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Characterization of Ca²⁺ Channels and G Proteins Involved in Arachidonic Acid Release by Endothelin-1/Endothelin_A Receptor

Departments of Neurosurgery (Y.K., K.N., N.H.) and Pharmacology (Y.K., T.M.), Kyoto University Graduate School of Medicine, Sakyo-ku, Kyoto, Japan

Received March 24, 2003; accepted May 14, 2003

	Abstract

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Endothelin-1 (ET-1) activates two types of Ca²⁺-permeable nonselective cation channels (designated NSCC-1 and NSCC-2) and a store-operated Ca²⁺ channel (SOCC) in Chinese hamster ovary cells expressing endothelin_A receptors (CHO-ET_AR). These channels can be distinguished by their sensitivity to Ca²⁺ channel blockers 1-(-[3-(4-methoxyphenyl) propoxy]-4-methoxyphenethyl)-1H-imidazole hydrochloride (SK&F 96365) and (R,S)-(3,4-dihydro-6,7-dimethoxy-isochinolin-1-yl)-2-phenyl-N,N-di[2-(2,3,4-trimethoxyphenyl)ethyl]acetamid mesylate (LOE 908). NSCC-1 is sensitive to LOE 908 and resistant to SK&F 96365; NSCC-2 is sensitive to both blockers, and SOCC is resistant to LOE 908 and sensitive to SK&F 96365. In this study, we examined the mechanism of ET-1–induced arachidonic acid (AA) release. Both SK&F 96365 and LOE 908 inhibited ET-1–induced AA release with the IC₅₀ values correlated to those of ET-1–induced Ca²⁺ influx. Moreover, combined treatment with these blockers abolished ET-1–induced AA release. Wortmannin and LY294002, inhibitors of phosphoinositide 3-kinase (PI3K), partially inhibited ET-1–induced AA release. LOE 908, but not SK&F 96365, inhibited ET-1–induced AA release in wortmannin-treated CHO-ET_AR. ET-1 also induced AA release in CHO cells expressing ET_AR truncated at the carboxyl terminal downstream of Cys385 (CHO-ET_AR385) or an unpalmitoylated (Cys³⁸³ Cys^385–388 Ser³⁸³Ser^385–388) ET_AR (CHO-SerET_AR), each of which is coupled with G_q or G_s/G₁₂, respectively. In CHO-SerET_AR, a dominant-negative mutant of G₁₂ inhibited AA release. SK&F 96365 inhibited ET-1–induced AA release in CHO-ET_AR385, whereas LOE 908 inhibited it in CHO-SerET_AR. These results indicate the following: 1) ET-1–induced AA release depends on Ca²⁺ influx through NSCC-1, NSCC-2, and SOCC in CHO-ET_AR; 2) G_q and G₁₂ mediate AA release through ET_AR in CHO cells; and 3) PI3K is involved in ET-1–induced AA release, which depends on NSCC-2 and SOCC.

Biological actions of ET-1 are mediated by two distinct receptor subtypes, ET_AR and ET_BR, that belong to a family of G protein-coupled receptors (Arai et al., 1990; Sakurai et al., 1990). ET_AR are functionally coupled with G_q, G_s, and G₁₂ in CHO cells (Aramori and Nakanishi, 1992; Kawanabe et al., 2002c). Therefore, in the present study, we investigated which G protein subtypes were involved in ET-1–induced AA release. For this purpose, we used a dominant-negative mutant of G₁₂ (G₁₂G228A) and two types of mutated ET_AR designated ET_AR385 and SerET_AR to clarify the involvement of G_q, G_s, and G₁₂ in ET-1–induced AA release. ET_AR385 lacks a C terminus downstream of Cys³⁸⁵ and couples only with G_q in CHO cells (Kawanabe et al., 2002c). SerET_AR is unpalmitoylated because of substitution of all of the cysteine-to-serine residues (Cys³⁸³Cys^385–388Ser³⁸³Ser^385–388) and couples with G_s and G₁₂ in CHO cells (Kawanabe et al., 2002c). Moreover, ET-1 activates SOCC in CHO-ET_AR385 and NSCC-1 in CHO-SerET_AR (Kawanabe et al., 2002b).

	Materials and Methods

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Cell Culture. We used CHO-ET_AR, CHO-ET_AR385, and CHO-SerET_AR, which were constructed as described previously (Kawanabe et al., 2002b,c). CHO cells were maintained in Ham's F-12 medium supplemented with 10% fetal calf serum under a humidified 5% CO₂/95% air atmosphere.

[³H]Arachidonic Acid Release. The level of [³H]arachidonic acid release was determined as described previously (Perez et al., 1993). Briefly, cells in 100-mm dishes were incubated overnight with [³H]arachidonic acid (final concentration, 1 µCi/ml). After washing, ET-1 was added for 5 min. The medium was then removed, acidified with 100 µl of 1 N formic acid, and extracted with 3 ml of chloroform. The extracts were evaporated to dryness, resuspended in 50 µl of chloroform, and applied to silica gel plates for thin-layer chromatography (Merck, Darmstadt, Germany). The plates were developed in heptane/diethyl ether/acetic acid/water (v/v, 75:25:4). The distance of movement was visualized with iodine vapor. The location of arachidonic acid was verified with the use of a purified arachidonic acid (PerkinElmer Life Sciences, Boston, MA). The plate was scraped, and the radioactivity was counted with use of a liquid scintillation counter.

Transfection of G₁₂G228A. We used G₁₂G228A, which was constructed as described previously (Kawanabe et al., 2002b,c). For transient expression, cells were transfected with plasmid (100 ng/µl) encoding for G₁₂G228A by the MBS Mammalian Transfection Kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. After 24 h of incubation, we used these cells for measurement of [³H]arachidonic acid release.

Drugs. LOE 908 was kindly provided by Boehringer Ingelheim GmbH (Ingelheim, Germany). All other chemicals were of reagent grade and were obtained commercially.

Statistical Analysis. All results were expressed as mean ± S.E.M. The data were subjected to a two-way analysis of variance. 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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Effects of ET-1 on AA Release in CHO-ET_AR. ET-1 induced AA release in a concentration-dependent manner with an EC₅₀ value of approximately 1 nM, and maximal effects were observed at concentrations 10 nM (Fig. 1A). In the absence of extracellular Ca²⁺, the magnitudes of ET-1–induced AA release were near the basal level (Fig. 1B). ET-1–induced AA release was abolished by BQ123, a specific antagonist of ET_AR, but it was unaffected by BQ788, a specific antagonist of ET_BR (Fig. 1B). Moreover, ET-1–induced AA release was inhibited by arachydonyl trifluoromethyl ketone (AACOCF₃), a selective inhibitor of cPLA₂.

View larger version (24K): [in this window] [in a new window]   Fig. 1. A, effects of various concentrations of ET-1 on AA release in CHO-ETAR. The cells were stimulated with increasing concentrations of ET-1 for 5 min. B, effects of extracellular Ca2+, BQ123, BQ788, and AACOCF3 on ET-1–induced AA release in CHO-ETAR. The cells were pretreated with or without 5 µM BQ123, 5 µM BQ788, or 50 µM AACOCF3 for 30 min and incubated with 10 nM ET-1 for 5 min. AA release was determined as described under Materials and Methods. Data presented are the mean ± S.E.M. of three determinations, each done in triplicate.

Effects of SK&F 96365 and LOE 908 on ET-1–Induced AA Release in CHO-ET_AR. SK&F 96365 inhibited ET-1–induced AA release in a concentration-dependent manner with IC₅₀ values of approximately 1 µM (Fig. 2A). Maximal inhibition was observed at concentrations 10 µM. The extent of maximal inhibition was approximately 80% of ET-1–induced AA release (Fig. 2B). Similarly, LOE 908 inhibited ET-1–induced AA release in a concentration-dependent manner with IC₅₀ values of approximately 1 µM, and maximal inhibition was observed at concentrations 10 µM (Fig. 2A). The extent of maximal inhibition was approximately 60% of ET-1–induced AA release (Fig. 2B). Moreover, the combined treatment with maximal effective concentration (10 µM) of SK&F 96365 and LOE 908 completely inhibited ET-1–induced AA release (Fig. 2B).

View larger version (22K): [in this window] [in a new window]   Fig. 2. A, effects of various concentrations of SK&F 96365 and LOE 908 on ET-1–induced AA release in CHO-ETAR. The cells were incubated for 15 min with various concentrations of SK&F 96365 () or LOE 908 () and then stimulated with 10 nM ET-1 for 5 min. B, effects of a maximal effective concentration (10 µM) of SK&F 96365 and LOE 908 on ET-1–induced AA release in CHO-ETAR. AA release was determined as described under Materials and Methods. Data presented are the mean ± S.E.M. of three determinations, each done in triplicate. #, P < 0.05, significantly different from the control values stimulated by ET-1 in each experiment.

Effects of PI3K Inhibitors on ET-1–Induced AA Release in CHO-ET_AR. Wortmannin inhibited ET-1–induced AA release in a concentration-dependent manner with IC₅₀ values of approximately 30 nM, and the maximal inhibition (80% of control) was seen at concentrations 1 µM (Fig. 3). ET-1–induced AA release in CHO-ET_AR preincubated with 1 µM wortmannin was inhibited by 10 µM LOE 908 (Fig. 3B). In contrast, 10 µM SK&F 96365 failed to inhibit ET-1–induced AA release in CHO-ET_AR preincubated with 1 µM wortmannin (Fig. 3B). We also used LY 294002, an inhibitor of PI3K, to evaluate the effects of PI3K on ET-1-induced AA release. LY 294002 at 50 µM also inhibited ET-1-induced AA release (Fig. 3B). ET-1-induced AA release was also sensitive to LOE 908 and resistant to SK&F 96365 in CHO-ET_AR preincubated with 50 µM LY 294002 (data not shown).

View larger version (23K): [in this window] [in a new window]   Fig. 3. A, effects of various concentrations of wortmannin on ET-1–induced AA release in CHO-ETAR. The cells were incubated for 15 min with various concentrations of wortmannin and then stimulated with 10 nM ET-1 for 5 min. B, effects of 50 µM LY294002 on ET-1–induced AA release in CHO-ETAR and maximal effective concentrations (10 µM) of SK&F 96365 and LOE 908 on ET-1–induced AA release in CHO-ETAR treated with 1 µM wortmannin. AA release was determined as described under Materials and Methods. Data presented are the mean ± S.E.M. of three determinations, each done in triplicate. #, P < 0.05, significantly different from the control values stimulated by ET-1 in each experiment. ##, P < 0.05, significantly different from the control values stimulated by ET-1 in the presence of wortmannin in each experiment.

Effects of ET-1 on AA Release in CHO-ET_AR385 and CHO-SerET_AR. ET-1 induced AA release in both CHO-ET_AR385 and CHO-SerET_AR (Fig. 4). However, the threshold concentrations of ET-1 for the induction of AA release were different. In CHO-ET_AR385, ET-1 induced AA release in a concentration-dependent manner with EC₅₀ values of between 1 and 10 nM, and maximal effects (approximately a 3.5-fold increase) were observed at concentrations 10 nM (Fig. 4). Because CHO-ET_AR385 couples with G_q but not with G_S or G₁₂ (Kawanabe et al., 2002c), G_q plays essential roles on ET-1–induced AA release in these cells. In CHO-SerET_AR, ET-1 induced AA release in a concentration-dependent manner with EC₅₀ values of between 0.01 and 0.1 nM, and maximal effects (approximately a 2-fold increase) were observed at concentrations 0.1 nM (Fig. 4).

View larger version (16K): [in this window] [in a new window]   Fig. 4. A, effects of various concentrations of ET-1 on AA release in CHO-ETAR385 or CHO-SerETAR. The cells were stimulated with increasing concentrations of ET-1 for 5 min. B, effects of a maximal effective concentration (10 nM) ET-1 on AA release in CHO-ETAR385 () and CHO-SerETAR (). AA release was determined as described under Materials and Methods. Data presented are the mean ± S.E.M. of three determinations, each done in triplicate.

Effects of G_s and G₁₂ in ET-1–Induced AA Release in CHO-SerET_AR. Because CHO-SerET_AR couples with G_S and G₁₂ (Kawanabe et al., 2002c), we examined the effects of G_S and G₁₂ on ET-1–induced AA release in these cells. Cholera toxin activates G_S via a receptor-independent mechanism (Belevych et al., 2001). Treatment with 1 µg/ml cholera toxin failed to induce AA release (Fig. 5A). Moreover, ET-1–induced AA release was not influenced by cholera toxin (Fig. 5A).

View larger version (23K): [in this window] [in a new window]   Fig. 5. A, effects of cholera toxin on AA release in resting CHO-SerETAR and CHO-SerETAR treated with ET-1. The cells were incubated for 60 min with 1 µg/ml cholera toxin and then stimulated with or without 10 nM ET-1 for 5 min. B, effects of G12G228A on ET-1–induced AA release in CHO-SerETAR. CHO-SerETAR cells were transfected with G12G228A transiently as described under Materials and Methods. The cells were incubated with 10 nM ET-1 for 5 min. AA release was determined as described under Materials and Methods. Data presented are the mean ± S.E.M. of three determinations, each done in triplicate. #, P < 0.05, significantly different from the control values stimulated by ET-1 in the absence of G12G228A in each experiment.

G₁₂G228A was transiently transfected to evaluate the role of G₁₂. For this purpose, we used the MBS Mammalian Transfection Kit (Stratagene). When we transfected green fluorescent protein with this method, approximately 65% of the cells were green fluorescent protein-positive (data not shown). The magnitudes of ET-1–induced AA release in CHO-SerET_AR transfected with G₁₂G228A were approximately 70% of those in CHO-SerET_AR (Fig. 5B). The magnitudes of ET-1–induced AA release in CHO-SerET_AR transfected with only vector were similar to those in CHO-SerET_AR (data not shown).

Effects of SK&F 96365, LOE 908, and Wortmannin on ET-1–Induced AA Release in CHO-ET_AR385 and CHO-SerET_AR. In CHO-ET_AR385, ET-1–induced AA release was inhibited by SK&F 96365 in a concentration-dependent manner with IC₅₀ values of approximately 1 µM, and complete inhibition was observed at concentrations 10 µM (Fig. 6). On the other hand, LOE 908 failed to inhibit ET-1– induced AA release in CHO-ET_AR385 (Fig. 6). In addition, ET-1 failed to induce AA release in CHO-ET_AR385 pretreated with 1 µM wortmannin (Fig. 6B). In CHO-SerET_AR, ET-1–induced AA release was inhibited by LOE 908 in a concentration-dependent manner with IC₅₀ values of approximately 1 µM, and a complete inhibition was observed at concentrations 10 µM (Fig. 7). On the other hand, SK&F 96365 failed to inhibit ET-1–induced AA release in CHO-ET_AR385 (Fig. 7). Moreover, the magnitudes of ET-1–induced AA release in CHO-SerET_AR pretreated with 1 µM wortmannin were similar to those observed in CHO-SerET_AR (Fig. 7B). LOE 908 also inhibited this wortmannin-resistant part of ET-1–induced AA release (Fig. 7B).

View larger version (25K): [in this window] [in a new window]   Fig. 6. A, effects of various concentrations of SK&F 96365 and LOE 908 on ET-1–induced AA release in CHO-ETAR385. The cells were incubated for 15 min with various concentrations of SK&F 96365 () or LOE 908 () and then stimulated with 10 nM ET-1 for 5 min. B, effects of a maximal effective concentration of SK&F 96365 (10 µM), LOE 908 (10 µM), and/or wortmannin (1 µM) on ET-1–induced AA release in CHO-ETAR385. AA release was determined as described under Materials and Methods. Data presented are the mean ± S.E.M. of three determinations, each done in triplicate.

View larger version (24K): [in this window] [in a new window]   Fig. 7. A, Effects of various concentrations of SK&F 96365 and LOE 908 on ET-1–induced AA release in CHO-SerETAR. The cells were incubated for 15 min with various concentrations of SK&F 96365 () or LOE 908 () and then stimulated with 10 nM ET-1 for 5 min. B, effects of a maximal effective concentration of SK&F 96365 (10 µM), LOE 908 (10 µM), and/or wortmannin (1 µM) on ET-1–induced AA release in CHO-SerETAR. AA release was determined as described under Materials and Methods. Data presented are the mean ± S.E.M. of three determinations, each done in triplicate.

	Discussion

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ET-1 induces AA release in CHO-ET_AR (Fig. 1A). BQ123 inhibited ET-1–induced AA release, whereas BQ788 failed to inhibit it (Fig. 1B). Therefore, ET-1–induced AA release is mediated by ET_AR. Based on the sensitivity to AACOCF₃ (Fig. 1B), ET-1 induces AA release through cPLA₂ activation. These results are in agreement with the observations in many cell types that agonist-induced AA release is mainly mediated by cPLA₂ (Ui et al., 1995; Wu-Wong et al., 1996; Kramer and Sharp, 1997; Trevisi et al., 2002). In the absence of extracellular Ca²⁺, the magnitudes of ET-1–induced AA release were near the basal level (Fig. 1B). Therefore, extracellular Ca²⁺ influx plays a critical role in ET-1–induced AA release in CHO-ET_AR as was also seen in vascular smooth muscle cells (Wu-Wong et al., 1996). With the use of SK&F 96365 and LOE 908, we attempted to determine the effects of extracellular Ca²⁺ influx through VICCs on ET-1–induced AA release. The inhibitory actions of SK&F 96365 and LOE 908 on ET-1–induced AA release are considered to be mediated by the blockade of Ca²⁺ entry through VICCs for the following two reasons. First, in our recent work using patch-clamp and [Ca²⁺]_i monitoring, ET-1 activates three types of VICCs in CHO-ET_AR, namely NSCC-1, NSCC-2, and SOCC. In addition, LOE 908 was able to block both NSCC-1 and NSCC-2, whereas SK&F 96365 blocked NSCC-2 and SOCC (Kawanabe et al., 2001). Second, the IC₅₀ values of these blockers for ET-1–induced AA release (Fig. 2A) correlated well with those for ET-1–induced extracellular Ca²⁺ influx (Kawanabe et al., 2001). Three types of VICC seem to be involved in ET-1–induced AA release in terms of its sensitivity to SK&F 96365 and LOE 908 (Fig. 2B): the first type of Ca²⁺ channel is sensitive to LOE 908 and is resistant to SK&F 96365; the second type is sensitive to both LOE 908 and SK&F 96365; and the third type is resistant to LOE 908 and sensitive to SK&F 96365. Because of their pharmacological characteristics, these channels are considered to be NSCC-1, NSCC-2, and SOCC, respectively. The magnitudes of ET-1–induced AA release that were inhibited by the combined treatment with SK&F 96365 and LOE 908 were similar to those in the absence of extracellular Ca²⁺ (Figs. 1B and 2B). Therefore, extracellular Ca²⁺ influx through NSCC-1, NSCC-2, and SOCC plays an important role in ET-1–induced AA release in CHO-ET_AR.

PI3K is involved in the activation of NSCC-2 and SOCC by ET-1 in CHO-ET_AR (Kawanabe et al., 2002a). Therefore, we investigated the effects of PI3K on ET-1–induced AA release in CHO-ET_AR. The inhibitory effects of wortmannin on ET-1–induced AA release may be caused by its inhibitory effects on PI3K, as determined 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, LY294002, also inhibited the wortmannin-sensitive ET-1–induced AA release (Fig. 3B); and 3) the IC₅₀ values (30 nM) and maximal effective concentration (1 µM) of wortmannin for ET-1–induced AA release (Fig. 3A) were similar to those for ET-1–induced phosphatidylinositol triphosphate formation, which was measured as an index of PI3K activity (Sugawara et al., 1996). Moreover, the IC₅₀ values and maximal effective concentration of wortmannin for ET-1–induced AA release (Fig. 3A) were also similar to those for ET-1–induced Ca²⁺ influx (Kawanabe et al., 2001). The wortmannin-resistant part of ET-1–induced AA release is dependent on extracellular Ca²⁺ influx through NSCC-1, which is determined by the sensitivity to SK&F 96365 and LOE 908 (SK&F 96365-resistant and LOE 908-sensitive) (Fig. 3B). Therefore, the wortmannin-sensitive part of ET-1–induced AA release is dependent on extracellular Ca²⁺ influx through NSCC-2 and SOCC. These results indicate that PI3K is involved in the ET-1–induced AA release, which depends on NSCC-2 and SOCC.

To identify the G proteins involved in the AA release by ET-1, we used CHO-ET_AR385 and CHO-SerET_AR. CHO-ET_AR385 and CHO-SerET_AR couple with G_q and with G_s/G₁₂, respectively (Kawanabe et al., 2002c). ET-1 induced AA release in CHO-ET_AR385 (Fig. 4). This result indicates that the G_q pathway is involved in ET-1–induced AA release. In addition, ET-1 also induced AA release in CHO-SerET_AR (Fig. 4). Therefore, either G_s and/or G₁₂ is required for ET-1–induced AA release. Cholera toxin had no effect on the resting AA release and in ET-1–induced AA release in CHO-SerET_AR (Fig. 5A). These results indicate that ET-1–induced AA release is not mediated by the G_s-dependent pathway. Disruption of signaling through endogenous G₁₂ by G₁₂G228A inhibited ET-1–induced AA release in CHO-SerET_AR (Fig. 5B), indicating that the activation of AA release is mediated by G₁₂. Therefore, G₁₂ and G_q play important roles in ET-1–induced AA release. These results are consistent with the previous report, which demonstrated that the GTPase-deficient activated mutant of G₁₂ stimulates AA release in NIH 3T3 cells (Dermott et al., 1999). ET-1–induced AA release was not inhibited completely by G₁₂G228A in this study (Fig. 5B). We believe that this is because G₁₂G228A is not transfected to all cells. However, another possibility is that ET-1 induces AA release with another unknown pathway in CHO-SerET_AR. Further research is necessary to confirm this. As determined from the sensitivity to SK&F 96365 and LOE 908 (SK&F 96365-sensitive and LOE 908-resistant), ET-1–induced AA release in CHO-ET_AR385 is dependent on extracellular Ca²⁺ influx through SOCC (Fig. 6). On the other hand, ET-1–induced AA release is dependent on extracellular Ca²⁺ influx through NSCC-1 in CHO-SerET_AR (SK&F 96365-resistant and LOE 908-sensitive) (Fig. 7). These results are in agreement with the previous observations that ET-1 activates SOCC in CHO-ET_AR385 or NSCC-1 in CHO-SerET_AR (Kawanabe et al., 2002b) and that ET-1–induced SOCC or NSCC-1 activation is dependent on the G_q-dependent pathway or the G₁₂-dependent pathway, respectively (Kawanabe et al., 2002b). The EC₅₀ values and maximal effects of ET-1 for AA release between CHO-ET_AR385 and CHO-SerET_AR are different (Fig. 4A). These differences seem to be the result of the sensitivity of NSCC-1 and SOCC to ET-1. NSCC-1 is activated by 0.1 nM ET-1, whereas SOCC is activated by 10 nM ET-1 in CHO-ET_AR (Kawanabe et al., 2001). These data also support the conclusion that extracellular Ca²⁺ influx plays an essential role in ET-1–induced AA release. Because both the G_q and G₁₂ pathways are necessary for NSCC-2 activation by ET-1 (Kawanabe et al., 2002b), ET-1 failed to activate NSCC-2 in CHO-ET_AR385 and CHO-SerET_AR. Therefore, the involvement of NSCC-2 in ET-1–induced AA release was not detected in these cells. However, taken from the data using CHO-ET_AR, we concluded that NSCC-2 was also involved in ET-1–induced AA release.

In conclusion, extracellular Ca²⁺ influx through NSCC-1, NSCC-2, and SOCC plays an essential role in ET-1–induced AA release in CHO-ET_AR. G_q and G₁₂ are involved in ET-1–induced AA release through ET_AR. In addition, PI3K acts as a regulator of ET-1–induced AA release, which depends on the extracellular Ca²⁺ influx through SOCC and NSCC-2.

	Acknowledgements

 
We thank Boehringer Ingelheim GmbH (Ingelheim, Germany) for the kind donation of LOE 908. We also thank Drs. Makoto Taketo, Masanobu Oshima, and Tomo-o Ishikawa (Department of Pharmacology, Kyoto University Graduate School of Medicine, Kyoto, Japan) for technical support.

	Footnotes

 
This study was supported by a grant from the Smoking Research Foundation, Japan, and by the Uehara Memorial Foundation Fellowship, Tokyo, Japan.

ABBREVIATIONS: AA, arachidonic acid; AACOCF₃, arachydonyl trifluoromethyl ketone; CHO, Chinese hamster ovary; [Ca²⁺]_i, intracellular free Ca²⁺ concentration; CHO-ET_AR, Chinese hamster ovary cells expressing endothelin_A receptors; CHO-ET_AR385, Chinese hamster ovary cells that express human endothelin_A receptor truncated at the carboxyl-terminal downstream of Cys385; CHO-SerET_AR, Chinese hamster ovary cells that express an unpalmitoylated (Cys³⁸³Cys^385–388 Ser³⁸³Ser^385–388) human endothelin_A receptor; cPLA₂, cytosolic phospholipase A₂; ET-1, endothelin-1; G₁₂G228A, dominant-negative mutant of G₁₂; NSCC, nonselective cation channel; PI3K, phosphoinositide 3-kinase; PLA₂, phospholipase A₂; SOCC, store-operated Ca²⁺ channel; VICC, voltage-independent Ca²⁺ channel; SK&F 96365, 1-(-[3-(4-methoxyphenyl) propoxy]-4-methoxyphenethyl)-1H-imidazole hydrochloride; LOE 908, (R,S)-(3,4-dihydro-6,7-dimethoxy-isochinolin-1-yl)-2-phenyl-N,N-di[2-(2,3,4-trimethoxyphenyl)ethyl]acetamid mesylate; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; BQ123, cyclo(D-Trp-D-Asp-Pro-D-Val-Leu-)Na⁺; BQ788, 2-6-dimethylpiperidinecarbonyl--methyl-Leu-N_in-[methoxycarbonyl]-D-Trp-D-Nle.

Address correspondence to: Yoshifumi Kawanabe, M.D., Ph.D., Renal Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Harvard Institutes of Medicine, Room 520, 77 Avenue Louis Pasteur, Boston, MA 02115. E-mail: ykawanabe{at}rics.bwh.harvard.edu

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