1 Department of Pharmacology and 2 Division of Enzyme Chemistry, The University of Tokushima School of Medicine, Tokushima 770-8503, Japan
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ABSTRACT |
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We have found that human chymase produces a
31-amino acid endothelin [ET-1-(131)] from the 38-amino
acid precursor (Big ET-1). We examined the mechanism of synthetic
ET-1-(1
31)-induced intracellular Ca2+ signaling in cultured human
coronary artery smooth muscle cells. ET-1-(1
31) increased the
intracellular free Ca2+
concentration
([Ca2+]i)
in a concentration-dependent manner
(10
14-10
10
M). This ET-1-(1
31)-induced
[Ca2+]i
increase was not affected by phosphoramidon, Bowman-Birk inhibitor, and
thiorphan. The ET-1-(1
31)-induced
[Ca2+]i
increase was not influenced by removal of extracellular
Ca2+ but was inhibited by
thapsigargin. ET-1-(1
31) at
10
12 M did not cause
Ca2+ influx, whereas
10
7 M ET-1-(1
31) evoked
marked Ca2+ influx, which was
inhibited by nifedipine. ET-1-(1
31) also increased inositol
trisphosphate formation. These results suggest that the ET-1-(1
31)-induced
[Ca2+]i
increase at relatively low concentrations is attributable to the
release of Ca2+ from inositol
trisphosphate-sensitive intracellular stores, whereas Ca2+ influx into the cells evoked
by high concentration of ET-1-(1
31) probably occurs through
voltage-dependent Ca2+ channels.
We concluded that the physiological activity of ET-1-(1
31) may be
attributable to Ca2+ mobilization
from intracellular stores rather than influx of Ca2+ from extracellular space.
human chymase; confocal laser microscopy
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INTRODUCTION |
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ENDOTHELIN-1 (ET-1) is a 21-amino acid polypeptide that exhibits various physiological actions, such as vascular contraction (40), cardiac hypertrophy, and mitogenesis (2). Human ET-1 is generated from the 38-amino acid precursor (Big ET-1), through a cleavage of the Trp21-Val22 bond via the action of endothelin (ET)-converting enzyme (ECE). Although ECE was originally shown to be a membrane-bound metalloprotease (40), several other metalloproteases have also been shown to catalyze the formation of ET-1 from Big ET-1. For example, an aspartic protease (7, 35), a metalloendopeptidase (19, 22), cathepsin D-like enzyme (27), and elastase (10) have been identified as putative converters of Big ET-1 to ET-1. Rat mast cell chymase has also been reported as a putative converter of Big ET-1 to ET-1 (36).
We have recently reported that human mast cell chymase, unlike rat mast
cell chymases, selectively cleaves Big ET-1 at the Tyr31-Gly32
bond to produce novel trachea-constricting 31-amino acid endothelins, ET-1-(131), without any further degradation products (18). ET-1-(1
31) may potentially be produced in the lung under certain circumstances, because in vitro study with human chymase, which is an
alternative angiotensin-converting enzyme in human tissue (32), has
shown that Big ET-1 can be converted to ET-1-(1
31). Because ET-1 has
been shown to play a significant role in human cardiovascular functions
(2), ET-1-(1
31) may possess biological activities as well in human
tissues. As it has been reported that chymase plays a significant role
in the foam cell formation in human coronary atheromas (14),
ET-1-(1
31) may be an atherogenetic substance in human tissues.
Recently, we have found that synthetic ET-1-(131) induces a rise in
intracellular free calcium concentration
([Ca2+]i)
in cultured human coronary artery smooth muscle cells (41). We also
observed that ET-1-(1
31) causes a contraction of microperfused rabbit
afferent and efferent arterioles (29). ET-1 has been shown to affect
voltage-dependent Ca2+ channels
and to cause Ca2+ influx into
vascular smooth muscle cells (3, 28). On the other hand, it has been
reported that ET-1 activates phospholipase C, which results in
Ca2+ mobilization from
intracellular stores (15, 17, 33). Therefore, we investigated in this
study the mechanism of ET-1-(1
31)-induced Ca2+ signaling with cultured human
coronary artery smooth muscle cells. Change in
[Ca2+]i
elicited by synthetic ET-1-(1
31) was measured by confocal laser
microscopy. The ET-1-(1
31)-induced
Ca2+ influx into the cells was
also examined with
45CaCl2.
In addition, the effects of known ET-receptor antagonists and various
protease inhibitors on change in ET-1-(1
31)-induced [Ca2+]i
were examined.
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METHODS |
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Cell preparation and culture. Human coronary artery smooth muscle cells were purchased from Clonetics (San Diego, CA). Cells were plated in 25-cm2 tissue culture flasks at a density of 5 × 103 cells/cm2 in MCDB-131 medium supplemented with 5% heat-inactivated fetal calf serum, 0.5 ng/ml epidermal growth factor, 1 ng/ml basic fibroblast growth factor, 5 µg/ml insulin, 50 µg/ml gentamicin, and 0.25 µg/ml amphotericin B. The cells were incubated at 37°C in 5% CO2, and the medium was replaced every other day until 60-80% confluent. Then, the cells were removed from the flasks with 0.025% trypsin plus 0.01% EDTA and washed twice with HEPES buffer solution (in mM: 30 HEPES, 130 NaCl, 3.0 KCl, 3.0 Na2HPO4, and 10 glucose, adjusted with NaOH to pH 7.40). Thereafter, the cells were seeded onto glass coverslips attached to 35-mm tissue culture dishes coated with poly-L-lysine. The dishes were purchased from MatTek (Ashland, MA). All experiments were performed with the cells in passages 5-15 and at 2-3 days postconfluence.
Loading of the Ca2+ indicator fluo 3 into cells. The culture medium was removed from the dishes and replaced with modified Krebs-Henseleit bicarbonate buffer solution (K-H solution; in mM: 135 NaCl, 5.6 KCl, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, 2.2 CaCl2, and 10 glucose, adjusted with HCl to pH 7.40) oxygenated with a 95% O2-5% CO2 gas mixture. Then, the cells were loaded with the ester form of the dye (fluo 3-acetoxymethyl ester). For this purpose, the cells were incubated at 37°C with a final concentration of 4 µM fluo 3-acetoxymethyl ester. After a loading period of 30 min, the solution was exchanged with a dye-free K-H solution, and the cells were allowed to deesterify the indicator for an additional 10 min. The low ester concentration was chosen to minimize problems arising from compartmentalization of the indicator (9).
Measurement of fluorescence intensity by confocal laser microscopy. We used the methods for confocal fluorescence measurements that have been reported previously (30). The confocal imaging system (RCM 8000, Nikon, Tokyo, Japan) with an argon-ion laser was attached to an inverted microscope (Nikon TMD300, Diaphot, Tokyo, Japan). Cells in the culture dish containing 1 ml of K-H solution were placed on the stage of the microscope, and the fluorescence in the cell was excited at 488 nm by the laser. Emission at wavelengths longer than 520 nm was then detected by a photomultiplier. The system scanned full-field images at 30 frames/s, but the images were obtained by averaging eight successive frames in order to improve the signal-to-noise ratio. The objective lens used was a Nikon CF Fluor ×20/NA0.75. After stable baseline fluorescence intensity was measured, 10 µl of an agent was added to extracellular medium to yield a 1/100 concentration, and the fluorescence intensity was recorded. The same cells were stimulated by 10 µM of ionomycin 1 min after test agent application, and the relative fluorescence intensity was calculated. For experiments of the inhibition of Ca2+ release from intracellular stores, thapsigargin, an inhibitor of sarcoplasmic reticulum Ca2+-ATPase, was added during dye loading. These experiments were performed under Ca2+-free conditions. Calibration of the fluo 3 fluorescence intensity to estimate [Ca2+]i was calculated from the difference between Fmax and Fmin. Fmin was defined as the minimum fluorescence intensity before each agonist stimulation. Fmax was estimated from the intensity at 1 min after addition of ionomycin in each experiment. Results were expressed as percentages of the difference between Fmax and Fmin. For data analysis, the images of confocal microscopic observation were stored on a Panasonic magnetico-optical disk (Matsushita Electric Industrial, Osaka, Japan). Sequences of digitized images were transferred to an IBM-Think Pad equipped with an image-processing software package. Data are presented as means ± SE.
Measurement of Ca2+ influx into the cells. The method for measurement of 45Ca2+ influx into the cells was as described previously (42). Briefly, the cultured cells were washed once with 1 ml of K-H solution and then incubated with test agents at 37°C for 10 min in K-H solution containing 45CaCl2 (4 µCi/ml). Then, the medium was discarded and the cells were washed three times with 1 ml of ice-cold Ca2+-free K-H solution. The cells were then solubilized by 1% Triton X-100, and the radioactivity in the cell lysate was measured in a liquid scintillation counter. The amount of 45Ca2+ taken up into the cells was calculated on the basis of the specific activities of radioactive 45Ca2+ in the reaction mixture and expressed in nanomoles/dish (1 × 106 cells). For experiments of the inhibition of Ca2+ influx into the cells, nifedipine, a typical voltage-dependent Ca2+ channel blocker, was added to the medium during incubation.
Measurement of inositol trisphosphate formation within
the cells. Agonist-induced inositol trisphosphate
(IP3) formation within the cells
was measured with a 3H assay
system kit (Amersham, Tokyo, Japan; Ref. 21). Briefly, the
cultured cells were washed once with 1 ml of K-H solution and then
incubated at 37°C for 15 s with or without test agents, and the
reaction was stopped by liquid nitrogen freezing. For experiments with
ET-receptor subtype (ETA;
ETB) antagonists, a selective
ETA antagonist, BQ123 (6), or a
selective ETB antagonist, BQ788
(8), was added to the medium during incubation. Then, both
the medium and cell lysate were centrifuged at 2,000 g for 5 min. The resultant supernatant
was further concentrated five times with a Speed-Vac concentrator (SVC
100, Savant, NY) and was stored frozen at 70°C until
assayed. The IP3 in the
supernatant was assayed with a
D-myo-[3H]inositol
1,4,5-trisphosphate assay system (7.5 nCi/ml). Results were expressed
as picomoles per dish (1 × 106 cells).
Statistics. One-way ANOVA was used to determine the significance among groups, after which the modified t-test with the Bonferroni correction was used for comparison between individual groups. A value of P < 0.05 was considered to be statistically significant.
Materials. Human ET-1, Big ET-1, and
phosphoramidon were obtained from Peptide Institute (Osaka, Japan).
ET-1-(131) was synthesized by solid-phase procedures by the Peptide
Institute. MCDB-131 medium, fetal calf serum, epidermal growth factor,
insulin, gentamicin, amphotericin B, and trypsin were obtained from
Clonetics. The D-myo-[3H]inositol
1,4,5-trisphosphate assay system and
45CaCl2
were obtained from Amersham. Fluo 3-acetoxymethyl ester, ionomycin, and
Triton X-100 were purchased from Wako Pure Chemical (Osaka, Japan).
BQ123 and BQ788 were gifts from Banyu Pharmaceutical (Tsukuba, Japan).
All other chemicals, including Bowman-Birk inhibitor (BBI), thapsigargin, and nifedipine were from Sigma (St.
Louis, MO).
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RESULTS |
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Concentration-dependent increase in
[Ca2+]i
induced by ET-1-(131). Figure
1 shows the time course of
10
12 M ET-1-(1
31)-induced
increase in
[Ca2+]i.
The ET-1-(1
31)-induced increase in
[Ca2+]i
reached a peak within 10 s and then gradually decreased to the baseline
resting value within 1 min. ET-1-(1
31) caused an increase in
[Ca2+]i
in a concentration-dependent manner from
10
14 M to
10
10 M. ET-1 also increased
[Ca2+]i;
however, it was ~10× more potent than ET-1-(1
31) at the
concentrations used. The concentration-response curve for Big ET-1 was
similar to that for ET-1-(1
31) (41). However, the concentrations of ET-1-(1
31) that caused half-maximal increases in
[Ca2+]i
were ~100× and 10,000× lower than those of angiotensin II
and norepinephrine, respectively (11).
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Possible conversion of ET-1-(131) to ET-1 by
ECE. To investigate the possibility that the
ET-1-(1
31)-induced increase in [Ca2+]i
is due to the degradation of ET-1-(1
31) to ET-1 by ECE in the
incubation medium or the cells, we examined the effects of an ECE
inhibitor and protease inhibitors on the increase. As shown in Table
1, phosphoramidon, a potent inhibitor of
ECE (16), at 10
5 and
10
4 M did not inhibit the
increase in
[Ca2+]i
caused by 10
12 M
ET-1-(1
31). Similar results were obtained when the cells were preincubated with BBI and thiorphan, known inhibitors of trypsin- or
chymotrypsin-type proteases and neutral endopeptidase 24.11 (22), in
the medium at concentrations of
10
5 M. Phosphoramidon at
10
4 M reduced the increase
in
[Ca2+]i
caused by 10
12 M Big ET-1.
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Ca2+
mobilization from intracellular stores and from the extracellular
space. Figure 2 shows the
responses of cultured coronary artery smooth muscle cells to
1012 M ET-1-(1
31) in the
presence or absence of extracellular
Ca2+. ET-1-(1
31)-induced
increase in
[Ca2+]i
was not affected by removal of
Ca2+ from the medium. However, it
was almost abolished by thapsigargin (10
5 M), a specific
inhibitor of the sarcoplasmic reticulum
Ca2+ pump. From these results, it
is assumed that a 10
12 M
ET-1-(1
31)-induced increase in
[Ca2+]i
is attributable to the release of
Ca2+ from intracellular stores. On
the other hand, ET-1-(1
31) at 10
7 M caused marked
45Ca2+
influx into the cells, as did ET-1
(10
7 M) and Big ET-1
(10
7 M; Table
2). The
10
7 M ET-1-(1
31)-induced
45Ca2+
influx was almost abolished by the addition of
10
5 M nifedipine, a
voltage-dependent Ca2+ channel
blocker, and nifedipine also abolished the
Ca2+ influx induced by ET-1 and
Big ET-1. ET-1-(1
31) at
10
12 M did not evoke
Ca2+ influx from the extracellular
space nor did ET-1 (10
12 M)
and Big ET-1 (10
12 M).
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IP3 formation
within the cells induced by ET-1-(131). As shown in
Fig. 3, application of
10
10 M ET-1-(1
31) caused
an ~3.2-fold increase of IP3
formation from the control value during 15 s of incubation. The
ET-1-induced IP3 level was similar
to that of ET-1-(1
31), whereas Big ET-1 was less potent than
ET-1-(1
31). Next, we examined the effects of known ET-receptor
antagonists. The ET-1-(1
31)-induced
IP3 formation was also inhibited
by the selective ETA antagonist
BQ123 (10
8 M) but not by
the selective ETB antagonist BQ788
(10
8 M).
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DISCUSSION |
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The present study demonstrated that ET-1-(131), which is cleaved at
the
Tyr31-Gly32
bond of Big ET-1 by human chymase, caused a rise in
[Ca2+]i
in cultured human coronary artery smooth muscle cells. The ET-1-(1
31)-induced rise in
[Ca2+]i
at relatively low concentrations may be attributable to the mobilization of Ca2+ from
intracellular stores, whereas ET-1-(1
31) causes marked Ca2+ influx into the cells at high
concentrations around the nanomolar range. We also demonstrated that
the ET-1-(1
31)-induced phenomena are mediated through
ETA or
ETA-like receptors of the cells,
which may be coupled to the
IP3-forming pathway.
Human chymase is highly efficient in converting angiotensin I to
angiotensin II (32). Urata et al. (31) reported that the
chymase-dependent angiotensin II-forming pathway is a major pathway for
angiotensin II formation in the failing human heart. We have recently
found that human mast cell chymase specifically converted Big ET-1 to
the novel trachea-constricting 31-amino acid peptide ET-1-(131),
which is different in amino acid length from the well-known 21-amino
acid ET-1 (18). It has also been reported that a serine protease in the
membrane fraction of human lung hydrolyzes Big ET-1 to ET-1-(1
31),
which showed contractile activity in the pulmonary artery (4). Because
ET-1 as well as angiotensin II has been shown to possess a wide variety
of biological actions contributing to vascular contraction, cardiac hypertrophy, or atherosclerosis (1, 2), ET-1-(1
31) may also be a
novel and alternative vasoactive peptide in the ET family. Recently, we
found that ET-1-(1
31) causes
[Ca2+]i
increase in cultured human coronary artery smooth muscle cells (41). We
also observed that ET-1-(1
31) induces contraction of rabbit afferent
and efferent arterioles, porcine coronary arteries, and rat aortae (12,
29). Because the increase in
[Ca2+]i
caused by ET-1 has been found to play a role in vascular contraction or
vascular smooth muscle cell proliferation (13), ET-1-(1
31) is an
alternative candidate for the causative substance for these events in
vivo. In the present study, we investigated the mechanism of
Ca2+ signaling elicited by
synthetic ET-1-(1
31) with cultured human coronary artery smooth
muscle cells.
The results shown in Fig. 1 revealed that ET-1-(131) in the picomolar
range causes a transient increase in
[Ca2+]i
in cultured human coronary artery smooth muscle cells. The effect of
Big ET-1 in vivo is comparable with that of ET-1, which is likely due
to the conversion of Big ET-1 to ET-1 (16). Therefore, it is important
to elucidate whether the response to ET-1-(1
31) is mediated by ET-1
after hydrolysis of ET-1-(1
31) or whether ET-1-(1
31) itself acts
directly on the receptors of the cells. As shown in Table 1,
phosphoramidon, an inhibitor of metalloendopeptidases and ECE (16), had
almost no effect on the increase in
[Ca2+]i
elicited by ET-1-(1
31). BBI and thiorphan, which are inhibitors of
trypsin- or chymotrypsin-type proteases and neutral endopeptidase 24.11 (18, 37), both failed to inhibit the ET-1-(1
31)-induced [Ca2+]i
increase. However, the Big ET-1-induced increase in
[Ca2+]i
was suppressed by phosphoramidon (Table 1). These results suggest that
the ability of ET-1-(1
31) to increase
[Ca2+]i
is not the consequence of its conversion to ET-1 by ECE, a chymotrypsin-type protease(s), or metalloendopeptidase(s). Moreover, it
is reported that ET-1-(1
31) is not a substrate for ECE purified from
bovine adrenal cortex (37). Nevertheless, it should be noted that
ET-1-(1
31) itself has biological activity in cultured human coronary
artery smooth muscle cells. Moreover, in a preliminary study, we have
detected ET-1-(1
31) not only in human lungs but also in human hearts,
and the amount of ET-1-(1
31) in autopsy specimens was similar to or
higher than that of ET-1. ET-1-(1
31) is thus a novel putative
vasoactive peptide member of the ET family and may play a significant
role in chymase-related pathophysiological processes in humans.
It has been reported that there are at least two main subtypes of
ET-receptors, termed ETA and
ETB (34). The
ETA receptor was originally found
in vascular smooth muscle cells (26), and recent studies revealed that
ETB receptors also exist in
vascular smooth muscle cells (24). These have been called
ETB2 receptors to distinguish them
from the ETB1 vasodilatory
receptors (20). In our study, the ET-1-(131)-induced increase in
[Ca2+]i
was inhibited by BQ123, but not by BQ788, known inhibitors of
ETA and
ETB receptors, respectively (41).
Although the vasoconstrictive actions of ET-1-(1
31) are blocked by
ETA-specific antagonists, it does
not necessarily mean that there are not specific receptor subtypes
through which these effects are mediated. Our results suggest that the
ET-1-(1
31)-induced response of the cells is mediated through
ETA or
ETA-like receptors. Further
studies are needed to clarify whether different receptors are present.
In this study, we investigated the characteristics of
Ca2+ signaling elicited by
ET-1-(131) in human coronary artery smooth muscle cells. As shown in
Fig. 2, the 10
12 M
ET-1-(1
31)-induced increase in
[Ca2+]i
was not affected by removal of extracellular
Ca2+ but was inhibited by
thapsigargin, a specific inhibitor of the sarcoplasmic reticulum
Ca2+ pump. The
[Ca2+]i
increase evoked by ET-1 at
10
13 M was also not
affected by removal of extracellular
Ca2+ but was inhibited by
thapsigargin (data not shown). From these results, it is concluded that
ET-1-(1
31) in the picomolar range causes
Ca2+ mobilization from
intracellular stores. ET-1 induces rapid and transient increase in
[Ca2+]i,
probably through the activation of
IP3 formation, in vascular smooth
muscle cells (38, 39). We also observed increase in IP3 formation after ET-1-(1
31)
treatment (Fig. 3). However, the above studies that examined
IP3 formation have used relatively high concentrations (nM range) of ET-1, and at these concentrations, significant Ca2+ influx after the
initial increase of
[Ca2+]i
may occur. Our results shown in Table 2 revealed that
10
12 M ET-1-(1
31) did not
evoke Ca2+ influx into the cells,
whereas ET-1-(1
31) at 10
7
M caused marked Ca2+ influx, as
did ET-1 and Big ET-1. Moreover, the
10
7 M ET-1-(1
31)-induced
Ca2+ influx was inhibited by
nifedipine, a typical voltage-dependent Ca2+ channel blocker. Taken
together, these data suggest that ET-1-(1
31) at low concentrations
(pM range) causes
[Ca2+]i
increase via mobilization from intracellular stores, whereas marked
Ca2+ influx occurs at relatively
high concentrations (nM range), probably through voltage-dependent
Ca2+ channels. These findings are
consistent with the concept that release of
Ca2+ from intracellular stores is
a necessary prerequisite for the operation of plasma membrane
Ca2+ channels (23). Considering
the findings that the tissue or blood concentrations of ET-1 are in the
picomolar range (5, 25), it is reasonable to speculate that the
physiological activity of ET-1-(1
31) is attributable to
Ca2+ mobilization from
intracellular stores rather than the influx of
Ca2+ from the extracellular space.
Our results also show that ET-1 and Big ET-1 mediate their responses at
picomolar concentrations via an intracellular mechanism and at
nanomolar concentrations via Ca2+
influx at least in cultured human coronary artery smooth muscle cells.
In summary, ET-1-(131) increased
[Ca2+]i
in a concentration-dependent manner
(10
14-10
10
M) in cultured human coronary artery smooth muscle cells. The ET-1-(1
31)-induced
[Ca2+]i
increase at relatively low concentrations (pM range) may be attributable to the release of
Ca2+ from
IP3-sensitive intracellular
stores, whereas Ca2+ influx into
the cells evoked by high concentration (nM range) of ET-1-(1
31) may
occur through voltage-dependent
Ca2+ channels. We conclude that
the physiological activity of ET-1-(1
31) may be attributable to
Ca2+ mobilization from
intracellular stores rather than influx of Ca2+ from the extracellular space.
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ACKNOWLEDGEMENTS |
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This work was supported in part by Grants-in-Aid for Scientific Research (no. 10770040 and no. 10670085) from the Ministry of Education, Science, Sports, and Culture, Japan. We are grateful to Keiko Tachibana for secretarial assistance.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: D. Inui, Dept. of Pharmacology, The Univ. of Tokushima School of Medicine, 3-18-15 Kuramoto, Tokushima 770-8503, Japan (E-mail: yakuri{at}basic.med.tokushima-u.ac.jp).
Received 14 July 1998; accepted in final form 3 March 1999.
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