Role of phosphoinositide 3-kinase in the nonselective cation channel activation by endothelin-1/endothelinB receptor

Yoshifumi Kawanabe1,2, Nobuo Hashimoto1, and Tomoh Masaki2

Departments of 1 Neurosurgery and 2 Pharmacology, Kyoto University Graduate School of Medicine, Sakyo-ku, Kyoto 606-8507, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 Galpha 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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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
[Ca<SUP>2+</SUP>]<SUB>i</SUB> = <IT>K</IT><SUB>d</SUB>(F − F<SUB>min</SUB>)/(F<SUB>max</SUB> − F)
where F is the experimental value of fluorescence and Kd was defined as 0.4 µM (10).

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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   A and B: original tracings illustrating the effects of wortmannin on the endothelin-1 (ET-1)-induced increase in intracellular free Ca2+ concentration ([Ca2+]i) in Chinese hamster ovarian cells expressing endothelinB receptor (CHO-ETBR). The cells loaded with fluo 3 were incubated with 1 µM wortmannin after (A) or before (B) 10 nM ET-1 stimulation. C and D: effects of wortmannin on the ET-1-induced transient (C) and sustained (D) increase in [Ca2+]i in CHO-ETBR. The cells loaded with fluo 3 were incubated with () or without (open circle ) 1 µM wortmannin before stimulation with various concentrations of ET-1. Each point represents mean ± SE of 5 experiments. #P < 0.05, ##P < 0.05, or ###P < 0.05; significantly different from the control values stimulated by 1, 10, or 100 nM ET-1, respectively, (in the absence of wortmannin) in each experiment.



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Fig. 2.   A: effects of various concentrations of wortmannin on the ET-1-induced transient (open circle ) and sustained () increase in [Ca2+]i in CHO-ETBR. The transient and sustained increase in [Ca2+]i in CHO-ETBR in the presence of wortmannin are presented as a percentage of values in its absence. B: effects of 1 µM wortmannin on the ET-1-induced sustained increase in [Ca2+]i in CHO-ETBR. Each point represents mean ± SE of 5 experiments. #P < 0.05, significantly different from the control values (in the absence of wortmannin) in each experiment.

In CHO-ETBR, wortmannin inhibited ET-1-induced sustained increase in [Ca2+]i in a concentration-dependent manner with an IC50 values of 28.3 ± 3.2 nM, and the maximal inhibition (~65% of control) was seen at concentrations >= 1 µM (Fig. 2). In contrast, wortmannin up to 1 µM failed to suppress ET-1-induced transient increase in [Ca2+]i (Fig. 2A).

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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Fig. 3.   A and B: original tracings illustrating the effects of LY-294002 on the ET-1-induced increase in [Ca2+]i in CHO-ETBR. The cells loaded with fluo 3 were incubated with 50 µM LY-294002 after (A) or before (B) 10 nM ET-1 stimulation. C and D: effects of 50 µM LY-294002 on the ET-1-induced transient (C) and sustained (D) increase in [Ca2+]i in CHO-ETBR. The transient and sustained increase in [Ca2+]i in CHO-ETBR in the presence of LY-294002 are presented as a percentage of values in its absence. Each point represents mean ± SE of 5 experiments. #P < 0.05, significantly different from the control values (in the absence of LY-294002) in each experiment.

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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Fig. 4.   A-C: original traces illustrating the effects of SK&F 96365 and/or LOE 908 on the ET-1-induced sustained increase in [Ca2+]i in CHO-ETBR (A) or CHO-ETBR pretreated with 0.1 µM (B) or 1 µM (C) wortmannin. The cells loaded with fluo 3 were incubated with wortmannin before 10 nM ET-1 stimulation. After [Ca2+]i reached a steady state, various concentrations of SK&F 96365 and/or LOE 908 were added at the time indicated by horizontal bars. D: effects of various concentrations of SK&F 96365 (open circle ) and LOE 908 () on the ET-1-induced sustained increase in [Ca2+]i in CHO-ETBR pretreated with 1 µM wortmannin. The sustained increases in [Ca2+]i in the presence of each drug are presented as a percentage of values in its absence. Each point represents mean ± SE of 5 experiments.

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 Cys405right-arrowSer402Ser403 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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Fig. 5.   A: original trace illustrating the effects of SK&F 96365 and LOE 908 on the ET-1-induced sustained increase in [Ca2+]i in Chinese hamster ovarian cells that express the unpalmitoylated (Cys402 Cys403 Cys405right-arrow Ser402 Ser403 Ser405) human endothelinB receptor (CHO-SerETBR) pretreated with wortmannin. The cells loaded with fluo 3 were incubated with 1 µM wortmannin before 10 nM ET-1 stimulation. After [Ca2+]i reached a steady state, 10 µM SK&F 96365 or 10 µM LOE 908 was added at the time indicated by horizontal bars. B: effects of wortmannin (1 µM), SK&F 96365 (10 µM), and/or LOE 908 (10 µM) on the ET-1-induced sustained increase in [Ca2+]i in CHO-SerETBR. The sustained increases in [Ca2+]i in the presence of each drug are presented as a percentage of values in its absence. Data are presented as mean ± SE of 5 experiments.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 Gbeta gamma is involved in PI3K activation (1, 15). Therefore, Gbeta gamma as well as Galpha 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.


    ACKNOWLEDGEMENTS

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


    FOOTNOTES

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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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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