Departments of 1 Neurosurgery and 2 Pharmacology, Kyoto University Graduate School of Medicine, Sakyo-ku, Kyoto 606-8507, Japan
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ABSTRACT |
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We recently demonstrated that endothelin-1 (ET-1) activates two types of Ca2+-permeable nonselective cation channel (designated NSCC-1 and NSCC-2) in Chinese hamster ovarian cells expressing endothelinB receptor (CHO-ETBR). These channels can be discriminated using the Ca2+ channel blockers, LOE 908 and SK&F 96365. LOE 908 is a blocker of NSCC-1 and NSCC-2, whereas SK&F 96365 is a blocker of NSCC-2. In this study, we investigated the possible role of phosphoinositide 3-kinase (PI3K) in the ET-1-induced activation of NSCCs in CHO-ETBR using wortmannin and LY-294002, inhibitors of PI3K. ET-1-induced Ca2+ influx was partially inhibited in CHO-ETBR pretreated with wortmannin or LY-294002. In contrast, addition of wortmannin or LY-294002 after stimulation with ET-1 did not suppress Ca2+ influx. The Ca2+ channels activated by ET-1 in wortmannin- or LY-294002-treated CHO-ETBR were sensitive to LOE 908 and resistant to SK&F 96365. In conclusion, NSCC-2 is stimulated by ET-1 via PI3K-dependent cascade, whereas NSCC-1 is stimulated independently of the PI3K pathway. Moreover, PI3K seems to be required for the initiation of the Ca2+ entry through NSCC-2 but not for its maintenance.
endothelin-1; endothelinB receptor; phosphoinositide 3-kinase; nonselective cation channel
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INTRODUCTION |
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ENDOTHELIN-1
(ET-1) was discovered as a potent vasoconstricting peptide
secreted from endothelial cells (17). It is generally accepted that ET-1 may play a role in the pathogenesis of certain clinical conditions such as hyperlipoproteinemia, atherosclerosis, stroke, cerebral vasospasm, and tumor growth (2, 9). We demonstrated recently that the extracellular Ca2+ influx is
required to ET-1-induced vascular contraction and mitogenesis (4,
6). These results indicate that if there exist pathways for
activation of Ca2+ channels with ET-1 to increase
Ca2+ influx, the blockade of these pathways may provide a
new strategy for treatment of some diseases involving ET-1 as a
pathogenesis. We recently demonstrated that the sustained increase in
intracellular free Ca2+ concentration
([Ca2+]i) caused by ET-1 results from
Ca2+ entry through two types of Ca2+-permeable
nonselective cation channel (designated NSCC-1 and NSCC-2) in Chinese
hamster ovarian (CHO) cells stably expressing human
endothelinB receptors (CHO-ETBR)
(7). Importantly, these channels can be distinguished in
terms of the sensitivity to the Ca2+ channel blockers SK&F
96365 and LOE 908. NSCC-1 is sensitive to LOE 908 and resistant to SK&F
96365, and NSCC-2 is sensitive to both LOE 908 and SK&F 96365 (7). The types of G protein involved in activation of
NSCC-1 and NSCC-2 are different in CHO-ETBR. NSCC-1 is
activated via the G13-dependent pathway, and NSCC-2 is
activated via both the Gq/phospholipase C (PLC)- and the
G13-dependent pathway (5). However, less is
known about intracellular signaling pathways that regulate the
activation of these Ca2+ channels. Previous reports have
demonstrated that phosphoinositide 3-kinase (PI3K) plays important
roles for stimulation of L-type voltage-operated Ca2+
channels with angiotensin (12, 16) and for T cell
Ca2+ signaling via the phosphatidylinositol
3,4,5-trisphosphate-sensitive Ca2+ entry pathway
(3). We thus examined whether the PI3K-dependent pathway
is involved in the activation of NSCC-1, NSCC-2, or both, with ET-1 in
CHO-ETBR.
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MATERIALS AND METHODS |
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Cell culture. Stable expression of ETBR or unpalmitoylated mutant ETBR (SerETBR) in CHO cells was accomplished as described previously (5, 7, 11). CHO-ETBR were routinely maintained in F-12 medium supplemented with 10% fetal calf serum under a humidified atmosphere of 5% CO2-95% air.
Monitoring of
[Ca2+]i.
The [Ca2+]i was monitored using the
fluorescent probe, fluo 3, as described previously (7).
Briefly, the cells 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% and 5 mM,
respectively, to obtain the maximum fluorescence (Fmax) and
the minimum fluorescence (Fmin). The
[Ca2+]i was determined by the equilibrium
equation
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Drugs. LOE 908 was kindly provided by Boehringer Ingelheim (Ingelheim, Germany). Other chemicals 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), wortmannin from Wako (Osaka, Japan), and LY-294002 from Sigma (St. Louis, MO).
Statistical analysis. All results were expressed as means ± SE. The data were subjected to a two-way analysis of variance, and 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.
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RESULTS |
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Effects of wortmannin on the ET-1-induced increase in
[Ca2+]i in
CHO-ETBR.
ET-1 at 10 nM induced a biphasic increase in
[Ca2+]i consisting of an initial transient
peak and a subsequent sustained increase in both CHO-ETBR
and CHO-ETBR preincubated with wortmannin (Fig. 1,
A and B). The
magnitude of the transient peak and that of the sustained increase in
[Ca2+]i depended on the concentration of ET-1
(Fig. 1, C and D). In experiments performed on
cells incubated in a bath in which the extracellular
Ca2+ had been removed, upon treatment with 10 nM ET-1, the
transient peak was not affected but the sustained increase was
abolished (data not shown). The EC50 values (0.63 ± 0.11 nM) of ET-1 for transient increase in
[Ca2+]i in CHO-ETBR preincubated
with 1 µM wortmannin was similar to those (0.64 ± 0.08 nM) in
CHO-ETBR (Fig. 1C). On the other hand, the
magnitude of sustained increase in [Ca2+]i
caused by 10 nM ET-1 in CHO-ETBR preincubated with
wortmannin was ~35% of that in CHO-ETBR (Figs.
1D and 2B). In contrast, addition of wortmannin
after stimulation with ET-1 did not affect to the sustained increase in
[Ca2+]i (Fig. 1A).
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Effects of LY-294002 on the ET-1-induced increase in
[Ca2+]i in
CHO-ETBR.
We also used LY-294002 to evaluate the involvement of PI3K on
ET-1-induced extracellular Ca2+ influx. LY-294002 at 50 µM inhibited PI3K activation completely in CHO cells
(8). The magnitudes of ET-1-induced transient increase in
[Ca2+]i in CHO-ETBR preincubated
with 50 µM LY-294002 were similar to those in CHO-ETBR
(Fig. 3, A-C).
On the other hand, 50 µM LY-294002 inhibited ET-1-induced sustained
increase in [Ca2+]i, and ~65% inhibition
was obtained (Fig. 3, B and D). Moreover, addition of LY-294002 after stimulation with ET-1 did not affect the
sustained increase in [Ca2+]i (Fig.
3A).
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Effects of SK&F 96365 and LOE 908 on ET-1-induced sustained
increase in [Ca2+]i in
CHO-ETBR preincubated with wortmannin.
SK&F 96365 inhibited ET-1-induced sustained increase in
[Ca2+]i partially in CHO-ETBR
(Fig. 4A). In
CHO-ETBR pretreated with 0.1 µM wortmannin, the SK&F
96365-sensitive part of ET-1-induced sustained increase in
[Ca2+]i was inhibited partially (Fig.
4B). The ET-1-induced sustained increase in
[Ca2+]i in CHO-ETBR preincubated
with 1 µM wortmannin was inhibited by LOE 908 in a
concentration-dependent manner, and complete inhibition was observed at
concentrations 10 µM (Fig. 4, C and D). In
contrast, SK&F 96365 up to 10 µM failed to inhibit ET-1-induced
sustained increase in [Ca2+]i in
CHO-ETBR preincubated with 1 µM wortmannin (Fig. 4,
C and D). These results suggest that activation
of NSCC-1 with ET-1 is wortmannin resistant, whereas that of NSCC-2 is
wortmannin sensitive. In CHO-ETBR preincubated with 50 µM
LY-294002, ET-1-induced sustained increase in
[Ca2+]i was also sensitive to LOE 908 and
resistant to SK&F 96365 (data not shown).
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Effects of wortmannin on ET-1-induced sustained increase in
[Ca2+]i in CHO cells
expressing unpalmitoylated mutant ETBR
(SerETBR).
To assess the effects of wortmannin on the activation of NSCC-1, we
used CHO-SerETBR. SerETBR is unpalmitoylated
because of substitution of all the cysteine residues to serine
(Cys402Cys403
Cys405Ser402Ser403
Ser405) and activates only NSCC-1 (5). Because
SerETBR does not couple with Gq, ET-1 failed to
induce an initial transient increase in [Ca2+]i, and it induced only a sustained
increase in [Ca2+]i (5).
Wortmannin at 1 µM did not affect ET-1-induced sustained increase in
[Ca2+]i in CHO-SerETBR (Fig.
5). LOE 908 at 10 µM inhibited
ET-1-induced sustained increase in [Ca2+]i
completely in wortmannin-treated CHO-SerETBR (Fig. 5). On
the other hand, SK&F 96365 at 10 µM failed to inhibit ET-1-induced sustained increase in [Ca2+]i in these cells
(Fig. 5).
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DISCUSSION |
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In this study, we focused on the involvement of PI3K in the Ca2+ channel activation caused by ET-1 in CHO-ETBR because PI3K plays important roles for stimulation of extracellular Ca2+ influx. From the data that addition of wortmannin or LY-294002 after stimulation with ET-1 did not suppress sustained increase in [Ca2+]i (Figs. 1A and 3A), wortmannin or LY-294002 seems not to directly act as Ca2+ channel blocker. Therefore, wortmannin and LY-294002 may be efficient tools for studying intracellular mechanisms involved in Ca2+ channel activation by ET-1. Moreover, PI3K seems to be required for the initiation of the Ca2+ entry but not for its maintenance. The inhibitory effects of wortmannin on ET-1-induced sustained increase in [Ca2+]i may be due to its inhibition of PI3K, judging from the following data: 1) wortmannin is generally accepted as a PI3K inhibitor (14). Moreover, at nanomolar concentrations, wortmannin acts specifically on PI3K (18); 2) another PI3K inhibitor, LY-294002, also inhibited the wortmannin-sensitive part of ET-1-induced sustained increase in [Ca2+]i; and 3) the IC50 values (~30 nM) and maximal effective concentration (1 µM) of wortmannin for ET-1-induced sustained increase in [Ca2+]i in CHO-ETBR (Fig. 2A) were similar to those for ET-1-induced phosphatidylinositol triphosphate (PIP3) formation as an index of PI3K activity (13).
As wortmannin and LY-294002 partially suppressed ET-1-induced sustained
increase in [Ca2+]i (Figs. 1-3), ET-1
induces extracellular Ca2+ influx through NSCCs via two
different pathways, PI3K-dependent and -independent, in
CHO-ETBR. On the basis of the sensitivity to SK&F 96365 and
LOE 908, the PI3K-inhibitor resistant part of sustained increase in
[Ca2+]i was due to Ca2+ influx
through NSCC-1 (LOE 908-sensitive and SK&F 96365-resistant) (Fig. 4,
C and D). Thus Ca2+ influx through
NSCC-2 was the PI3K inhibitor-sensitive part. These results indicate
that PI3K may play important roles for ET-1-induced activation of
NSCC-2. The result that wortmannin failed to inhibit the activation of
NSCC-1 by ET-1 in CHO-SerETBR (Fig. 5) is consistent with
this indication. NSCC-2 activation by ET-1 involves
Gq/PLC-dependent cascade and depends on mobilization of
Ca2+ from the intracellular Ca2+ store in
CHO-ETBR (5). Moreover, it is generally
accepted that G is involved in PI3K activation (1,
15). Therefore, G
as well as G
may play important roles
for NSCC-2 activation by ET-1.
In conclusion, NSCC-2 are stimulated by ET-1 via PI3K-dependent cascade, whereas NSCC-1 is stimulated via PI3K-independent cascade. Moreover, PI3K seems to be required for the initiation of the Ca2+ entry but not for its maintenance.
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ACKNOWLEDGEMENTS |
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We thank Boehringer Ingelheim for the kind donation of LOE 908.
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FOOTNOTES |
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This work was supported by Grants-in-Aid from the Ministry of Education, Science, Sports, and Culture of Japan and by the Uehara Memorial Foundation Fellowship, Tokyo, Japan.
Address for reprint requests and other correspondence: Y. Kawanabe, Renal Division, Dept. of Medicine, Brigham and Women's Hospital and Harvard Medical School, Harvard Institutes of Medicine, Rm. 520, 77 Ave. Louis Pasteur, Boston, MA 02115 (E-mail: ykawanabe{at}rics.bwh.harvard.edu).
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.00384.2002
Received 1 October 2002; accepted in final form 10 October 2002.
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