Effects of nonselective cation channels and PI3K on endothelin-1-induced PYK2 tyrosine phosphorylation in C6 glioma cells

Yoshifumi Kawanabe,1,2,3 Nobuo Hashimoto,1 and Tomoh Masaki2

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


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We recently demonstrated that endothelin-1 (ET-1) activates two types of Ca2+-permeable nonselective cation channels (designated NSCC-1 and NSCC-2) in C6 glioma cells. In the present study, we investigated the effects of NSCCs on the ET-1-induced proline-rich tyrosine kinase 2 (PYK2) phosphorylation in C6 glioma cells. In addition, we examined the effects of phosphoinositide 3-kinase (PI3K) on the ET-1-induced NSCCs activation and PYK2 phosphorylation. The PI3K inhibitors wortmannin and LY-294002 inhibited ET-1-induced Ca2+ influx through NSCC-2 but not NSCC-1. On the other hand, addition of these inhibitors after stimulation with ET-1 failed to suppress Ca2+ influx through NSCC-2. PYK2 phosphorylation was abolished by blocking Ca2+ influx through NSCCs. The PI3K inhibitors blocked the NSCC-2-dependent part of ET-1-induced PYK2 phosphorylation. These results indicate that 1) NSCC-2 is stimulated by ET-1 via a PI3K-dependent cascade, whereas NSCC-1 is stimulated via a PI3K-independent cascade; 2) PI3K seems to be required for the activation of the Ca2+ entry, but not for its maintenance; 3) Ca2+ influx through NSCC-1 and NSCC-2 plays an essential role in ET-1-induced PYK2 phosphorylation; and 4) PI3K is involved in the ET-1-induced PYK2 phosphorylation that depends on the Ca2+ influx through NSCC-2.

endothelin; phosphoinositide 3-kinase; nonselective cation channel; proline-rich tyrosine kinase 2; glioma cell


ENDOTHELIN-1 (ET-1) is a 21-amino acid peptide, and it is one of the most potent endogenous vasoconstricting agents yet discovered (26). The biochemical characterization of ET-1 has attracted intensive research, and it has been shown that ET-1 exerts a wide spectrum of effects on various nonvascular tissues, including those of the central nervous system (17). The variety of described actions of ET-1 on the central nervous system has led to the recognition of ET-1 as a neuropeptide (15). ET-1 is produced in neurons in the brain and spinal cord (5, 16), in the endothelium of cerebral microvessels (27), and in glial cells (17). ET-1 is a growth factor for some tumor cell lines, including C6 glioma cells (21, 23). We have recently shown that a 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 channels (designated NSCC-1 and NSCC-2) in C6 glioma cells (7). In particular, these channels can be distinguished by using Ca2+ channel blockers such as SK&F 96365 and LOE 908. NSCC-1 is sensitive to LOE 908 and resistant to SK&F 96365, whereas NSCC-2 is sensitive to both LOE 908 and SK&F 96365 (7). Moreover, Ca2+ influx through these NSCCs plays a critical role in ET-1-induced mitogenesis in C6 glioma cells (7). These results indicate that if the activation pathways of NSCCs are revealed, blockade of these pathways may prevent the growth of glioma cells caused by ET-1. However, less is known about the intracellular signaling pathways that regulate the activation of NSCCs. ET-1 stimulates phosphoinositide 3-kinase (PI3K) (24). PI3K plays an important role for stimulation of L-type voltage-dependent Ca2+ channels by angiotensin (22, 25) and T-cell Ca2+ signaling via a phosphatidylinositol 3,4,5-triphosphate-sensitive Ca2+ entry pathway (6). In addition, we demonstrated that NSCC-2 is stimulated by ET-1 via a PI3K-dependent cascade, whereas NSCC-1 is stimulated via a PI3K-independent cascade in Chinese hamster ovary cells stably expressing endothelinA or endothelinB receptors (CHO-ETAR or CHO-ETBR, respectively) (8, 11). Therefore, we investigated the effects of PI3K on the ET-1-induced activation of NSCCs in C6 glioma cells in this study. Moreover, it remains to be determined which intracellular mitogenic cascades require extracellular Ca2+ influx through NSCCs. Extracellular Ca2+ influx through NSCCs has an important role in the ET-1-induced extracellular signal-regulated kinase 1 and 2 (ERK1/2) (9). The cloning of the Ca2+-regulated cytoplasmic proline-rich tyrosine kinase (PYK2) suggested a link between G protein-coupled receptors and the induction of tyrosine phosphorylation via mobilization of intracellular Ca2+ (1, 20). PYK2 plays a critical role in G protein-coupled receptor-mediated activation of mitogen-activated protein kinase cascades (4). Especially, ET-1-induced ERK1/2 activation was found to coincide with PYK2 tyrosine phosphorylation in primary astrocytes (3). Therefore, it is important to investigate the activation mechanism of PYK2. In this study, we examined whether and which NSCCs are involved in ET-1-induced PYK2 phosphorylation. We also investigated the effects of PI3K on ET-1-induced PYK2 phosphorylation.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture. C6 glioma cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum supplemented with 100 u/ml penicillin G and 100 ug/ml streptomycin under a humidified 5% CO2-95% air atmosphere.

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.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Inhibition of PI3K attenuates ET-1-induced increase in [Ca2+]i. ET-1 at 10 nM induced a biphasic increase in [Ca2+]i consisting of an initial transient peak and a subsequent sustained increase in C6 glioma cells preincubated with 1 µM wortmannin (Fig. 1A). The maximal effective concentration (10 nM) of ET-1 for transient increase in [Ca2+]i in C6 glioma cells preincubated with wortmannin was similar to those in C6 glioma cells (Fig. 1, AC). In contrast, wortmannin inhibited ET-1-induced sustained increase in [Ca2+]i in a concentration-dependent manner with an IC50 values of ~30 nM, and the maximal inhibition (~65% of control) was seen at concentrations >=1 µM (Fig. 1, C and D). On the other hand, addition of wortmannin after stimulation with ET-1 did not affect the sustained increase in [Ca2+]i (Fig. 1B). We also used LY-294002 to evaluate the effects of PI3K on ET-1-induced extracellular Ca2+ influx. LY-294002 at 50 µM inhibited PI3K activation completely (14). The magnitudes of ET-1-induced transient increase in [Ca2+]i in LY-294002-pretreated C6 glioma cells were similar to those in nontreated cells (Fig. 2, AC). On the other hand, LY-294002 inhibited ET-1-induced sustained increase in [Ca2+]i, and ~65% inhibition was obtained (Fig. 2, A and D). Moreover, addition of LY-294002 after stimulation with ET-1 did not affect the sustained increase in [Ca2+]i (Fig. 2B).



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Fig. 1. Original tracings illustrating the effects of wortmannin on the increase in [Ca2+]i by endothelin-1 (ET-1) in C6 glioma cells. A: the cells preincubated with 1 µM wortmannin were stimulated by 10 nM ET-1. B: after [Ca2+]i reached a steady state, 1 µM wortmannin was added at the time indicated by the horizontal bar. C: effects of various concentrations of wortmannin on ET-1-induced transient ({circ}) and sustained ({bullet}) increase in [Ca2+]i in C6 glioma cells. D: effects of 1 µM wortmannin on ET-1-induced sustained increase in [Ca2+]i in C6 glioma cells. Data are means ± SE of 5 determinations, each done in triplicate.

 


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Fig. 2. Original tracings illustrating the effects of LY-294002 on the increase in [Ca2+]i by ET-1 in C6 glioma cells. A: the cells preincubated with 50 µM LY-294002 were stimulated by 10 nM ET-1. B: after [Ca2+]i reached a steady state, 50 µM LY-294002 was added at the time indicated by the horizontal bar. C and D: effects of 50 µM LY-294002 on ET-1-induced transient (C) and sustained (D) increase in [Ca2+]i in C6 glioma cells. Data are means ± SE of 5 determinations, each done in triplicate.

 

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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Fig. 3. A: original tracings illustrating the effects of LOE 908 and SK&F 96365 on the sustained increase in [Ca2+]i by ET-1 in C6 glioma cells treated with wortmannin. The cells pretreated with 1 µM wortmannin were stimulated by 10 nM ET-1. After [Ca2+]i reached a steady state, 10 µM SK&F 96365 and various concentrations of LOE 908 were added at the time indicated by the horizontal bars and the arrows. B: effects of various concentrations of LOE 908 ({bullet}) and SK&F 96365 ({circ}) on the sustained increase in [Ca2+]i by 10 nM ET-1. Data are means ± SE of 5 determinations, each done in triplicate.

 

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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Fig. 4. A: effects of various concentrations of ET-1 on the sustained increase in [Ca2+]i in C6 glioma cells in the absence ({bullet}) or presence of 1 µM wortmannin ({circ}), 10 µM SK&F 96365 ({blacksquare}), or 10 µM LOE 908 ({square}). The sustained increase in [Ca2+]i after stimulation with 10 nM ET-1 was set at 100%, and the sustained increase in [Ca2+]i before stimulation with ET-1 was set at 0%. Data are means ± SE of 5 determinations, each done in triplicate. B: original tracings illustrating the effects of 2-aminoethoxyphenyl borate (2-APB) on the increase in [Ca2+]i by ET-1 in C6 glioma cells. C6 glioma cells were incubated with 75 µM 2-APB for 15 min before 10 nM ET-1 stimulation. ET-1 was added at the time indicated by the horizontal bar. After [Ca2+]i reached a steady state, 10 µM LOE 908 or 10 µM SK&F 96365 was added.

 

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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Fig. 5. A: effects of various concentrations of wortmannin on ET-1-induced mitogenic response in C6 glioma cells. B: effects of 1 µM wortmannin, 10 µM LOE 908, and/or 10 µM SK&F 96365 on 10 nM ET-1-induced mitogenic response in C6 glioma cells. [3H]thymidine incorporation was determined as described in MATERIALS AND METHODS. Data are means ± SE of 3 determinations, each done in triplicate.

 

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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Fig. 6. Effects of SK&F 96365 and LOE 908 on ET-1-induced proline-rich tyrosine kinase 2 (PYK2) phosphorylation in C6 glioma cells. A and B: effects of a maximal effective concentration (10 µM) of SK&F 96365 or LOE 908 on ET-1-induced PYK2 phosphorylation in C6 glioma cells. PYK2 was immunoprecipitated from cell lysates and analyzed by immunoblotting with either anti-phosphotyrosine (PY20) (top) or monoclonal anti-PYK2 antibody (bottom). C and D: effects of various concentrations (C) or a maximal effective concentration (10 µM) (D) of SK&F 96365 or LOE 908 on ET-1-induced PYK2 phosphorylation in C6 glioma cells. Starved cells were incubated for 15 min with various concentrations of SK&F 96365 ({circ}) or LOE 908 ({bullet}) and then stimulated with 10 nM ET-1 for 3 min. PYK2 phosphorylation was determined using a universal tyrosine kinase assay kit as described in MATERIALS AND METHODS. Data presented are means ± SE of 5 determinations, each done in triplicate.

 

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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Fig. 7. Effects of wortmannin and LY-294002 on ET-1-induced PYK2 phosphorylation in C6 glioma cells. Starved cells were incubated for 15 min with 1 µM wortmannin (A) or 50 µM LY-294002 (B) and then stimulated with 10 nM ET-1 for 3 min. PYK2 was immunoprecipitated from cell lysates and analyzed by immunoblotting with either anti-phosphotyrosine (PY20) (top) or monoclonal anti-PYK2 antibody (bottom). C: effects of various concentrations of wortmannin on ET-1-induced PYK2 phosphorylation. D: effects of a maximal effective concentration (10 µM) of SK&F 96365 or LOE 908 on ET-1-induced PYK2 phosphorylation. Starved cells were incubated for 15 min with SK&F 96365 or LOE 908 with 1 µM wortmannin and then stimulated with 10 nM ET-1 for 3 min. PYK2 phosphorylation was determined using a universal tyrosine kinase assay kit as described in MATERIALS AND METHODS. Data presented are means ± SE of 3 determinations, each done in triplicate.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ET-1 evoked an increase in [Ca2+]i consisting of two components: a rapid initial transient peak and a sustained phase (Fig. 1) as described in previous report (7). The sustained phase was abolished by the removal of extracellular Ca2+, whereas the transient peak remained unaffected (7). These results indicate that the sustained phase is due to a transmembrane Ca2+ influx, whereas the transient phase is the result of Ca2+ mobilization from the intracellular stores. The ET-1-induced sustained increase in [Ca2+]i results from extracellular Ca2+ influx through two types of Ca2+-permeable NSCC: NSCC-1 and NSCC-2 (7). On the basis of the sensitivity to LOE 908 and SK&F 96365 (sensitive to LOE 908 and resistant to SK&F 96365; Fig. 4A), 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 NSCC-1. On the other hand, the increase in [Ca2+]i caused by higher concentrations (>=1 nM) of ET-1 seems to involve Ca2+ entry through two types of Ca2+-permeable NSCCs in terms of its sensitivity to LOE 908 and SK&F 96365. 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. 4A). 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. Because NSCC-1 is activated by lower concentration of ET-1, the roles of NSCC-1 for some intracellular signaling pathways may be more important than those of NSCC-2. On the basis of the data using endothelinA or endothelinB receptor blocker (7), endothelinA receptor is mainly linked to NSCCs activation. Nifedipine, a specific blocker of voltage-operated Ca2+ channels, failed to inhibit ET-1-induced sustained increase in [Ca2+]i in C6 glioma cells (7). This result indicates that voltage-operated Ca2+ channels are not activated by ET-1 in C6 glioma cells. Extracellular Ca2+ influx through these NSCCs plays critical roles for ET-1-induced C6 glioma cell proliferation (7). Therefore, it is important to reveal the mechanism of each Ca2+ channel activation by ET-1 in C6 glioma cells. On the basis of the data using IP3 blocker (Fig. 4B) and phospholipase C inhibitor (12), ET-1 activates NSCC-1 via a Gq/phospholipase C/IP3-independent pathway. In contrast, ET-1 activates NSCC-2 via a Gq/phospholipase C-dependent pathway (12). In addition, NSCC-2 may consist of two different channels. One is activated by ET-1 via an IP3-dependent pathway, and the other is activated via an IP3-independent pathway. PI3K is involved in the activation of NSCC-2, but not NSCC-1, in CHO-ETAR or CHO-ETBR (8, 11). In addition, PI3K is also involved in the ET-1-induced cell proliferation (24). Thus, at first, we examined the effects of PI3K on ET-1-induced extracellular Ca2+ influx and cell proliferation in C6 glioma cells. The IC50 values (~30 nM) and maximal effective concentration (1 µM) of wortmannin for ET-1-induced sustained increase in [Ca2+]i in C6 glioma cells (Fig. 1C) were similar to those in CHO-ETAR and CHO-ETBR (8, 11). Moreover, the IC50 values and maximal effective concentration of wortmannin for ET-1-induced sustained increase in [Ca2+]i (Fig. 1) were similar to those for ET-1-induced phosphatidylinositol triphosphate formation as an index of PI3K activity (24). These results indicate that the inhibitory effects of wortmannin on ET-1-induced sustained increase in [Ca2+]i may be due to its inhibitory effects on PI3K. Moreover, on the basis of the data that addition of wortmannin or LY-294002 after stimulation with ET-1 did not inhibit sustained increase in [Ca2+]i (Figs. 1B and 2B), PI3K seems to be required for the activation of the Ca2+ entry, but not for its maintenance. On the other hand, judging from the data that wortmannin and LY-294002 failed to affect ET-1-induced transient increase in [Ca2+]i (Figs. 1 and 2), PI3K is not involved in Ca2+ mobilization from the intracellular stores.

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 1–100 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.


    DISCLOSURES
 
This study was supported by the Banyu Fellowship Awards in Cardiovascular Medicine, which are sponsored by Banyu Pharmaceutical Co., Ltd. and The Merck Foundation, by a grant from the Smoking Research Foundation, Japan, and by the Uehara Memorial Foundation Fellowship, Tokyo, Japan.


    ACKNOWLEDGMENTS
 
We thank Boehringer Ingelheim for the kind donation of LOE 908.


    FOOTNOTES
 

Address for reprint requests and other correspondence: Y. Kawanabe, Renal Division, Dept. of Medicine, Brigham and Women's Hospital and Harvard Medical School, Harvard Institute of Medicine, Rm. 520, 77 Ave. Louis Pasteur, Boston, MA (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.


    REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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