Ca2+ channels activated by endothelin-1 in CHO
cells expressing endothelin-A or endothelin-B receptors
Yoshifumi
Kawanabe1,2,
Yasuo
Okamoto1,
Taijiro
Enoki3,
Nobuo
Hashimoto2, and
Tomoh
Masaki1
Departments of 1 Pharmacology, 2 Neurosurgery, and
3 Anesthesiology, Kyoto University Faculty of Medicine,
Kyoto 606-8507, Japan
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ABSTRACT |
We compared
the Ca2+ channels activated by endothelin-1 (ET-1) in
Chinese hamster ovary (CHO) cells stably expressing endothelin type A
(ETA) or endothelin type B (ETB) receptors
using the Ca2+ channel blockers LOE-908 and SK&F-96365. In
both CHO-ETA and CHO-ETB, ET-1 at 0.1 nM
activated the Ca2+-permeable nonselective cation channel-1
(NSCC-1), which was sensitive to LOE-908 and resistant to SK&F-96365.
ET-1 at 1 nM activated NSCC-2 in addition to NSCC-1; NSCC-2 was
sensitive to both LOE-908 and SK&F-96365. ET-1 at 10 nM activated the
same channels as 1 nM ET-1 in both cell types, but in
CHO-ETA, it additionally activated the store-operated
Ca2+ channel (SOCC), which was resistant to LOE-908 and
sensitive to SK&F-96365. Up to 1 nM ET-1, the level of the formation of inositol phosphates (IPs) was low and similar in both cell types, but,
at 10 nM ET-1, it was far greater in CHO-ETA than in
CHO-ETB. These results show that, in CHO-ETA
and CHO-ETB, ET-1 up to 10 nM activated the same
Ca2+ entry channels: 0.1 nM ET-1 activated NSCC-1, and
ET-1
1 nM activated NSCC-1 and NSCC-2. Notably, in
CHO-ETA, 10 nM ET-1 activated SOCCs because of the higher
formation of IPs.
endothelin-1; endothelin receptor; calcium channel; calcium ion; Chinese hamster ovary
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INTRODUCTION |
ENDOTHELIN-1 (ET-1)
is a 21-amino-acid peptide and is one of the most potent endogenous
vasoconstricting agents discovered thus far (26).
Subsequent studies have described its multiple, wide-ranging biological
activities. The mitogenic activities of ET-1 indicate that it may play
a role in the pathogenesis of certain clinical conditions, such as
hyperlipoproteinemia and atherosclerosis (7, 12). ET-1 has
also been identified as an autocrine/paracrine growth factor in some
human cancer cell lines (21).
Recent reports have demonstrated that ET-1 induces contraction of rat
aorta and cell proliferation by inducing extracellular Ca2+
influx (21, 27). The increase in intracellular free
Ca2+ concentration ([Ca2+]i)
induced by ET-1 usually consists of the following two phases: an
initial transient increase and a subsequent sustained increase (4, 10, 11, 15). It is generally accepted that the initial transient increase results from mobilization of Ca2+ from
the intracellular store, whereas the sustained increase results from
entry of extracellular Ca2+ (6). We recently
showed that the ET-1-induced sustained increase in
[Ca2+]i in A7r5 cells (a cell line derived
from rat thoracic aortic smooth muscle cells) mainly results from
Ca2+ entry through several Ca2+ channels other
than the voltage-operated Ca2+ channel (VOCC). These other
Ca2+ channels include two types of
Ca2+-permeable nonselective cation channels (designated
NSCC-1 and NSCC-2) and the store-operated Ca2+ channel
(SOCC; see Refs. 10 and 11). The NSCCs possess a permeability to Ca2+ that is approximately twofold higher
than that to monovalent cations (11). The SOCC is highly
specific for Ca2+ and is activated by depletion of the
intracellular Ca2+ store (5). Of importance,
it has been demonstrated that these channels can be distinguished using
various Ca2+ channel blockers, such as SK&F-96365 and
LOE-908 (3, 14). 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). In other words, LOE-908 is a blocker of
NSCCs, whereas SK&F-96365 is a blocker of SOCC and NSCC-2. Moreover,
the increase in [Ca2+]i via these channels
plays a critical role in ET-1-induced vasoconstriction (27). Therefore, it is important to clarify the mechanisms
through which ET-1 activates voltage-independent Ca2+
channels (VICCs). However, vascular smooth muscle cells (VSMCs) express
both endothelin type A receptor (ETA) and endothelin type B
receptor (ETB; see Refs. 1 and 2). Therefore,
it is not known whether stimulation of ETA or
ETB on VSMCs results in the activation of different types
of Ca2+ channels. If different types of Ca2+
channels are activated, the mechanisms responsible for that difference are not known.
The transfection and functional expression of the cDNA clone for
ETA or ETB into the same cell type provide a
model system to study the precise signal transduction of a single
receptor subtype without any ambiguity resulting from the presence of
multiple receptor subtypes. Moreover, several mutant ETA
and ETB clones (8, 18) may be useful for
studying the mechanisms of the activation of Ca2+ channels
by ET-1 in this system. We used Chinese hamster ovary (CHO) cells
stably expressing ETA or ETB in the present
study. The purpose of the present study was to identify and compare
which Ca2+ channels are activated by ET-1 upon binding to
ETA or ETB in CHO-ETA or
CHO-ETB, respectively, using whole cell recordings of the
patch-clamp technique and monitoring of
[Ca2+]i, combined with the use of specific
Ca2+ channel blockers such as LOE-908 and SK&F-96365. Next,
we tried to find the cause of the differential activation of
Ca2+ channels by ET-1 between CHO-ETA and
CHO-ETB. The results of the present study clarified the
mechanisms of the activation of VICCs by ET-1.
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MATERIALS AND METHODS |
Cell culture.
CHO cells were maintained in Ham's F-12 medium supplemented with 10%
FCS under a humidified 5% CO2-95% air atmosphere.
Stable expression of ETA or ETB in CHO
cells.
To obtain a cell line stably expressing ETA or
ETB (CHO-ETA and CHO-ETB,
respectively), we used a mammalian expression vector, pME18Sf, that
carried a cDNA construct encoding human recombinant ETA
receptor or ETB receptor. Construction and subcloning of
receptor cDNAs were performed as described by Sakamoto et al.
(19). Briefly, each expression vector was cotransfected
with pSVbsrr plasmid into CHO cells by lipofection using
Lipofectamine (Life Technologies, Tokyo, Japan) according to the
manufacturer's instructions. Cell populations expressing the
bsrr gene product were selected in Ham's F-12
medium supplemented with 10% FCS and 0.5 µg/ml blasticidin. From
these selected populations, clonal cell lines were isolated by colony
lifting and were maintained in the same selection medium.
Formation of inositol phosphates.
The level of the formation of inositol phosphates (IPs) was determined
as described previously (22). Briefly, CHO-ETA
or CHO-ETB in 24-well plates were incubated with
myo-[3H]inositol (final concentration, 5 µCi/ml) in 0.3 ml of Ham's F-12 medium supplemented with 10% FCS
for 18 h. After being washed, the cells were incubated with or
without various concentrations of ET-1 for 30 min, and the reaction was
terminated by adding ice-cold perchloric acid. After neutralization
with KOH and Tris, the samples were applied to small columns of AG1X8
(100-200 mesh, Cl
form; Bio-Rad, Hercules, CA) to
separate the total IPs from the myo-[3H]inositol. The 3H-labeled
IPs were eluted with 1 N HCl, and the radioactivity was counted with a
liquid scintillation counter.
Monitoring of [Ca2+]i
in CHO-ETA and CHO-ETB.
The [Ca2+]i was monitored using the
fluorescent probe fluo 3, as described previously (4).
Briefly, CHO-ETA or CHO-ETB were loaded with
fluo 3 by incubating the cells with 10 µM fluo 3-AM at 37°C under
reduced light for 30 min. After being washed, the cells were suspended
at a density of ~2 × 107 cells/ml, and 0.5-ml
aliquots were used for measurement of fluorescence by a CAF 110 spectrophotometer (JASCO, Tokyo, Japan) with an excitation wavelength
of 490 nm and an emission wavelength of 540 nm. At the end of the
experiment, Triton X-100 and subsequently EGTA were added at a final
concentration of 0.1% or 5 mM, respectively, to obtain the maximum
fluorescence (Fmax) and the minimum fluorescence (Fmin). The [Ca2+]i was
determined by the equilibrium equation
[Ca2+]i = Kd(F
Fmin)/(Fmax
F), where F is the
experimental value of fluorescence and the dissociation constant
(Kd) was defined as 0.4 µM (16).
Electrophysiology.
CHO-ETA or CHO-ETB were perfused with
Krebs-HEPES solution and visualized with Nomarski optics (Zeiss, Tokyo,
Japan). Whole cell recordings were made with thin-wall borosilicate
glass patch pipettes (resistance, 3-5 M
) as previously
described (4). The Krebs-HEPES solution contained (in mM)
140 NaCl, 3 KCl, 2 CaCl2, 1 MgCl2, 11 glucose,
and 10 HEPES (adjusted to pH 7.3 with NaOH). The pipettes were filled
with cesium aspartate solution containing (in mM) 120 cesium aspartate,
20 CsCl, 2 MgCl2, 10 HEPES, and 10 EGTA (adjusted to pH 7.3 with CsOH). EGTA was added to the pipette solution at a final
concentration of 10 mM; this concentration of EGTA had enough buffering
capacity for Ca2+ to prevent a transient increase in
[Ca2+]i (17), and the
concentration of Ca2+ in the solution was maintained at 100 nM by adding the appropriate amount of CaCl2 as described
previously (24). Tight-seal whole cell currents were
recorded with a EPC7 patch-clamp amplifier (List, Darmstadt, Germany).
The perfusion rate was maintained at 2.2 ~ 2.5 ml/min, and the
bath volume was ~1.0 ml. All experiments were performed under voltage
clamp at a holding potential of
60 mV at room temperature (22 ~ 24°C). To test the contribution of the Cl
current,
the bath solution was switched from Krebs-HEPES to a solution with a
low Cl
concentration that contained (in mM) 140 sodium
gluconate, 3 KCl, 2 CaCl2, 1 MgCl2, 11 glucose,
and 10 HEPES (pH 7.3). The permeability of Ca2+ through
channels was measured in a
Ca2+-N-methyl-D-glucamine (NMDG)
solution containing (in mM) 30 CaCl2, 100 NMDG chloride, 11 glucose, and 10 HEPES (adjusted to pH 7.3 with Tris). Current-voltage
relationships were obtained by applying voltage steps of 100-ms
duration ranging from
100 to +80 mV in 20-mV increments, before and
after application of ET-1 or channel blocker(s). The ET-1-induced
current at each membrane potential was determined by subtracting the
current before application of ET-1 from the current after its
application. The drug-inhibited current was determined by subtracting
the current after application of the drug from the current before its application.
Drugs.
LOE-908 was kindly provided by Boehringer Ingelheim (Ingelheim,
Germany). Other chemicals were obtained commercially 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); and 125I-labeled ET-1 and
myo-[3H]inositol from Amersham Pharmacia
Biotech (Buckinghamshire, UK).
Statistical analysis.
All results were expressed as means ± SE.
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RESULTS |
Stable expression of ETA or ETB in CHO
cells.
By cotransfecting CHO cells with each expression plasmid and
pSVbsrr and then selecting for resistance against
blasticidin, we obtained eight individual clonal cell lines that stably
expressed ETA and 10 individual clonal cell lines that
stably expressed ETB. 125I-labeled ET-1 binding
assays using membrane preparations from each clone gave
Kd values of 30 ~ 150 pM and maximal
binding (Bmax) values of 0.5 ~ 1.7 pmol/mg protein.
Cell clones with a similar level of affinity for ET-1 and receptor
density were used in the subsequent studies (the
Kd and Bmax values of these cell
clones were 52.8 ± 2.4 pM and 1.08 ± 0.16 pmol/mg protein,
respectively, for CHO-ETA and 46.8 ± 5.3 pM and
0.97 ± 0.08 pmol/mg protein, respectively, for
CHO-ETB).
Characterization of the currents induced by ET-1 in
CHO-ETA and CHO-ETB with whole cell recordings
of the patch clamp.
To elucidate the ionic channels in CHO-ETA and
CHO-ETB that were activated by ET-1, whole cell recordings
of CHO-ETA and CHO-ETB were performed.
Stimulation with various concentrations of ET-1 (0.1, 1, or 10 nM)
induced an inward current in CHO-ETA and
CHO-ETB held at
60 mV (Fig.
1, A-D). The currents induced
by 0.1, 1, or 10 nM of ET-1 showed linear current-voltage relationships
in both CHO-ETA and CHO-ETB with a reversal
potential of
4.4 ± 1.5 mV (n = 15),
1.7 ± 2.7 mV (n = 15), or
0.8 ± 2.2 mV
(n = 15), respectively, in CHO-ETA (Fig.
1E) and
5.2 ± 2.8 mV (n = 15),
1.0 ± 2.4 mV (n = 15), or
0.6 ± 3.3 mV
(n = 15), respectively, in CHO-ETB (Fig.
1F).

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Fig. 1.
Whole cell recordings of the currents in Chinese hamster
ovary (CHO) cells stably expressing endothelin type A or endothelin
type B receptor (CHO-ETA or CHO-ETB,
respectively) induced by incubation with various concentrations of
endothelin-1 (ET-1). A-D: original tracings showing
whole cell currents in CHO-ETA (A and
B) and CHO-ETB (C and D)
that were treated with ET-1. The cells were clamped at a holding
potential of 60 mV with the whole cell configuration, and ET-1 (final
concentration 0.1 or 10 nM) was added to the bath solution at the time
indicated by the arrow. E and F: current-voltage
relationships of the currents induced by 0.1, 1, or 10 nM ET-1 in
CHO-ETA (E) and CHO-ETB
(F). Cells were treated with 0.1, 1.0, or 10 nM ET-1. We
applied a 100-ms voltage step at time x before ET-1
treatment and at time y after the cells had reached a steady
state after ET-1 was added. The current was measured at time
x and at time y, and the current at x was
subtracted from the current at y. We applied voltage steps
ranging from 100 to +80 mV in 20-mV increments. G and
H: current-voltage relationships of the whole cell
Ca2+ current in CHO-ETA (G) and
CHO-ETB (H) that were induced by ET-1. Whole
cell recordings of the patch clamp and application of voltage steps
were performed using
Ca2+-N-methyl-D-glucamine (NMDG)
solution. , 0.1 nM ET-1; , 1 nM ET-1;
, 10 nM ET-1.
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The current-voltage relationships of the currents induced by any of the
three concentrations of ET-1 in either cell type were not affected by
reducing the concentration of Cl
in the bath solution
from 149 to 9 mM (data not shown). The reversal potential for 0.1, 1, or 10 nM ET-1 in a solution with a low Cl
concentration
(9 mM) was
3.6 ± 2.2 mV (n = 6),
1.2 ± 1.4 mV (n = 6), and
0.5 ± 2.4 mV
(n = 6), respectively, in CHO-ETA and
4.4 ± 1.8 mV (n = 6),
0.8 ± 2.3 mV
(n = 6), and
0.4 ± 1.4 mV (n = 6), respectively, in CHO-ETB. These values did not
significantly differ from those obtained in the cells incubated in
Krebs-HEPES solution containing the normal concentration of
Cl
.
To test whether the channels activated by ET-1 are permeable to
Ca2+, all other cations in the bath solution were replaced
with the nonpermeant cation NMDG while the concentration of
Ca2+ was increased from 1 to 30 mM. Even under such
conditions, ET-1 at 0.1, 1, or 10 nM induced an inward current in
CHO-ETA and CHO-ETB held at
60 mV (Fig. 1,
G and H). The reversal potential for 0.1, 1, or
10 nM ET-1 was
12.3 ± 1.6 mV (n = 6),
11.5 ± 1.2 mV (n = 6), and
10.8 ± 2.2 mV (n = 6), respectively, in CHO-ETA (Fig. 1G) and
12.1 ± 1.8 mV (n = 6),
11.5 ± 1.3 mV (n = 6), and
10.3 ± 1.4 mV (n = 6), respectively, in CHO-ETB (Fig.
1H).
Pharmacological properties of the whole cell currents induced by
ET-1 in CHO-ETA and CHO-ETB.
To determine the maximally effective concentration of various
Ca2+ channel blockers, we first examined the effect of
various concentrations (30 nM ~ 30 µM) of SK&F-96365 or
LOE-908 on the whole cell currents in CHO-ETA and
CHO-ETB induced by 0.1, 1, or 10 nM ET-1. SK&F-96365 and
LOE-908 each inhibited ET-1-induced whole cell currents in a
concentration-dependent manner, and the maximal effect was seen at
concentrations
10 µM (data not shown). On the basis of these data,
we decided to use 10 µM as the concentration of SK&F-96365 and
LOE-908 in the following experiments.
In CHO-ETA, the current induced by 0.1 nM ET-1 was
abolished by 10 µM LOE-908 (Fig.
2B), whereas it was not
affected by 10 µM SK&F-96365 (Fig. 2A). The current
inhibited by LOE-908 showed a linear current-voltage relationship and a
reversal potential of
3.9 ± 3.0 mV (n = 6; Fig.
2E), indicating that LOE-908 suppressed the same current
activated by ET-1. The currents induced by 1 and 10 nM ET-1 in both
cell types were also abolished by 10 µM LOE-908 (10 nM; Fig.
2D); a major portion (~65%) of the current was suppressed
by 10 µM SK&F-96365 (10 nM ET-1 in CHO-ETA, Fig. 2C). The characteristics of the currents inhibited by
SK&F-96365 or LOE-908 were similar to those of the ET-1-induced
currents in terms of the linear current-voltage relationship and the
reversal potential of
2.6 ± 2.2 mV (n = 6) or
3.6 ± 2.3 mV (n = 6), respectively (Fig.
2F), indicating that both drugs suppressed the same currents activated by ET-1. The pharmacological properties of the whole cell
currents induced by ET-1 in CHO-ETB were similar to those in CHO-ETA (Fig. 2, G and H). On the
basis of these results, the current induced by 1 or 10 nM ET-1 was
divided into two components. Namely, one component is sensitive to
LOE-908 and resistant to SK&F-96365, and the second component is
sensitive to both LOE-908 and SK&F-96365.

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Fig. 2.
Effect of SK&F-96365 and LOE-908 on the whole cell
currents induced by various concentrations of ET-1 in
CHO-ETA and CHO-ETB. A-D: original
tracings showing the whole cell current in CHO-ETA that had
been activated by 0.1 nM (A and B) or 10 nM
(C and D) ET-1 and then treated with SK&F-96365
or LOE-908. The experimental protocol was the same as that described in
Fig. 1. After the ET-1-induced current had reached a steady state,
either SK&F-96365 (A and C) or LOE-908
(B and D) was added to the bath solution at a
final concentration of 10 µM. E-H: current-voltage
relationships of the currents induced by 0.1 or 10 nM ET-1 in
CHO-ETA (E and F) and
CHO-ETB (G and H). At the times
indicated by x, y, and z, when the
cells were in a stable condition, a voltage step of 100-ms duration was
applied. Voltage steps ranging from 100 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 x from that at
y or the current at z from that at y,
and these were plotted against the membrane potential. E and
G: , current obtained by subtracting the
current at x from that at y; ,
current obtained by subtracting the current at z from that
at y. F and H: circles, subtracted
currents in the presence of LOE-908; squares, subtracted currents in
the presence of SK&F-96365; , current obtained by
subtracting the current at x from that at y;
, current obtained by subtracting the current at
z from that at y; , current
obtained by subtracting the current at x from that at
y; , current obtained by subtracting the
current at z from that at y.
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Pharmacological properties of the SOCC in CHO cells.
Treatment of cells with thapsigargin (an inhibitor of
Ca2+-pump ATPase on the membrane of the
sarcoplasmic/endoplasmic reticulum) depletes the intracellular store of
Ca2+ and thereby activates the Ca2+ channels on
the plasma membrane called SOCCs or capacitative Ca2+ entry
channels, causing a sustained increase in
[Ca2+]i (23). Therefore, a
sustained increase in [Ca2+]i is regarded as
an index of the activity of SOCC. Our recent studies showed that the
thapsigargin-induced increase in [Ca2+]i in
A7r5 cells and in VSMCs in primary culture was abolished by SK&F-96365
but was not affected by nifedipine or LOE-908 (11, 27). We
characterized the pharmacological properties of the SOCCs in CHO cells
using thapsigargin.
The sustained increase in [Ca2+]i in
wild-type CHO cells that had been induced by 0.1 µM thapsigargin was
suppressed by SK&F-96365 in a concentration-dependent manner with an
IC50 value of ~2 µM and was abolished at concentrations
10 µM (Fig. 3). However, the
thapsigargin-induced increase in [Ca2+]i was
not affected by LOE-908 (Fig. 3B) or nifedipine (data not shown) up to the concentration of 30 or 10 µM, respectively. These results demonstrate that the SOCCs in CHO cells have the same pharmacological properties as those in VSMCs in primary culture and
A7r5 cells.

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Fig. 3.
Effects of various concentrations of SK&F-96365 or
LOE-908 on the thapsigargin-induced increase in intracellular
Ca2+ concentration ([Ca2+]i) in
wild-type CHO cells as an index of the activity of store-operated
Ca2+ channels. Wild-type CHO cells were loaded with fluo 3 and subjected to monitoring of [Ca2+]i. The
cells were exposed to 0.1 µM thapsigargin during the period of time
indicated by the horizontal hatched bar. After the
[Ca2+]i reached a steady state, increasing
concentrations of either SK&F-96365 or LOE-908 were added to the bath
solution. A: original tracing showing the inhibitory effect
of increasing concentrations of SK&F-96365 on the thapsigargin-induced
increase in [Ca2+]i. B:
concentration-response curves of the effect of SK&F-96365
( ) or LOE-908 ( ) on the
thapsigargin-induced increase in [Ca2+]i. The
level of inhibition of the increase by a drug was represented as the
percentage of the level of [Ca2+]i after
treatment with thapsigargin and just before the addition of the drug
(control). Each point represents the mean ± SE of 5 experiments.
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Basic properties of the ET-1-induced increase in
[Ca2+]i in
CHO-ETA and CHO-ETB.
ET-1 at 0.1 nM induced a monophasic increase in
[Ca2+]i in CHO-ETA and
CHO-ETB, as monitored by a Ca2+ indicator, fluo
3 (Fig. 4, A and
E). In contrast, higher concentrations (1 and 10 nM) of ET-1
induced a biphasic increase in [Ca2+]i
consisting of an initial transient peak and a subsequent sustained increase (Fig. 4, B, C, F, and
G). In experiments performed on cells incubated in a bath in
which the extracellular Ca2+ had been removed, upon
treatment with 1 or 10 nM ET-1, the transient peak was not affected,
but the sustained increase induced by either concentration of ET-1 was
abolished (data not shown), indicating that only the sustained increase
in [Ca2+]i results from Ca2+
influx.

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Fig. 4.
A-C and E-G: original tracings
showing the effect of treatment with various concentrations of ET-1 on
the [Ca2+]i in CHO-ETA and
CHO-ETB. CHO-ETA (A-C) and
CHO-ETB (E-G) cells were loaded with a
Ca2+ indicator, fluo 3, and subjected to monitoring of
[Ca2+]i. ET-1 at 0.1, 1, or 10 nM was added
to the bath solution at the time indicated by the arrow. D
and H: concentration-response curves of the ET-1-induced
increase in [Ca2+]i in CHO-ETA
(D) and CHO-ETB (H). The transient
increase ( ) and the sustained increase
( ) of [Ca2+]i were plotted
separately against the concentration of ET-1. Each point represents the
mean ± SE of 5 experiments.
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The magnitude of the transient peak and that of the sustained increase
in [Ca2+]i depended on the concentration of
ET-1 in both cell types (Fig. 4). However, the threshold concentration
of ET-1 for induction of the transient peak or the sustained increase
differed; among the tested concentrations of ET-1, the lowest
concentration at which the transient increase in
[Ca2+]i was seen was 1 nM ET-1, whereas the
lowest concentration at which the sustained increase in
[Ca2+]i was seen was 0.1 nM in both cell
types (Fig. 4).
The concentration of ET-1 at which the magnitude of the increase in
[Ca2+]i reached the maximum differed
between CHO-ETA and CHO-ETB. In CHO-ETA, both the transient peak and the sustained increase
reached a plateau at 10 nM ET-1 (Fig. 4D), whereas in
CHO-ETB they reached a plateau at 1 nM ET-1 (Fig.
4H). Furthermore, the magnitude of the transient peak and
that of the sustained increase in CHO-ETA were greater than
those in CHO-ETB (Fig. 4).
Pharmacological analysis of the increase in
[Ca2+]i induced by various
concentrations of ET-1.
In CHO-ETA, the sustained increase in
[Ca2+]i upon treatment with ET-1 was
suppressed by LOE-908 in a concentration-dependent manner with an
IC50 value of ~3 µM, and maximal inhibition was observed at concentrations
10 µM (Figs.
5, B and D, and
Fig. 7B). However, the
extent of the maximal inhibition of LOE-908 differed depending on the
concentration of ET-1; the inhibition amounted to 100% at 0.1 and 1 nM
ET-1, whereas ~40% of the ET-1-induced increase in
[Ca2+]i was left unsuppressed at 10 nM ET-1
(Fig. 7B). In CHO-ETB, the sustained increase in
[Ca2+]i upon treatment with ET-1 was also
suppressed by LOE-908 in a concentration-dependent manner with an
IC50 value of ~3 µM, and maximal inhibition was
observed at concentrations
10 µM (Figs. 6, B and D, and
Fig. 7D). In this case, the inhibition by LOE-908 was
virtually complete regardless of the concentration of ET-1 (Fig.
7D).

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Fig. 5.
Original tracings showing the effects of various concentrations of
LOE-908 or SK&F-96365 on the [Ca2+]i in
CHO-ETA that had been treated with 0.1 or 10 nM ET-1. Cells
loaded with fluo 3 were stimulated with either 0.1 nM (A and
B) or 10 nM (C and D) ET-1. After the
[Ca2+]i reached a steady state, increasing
concentrations of either SK&F-96365 (A and C) or
LOE-908 (B and D) were added at the times
indicated by the arrow.
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Fig. 6.
Original tracings showing the effect of various concentrations of
SK&F-96365 (A and C) or LOE-908 (B and
D) on the [Ca2+]i in
CHO-ETB that had been treated with 0.1 nM (A and
B) or 10 nM (C and D) ET-1. The
experiments were performed as described in the legend for Fig. 5, using
CHO-ETB instead of CHO-ETA.
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Fig. 7.
Concentration-response curves of the inhibition of the
ET-1-induced increase in [Ca2+]i by
SK&F-96365 or LOE-908 in CHO-ETA and CHO-ETB.
CHO-ETA (A and B) and
CHO-ETB (C and D) cells were loaded
with a Ca2+ indicator, fluo 3, and subjected to monitoring
of [Ca2+]i. The cells were stimulated with
0.1 nM ( ), 1 nM ( ), or 10 nM
( ) ET-1. After the [Ca2+]i
reached a steady state, increasing concentrations of either SK&F-96365
(A and C) or LOE-908 (B and
D) were added. The [Ca2+]i after
stimulation with 0.1, 1, or 10 nM ET-1 was set at 100%, and the
[Ca2+]i before stimulation with ET-1 was set
at 0%. The [Ca2+]i after the addition of
SK&F-96365 or LOE-908 was represented on this scale. Each point
represents the mean ± SE of 5 experiments.
|
|
Different responses were obtained in the cells treated with SK&F-96365.
That is, in CHO-ETA, the sustained increase in
[Ca2+]i induced by 0.1 nM ET-1 was virtually
resistant to SK&F-96365 (Figs. 5A and 7A).
However, the increase induced by 1 or 10 nM ET-1 was suppressed by
SK&F-96365 in a concentration-dependent manner, and maximal inhibition
was observed at concentrations
10 µM (Figs. 5C and
7A); the extent of the inhibition by SK&F-96365 was larger
at higher concentrations of ET-1 (the inhibition against 1 and 10 nM
ET-1 amounted to 65 and 80%, respectively; Fig. 7A). In
CHO-ETB, similar results were obtained except that the
extent of the inhibition against 1 and 10 nM ET-1 did not differ (Fig. 6, A, and C, and Fig. 7C).
Summary of the inhibitory effects of the maximally effective
concentration of LOE-908, SK&F-96365, and their combination on the
ET-1-induced increase in
[Ca2+]i.
Table 1 summarizes the inhibitory effect
of the maximally effective concentration (10 µM) of LOE-908,
SK&F-96365, and their combination on the sustained increase in
[Ca2+]i induced by ET-1.
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|
Table 1.
Inhibitory effects of SK&F-96365, LOE-908, or their combination on the
sustained increase in
[Ca2+]i induced by various
concentrations of ET-1 in CHO cells expressing ETA or
ETB
|
|
In CHO-ETA and CHO-ETB, the sustained increase
in [Ca2+]i induced by 0.1 nM ET-1 was not
affected by SK&F-96365 but was abolished by LOE-908. In contrast, the
sustained increase in [Ca2+]i induced by 1 nM
ET-1 was partially suppressed by SK&F-96365, and it was abolished by
LOE-908. The SK&F-96365-resistant part was abrogated by the combined
treatment with LOE-908.
The increase in [Ca2+]i induced by 10 nM ET-1
in CHO-ETA and CHO-ETB showed different
responses to these blockers. That is, in CHO-ETB, the
responses to these blockers were the same as those that had been
induced by 1 nM ET-1. However, in CHO-ETA the increase in
[Ca2+]i was partially resistant to both
SK&F-96365 and LOE-908, and the SK&F-96365-resistant part or
LOE-908-resistant part was abrogated by the combined treatment with
LOE-908 or SK&F-96365, respectively.
Formation of IPs in CHO-ETA and CHO-ETB
after stimulation with ET-1.
To gain insight into the mechanisms underlying the differential
activation of Ca2+ channels by 10 nM ET-1 in
CHO-ETA and CHO-ETB, we measured the level of
the formation of IPs in CHO-ETA and CHO-ETB
that had been treated with various concentrations of ET-1 (Fig.
8).

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|
Fig. 8.
Formation of total inositol phosphates (IPs) in
CHO-ETA and CHO-ETB after stimulation with
various concentrations of ET-1. Cells that had been incubated with
myo-[3H]inositol for 18 h were stimulated
by various concentrations of ET-1 for 30 min. The level of total IPs in
the cell extract was determined as described in MATERIALS AND
METHODS. , CHO-ETA; ,
CHO-ETB; , wild-type CHO cells. Each point
represents the mean ± SE of 5 experiments.
|
|
In both cell types, stimulation with ET-1 increased the formation of
IPs in a concentration-dependent manner with an EC50 value
of ~1 nM, and the maximal response was seen at a concentration of
ET-1 of 10 nM. The level of formation of IPs in CHO-ETA was comparable to that in CHO-ETB up to 1 nM ET-1, but at
concentrations of ET-1
10 nM, it was approximately threefold higher
than that in CHO-ETB (Fig. 8).
Effect of thapsigargin on the
[Ca2+]i after stimulation
with ET-1 in CHO-ETA and CHO-ETB.
In CHO-ETA, thapsigargin induced a further increase in
[Ca2+]i when it was added during the
sustained increase in [Ca2+]i that had been
induced by 1 nM ET-1 (Fig. 9A)
or 0.1 nM ET-1 (data not shown), but it did not induce a further
increase after stimulation with 10 nM ET-1 (Fig. 9B). In
contrast, thapsigargin induced a further increase in
[Ca2+]i in CHO-ETB regardless of
whether the concentration of ET-1 was 1 or 10 nM (Fig. 9, C
and D).

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Fig. 9.
Effect of thapsigargin on the
[Ca2+]i in CHO-ETA and
CHO-ETB that had been treated with 1 or 10 nM ET-1.
CHO-ETA (A and B) and
CHO-ETB (C and D) loaded with fluo 3 were stimulated with either 1 nM (A and C) or 10 nM (B and D) ET-1. After the
[Ca2+]i reached a steady state, 0.1 µM
thapsigargin was added at the time indicated by the arrow.
|
|
 |
DISCUSSION |
Characterization of the Ca2+ channels
activated by ET-1 in CHO-ETA and CHO-ETB.
In CHO-ETA and CHO-ETB, the whole cell currents
induced by ET-1 at 0.1, 1, and 10 nM are considered to be conducted
through NSCCs for the following reasons: 1) the
current-voltage relationships are linear (Fig. 1); 2) the
reversal potentials are close to 0 mV (Fig. 1); and 3) the
reversal potentials were not affected by reducing the concentration of
Cl
in the bath solution (see RESULTS).
Furthermore, the channels are considered to be permeable to
Ca2+ because the current could be induced by ET-1 even in a
bath solution containing only Ca2+ as the movable cation;
the permeability ratio of Ca2+ to Cs+ is ~1.6
(Fig. 1).
The current activated by 0.1 nM ET-1 in CHO-ETA and
CHO-ETB consists of only one component in terms of its
pharmacology. Judging from the findings that it was sensitive to
LOE-908 and insensitive to SK&F-96365 (Fig. 2), the current is
considered to be conducted through NSCC-1. In contrast, the current
activated by 1 or 10 nM ET-1 can be divided into two components
according to their sensitivity to the blockers. One component is
sensitive to LOE-908 and resistant to SK&F-96365, whereas the second
component is sensitive to both LOE-908 and SK&F-96365 (Fig. 2). The
pharmacological criteria indicated that the former component is carried
through NSCC-1, whereas the latter is carried through NSCC-2.
As reported previously (11), the Ca2+ current
through SOCCs cannot be monitored under our conditions of whole cell
recordings. This is probably because of latent activation of SOCCs
under the basal condition, which results from the presence of a
Ca2+ chelator, EGTA, in the pipette solution (to prevent
Ca2+-activated currents). EGTA has been reported to deplete
the intracellular Ca2+ store and activate SOCCs
(9). In the present study, the activity of SOCCs was
monitored by measuring the increase in
[Ca2+]i after the addition of thapsigargin
(Fig. 3). As reported for A7r5 cells and VSMCs in primary culture
(11, 27), the SOCCs in CHO cells are sensitive to
SK&F-96365 and resistant to LOE-908 (Fig. 3).
Taken together, these data show that the NSCC-1, NSCC-2, and SOCC in
CHO cells possess the same pharmacology (i.e., sensitivity to LOE-908
and SK&F-96365) as the respective channel in A7r5 cells and VSMCs in
primary culture (11, 27).
Ca2+ channels involved in the
increase in [Ca2+]i induced
by various concentrations of ET-1 in CHO-ETA and
CHO-ETB.
In CHO cells, VOCCs do not seem to be involved in the ET-1-induced
increase in [Ca2+]i for the following
reasons: 1) CHO cells are nonexcitable cells that usually
lack VOCCs; 2) depolarization by high K+
stimulation did not elevate the [Ca2+]i (data
not shown); and 3) the ET-1-induced increase in
[Ca2+]i was resistant to specific blockers of
L-type VOCC such as nifedipine (data not shown). Therefore, VICCs play
a critical role in the ET-1-induced increase in
[Ca2+]i.
In CHO-ETA and CHO-ETB, the increase in
[Ca2+]i induced by the lowest effective
concentration (0.1 nM) of ET-1 is considered to result from
Ca2+ entry through only one type of
Ca2+-permeable NSCC (Figs. 5-7). On the basis of its
pharmacology (sensitive to LOE-908 and resistant to SK&F-96365; Fig. 7
and Table 1), this channel is regarded to be NSCC-1.
At 1 nM ET-1, the increase in [Ca2+]i in both
cell types seemed to involve Ca2+ entry through two types
of Ca2+-permeable NSCCs in terms of its sensitivity to
channel blockers. That is, the major portion of the increase in
[Ca2+]i (65%) was sensitive to both LOE-908
and SK&F-96365, whereas the remaining portion (35%) was sensitive to
LOE-908 and resistant to SK&F-96365 (Fig. 7 and Table 1). From the
pharmacological point of view, the former is mediated by
Ca2+ entry through NSCC-2, whereas the latter is mediated
by Ca2+ entry through NSCC-1.
Notably, different Ca2+ channels seemed to be activated by
the saturating concentration (10 nM) of ET-1 in CHO-ETA and
CHO-ETB. That is, the increase in
[Ca2+]i in CHO-ETB induced by 10 nM ET-1 showed the same pharmacology as that induced by 1 nM ET-1 (Fig.
7, C and D, and Table 1), indicating that the
increase involved Ca2+ entry through NSCC-1 and NSCC-2,
which contributed ~35 and 65%, respectively, of the total
Ca2+ entry. However, in CHO-ETA, the increase
in [Ca2+]i consisted of three components in
terms of the sensitivity to SK&F-96365 and LOE-908 (Fig. 7,
A and C, and Table 1). One component, which
contributed ~20% of the total increase in
[Ca2+]i, was resistant to SK&F-96365 and
sensitive to LOE-908, and the second component, which contributed
~40% of the total increase in [Ca2+]i, was
resistant to LOE-908 and sensitive to SK&F-96365 (Table 1). According
to the pharmacological criteria, the first component is NSCC-1, and the
second component is SOCC. Because the LOE-908-sensitive portion
(contributing ~60%) of the total 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%. Thus, in CHO-ETA treated with 10 nM ET-1, Ca2+ influx through NSCC-1,
NSCC-2, and SOCC contributes ~20, 40, and 40%, respectively, of the
total increase in [Ca2+]i. Moreover, on the
basis of sensitivity to SK&F-96365 and LOE-908, the VICCs activated by
ET-1 in CHO-ETA may be the same channels activated by ET-1
in A7r5 cells.
Mechanisms of the differential activation of SOCCs.
The differential activation of SOCCs by 10 nM ET-1 seems to be the
result of the larger amount of IP3 produced and hence
depletion of the intracellular Ca2+ store in
CHO-ETA for the following reasons: 1) when the
level of the formation of IPs, which was used as an index of
IP3 formation, was low and comparable between
CHO-ETA and CHO-ETB (
1 nM ET-1), NSCCs but
not SOCCs were activated in both cell types (Fig. 8 and Table 1);
2) when the level of formation of IPs was higher in
CHO-ETA than in CHO-ETB (at 10 nM ET-1), SOCCs
were activated only in CHO-ETA (Fig. 8 and Table 1);
3) thapsigargin, which specifically activates SOCCs by
depleting the intracellular Ca2+ store, further enhanced
the increase in [Ca2+]i induced by ET-1 only
when the level of the formation of IPs was low (0.1 and 1 nM ET-1 for
CHO-ETA; 0.1, 1, and 10 nM ET-1 for CHO-ETB;
Fig. 9, A, C, and D). In contrast,
thapsigargin did not further increase the
[Ca2+]i in CHO-ETA that had been
induced by 10 nM ET-1 (Fig. 9B).
To exclude the possibility that heterogeneity in the coupling of
ETA to SOCC within CHO cells exists, we used different
CHO-ETA and CHO-ETB clones to look for the
absence or gain of SOCC coupling to endothelin receptors. Assays on
several independent clones with various receptor densities suggested
that, within the range of receptor densities that we could obtain,
these differences were independent of the differences in receptor
density. All clones expressing ETA or ETB gave
essentially the same results (data not shown). Thus the difference in
SOCC activation between ETA and ETB is the
result of intrinsic differences between the two receptor subtypes.
Mechanisms of the activation of VICCs.
The mechanisms underlying the activation of VICCs are presently not
known. However, different mechanisms seem to be involved in the
activation of these channels, because the concentration of ET-1
required for their activation differed. Several lines of evidence
suggest that the
-subunit or 
-subunit of G proteins is
involved in the Ca2+ influx activated by stimulation of G
protein-coupled receptors (13, 20, 25). The mechanisms
underlying the activation of VICCs are now under investigation in our
laboratory using CHO cells stably expressing mutant ETA and
ETB with antisense oligonucleotides against G proteins and
dominant-negative mutants of G proteins.
 |
ACKNOWLEDGEMENTS |
We thank Boehringer Ingelheim (Ingelheim, Germany) for the kind
donation 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.
Received 26 April 2001; accepted in final form 10 July 2001.
 |
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