Departments of 1Neurosurgery and 2Pharmacology, Kyoto University Graduate School of Medicine, Kyoto 606-8507, Japan; 3Renal Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115
Submitted 11 December 2002 ; accepted in final form 24 April 2003
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ABSTRACT |
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endothelin; phosphoinositide 3-kinase; nonselective cation channel; proline-rich tyrosine kinase 2; glioma cell
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MATERIALS AND METHODS |
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Measurement of [Ca2+]i. [Ca2+]i was measured using a fluorescent probe fluo 3. We did not use fura 2 in this experiment because LOE 908 influenced fura 2 directly (data not shown). The measurement of fluorescence by a CAF 110 spectrophotometer (JASCO, Tokyo, Japan) was performed exactly as described in a previous report (13).
[3H]thymidine incorporation. [3H]thymidine incorporation was performed as described previously (13).
Measurement of PYK2 phosphorylation. Measurement of PYK2 phosphorylation was performed using a universal tyrosine kinase assay kit (Takara, Tokyo, Japan) as described previously (10). Extraction buffer and kinase reacting solution were provided in this kit. Cells seeded at 5 x 106 cells/well in six-well plates were starved for 24 h and then stimulated with ET-1 for 3 min (2). Reaction was terminated by washing cells three times with PBS. After the addition of 1 ml of extraction buffer, the cells were scraped off and centrifuged at 14,500 rpm for 10 min at 4°C. The supernatant was incubated with mouse monoclonal anti-PYK2 antibody (Upstate Biotechnology) for 2 h at room temperature and subsequently incubated with Protein A-agarose for an additional 2 h. The mixture was centrifuged at 10,000 g for 1 min at 4°C, and the pellets were washed three times with PBS. The washed pellets were resuspended in 150 µl of kinase reaction buffer. PYK2 phosphorylation was determined according to the manufacturer's instructions. The absorbance of the lysate at 450 nm was measured with an EL340 Microtiter plate reader (Bio-Tek Instruments, Winooski, VT).
Immunoblotting. Samples resuspended in kinase reaction buffer were analyzed by SDS-PAGE and transferred electrophoretically to polyvinylidene difluoride membranes (15 V, 90 min). After being blocked with 5% bovine serum albumin for 1 h, membranes were reacted with anti-phosphotyrosine monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or mouse monoclonal anti-PYK2 antibody for 1 h. The blots were washed and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h. After further washing, immunoreactive proteins were detected by the enhanced chemiluminescence (ECL) system.
Drugs. Boehringer Ingelheim (Ingelheim, Germany) kindly provided LOE 908. Other chemicals were obtained commercially from the following sources: ET-1 from Peptide Institute (Osaka, Japan), SK&F 96365 from Biomol (Plymouth Meeting), fluo 3-AM from Dojindo Laboratories (Kumamoto, Japan), [3H]thymidine from NEN (Boston, MA), wortmannin from Wako (Osaka, Japan), and LY-294002 from Sigma (St. Louis, MO). All other chemicals were of reagent grade and were obtained commercially.
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RESULTS |
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NSCC-1 blockade abolishes residual Ca2+
influx after PI3K inhibition. The ET-1-induced sustained increase in
[Ca2+]i in C6 glioma cells preincubated with
wortmannin was inhibited by LOE 908 in a concentration-dependent manner, and
complete inhibition was observed at concentrations 3 µM
(Fig. 3). In contrast, SK&F
96365 up to 10 µM failed to inhibit ET-1-induced sustained increase in
[Ca2+]i in C6 glioma cells preincubated with
wortmannin (Fig. 3). In
LY-294002-pretreated C6 glioma cells, 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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Concentration-response pattern of ET-1 for the sustained increase in
[Ca2+]i. We also
characterize the concentration-response pattern of ET-1 for the sustained
increase in [Ca2+]i in the absence or
presence of LOE 908, SK&F 96365, or wortmannin. ET-1 induced sustained
increase in [Ca2+]i in a
concentration-dependent manner with an EC50 value of between 0.1
and 1 nM, and maximal effects were observed at concentrations of 10 nM
(Fig. 4A). In the
presence of 10 µMLOE 908, ET-1 failed to induce sustained increase in
[Ca2+]i
(Fig. 4A). The
magnitudes of ET-1-induced sustained increase in
[Ca2+]i in C6 glioma cells-treated with 10
µM SK&F 96365 or 1 µM wortmannin were around 35% of those in control
(Fig. 4A). The maximal
sustained increases in [Ca2+]i in C6 glioma
cells treated with SK&F 96365 or wortmannin reached a plateau at 0.1 nM
ET-1 (Fig. 4A).
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Blockade of IP3 receptor inhibits ET-1-induced sustained increase in [Ca2+]i partially. Involvement of inositol 1,4,5-triphosphate (IP3) receptor in ET-1-induced sustained increase in [Ca2+]i was examined by using IP3 receptor blocker, 2-aminoethoxydiphenyl borate (2-APB). In C6 glioma cells pretreated with 75 µM 2-APB, ET-1 failed to induce transient increase in [Ca2+]i (Fig. 4B). On the other hand, extracellular Ca2+ influx caused by ET-1 was inhibited partially in these cells (Fig. 4B). The ET-1-induced sustained increase in [Ca2+]i was abolished upon treatment of 10 µM LOE 908 (Fig. 4B). SK&F 96365 at 10 µM inhibited ET-1-induced sustained increase in [Ca2+]i slightly (Fig. 4B).
Inhibition of PI3K attenuates ET-1-induced cell proliferation.
Extracellular Ca2+ influx through NSCCs plays an
essential role in ET-1-induced mitogenesis in C6 glioma cells
(7). Therefore, we examined
whether PI3K was involved in the NSCC-2-dependent part of ET-1-induced
mitogenesis. Wortmannin inhibited ET-1-induced mitogenesis in a
concentration-dependent manner with an IC50 value of 30 nM.
Maximal inhibition was observed at concentrations
1 µM
(Fig. 5A). The extent
of maximal inhibition was
65% (Fig.
5B). The wortmannin-resistant part of mitogenesis caused
by ET-1 was abolished by 10 µM LOE 908
(Fig. 5B). In
contrast, SK&F 96365 up to 10 µM did not affect the
wortmannin-resistant part of mitogenesis caused by ET-1
(Fig. 5B).
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NSCCs blockade abolishes ET-1-induced PYK2 phosphorylation. ET-1
stimulates PYK2 phosphorylation (Fig. 6,
A and B). Using SK&F 96365 and LOE 908, we
attempted to determine the effects of extracellular Ca2+
influx through NSCCs on ET-1-induced PYK2 phosphorylation. The ET-1-induced
PYK2 phosphorylation was suppressed by LOE 908 or SK&F 96365 in a
concentration-dependent manner with IC50 values of 3 µM
(Fig. 6C). LOE 908, at
concentrations
10 µM, completely suppressed this response, whereas the
maximal effect of SK&F 96365 was incomplete: about 35% was unsuppressed at
concentrations
10 µM (Fig. 6,
C and D).
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Inhibition of PI3K attenuates ET-1-induced PYK2 phosphorylation.
The magnitudes of PYK2 phosphorylation caused by 10 nM ET-1 in C6 glioma cells
preincubated with wortmannin or LY-294002 were smaller than those in control
cells (Fig. 7, A and
B). Wortmannin inhibited ET-1-induced PYK2
phosphorylation in a concentration-dependent manner with an IC50
value of 30 nM (Fig.
7C). Maximal inhibition was observed at concentrations
1 µM (Fig. 7C).
The extent of maximal inhibition was around 65% of PYK2 phosphorylation
(Fig. 7D). The
ET-1-induced PYK2 phosphorylation in C6 glioma cells preincubated with 1 µM
wortmannin was abolished by LOE 908, concentrations
10 µM effecting
complete inhibition (Fig.
7D). In contrast, SK&F 96365 up to 10 µM failed to
inhibit ET-1-induced PYK2 phosphorylation in C6 glioma cells preincubated with
1 µM wortmannin (Fig.
7D).
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DISCUSSION |
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Because wortmannin and LY-294002 partially inhibited ET-1-induced sustained
increase in [Ca2+]i (Figs.
1D and
2D), ET-1 seems to
activate both PI3K-dependent and PI3K-independent Ca2+
channels. On the basis of the data that the wortmanninor LY-294002-resistant
part of ET-1-induced sustained increase in
[Ca2+]i was sensitive to LOE 908 and
resistant to SK&F 96365 (Fig.
3), Ca2+ influx through NSCC-1 or NSCC-2 is
composed of a wortmannin-resistant or -sensitive part, respectively. Thus PI3K
may play an important role for NSCC-2 activation by ET-1 in C6 glioma cells
like CHO-ETAR or CHO-ETBR
(8,
11). The IC50
values and maximal inhibition of wortmannin to ET-1-induced sustained increase
in [Ca2+]i are similar to those of SK&F
96365 (Fig. 4A).
Neither SK&F 96365 nor wortmannin blocked sustained increase in
[Ca2+]i caused by 0.1 nM ET-1. The magnitudes
of 0.1 nM ET-1-induced sustained increase in
[Ca2+]i in C6 glioma cells treated with
SK&F 96365 or wortmannin were similar to those in control cells
(Fig. 4A). On the
basis of the sensitivity to LOE 908 and SK&F 96365
(Fig. 4A), 0.1 nM ET-1
induces only sustained increase in [Ca2+]i
through NSCC-1 in C6 glioma cells, as described previously
(7). Moreover, the result that
wortmannin failed to inhibit 0.1 nM ET-1-induced sustained increase in
[Ca2+]i
(Fig. 4A) is in
agreement with the observation that PI3K is not involved in NSCC-1 activation
by ET-1. On the other hand, SK&F 96365 and wortmannin inhibited sustained
increase in [Ca2+]i caused by higher
concentrations (1 nM) of ET-1 (Fig.
4A). A SK&F 96365- or wortmannin-resistant part of
1100 nM ET-1-induced sustained increases in
[Ca2+]i was inhibited by LOE 908
(Fig. 4A). Therefore,
SK&F 96365- or wortmannin-resistant parts of sustained increases in
[Ca2+]i consist of extracellular
Ca2+ influx through NSCC-1. These data are in agreement
with the conclusion that SK&F 96365 and wortmannin modulate only NSCC-2
activation. Because PI3K failed to inhibit ET-1-induced transient increase in
[Ca2+]i
(Fig. 1), PI3K might modulate
NSCC-2 activation at the points after intracellular Ca2+
release or by different pathway. It may be worthwhile to compare the time
course for activation of NSCC and PI3K. However, it is difficult to
distinguish these time courses. PI3K is stimulated rapidly, and maximal
activation is observed within 5 s
(19). Activation of NSCC is
also started soon after stimulation with ET-1
(Fig. 4B). Wortmannin
also inhibits NSCC-2 activation caused by vasopressin in C6 glioma cells
(unpublished data). Therefore, we believe that NSCC-2 is activated by several
agonists by the PI3K-dependent pathway. The inhibitory effects of wortmannin
on ET-1-induced mitogenesis may be mediated by blockade of
Ca2+ entry through NSCC-2 for the following reasons.
1) The IC50 values of wortmannin for ET-1-induced
mitogenesis (Fig. 5A)
correlated well with those for ET-1-induced
[Ca2+]i response
(Fig. 1C). 2)
The wortmannin-resistant part of ET-1-induced mitogenesis was dependent on the
extracellular Ca2+ influx through NSCC-1, on the basis
of the sensitivity to SK&F 96365 and LOE 908 (SK&F 96365-resistant and
LOE 908-sensitive) (Fig.
5B). Moreover, these results indicate that PI3K may be
involved in the ET-1-induced mitogenesis by activating extracellular
Ca2+ influx through NSCC-2.
The magnitudes of ET-1-induced PYK2 phosphorylation in the absence of
extracellular Ca2+ were near the basal level (data not
shown). These results indicate that extracellular Ca2+
influx plays an important role in ET-1-induced PYK2 phosphorylation. Thus we
examined the involvement of NSCC-1 and NSCC-2 in ET-1-induced PYK2
phosphorylation using SK&F 96365 and LOE 908. The inhibitory actions of
SK&F 96365 and LOE 908 on ET-1-induced PYK2 phosphorylation seem to be
mediated by blockage of Ca2+ entry through NSCCs for the
following reasons. 1) The IC50 values of these blockers
for ET-1-induced PYK2 phosphorylation (Fig.
6C) correlated well with those for ET-1-induced
extracellular Ca2+ influx
(7). 2) On the basis
of the sensitivity to SK&F 96365 and LOE 908, one type of
Ca2+ channel is sensitive to LOE 908 and resistant to
SK&F 96365, and another type is sensitive to both LOE 908 and SK&F
96365. On the basis of pharmacological criteria, these channels are considered
to be NSCC-1 and NSCC-2, respectively. Because PI3K plays important roles for
NSCC-2 activation by ET-1, we investigated the effects of PI3K on ET-1-induced
PYK2 phosphorylation. As described in Fig.
7, wortmannin and LY-294002 inhibited ET-1-induced PYK2
phosphorylation. The IC50 values (30 nM) and maximal effective
concentration (1 µM) of wortmannin for the ET-1-induced PYK2
phosphorylation (Fig.
7C) were similar to those for ET-1-induced sustained
increase in [Ca2+]i
(Fig. 3). Therefore, the
inhibitory effects of wortmannin on ET-1-induced PYK2 phosphorylation may also
be due to its inhibitory effects on PI3K. The wortmannin-resistant part of
ET-1-induced PYK2 phosphorylation was dependent on extracellular
Ca2+ influx through NSCC-1, on the basis of the
sensitivity to SK&F 96365 and LOE 908 (SK&F 96365-resistant and LOE
908-sensitive) (Fig.
7D). These results indicate that the inhibitory effects
of wortmannin on ET-1-induced PYK2 phosphorylation might be mediated by
blockage of Ca2+ entry through NSCC-2.
In conclusion, NSCC-2 is stimulated by ET-1 via a PI3K-dependent cascade, whereas NSCC-1 is stimulated via a PI3K-independent cascade in C6 glioma cells. Extracellular Ca2+ influx through NSCC-1 and NSCC-2 plays an essential role for ET-1-induced PYK2 phosphorylation. In addition, PI3K is involved in the NSCC-2-dependent part of cell proliferation and PYK2 phosphorylation by ET-1.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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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. Section 1734 solely to indicate this fact.
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