Introduction

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Endothelin-1 (ET-1) was discovered as a potent vasoconstricting peptide secreted from endothelial cells (Yanagisawa et al., 1988). 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 (Lerman et al., 1991; Haak et al., 1994). Moreover, recent reports showed that the extracellular Ca²⁺ influx is required for ET-1-induced vascular contraction and mitogenesis (Zhang et al., 1999; Kawanabe et al., 2002a). These results indicate that if the activation pathways of Ca²⁺ channels involved in the extracellular Ca²⁺ influx caused by ET-1 are revealed, blockade of these pathways may become a new treatment for ET-1-induced clinical conditions described above. We recently demonstrated that the sustained increase in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) caused by ET-1 results from Ca²⁺ entry through three types of VICC in CHO cells stably expressing human endothelin_A receptors (CHO-ET_AR), two types of Ca²⁺-permeable nonselective cation channel (designated NSCC-1 and NSCC-2), and a store-operated Ca²⁺ channel (SOCC) (Kawanabe et al., 2001). Importantly, these channels can be distinguished in terms of the sensitivity to Ca²⁺ channel blockers SK&F 96365 and LOE 908. 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 but sensitive to SK&F 96365 (Kawanabe et al., 2001). VICCs activated by ET-1 in CHO-ET_AR seem to be pharmacologically similar to those in vascular smooth muscle cells, which predominantly express ET_ARs (Kawanabe et al., 2002a). Therefore, our findings on CHO cells may give some insights into the functional roles of ET_ARs related to ET-1-induced Ca²⁺ channel activation on vascular smooth muscle cells.

The types of G protein involved in activation of NSCC-1, NSCC-2, and SOCC are different in CHO-ET_AR. NSCC-1 is activated via a G₁₂-dependent pathway, NSCC-2 is activated via both a G_q/phospholipase C (PLC)- and a G₁₂-dependent pathway, and SOCC is activated via a G_q/PLC-dependent pathway (Kawanabe et al., 2002c). However, less is known about intracellular signaling pathways regulate the activation of these Ca²⁺ channels. Previous reports demonstrate that phosphoinositide 3-kinase (PI3K) plays important roles for stimulation of L-type voltage-dependent Ca²⁺ channels by angiotensin (Seki et al., 1999; Viard et al., 1999) and T-cell Ca²⁺ signaling via phosphatidylinositol 3,4,5-triphosphate-sensitive Ca²⁺ entry pathway (Hsu et al., 2000). ET-1 activates PI3K in CHO-ET_AR from the data using the PI3K inhibitor wortmannin (Sugawara et al., 1996). Therefore, at first, we examined whether and which VICCs are activated by ET-1 in CHO-ET_AR via PI3K-dependent pathway.

ET-1 induces mitogenic response in CHO-ET_AR (Sugawara et al., 1996). However, it remains unclear whether Ca²⁺ influx is essential for ET-1-induced mitogenesis of CHO-ET_AR, and it is equally unclear what types of Ca²⁺ channels are involved in mitogenesis in CHO-ET_AR. We attempted to pharmacologically characterize the Ca²⁺ channels involved in ET-1-induced mitogenesis in CHO-ET_AR using SK&F 96365 and LOE 908. We also investigated the effects of PI3K on the ET-1-induced mitogenesis that depends on extracellular Ca²⁺ influx.

	Materials and Methods

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Cell Culture. Stable expression of ET_ARs in CHO cells was accomplished as described previously (Kawanabe et al., 2001). CHO-ET_AR were routinely maintained in Ham's F12 medium supplemented with 10% fetal calf serum under a humidified atmosphere of 5% CO₂/95% air.

Monitoring of [Ca²⁺]_i in CHO-ET_AR. The [Ca²⁺]_i was monitored using the fluorescent probe fluo-3 as described previously (Kawanabe et al., 2001).

MTT assay. Cells were seeded into 96-well plates at 5 × 10³ cells/well for the assay using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT). They were incubated overnight in Ham's F12 medium supplemented with 10% fetal calf serum at 37°C. The cells were deprived of serum for 24 h, washed with phosphate-buffered saline, and incubated with ET-1 for a further 48 h in serum-free Ham's F12 medium with or without Ca²⁺ channel blockers. MTT assay was performed as described previously (Kawanabe et al., 2002a).

Drugs. LOE 908 was kindly provided by Boehringer Ingelheim K.G. (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/acetoxymethyl ester from Dojindo Laboratories (Kumamoto, Japan); wortmannin from Wako (Osaka, Japan); and MTT and LY 294002 from Sigma-Aldrich (St. Louis, MO)

Statistical analysis. All results were expressed as mean ± S.E.M.

	Results

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Effects of Wortmannin on the ET-1-Induced Increase in [Ca²⁺]_i in CHO-ET_AR. 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-ET_AR and CHO-ET_AR 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, the transient peak was not affected on treatment with 10 nM ET-1, but the sustained increase was abolished (data not shown). The EC₅₀ values (~1 nM) and the maximal effective concentration (10 nM) of ET-1 for transient increase in [Ca²⁺]_i in CHO-ET_AR preincubated with 1 µM wortmannin was similar to those in CHO-ET_AR (Fig. 1C). On the other hand, the magnitude of sustained increase in [Ca²⁺]_i caused by 10 nM ET-1 in CHO-ET_AR preincubated with wortmannin was ~20% of that in CHO-ET_AR (Fig. 1D). In contrast, addition of wortmannin after stimulation with ET-1 did not affect the sustained increase in [Ca²⁺]_i (Fig. 1A).

View larger version (27K): [in this window] [in a new window]   Fig. 1.   A and B, original tracings illustrating the effects of wortmannin on the ET-1-induced increase in [Ca2+]i in CHO-ETAR. 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-ETAR. The cells loaded with fluo-3 were incubated with () or without () 1 µM wortmannin before stimulation with various concentrations of ET-1. Each point represents the mean ± S.E.M. of five experiments.

View larger version (30K): [in this window] [in a new window]   Fig. 2.   A, effects of various concentrations of wortmannin on the ET-1-induced transient () and sustained () increase in [Ca2+]i in CHO-ETAR. B and C, effects of maximal concentration (1 µM) of wortmannin on the ET-1-induced transient (B) and sustained (C) increase in [Ca2+]i in CHO-ETAR. The transient and sustained increase in [Ca2+]i in CHO-ETAR in the presence of wortmannin are presented as a percentage of values in its absence. Each point represents the mean ± S.E.M. of five experiments.

Effects of LY 294002 on the ET-1-Induced Increase in [Ca²⁺]_i in CHO-ET_AR. 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 in CHO cells (Kurashima et al., 1998). The magnitudes of ET-1-induced transient increase in [Ca²⁺]_i in CHO-ET_AR preincubated with 50 µM LY 294002 were similar to those in CHO-ET_AR (Fig. 3, B and C). On the other hand, 50 µM LY 294002 inhibited ET-1-induced sustained increase in [Ca²⁺]_i, and ~80% 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).

View larger version (40K): [in this window] [in a new window]   Fig. 3.   A and B, original tracings illustrating the effects of LY 294002 on the ET-1-induced increase in [Ca2+]i in CHO-ETAR. 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-ETAR. The transient and sustained increase in [Ca2+]i in CHO-ETAR in the presence of LY 294002 are presented as a percentage of values in its absence. Each point represents the mean ± S.E.M. of five experiments.

Effects of SK&F 96365 and LOE 908 on ET-1-Induced Sustained Increase in [Ca²⁺]_i in CHO-ET_AR Preincubated with Wortmannin. The ET-1-induced sustained increase in [Ca²⁺]_i in CHO-ET_AR 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). In contrast, SK&F 96365 up to 10 µM failed to inhibit ET-1-induced sustained increase in [Ca²⁺]_i in CHO-ET_AR preincubated with 1 µM wortmannin (Fig. 4). These results suggest that NSCC-1 is activated by ET-1 via wortmannin-independent pathway, whereas NSCC-2 and SOCC are activated via wortmannin-dependent pathway. In CHO-ET_AR 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).

View larger version (20K): [in this window] [in a new window]   Fig. 4.   A, original tracings illustrating the effects of SK&F 96365 and LOE 908 on the ET-1-induced sustained increase in [Ca2+]i in CHO-ETAR 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 various concentrations of SK&F 96365 () and LOE 908 () on the ET-1-induced sustained increase in [Ca2+]i in CHO-ETAR pretreated with 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 the mean ± S.E.M. of five experiments.

Effects of Wortmannin on ET-1-Induced Sustained Increase in [Ca²⁺]_i in CHO Cells Expressing Unpalmitoylated Mutant ET_AR (SerET_AR). To assess the effects of wortmannin on the activation of NSCC-1, we used CHO-SerET_AR. SerET_AR is unpalmitoylated because of substitution of all the cysteine residues to serine (Cys³⁸³Cys^385-388Ser³⁸³Ser^385-388) and activates only NSCC-1 (Kawanabe et al., 2002b,c). Wortmannin at 1 µM did not affect ET-1-induced sustained increase in [Ca²⁺]_i in CHO-SerET_AR (Fig. 5). LOE 908 at 10 µM inhibited ET-1-induced sustained increase in [Ca²⁺]_i completely in wortmannin-treated CHO-SerET_AR (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).

View larger version (42K): [in this window] [in a new window]   Fig. 5.   A, original tracings illustrating the effects of SK&F 96365 and LOE 908 on the ET-1-induced sustained increase in [Ca2+]i in CHO-SerETAR 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-SerETAR. 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 ± S.E.M. of five experiments.

Effects of ET-1 on Mitogenic Response in CHO-ET_AR. After stimulation with ET-1, the number of viable cells as estimated by the MTT assay increased with time up to 48 h in CHO-ET_AR (Fig. 6A). Therefore, in subsequent experiments, the stimulation time was set at 48 h.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   A, time course of the mitogenic response of CHO-ETAR following stimulation with ET-1. After cells had been cultured in serum-free medium for 24 h, they were stimulated with 10 nM ET-1 for the indicated time. Four hours before the end of the incubation with ET-1, MTT was added to the incubation medium to estimate the number of viable cells. B, effects of various concentrations of ET-1 on the number of viable cells in CHO-ETAR. After cells had been deprived of serum for 24 h, they were stimulated with increasing concentrations of ET-1 for a further 48 h. Data are presented as mean ± S.E.M. of three determinations, each done in triplicate.

Effects of SK&F 96365 and LOE 908 on ET-1-induced mitogenesis in CHO-ET_AR. Using SK&F 96365 and LOE 908, we attempted to determine the effects of Ca²⁺ influx through VICCs on the ET-1-induced mitogenic response in CHO-ET_AR. SK&F 96365 inhibited ET-1-induced mitogenesis in a concentration-dependent manner with an IC₅₀ value of ~3 µM. Maximal inhibition was observed at concentrations 10 µM (Fig. 7A). The extent of maximal inhibition was ~80% (Fig. 7B). Similarly, the IC₅₀ values of LOE 908 for inhibition of ET-1-induced mitogenesis were ~3 µM, and maximal inhibition was observed at concentrations 10 µM (Fig. 7A). The extent of maximal inhibition was ~60% (Fig. 7B). Notably, the ET-1-induced mitogenesis was abolished by combined treatment with the maximally effective concentration (10 µM) of LOE 908 and SK&F 96365 (Fig. 7B). In contrast, neither SK&F 96365 nor LOE 908 had any effects at concentrations up to 30 µM on the number of cells in the absence of ET-1 (Fig. 7A).

View larger version (27K): [in this window] [in a new window]   Fig. 7.   A, effects of various concentrations of SK&F 96365 and LOE 908 on the ET-1-induced mitogenic response in CHO-ETAR. Starved cells were incubated for 15 min with increasing concentrations of SK&F 96365 (triangles) or LOE 908 (circles), and then they were stimulated with (open symbols) or without (closed symbols) 10 nM endothelin-1. B, effects of a maximally effective concentration of SK&F 96365 (10 µM) and/or LOE 908 (10 µM) on the ET-1-induced mitogenic response. The ET-1-induced mitogenic responses in the presence of SK&F 96365 and/or LOE 908 are represented as a percentage of values in its absence. Data are presented as mean ± S.E.M. of three experiments.

Effects of Wortmannin on ET-1-Induced Mitogenesis in CHO-ET_AR. 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. 8A). The extent of maximal inhibition was ~80% (Fig. 8B). The wortmannin-resistant part of mitogenesis caused by ET-1 was abolished by 10 µM LOE 908 (Fig. 8B). In contrast, SK&F 96365 up to 10 µM did not affect the wortmannin-resistant part of mitogenesis caused by ET-1 (Fig. 8B).

View larger version (25K): [in this window] [in a new window]   Fig. 8.   A, effects of various concentrations of wortmannin on the ET-1-induced mitogenic response in CHO-ETAR. Starved cells were incubated for 15 min with increasing concentrations of wortmannin, and then they were stimulated with 10 nM ET-1. B, effects of a maximally effective concentration of wortmannin (1 µM), SK&F 96365 (10 µM), and/or LOE 908 (10 µM) on the ET-1-induced mitogenic response. The ET-1-induced mitogenic response in the presence of wortmannin, SK&F 96365, and/or LOE 908 are represented as a percentage of values in its absence. Data are presented as mean ± S.E.M. of three experiments.

	Discussion

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It is important to reveal the intracellular activation mechanisms of VICCs by ET-1. In this study, we focused on the effects of PI3K on these Ca²⁺ channels activation caused by ET-1 in CHO-ET_AR because PI3K plays important roles for stimulation of extracellular Ca²⁺ influx. Given 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 and LY 294002 seem not to have functions as Ca²⁺ channel blockers. Therefore, wortmannin and LY 294002 may be effectors for intracellular mechanisms involved in Ca²⁺ channel activation by ET-1. Moreover, PI3K seems to be required for the activation 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 inhibitory effects on PI3K, judging from the following data: 1) Wortmannin is generally accepted as a PI3K inhibitor (Ui et al., 1995). Moreover, at nanomolar concentrations, wortmannin acts specifically on PI3K (Yano et al., 1993). 2) Another PI3K inhibitor, LY 294002, also inhibited the wortmannin-sensitive part of ET-1-induced sustained increase in [Ca²⁺]_i. 3) In CHO-ET_AR, the IC₅₀ values (~30 nM) and maximal effective concentration (1 µM) of wortmannin for ET-1-induced sustained increase in [Ca²⁺]_i (Fig. 2) were similar to those for ET-1-induced phosphatidylinositol triphosphate formation as an index of PI3K activity (Sugawara et al., 1996).

Because wortmannin partially suppressed ET-1-induced sustained increase in [Ca²⁺]_i (Figs. 1 and 2), ET-1 induces extracellular Ca²⁺ influx through VICCs via both PI3K-dependent and -independent pathways in CHO-ET_AR. Based on the sensitivity to SK&F 96365 and LOE 908, the wortmannin-resistant part of sustained increase in [Ca²⁺]_i was caused by Ca²⁺ influx through NSCC-1 (LOE 908-sensitive and SK&F 96365-resistant) (Fig. 4). Therefore, Ca²⁺ influx through NSCC-2 and SOCC are composed of wortmannin sensitive part. These results indicate that PI3K may play important roles for ET-1-induced activation of NSCC-2 and SOCC. The result that wortmannin failed to inhibit the activation of NSCC-1 by ET-1 in CHO-SerET_AR (Fig. 5) is consistent with this indication. Both NSCC-2 and SOCC activations by ET-1 involve G_q/PLC-dependent cascade and depend on mobilization of Ca²⁺ from the intracellular Ca²⁺ store in CHO-ET_AR (Kawanabe et al., 2002c). Moreover, it is generally accepted that G is involved in PI3K activation (Clapham and Neer, 1997; Vanhaesebroeck et al., 1997). Therefore, G as well as G may play important roles for NSCC-2 and SOCC activation by ET-1.

ET-1 induces mitogenic response in CHO-ET_AR, judging from results of MTT assay as described previously (Sugawara et al., 1996). The inhibitory action of SK&F 96365 or LOE 908 on ET-1-induced mitogenesis may be mediated by blockade of Ca²⁺ entry through VICCs for the following reasons: the IC₅₀ values of SK&F 96365 and LOE 908 for ET-1-induced mitogenesis and the extent of inhibition of the response by these blockers (Fig. 7) correlated well with those for the ET-1-induced [Ca²⁺]_i response (Kawanabe et al., 2001), and neither SK&F 96365 nor LOE 908 is considered to exert cytotoxic effects on quiescent cells, judging from data from MTT assay (Fig. 7). The ET-1-induced mitogenesis can be divided into three parts based on its pharmacology. The first part is sensitive to LOE 908 and resistant to SK&F 96365, the second is sensitive to both LOE 908 and SK&F 96365, and the third is resistant to LOE 908 and sensitive to SK&F 96365 (Fig. 7B). The pharmacological criteria indicate that the first part involves Ca²⁺ entry through NSCC-1, the second involves Ca²⁺ entry through NSCC-2, and the third involves Ca²⁺ entry through SOCC. Furthermore, the percentage contribution of Ca²⁺ entry through NSCC-1, NSCC-2, and SOCC to the mitogenesis is calculated to be 20, 40, and 40%, respectively (Fig. 7B). The inhibitory effects of wortmannin on ET-1-induced mitogenesis may be mediated by blockade of Ca²⁺ entry through NSCC-2 and SOCC for the following reasons: the IC₅₀ values of wortmannin for ET-1-induced mitogenesis (Fig. 8A) correlated well with those for the ET-1-induced [Ca²⁺]_i response (Fig. 2A), and the wortmannin-resistant part of ET-1-induced mitogenesis was dependent on the extracellular Ca²⁺ influx through NSCC-1, based on the sensitivity to SK&F 96365 and LOE 908 (SK&F 96365-resistant and LOE 908-sensitive) (Fig. 8B). Moreover, these results indicate that PI3K may be involved in the ET-1-induced mitogenesis by activating extracellular Ca²⁺ influx through NSCC-2 and SOCC.

In conclusion, NSCC-2 and SOCC are stimulated by ET-1 via PI3K-dependent cascade, whereas NSCC-1 is stimulated via PI3K-independent cascade. Moreover, PI3K is involved in the ET-1-induced mitogenesis that depends on the extracellular Ca²⁺ influx through SOCC and NSCC-2.