) in the presence of S-petasin (10, 100
M) or vehicle for 10 min and
the peak increment in [Ca
2⫹
]
i
was determined. Values are mean ⫾S.E.;
n⫽3 to 6 separate experiments. ***Statistically significant difference
(P⬍0.001) between the vehicle and the S-petasin-treated group.
S-Petasin Blocks Voltage-Dependent Ca
2ⴙ
Channels 245
Huang for excellent technical assistance in the performance of some
of these studies.
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Send reprint requests to: Dr. Guei-Jane Wang, National Research Institute
of Chinese Medicine, Rm. 355, 155-1, Sec. 2, Li-Nong St., Pei-tou Dist. (112),
Taipei, Taiwan, Republic of China. E-mail: jennyw@cma23.nricm.edu.tw
246 Wang et al.
