Introduction

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Endothelin-1 (ET-1), a 21-amino acid peptide, is one of the most potent endogenous vasoconstricting agents (Yanagisawa et al., 1988). Subsequent studies have described its multiple and wide-ranging biological activities, including modulation of neurotransmission (Koseki et al., 1989) and stimulation of cell proliferation (Komuro et al., 1988; Shichiri et al., 1991). Recent reports have demonstrated that extracellular Ca²⁺ influx through voltage-independent Ca²⁺ channels (VICCs) plays a critical role for ET-1-induced contraction of rat aorta (Zhang et al., 1999) and ET-1-induced proliferation of vascular smooth muscle cells (VSMCs) (Kawanabe et al., 2002a). Thus, it is important to elucidate activation mechanisms of VICCs by ET-1. Biological actions of ET-1 are mediated by two distinct receptor subtypes: endothelin_A and endothelin_B receptors (ET_ARs and ET_BRs, respectively), which belong to a family of G protein-coupled receptors (Arai et al., 1990; Sakurai et al., 1990). Therefore, in the present study, we focused on investigating which G protein subtypes were involved in the activation of each Ca²⁺ channel by ET-1.

Transfection and functional expression of wild-type or mutant ET_AR cDNAs into the same cell type provides a model system for study of the precise characteristics of signal transduction by a single receptor subtype. We used Chinese hamster ovary (CHO) cells stably expressing wild-type or mutant ET_ARs in this study. We have recently shown that a sustained increase in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) caused by ET-1 results from Ca²⁺ entry through three types of VICCs into CHO cells stably expressing wild-type ET_AR (CHO-ET_AR): two types of Ca²⁺-permeable nonselective cation channels (designated NSCC-1 and NSCC-2) and a store-operated Ca²⁺ channel (SOCC) (Kawanabe et al., 2001). In particular, these channels can be distinguished using Ca²⁺ channel blockers such as SK&F 96365 and LOE 908. Thus, 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 the SOCC is resistant to LOE 908 and sensitive to SK&F 96365 (Kawanabe et al., 2001). The VICCs activated by ET-1 in CHO-ET_AR are pharmacologically identical to those in VSMCs (Kawanabe et al., 2002a). Therefore, CHO-ET_AR may be a good model for studying the mechanism of activation of VICCs. ET_ARs are functionally coupled with G_q and G_s in CHO cells (Aramori and Nakanishi, 1992). Activation of G_q and G_s causes stimulation of phospholipase C (PLC) and adenylyl cyclase, respectively (Aramori and Nakanishi, 1992). In addition, ET_ARs also couple with G₁₂ via its C terminus to induce actin stress fiber formation in CHO cells (Kawanabe et al., 2002b). In the present study, we used a dominant-negative mutant of G₁₂ and two types of mutated ET_ARs designated ET_AR385 and SerET_AR to clarify the involvement of G_q, G_s, and G₁₂ for Ca²⁺ channel activation by ET-1. ET_AR385 lacks a C terminus downstream of Cys³⁸⁵ and couples only with G_q in CHO cells (Kawanabe et al., 2002b). SerET_AR is unpalmitoylated because of substitution of all the cysteine residues to serine (Cys³⁸³Cys^385-388Ser³⁸³Ser^385-388) and couples with G_s and G₁₂ in CHO cells (Kawanabe et al., 2002b).

	Experimental Procedures

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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). The K_d (picomolar) and B_max (picomoles per milligram of protein) values for CHO-ET_AR, CHO-ET_AR385, and CHO-SerET_AR were 52.8 ± 2.4 and 1.08 ± 0.16, 49.5 ± 4.3 and 1.12 ± 0.08, and 70.2 ± 4.4 and 1.04 ± 0.14, respectively. CHO cells were maintained in Ham's F12 medium supplemented with 10% fetal calf serum under a humidified 5% CO₂/95% air atmosphere.

Measurement of Intracellular Free Ca²⁺ Concentration. [Ca²⁺]_i was measured using the fluorescent probe fluo-3. The measurement of fluorescence by a CAF 110 spectrophotometer (Jasco, Tokyo, Japan) was performed as described previously (Kawanabe et al., 2001).

Microinjection. For microinjection, cells were seeded onto glass coverslips coated with fibronectin (Iwaki Glass, Chiba, Japan), which were marked with a cross to facilitate the localization of injected cells, and were incubated overnight in Ham's F12 medium containing 1% fetal calf serum. Microinjection of G₁₂G228A, constructed as described previously (Kawanabe et al., 2002b), was performed using a Zeiss microinjection system (Carl Zeiss). Plasmid (100 ng/µl) was used for microinjection into the cell nuclei.

Materials. Boehringer Ingelheim GmbH (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 Research Laboratories (Plymouth Meeting, PA); fluo-3/AM from Dojin Laboratories (Kumamoto, Japan); and U73122 from Funakoshi (Tokyo, Japan).

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

	Results

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Basic Properties of the ET-1-Induced Increase in [Ca²⁺]_i in CHO-ET_AR, CHO-ET_AR385, and CHO-SerET_AR. ET-1 induced a biphasic increase in [Ca²⁺]_i in CHO-ET_AR, consisting of an initial transient phase and a subsequent sustained phase (Fig. 1A). Both the transient and sustained increase in [Ca²⁺]_i were dependent on the concentrations of ET-1 with EC₅₀ values of approximately 1 nM, and they reached the maximal value at concentrations 10 nM (Fig. 1, D and E).

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Original tracings illustrating the effects of ET-1 on the increase in [Ca2+]i in CHO-ETAR (A), CHO-ETAR385 (B), and CHO-SerETAR (C). The cells were loaded with fluo-3 and stimulated with 10 nM ET-1 at the time indicated by horizontal bars. Effects of various concentrations of ET-1 on the transient increase in [Ca2+]i (D) and the sustained increase in [Ca2+]i (E) in CHO-ETAR, CHO-ETAR385, and CHO-SerETAR. , CHO-ETAR; , CHO-ETAR385, and , CHO-SerETAR. Each point represents the mean ± S.E.M. of five experiments.

View larger version (36K): [in this window] [in a new window]   Fig. 2.   Original tracings illustrating the effects of maximally effective concentration of LOE 908 and SK&F 96365 on the ET-1-induced sustained increase in [Ca2+]i in CHO-ETAR (A), CHO-ETAR385 (B), and CHO-SerETAR (C). The cells were loaded with fluo-3 and stimulated with 10 nM ET-1 at the time indicated by horizontal bars. After [Ca2+]i reached a steady-state, 10 µM LOE 908 or 10 µM SK&F 96365 was sequentially added, as indicated by horizontal bars. Effects of maximally effective concentration of LOE 908, SK&F 96365, and their combination on the ET-1-induced sustained increase in [Ca2+]i in CHO-ETAR (D), CHO-ETAR385 (E), and CHO-SerETAR (F). The experimental protocols were described under Materials and Methods, and the values of [Ca2+]i after the addition of 10 µM LOE 908 and/or 10 µM SK&F 96365 were determined. Each point represents the mean ± S.E.M. of five experiments.

Pharmacological Identification of Ca²⁺ Channels Activated by ET-1 in CHO-ET_AR, CHO-ET_AR385, and CHO-SerET_AR. As described previously (Kawanabe et al., 2001), in CHO-ET_AR, the ET-1-induced sustained increase in [Ca²⁺]_i was partially suppressed by the maximally effective concentration (10 µM) of either SK&F 96365 or LOE 908, and it was abolished by combined treatment with both blockers (Fig. 2, A and D). In CHO-ET_AR385, the ET-1-induced sustained increase in [Ca²⁺]_i was completely inhibited by 10 µM SK&F 96365, whereas LOE 908 at concentrations up to 10 µM had no effects (Fig. 2, B and E). In CHO-SerET_AR, the ET-1-induced sustained increase in [Ca²⁺]_i was completely inhibited by 10 µM LOE 908, whereas SK&F 96365 at concentrations up to 10 µM had no effects (Fig. 2, C and F).

Effects of Inhibition of PLC on the Species of ET-1-Activated Ca²⁺ Channels in CHO-ET_AR. In CHO-SerET_AR, coupling between the receptor and G_q is missing, and hence, PLC as an effector of G_q cannot be activated upon stimulation of the receptor. To mimic the stimulation in CHO-SerET_AR and confirm that PLC actually acts as an effector for activation of Ca²⁺ channels, we used U73122, a PLC blocker. Previous reports demonstrated that 5 to 10 µM U73122 inhibits PLC activation completely (Okamoto et al., 1995; Kanki et al., 2001). ET-1 stimulated [³H]inositol phosphates (IPs) formation in CHO-ET_AR (Kawanabe et al., 2001). On the other hand, ET-1 failed to induce [³H]IPs formation in CHO-ET_AR treated with 5 µM U73122 (data not shown). ET-1 at 10 nM induced only the sustained increase in [Ca²⁺]_i in CHO-ET_AR treated with 5 µM U73122 (Fig. 3, A and B). The magnitude of the sustained increase in [Ca²⁺]_i was approximately 20% of that in the absence of U73122. This sustained increase in [Ca²⁺]_i was completely inhibited by 10 µM LOE 908, whereas SK&F 96365 at concentrations up to 10 µM had no effects (Fig. 3, A and B).

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Original tracings illustrating the effects of maximally effective concentration of LOE 908 (A) and SK&F 96365 (B) on the ET-1-induced sustained increase in [Ca2+]i in CHO-ETAR pretreated with U73122. The cells loaded with fluo-3 were incubated with 5 µM U73122 for 10 min before stimulation with 10 nM ET-1. After [Ca2+]i reached a steady state, 10 µM LOE 908 or 10 µM SK&F 96365 was added at the time indicated by horizontal bars. Original tracings illustrating the effects of dibutyryl cAMP on the resting [Ca2+]i (C) and the ET-1-induced sustained increase in [Ca2+]i (D) in CHO-SerETAR. C, the cells loaded with fluo-3 were stimulated by 1 mM dibutyryl cAMP at the time indicated by the horizontal bar. D, the cells loaded with fluo-3 were stimulated by 10 nM ET-1. After [Ca2+]i reached a steady state, 1 mM dibutyryl cAMP was added at the time indicated by the horizontal bar.

Effects of Dibutyryl cAMP on the Resting [Ca²⁺]_i and the ET-1-Induced Increase in [Ca²⁺]_i in CHO-SerET_AR. Because CHO-SerET_AR is coupled with G_s and G₁₂ (Kawanabe et al., 2002b), it is unknown which of the G proteins is involved in the activation of Ca²⁺ channels. To clarify whether G_s was involved in Ca²⁺ channel activation, we examined the effects of dibutyryl cAMP on the resting [Ca²⁺]_i and the ET-1-induced increase in [Ca²⁺]_i. A previous report demonstrated that 1 mM dibutyryl cAMP activates protein kinase A in CHO cells (Lee and Fraser, 1993). Dibutyryl cAMP at 1 mM failed to evoke an increase in [Ca²⁺]_i in CHO-SerET_AR (Fig. 3C). Moreover, the ET-1-induced sustained increase in [Ca²⁺]_i was not affected by 1 mM dibutyryl cAMP in CHO-SerET_AR (Fig. 3D). Dibutyryl cAMP also failed to affect the resting [Ca²⁺]_i and the ET-1-induced increase in [Ca²⁺]_i in CHO-ET_AR (data not shown).

Effects of G₁₂ on the ET-1-Induced Sustained Increase in [Ca²⁺]_i in CHO-ET_AR or CHO-SerET_AR. To confirm that G₁₂ is involved in the activation of Ca²⁺ channels, we investigated the effects of G₁₂G228A on the ET-1-induced increase in [Ca²⁺]_i in CHO-ET_AR and CHO- SerET_AR. In this experiment, G₁₂G228A was microinjected into CHO-ET_AR and CHO-SerET_AR, and the ET-1-induced increase in [Ca²⁺]_i in these cells was analyzed using microfluorimetry.

View larger version (39K): [in this window] [in a new window]   Fig. 4.   A, effects of a maximally effective concentration of SK&F 96365 (10 µM) or LOE 908 (10 µM) on ET-1-induced sustained increase in [Ca2+]i in CHO-ETAR microinjected with G12G228A. The sustained increase in [Ca2+]i in the presence of SK&F 96365 or LOE 908 is represented as a percentage of values in its absence. B, effects of G12G228A on ET-1-induced sustained increase in [Ca2+]i in CHO-SerETAR. The cells loaded with fluo-3 were stimulated by 10 nM ET-1. The sustained increase in [Ca2+]i in the presence of G12G228A is represented as a percentage of values in its absence. Data are presented as the mean ± S.E.M. of three experiments.

	Discussion

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As reported previously (Kawanabe et al., 2001), the ET-1-induced sustained increase in [Ca²⁺]_i in CHO-ET_AR results from extracellular Ca²⁺ influx through three types of VICCs: NSCC-1, NSCC-2, and SOCC. Pharmacological identification of these Ca²⁺ channels and calculation for contribution of Ca²⁺ influx through each channel are explained schematically in Fig. 5. We have pharmacologically defined these channels in CHO cells expressing human recombinant ET_AR and VSMCs expressing endogenous ET_AR and have found that the same Ca²⁺ channels are activated in these cells (Kawanabe et al., 2001, 2002a).

View larger version (37K): [in this window] [in a new window]   Fig. 5.   A and B, pharmacological identification of three types of voltage-independent Ca2+ channels activated by 10 nM ET-1 and calculation for contribution of Ca2+ influx through each channel in CHO-ETAR. The ET-1-induced sustained increase in [Ca2+]i in the presence of 10 µM LOE 908 and/or 10 µM SK&F 96365 are represented as a percentage of values in its absence. The contributions of SOCC and NSCC-1 are represented as X and Z, respectively. The contribution of NSCC-2 is represented as W-Z or Y-X.

In CHO-ET_AR385, the only Ca²⁺ channels activated by ET-1 are SOCCs, judging from the sensitivity of the ET-1-induced sustained increase in [Ca²⁺]_i to SK&F 96365 and LOE 908 (Fig. 2, B and E). Because CHO-ET_AR385 is coupled with G_q but not with G_s or G₁₂ (Kawanabe et al., 2002b), this result indicates that G_q is required (sufficient) for activation of SOCC. Furthermore, activation of SOCC in CHO-ET_AR is lost after treatment with U73122 (Fig. 3, A and B), indicating that PLC acts as an effector downstream of G_q. These results, taken together, show that SOCC is activated via a G_q/PLC-dependent pathway.

Conversely, the results obtained from CHO-ET_AR385 suggest that the activation of NSCC-1 and NSCC-2 requires G proteins other than G_q. Because CHO-ET_AR is coupled with G_s and G₁₂ in addition to G_q (Aramori and Nakanishi, 1992; Kawanabe et al., 2002b), activation of NSCC-1 and NSCC-2 might be mediated by either G_s or G₁₂. To address this point, we used CHO-SerET_AR, which couples with G_s and G₁₂ but not with G_q (Kawanabe et al., 2002b). In CHO-SerET_AR, ET-1 activated NSCC-1 but not NSCC-2, because of the pharmacology of the sustained increase in [Ca²⁺]_i (sensitive to LOE 908 and resistant to SK&F 96365) (Fig. 3, C and D). These results indicate that either G_s or G₁₂ is required for activation of NSCC-1, whereas either G_s, G₁₂, or both are not sufficient for activation of NSCC-2. Regarding a mechanism for activation of NSCC-1, dibutyryl cAMP alone was without effect in the resting [Ca²⁺]_i and the ET-1-induced sustained increase in [Ca²⁺]_i in CHO-SerET_AR (Fig. 3), excluding the possibility that activation of NSCC-1 is mediated by cAMP, a product of G_s/adenylate cyclase-dependent pathway. Moreover, disruption of signaling through endogenous G₁₂ by its dominant-negative mutant (G₁₂G228A) abrogated activation of NSCC-1 in CHO-SerET_AR as well as CHO-ET_AR (Fig. 4), indicating that activation of NSCC-1 is mediated by G₁₂. Taken together, these results strongly demonstrate that NSCC-1 is activated via a G₁₂-dependent pathway.

As for a mechanism for activation of NSCC-2, the channel was not activated in CHO-ET_AR pretreated with U73122 or in CHO-ET_AR microinjected with G₁₂G228A (Figs. 3 and 4). These results indicate that both a G_q/PLC-dependent pathway and a G₁₂-dependent pathway are required for activation of NSCC-2. In accordance with this conclusion, NSCC-2 was not activated after the stimulation of ET_AR lacking coupling with either G_q or G₁₂, i.e., SerET_AR and ET_AR385.

Signaling mechanisms downstream of G proteins are presently unknown. In the case of G_q, it is very likely that PLC is activated downstream of G_q, considering that CHO-ET_AR treated with U73122 (an inhibitor of PLC) mimicked CHO-SerET_AR (which lost the ability to couple with G_q) in terms of temporal pattern and magnitude of the ET-1-induced changes in [Ca²⁺]_i and species of activated channels. Stimulation of PLC leads to increased formation of inositol-1,4,5-trisphosphate and diacylglycerol (DAG). Inositol-1,4,5-trisphosphate acts on its receptor on sarcoplasmic reticulum as an intracellular Ca²⁺ store to release Ca²⁺ and subsequently deplete the store (Berridge, 1993). Thus, the store depletion could be a trigger for activation of the Ca²⁺ channels on the plasma membrane called capacitative Ca²⁺ channel (Thasreup et al., 1990). In fact, SOCC is activated in CHO-ET_AR and VSMCs, after depletion of the Ca²⁺ store by treatment with thapsigargin (an inhibitor of Ca²⁺-pump ATPase on the membrane of sarcoplasmic reticulum) (Kawanabe et al., 2001, 2002a). The concentrations of ET-1 that failed to induce a transient increase in [Ca²⁺]_i and IP accumulation did not induce SOCC activation (Kawanabe et al., 2001). Judging from these data, we concluded that the transient increase in [Ca²⁺]_i (depletion of Ca²⁺ from intracellular Ca²⁺ store) essentially follows the characteristics of SOCC. On the other hand, another second messenger, DAG, can directly activate Ca²⁺ channels such as transient receptor potential (Hofmann et al., 1999). Thus, DAG might be an effector downstream of G_q. In the case of G₁₂, several kinds of proteins are reported to be its effectors (Seasholtz et al., 1999). We have recently shown that stimulation of CHO-ET_AR with ET-1 induces actin stress fiber formation through G₁₂, and that downstream of G₁₂, a small GTP-binding protein Rho and -associated coiled-coil-forming protein kinase is activated (Kawanabe et al., 2002b). Thus Rho/Rho-associated coiled-coil-forming protein kinase might be a signal downstream of G₁₂. Additional study is needed to identify the effectors downstream of G_q and G₁₂ for activation of NSCC-1, NSCC-2, and SOCC.

In summary, stimulation of ET_AR with ET-1 activates NSCC-1, NSCC-2, and SOCC in CHO-ET_AR. NSCC-1 is activated via a G₁₂-dependent pathway, NSCC-2 is activated via G_q- and G₁₂-dependent pathways, and SOCC is activated via a G_q-dependent pathway.