Ca2+ channels involved in endothelin-induced
mitogenic response in carotid artery vascular smooth muscle cells
Yoshifumi
Kawanabe1,2,
Nobuo
Hashimoto1, and
Tomoh
Masaki2
Departments of 1 Neurosurgery and 2 Pharmacology,
Kyoto University Faculty of Medicine, Kyoto 606-8507, Japan
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ABSTRACT |
Endothelin (ET)-1 activates two
types of Ca2+-permeable nonselective cation channels
(NSCC-1 and NSCC-2) and a store-operated Ca2+ channel
(SOCC) in rabbit internal carotid artery (ICA) vascular smooth muscle
cells (VSMCs) in addition to the voltage-operated Ca2+
channel (VOCC). These channels can be discriminated using the Ca2+ channel blockers SK&F-96365 and LOE-908. SK&F-96365 is
sensitive to NSCC-2 and SOCC, and LOE-908 is sensitive to NSCC-1 and
NSCC-2. On the basis of sensitivity to nifedipine, a specific blocker of the L-type VOCC, VOCCs have a minor role in ET-1-induced
mitogenesis. Both LOE-908 and SK&F-96365 inhibited ET-1-induced
mitogenesis in a concentration-dependent manner, and the combination of
LOE-908 and SK&F-96365 abolished it. The IC50 values of
these blockers for ET-1-induced mitogenesis correlated well with those
of the ET-1-induced intracellular free Ca2+
concentration responses. These results indicate that the inhibitory action of these blockers on ET-1-induced mitogenesis may be
mediated by blockade of NSCC-1, NSCC-2, and SOCC. Collectively,
extracellular Ca2+ influx through NSCC-1, NSCC-2, and SOCC
may be essential for ET-1-induced mitogenesis in ICA VSMCs.
endothelin; calcium ion channel; cell proliferation
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INTRODUCTION |
ENDOTHELIN (ET)-1 is
a potent vasoconstricting peptide with a long duration of action
(20). However, studies have indicated that it also
possesses multiple additional biological activities. Among the diverse
actions of ET-1, its mitogenic properties have attracted much attention
because they indicate a possible role for this peptide in the
pathogenesis of certain clinical conditions such as
hyperlipoproteinemia or atherosclerosis (8, 13). Moreover,
ET-1 plays an important role in neointimal hyperplasia after
balloon-induced vascular injury, including that of the carotid artery
(1, 3). The ability of an ET type A receptor
(ETAR)-specific antagonist to block the mitogenic activity
of ET-1 has been demonstrated (1, 17), suggesting that
ET-1-ETAR interaction is necessary for the mitogenic action
of ET-1.
It is generally accepted that extracellular Ca2+
influx plays a critical role in growth factor-induced cell
proliferation (18). Moreover, extracellular
Ca2+ influx via nonselective cation channels (NSCCs) is
important for the growth factor-induced mitogenic response in mouse
fibroblasts (12). However, it remains unclear whether
Ca2+ influx is essential for ET-1-induced mitogenesis of
native vascular smooth muscle cells (VSMCs), and it is equally unclear
what types of Ca2+ channels are involved in mitogenesis in
native VSMCs. These uncertainties are mainly attributable to the lack
of specific Ca2+ channel blockers. Therefore, we attempted
to pharmacologically characterize the Ca2+ channels
activated by ET-1 in internal carotid artery (ICA) VSMCs using the
Ca2+ channel blockers SK&F-96365 and LOE-908 (4,
14). ET-1 binds to its receptors and induces a biphasic increase
in intracellular free Ca2+ concentration
([Ca2+]i) consisting of a transient peak and
a subsequent sustained increase (11, 15). It is generally
accepted that the sustained increase in
[Ca2+]i requires the persistent entry of
extracellular Ca2+, whereas the transient increase results
from mobilization of Ca2+ from the intracellular
Ca2+ store (6). We recently showed
(11) that the ET-1-induced sustained increase in
[Ca2+]i within A7r5 cells, which are cultured
thoracic aorta VSMCs that predominantly express ETARs, is
the result of Ca2+ influx through voltage-independent
Ca2+ channels (VICCs) in addition to voltage-operated
Ca2+ channels (VOCCs). These VICCs (associated with
ETARs) consist of two types of Ca2+-permeable
NSCCs (designated NSCC-1 and NSCC-2) and one store-operated Ca2+ channel (SOCC) (11). In particular, we
demonstrated that these channels can be discriminated by using
SK&F-96365 and LOE-908. That is, NSCC-1 is sensitive to LOE-908 and
resistant to SK&F-96365, NSCC-2 is sensitive to both LOE-908 and
SK&F-96365, and SOCC is resistant to LOE-908 and sensitive to
SK&F-96365 (11). Thus SK&F-96365 and LOE-908 may be useful
to identify which Ca2+ channels are activated by ET-1 and
which Ca2+ channels are involved in ET-1-induced
mitogenesis in ICA VSMCs.
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MATERIALS AND METHODS |
Preparation and primary culture of ICA VSMCs for whole cell
recording and monitoring of
[Ca2+]i.
Isolated VSMCs were prepared from rabbit ICA as described previously
(5, 10, 15). Briefly, male Japanese White rabbits, each
weighing 2-3 kg, were anesthetized by an intravenous injection of
thiopental sodium (20 mg/kg) and killed by exsanguination. The
ICA was removed, cleaned of surrounding tissues, dissected into small
strips (2 mm × 5 mm), and kept in Ca2+-free
Krebs-HEPES solution containing (in mM) 140 NaCl, 3 KCl, 1 MgCl2, 11 glucose, and 10 HEPES (pH 7.3, adjusted with
NaOH). The strips were incubated overnight (12-24 h) at 4°C in
Ca2+-free Krebs-HEPES solution containing papain
(0.2-0.3 mg/ml) and 0.5 mM dithiothreitol. Thereafter, the strips
were resuspended and incubated in Ca2+-free Krebs-HEPES
solution containing collagenase (0.25-0.5 mg/ml) at 35°C for 10 min. The digested strips were cut into pieces with fine scissors and
triturated with a blunt-tipped pipette until a sufficient number of
single cells were released. The freshly dispersed cells were used for
electrophysiological experiments. For measurement of
[Ca2+]i, dispersed VSMCs were seeded on 60-mm
tissue culture dishes (Falcon) and cultured in Dulbecco's modified
Eagle's medium (DMEM) containing 10% FCS supplemented with 100 U/ml
penicillin G and 100 µg/ml streptomycin at 37°C in a humidified 5%
CO2-95% air atmosphere.
Electrophysiology and measurement of
[Ca2+]i.
Whole cell recordings and measurement of
[Ca2+]i were performed as previously
described (5, 11).
MTT assay and [3H]thymidine incorporation.
Cells were seeded into 96-well plates at 5 × 103
cells/well for the assay with
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)
and into 24-well plates at 4 × 104 cells/well for
[3H]thymidine incorporation. They were incubated
overnight in DMEM supplemented with 10% FCS at 37°C. The cells were
deprived of serum for 24 h, washed with phosphate-buffered saline,
and incubated with ET-1 for a further 48 h in serum-free DMEM with
or without Ca2+ channel blockers. MTT assay and
[3H]thymidine incorporation were performed as described
previously (17).
Statistical analysis.
Results are expressed as means ± SE. Data were subjected to a
two-way analysis of variance. When a significant F-value was encountered, the Newman-Keuls multiple-range test was used to test for
significant differences between treatment groups. A probability level
of P < 0.05 was considered statistically significant.
Drugs.
Boehringer Ingelheim (Ingelheim, Germany) kindly provided LOE-908.
Other reagents were commercially obtained from the following sources:
ET-1 from Peptide Institute (Osaka, Japan), SK&F-96365 from Biomol
(Plymouth Meeting, PA), fluo 3-AM from Dojindo Laboratories (Kumamoto, Japan), nifedipine and MTT from Sigma (St. Louis, MO), and [3H]thymidine from NEN (Boston, MA).
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RESULTS |
Effects of ET-1 on VOCC activation.
We first attempted to determine the maximal concentration of nifedipine
for complete inhibition of the L-type VOCC. We tested the effects of
various concentrations of nifedipine on an increase in
[Ca2+]i induced by high-K+ (50 mM) stimulation, which causes depolarization of the plasma membrane and
subsequent activation of the VOCC. Nifedipine completely inhibited the
high K+-induced increase in
[Ca2+]i at concentrations
1 µM (data not
shown). Thus, in the following experiments, we added 1 µM nifedipine
to the bath solution to block the VOCC completely.
ET-1 at 10 nM induced a biphasic increase in
[Ca2+]i consisting of an initial transient
peak and a subsequent sustained phase (Fig.
1A). On the other hand, 0.1 nM
ET-1 evoked only a sustained increase in
[Ca2+]i (Fig. 1C). After removal
of extracellular Ca2+, the transient peak was unaffected
but the sustained increase evoked by 10 nM ET-1 was abolished (data not
shown), indicating that only a sustained increase results from
Ca2+ influx. Nifedipine suppressed the 10 nM ET-1-induced
sustained increase in [Ca2+]i by a maximum of
no more than 10% at concentrations
1 µM (Fig. 1B). In
contrast, the 0.1 nM ET-1-induced sustained increase in [Ca2+]i was not affected by nifedipine up to
1 µM (Fig. 1D).

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Fig. 1.
A and C: original tracings showing
the effects of nifedipine on the increase in intracellular free
Ca2+ concentration ([Ca2+]i) in
internal carotid artery (ICA) vascular smooth muscle cells (VSMCs)
induced by endothelin (ET)-1. The fluo 3-loaded cells were stimulated
by 10 (A) or 0.1 nM ET-1 (C) in normal
Krebs-HEPES solution. Preparation of ICA VSMCs and monitoring of
[Ca2+]i were performed as described in
MATERIALS AND METHODS. During the plateau phase of the
[Ca2+]i increase, various concentrations of
nifedipine were added to the medium. B and D:
inhibitory effects of the maximally effective concentration of
nifedipine (1 µM) on the sustained increase in
[Ca2+]i induced by 10 (B) or 0.1 nM ET-1 (D). Increases in [Ca2+]i
are represented as percentages of the value in the absence of
nifedipine. Each bar represents mean ± SE of 5 experiments.
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Characterization of currents induced by ET-1 in ICA VSMCs with
whole cell recordings by patch clamp.
In the presence of 1 µM nifedipine, 10 nM ET-1 induced inward
currents in ICA VSMCs held at
60 mV (Fig.
2A). Currents induced by ET-1
showed linear current-voltage relationships with reversal potentials of
5.4 ± 1.2 mV (n = 10; Fig. 2B).
Current-voltage relationships induced by ET-1 were not affected by the
reduction of the Cl
concentration in the bath solution
from 149 mM to 9 mM (data not shown). The reversal potential was
3.6 ± 1.4 mV (n = 10). To test whether channels
activated by ET-1 were permeable to Ca2+, all monovalent
cations in the bath solution were replaced with nonpermeant cation
N-methyl-D-glucamine (NMDG) while the
concentration of Ca2+ was elevated from 1 mM to 30 mM. Even
under such conditions, ET-1 induced inward currents in ICA VSMCs held
at
60 mV (Fig. 2C). The reversal potential was
10.3 ± 1.6 mV (n = 10; Fig. 2D).

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Fig. 2.
A and C: original tracings
illustrating whole cell currents induced by 10 nM ET-1 in Krebs-HEPES
(A) or
Ca2+-N-methyl-D-glucamine (NMDG)
(C) solution. Cells were clamped at a holding potential of
60 mV with the whole cell configuration, and ET-1 was added to the
bath solution. B and D: current-voltage
relationships for currents induced by ET-1 in Krebs-HEPES
(B) or Ca2+-NMDG (D) solution. At the
times indicated by v, w, x, and
y, voltage steps of 100-ms duration ranging from 100 mV to
+80 mV in 20-mV increments were applied. The ET-1-induced current at
each membrane potential was obtained by subtracting the current at
v from that at w (B) or the current at
x from that at y (D) and plotted
against the membrane potential.
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Pharmacological properties of whole cell currents induced by ET-1
in ICA VSMCs.
To determine the maximally effective concentration of Ca2+
channel blockers such as SK&F-96365 or LOE-908, we first examined the
effects of various concentrations (~30 nM-30 µM) of these blockers on whole cell currents in ICA VSMCs induced by ET-1. SK&F-96365 and LOE-908 inhibited ET-1-induced currents in a
concentration-dependent manner with IC50 values of 2.1 ± 0.3 and 1.9 ± 0.3 µM, respectively, and maximal inhibition
was observed at concentrations
10 µM (data not shown). On the basis
of these data, we used 10 µM as the maximally effective concentration
of SK&F-96365 and LOE-908 in the following experiments.
ET-1-induced currents were abolished by 10 µM LOE-908 (Fig.
3A), and a major portion
(~65%) of these currents were suppressed by 10 µM SK&F-96365 (Fig.
3C). The characteristics of the currents inhibited by
LOE-908 or SK&F-96365 were essentially similar to those of ET-1-induced
currents in terms of linear current-voltage relationships and reversal
potentials of approximately
2 to
5 mV (Fig. 3, B and
D). ET-1 failed to induce currents in ICA VSMCs pretreated
with 10 µM LOE-908, whereas ET-1 induced currents in ICA VSMCs
preincubated with 10 µM SK&F-96365 (data not shown). The magnitude of
the current in SK&F-96365-treated ICA VSMCs was ~35% of that in
nontreated ICA VSMCs.

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Fig. 3.
Effects of SK&F-96365 or LOE-908 on whole cell currents
in ICA VSMCs induced by 10 nM ET-1. The experimental protocols were the
same as those in Fig. 2. After ET-1-induced currents had reached a
steady state, either SK&F-96365 (C) or LOE-908
(A) was added to the bath solution at a final concentration
of 10 µM. The current induced by ET-1 at each membrane potential was
obtained by subtracting the current before application of ET-1 from
that after its application and plotted against the membrane potential
(LOE-908, B; SK&F-96365, D).
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Pharmacological properties of SOCC in ICA VSMCs.
Generally, treatment of cells with thapsigargin (an inhibitor of
Ca2+-pump ATPase on the membrane of
sarcoplasmic/endoplasmic reticulum membrane) depletes the intracellular
Ca2+ store and thereby activates the Ca2+
channel on the plasma membrane called the SOCC or capacitative Ca2+ entry channel, causing a sustained increase in
[Ca2+]i (19). Therefore, the
sustained increase in [Ca2+]i is regarded as
an index of SOCC activity. A recent study showed that the
thapsigargin-induced increase in [Ca2+]i in
A7r5 cells was abolished by SK&F-96365, whereas it was unaffected by
nifedipine and LOE-908 (11). Thus we characterized the
pharmacological properties of SOCC in ICA VSMCs by using thapsigargin.
The sustained increase in [Ca2+]i in ICA
VSMCs induced by 0.1 µM thapsigargin was suppressed by SK&F-96365 in
a concentration-dependent manner, with an IC50 value of
2.7 ± 0.4 µM, and abolished at concentrations
10 µM (Fig.
4A). However, the increase in
[Ca2+]i was not affected by LOE-908 up to a
concentration of 30 µM (Fig. 4B).

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Fig. 4.
Original tracings illustrating the effects of SK&F-96365
(A) or LOE-908 (B) on the 0.1 µM
thapsigargin-induced increase in [Ca2+]i in
ICA VSMCs as an index of the activity of the store-operated
Ca2+ channel (SOCC). ICA VSMCs were loaded with fluo 3 and
subjected to monitoring of [Ca2+]i. Cells
were stimulated with thapsigargin at the time indicated by arrows.
After [Ca2+]i reached a steady state, various
concentrations of SK&F-96365 or LOE-908 were added to the bath solution
at the times indicated by arrows.
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Pharmacological analysis of ET-1-induced increase in
[Ca2+]i.
The sustained increase in [Ca2+]i evoked by
10 nM ET-1 was suppressed by LOE-908 in a concentration-dependent
manner, and maximal inhibition was observed at concentrations
10 µM
(Fig. 5A). The extent of
maximal inhibition was ~60%. On the other hand, the sustained
increase in [Ca2+]i was suppressed by
SK&F-96365 in a concentration-dependent manner, and maximal inhibition
was observed at concentrations
10 µM (Fig. 5B). The
extent of maximal inhibition was ~80%. The sustained increase in
[Ca2+]i caused by 0.1 nM ET-1 was abolished
by 10 µM LOE-908 (Fig. 5C), whereas it was not affected by
10 µM SK&F-96365 (Fig. 5D).

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Fig. 5.
Original tracings illustrating the effects of LOE-908 (A
and C) and SK&F-96365 (B and D) on the
increase in [Ca2+]i in ICA VSMCs induced by
10 (A and B) or 0.1 nM ET-1 (C and
D). Cells loaded with fluo 3 were stimulated by ET-1. After
[Ca2+]i reached a steady state, various
concentrations of LOE-908 or SK&F-96365 were added at the times
indicated by arrows.
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ET-1 induced a transient increase in [Ca2+]i
in a concentration-dependent manner with EC50 values of
~2 nM in ICA VSMCs treated with or without 10 µM LOE-908 or 10 µM
SK&F-96365 before stimulation with ET-1 (Fig.
6A). ET-1 also induced a
sustained increase in [Ca2+]i in these cells.
However, the EC50 values of ET-1 differed between these
cells. In ICA VSMCs, the EC50 values of ET-1 were 0.6 ± 0.3 nM and the maximal effect was observed at concentrations
10 nM
(Fig. 6B). In ICA VSMCs treated with LOE-908 before
stimulation with ET-1, the EC50 values were within 0.1 nM
and the maximal effect was observed at concentrations
0.1 nM (Fig.
6B). In ICA VSMCs treated with SK&F-96365 before stimulation
with ET-1, the EC50 values were 2.7 ± 0.3 nM and the
maximal effect was observed at concentrations
10 nM (Fig.
6B).

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Fig. 6.
Concentration-response curves for transient (A) or
sustained (B) increases in [Ca2+]i
induced by various concentrations of ET-1 in ICA VSMCs
( ) and ICA VSMCs pretreated with 10 µM LOE-908
( ) or 10 µM SK&F-96365 ( ) for 10 min.
Monitoring of [Ca2+]i was performed as
described in MATERIALS AND METHODS. C:
inhibitory effects of pretreatment for 10 min with various
concentrations of LOE-908 ( ) and SK&F-96365
( ) on the 10 nM ET-1-induced sustained increase in
[Ca2+]i in ICA VSMCs. D:
inhibitory effects of 10 µM SK&F-96365 and/or 10 µM LOE-908
pretreatment for 10 min on ET-1-induced sustained increase in
[Ca2+]i in ICA VSMCs. Increases in
[Ca2+]i in the presence of drugs are
represented as percentages of the increase in
[Ca2+]i induced by ET-1. Data are means ± SE of 5 experiments, each done in triplicate.
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LOE-908 and SK&F-96365 inhibited the 10 nM ET-1-induced sustained
increase in [Ca2+]i in a
concentration-dependent manner, with IC50 values of
2.6 ± 0.2 and 2.3 ± 0.3 µM, respectively, and maximal
inhibition was observed at concentrations
10 µM (Fig.
6C). The extent of maximal inhibition of the sustained
increase in [Ca2+]i in ICA VSMCs treated with
10 µM LOE-908 or 10 µM SK&F-96365 before stimulation with ET-1 was
similar to that in ICA VSMCs treated with 10 µM LOE-908 or 10 µM
SK&F-96365 after stimulation with ET-1 (~60% for LOE-908 and ~80%
for SK&F-96365; Fig. 6D). Moreover, the sustained increase
in [Ca2+]i was abolished by combined
treatment with 10 µM SK&F-96365 and 10 µM LOE-908 (Fig.
6D).
Effects of SK&F-96365 or LOE-908 on 10 nM ET-1-induced mitogenesis.
After stimulation with 10 nM ET-1, both the number of viable cells as
estimated by the MTT assay and mitogenic activity as estimated by
[3H]thymidine incorporation increased with time up to
48 h (data not shown). Therefore, in subsequent experiments the
stimulation time was set at 48 h.
ET-1 stimulated mitogenesis in ICA VSMCs in a concentration-dependent
manner, with EC50 values of 0.7 ± 0.2 and 0.8 ± 0.1 nM for the MTT assay and [3H]thymidine incorporation,
respectively. Maximal effects, ~5-fold increase in the MTT assay and
~4.2-fold increase for [3H]thymidine incorporation,
were obtained at concentrations
10 nM (Fig.
7).

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Fig. 7.
Effects of various concentrations of ET-1 on the number of viable
cells (A) and amount of DNA synthesis (B) in ICA
VSMCs. After cells had been deprived of serum for 24 h, they were
stimulated with increasing concentrations of ET-1 for a further 48 h. The number of viable cells
{3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)
assay; A} and [3H]thymidine incorporation
(B) were determined as described in MATERIALS AND
METHODS. Data are means ± SE of 3 determinations, each done
in triplicate.
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We next examined the effects of Ca2+ influx through VOCCs
on 10 nM ET-1-induced mitogenesis with nifedipine. Nifedipine up to 1 µM inhibited only ~10% of ET-1-induced mitogenesis (data not shown). Using SK&F-96365 and LOE-908, we then attempted to determine the effects of Ca2+ influx through VICCs on ET-1-induced
mitogenesis. In the following experiments, nifedipine was added to the
incubation media at a final concentration of 1 µM. SK&F-96365
inhibited a 10 nM ET-1-induced mitogenesis in a concentration-dependent
manner with IC50 values of ~2.9 ± 0.2 µM and
~3.0 ± 0.2 µM in the MTT and [3H]thymidine
incorporation assays, respectively. Maximal inhibition was
observed at concentrations
10 µM (Fig.
8). The extent of maximal inhibition was
~80% (Fig. 9). Similarly, the
IC50 values of LOE-908 for inhibition of 10 nM ET-1-induced
mitogenesis were 3.2 ± 0.3 and 3.0 ± 0.2 µM for the MTT
assay and [3H]thymidine incorporation, respectively, with
maximal inhibition being observed at concentrations
10 µM (Fig. 8).
The extent of maximal inhibition was ~60% (Fig. 9). In contrast,
neither SK&F-96365 nor LOE-908 had any effect at concentrations up to
30 µM on the number of cells in the absence of ET-1 (Fig. 8).
Notably, 10 nM ET-1-induced mitogenesis was abolished by combined
treatment with maximally effective concentrations (10 µM) of LOE-908
and SK&F-96365 (Fig. 9).

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Fig. 8.
Inhibitory effects of SK&F-96365 or LOE-908 on ET-1-induced
increases in the number of viable cells (A) and DNA
synthesis (B). Starved cells were incubated for 15 min with
increasing concentrations of SK&F-96365 (triangles) or LOE-908
(circles) and were then stimulated with (open symbols) or without
(closed symbols) 10 nM ET-1. The number of viable cells (MTT assay) and
[3H]thymidine incorporation were determined as described
in MATERIALS AND METHODS. Data are means ± SE of 3 determinations, each done in triplicate.
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Fig. 9.
Inhibitory effects of the maximally effective
concentrations of SK&F-96365 (10 µM) and/or LOE-908 (10 µM) on the
number of viable cells (A) and DNA synthesis (B)
induced by 10 nM ET-1. The MTT and [3H]thymidine
incorporation assays were performed as described in MATERIALS AND
METHODS. The number of viable cells and
[3H]thymidine incorporation in the presence of SK&F-96365
and/or LOE-908 are represented as percentages of the values in the
absence of drug. Data are means ± SE of 3 experiments.
#Significantly different from control values in each experiment
(P < 0.01). NIF, nifedipine.
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DISCUSSION |
Characterization of Ca2+ channels
activated by ET-1 in ICA VSMCs.
On the basis of the sensitivity of the ET-1-induced sustained increase
in [Ca2+]i to nifedipine, involvement of the
VOCC in this response is estimated to be minor, within 10% (Fig. 1).
This is in agreement with previous reports that Ca2+ influx
through VOCCs has a minor role in ET-1-induced sustained increase in
[Ca2+]i (16, 21). Moreover, a
recent report demonstrated that the ETAR is negatively
coupled to the L-type VOCC and is positively coupled to
receptor-operated Ca2+-permeable channels in rabbit
cerebral cortex arterioles (7). Therefore,
Ca2+ channels other than the VOCC may play important roles
in ET-1-induced sustained increase in [Ca2+]i
in ICA VSMCs.
The whole cell currents induced by ET-1 are considered to be conducted
through NSCCs for the following reasons. 1) Current-voltage relationships were linear, and the reversal potentials were close to 0 mV (Fig. 2B), indicating that the currents are conducted through either NSCCs or Cl
channels. 2)
Reversal potentials of the ET-1-induced currents were unaffected by
changes in Cl
concentration in the bath solution (see
RESULTS), indicating that the current is carried through
NSCCs. Furthermore, these NSCCs are permeable to Ca2+,
because currents can be induced in a bath solution containing only
Ca2+ as a movable cation (Fig. 2, C and
D). These data are in agreement with the previous report
that ET-1 can activate nifedipine-insensitive noncation channels in
VSMCs (2). The patch-clamp study showed that two
types of Ca2+-permeable NSCCs are activated by ET-1 in ICA
VSMCs: one type was sensitive to LOE-908 and resistant to SK&F-96365,
whereas the other was sensitive to both drugs (Fig. 3). From a
pharmacological viewpoint, these channels correspond to NSCC-1 and
NSCC-2, respectively, as defined in A7r5 cells (11).
ICA VSMCs are considered to possess SOCCs, because an increase in
[Ca2+]i as an index of Ca2+
influx through SOCCs could be induced by thapsigargin (Fig. 4). As
reported previously (11), Ca2+ current through
SOCCs cannot be monitored under our conditions of whole cell
recordings. This is probably because of latent activation of SOCCs
under basal conditions, which results from the presence of the
Ca2+ chelator EGTA in the pipette solution (to prevent
Ca2+-activated currents). EGTA is reported to deplete the
intracellular Ca2+ store and to activate SOCCs
(9). The SOCC thus defined in ICA VSMCs is sensitive to
SK&F-96365 and resistant to LOE-908, indicating that the
pharmacological properties of SOCCs in ICA VSMCs are the same as those
in A7r5 cells (11).
Given that the pharmacological properties of these Ca2+
channels have been clarified, we analyzed the involvement of
Ca2+ channels in ET-1-induced increase in
[Ca2+]i in the presence of nifedipine. The
ET-1-induced sustained increase in [Ca2+]i
consisted of three components in terms of sensitivity to SK&F-96365 and
LOE-908 in ICA VSMCs (Figs. 5 and 6; Table
1). NSCC-1 contributed ~20% to the
increase in [Ca2+]i and was resistant to
SK&F-96365 and sensitive to LOE-908. SOCC contributed ~40% to the
increase in [Ca2+]i and was resistant to
LOE-908 and sensitive to SK&F-96365 (Table 1). Because the LOE
908-sensitive part (contributing ~60%) of the increase in
[Ca2+]i consisted of Ca2+ influx
through NSCC-1 and NSCC-2, the contribution of Ca2+ influx
through NSCC-2 was calculated to be 40%. In conclusion, Ca2+ influx through NSCC-1, NSCC-2, and SOCC contributes
~20%, 40%, and 40%, respectively, to the increase in
[Ca2+]i induced by ET-1 in ICA VSMCs (Table
1). Moreover, the VICCs activated by ET-1 in ICA VSMCs may be
pharmacologically similar to those in A7r5 cells (11). In
ICA VSMCs, NSCC-1 is activated by 0.1 nM ET-1, whereas NSCC-2 and SOCC
are activated by ET-1 at concentrations
1 nM (Figs. 5 and 6).
Therefore, the sensitivity of NSCC-1 to ET-1 is higher than that of
NSCC-2 or SOCC to ET-1. These results indicate that the sensitivity
pattern of VICCs to ET-1 in ICA VSMCs may be similar to that in A7r5
cells (11).
View this table:
[in this window]
[in a new window]
|
Table 1.
Contribution of NSCC-1, NSCC-2, and SOCC to ET-1-induced ICA VSMC
proliferation and a sustained increase in
[Ca2+]i
|
|
Characterization of Ca2+ channels
involved in ET-1-induced mitogenesis in ICA VSMCs.
ET-1 induces mitogenic response in ICA VSMCs, judging from results of
MTT assay and [3H]thymidine incorporation (Fig. 7). As
demonstrated by sensitivity to BQ-123 and BQ-788, ET-1 mediates
increases in mitogenesis through ETARs (data not shown).
In light of the nifedipine sensitivity of ET-1-induced mitogenesis,
Ca2+ channels other than VOCCs may play important roles in
ET-1-induced mitogenesis in ICA VSMCs. Three types of VICCs seem to be
involved in ET-1-induced mitogenesis in terms of its sensitivity to
LOE-908 and SK&F-96365 (Figs. 8 and 9). One type of Ca2+
channel is sensitive to LOE-908 and resistant to SK&F-96365, another
type is sensitive to both LOE-908 and SK&F-96365, and the third type is
resistant to LOE-908 and sensitive to SK&F-96365. On the basis of
pharmacological criteria, these channels are considered to be NSCC-1,
NSCC-2, and SOCC, respectively. Moreover, the percent contribution of
NSCC-1, NSCC-2, and SOCC to ET-1-induced mitogenesis is calculated to
be ~20%, 40%, and 40%, respectively (Table 1). The inhibitory
action of SK&F-96365 or LOE-908 on ET-1-induced mitogenesis may be
mediated by blockade of Ca2+ entry through VICCs for the
following reasons. 1) From the results of patch clamp and
[Ca2+]i monitoring, ET-1 was found to
activate three types of VICCs in ICA VSMCs, namely, NSCC-1, NSCC-2, and
SOCC (Table 1). 2) The IC50 values of SK&F-96365
and LOE-908 for ET-1-induced mitogenesis and the extent of inhibition
of the response by these blockers (Fig. 8; Table 1) correlated well
with those for the ET-1-induced [Ca2+]i
response (Fig. 3). 3) Neither SK&F-96365 nor LOE-908 is
considered to exert cytotoxic effects on quiescent cells, judging from
data from MTT and [3H]thymidine incorporation assays
(Fig. 8).
In conclusion, ET-1 activates VICCs such as NSCC-1, NSCC-2, and SOCC in
ICA VSMCs. Notably, Ca2+ influx through these channels
plays an essential role in ET-1-induced mitogenesis in ICA VSMCs. We
recently showed (17) that the ET-1-induced mitogenic
response in Chinese hamster ovary cells expressing recombinant ETARs involves a mitogen-activated protein kinase cascade,
the activation of which is dependent on both protein kinase C and phosphatidylinositol 3-kinase. However, it is not known whether the
same signaling pathways operate in ICA VSMCs. It remains to be
determined which signaling pathways are involved in the ET-1-induced mitogenic response and which step(s) of the intracellular signaling pathways requires Ca2+.
 |
ACKNOWLEDGEMENTS |
We thank Boehringer Ingelheim (Ingelheim, Germany) for the kind
gift of LOE-908.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: Y. Kawanabe, Dept. of Neurosurgery, Kyoto Univ. Faculty of Medicine, 54 Shougoin-Kawaharachou, Sakyo-ku, Kyoto 606-8507, Japan (E-mail: kawanabe{at}kuhp.kyoto-u.ac.jp).
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. Section 1734 solely to indicate this fact.
10.1152/ajpcell.00227.2001
Received 3 August 2001; accepted in final form 1 October 2001.
 |
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