Characterization of G proteins involved in activation of nonselective cation channels by endothelin_B receptor

Abstract

1. We recently demonstrated that endothelin-1 (ET-1) activates two types of Ca²⁺-permeable nonselective cation channels (NSCC-1 and NSCC-2) in Chinese hamster ovary cells expressing endothelin_B receptors (CHO-ET_BR) that couple with G_q and G_i. The purpose of the present study was to identify the G proteins involved in the activation of these Ca²⁺ channels by ET-1. For this purpose, we constructed CHO cells expressing an unpalmitoylated (Cys⁴⁰²Cys⁴⁰³ Cys⁴⁰⁵→Ser⁴⁰²Ser⁴⁰³Ser⁴⁰⁵) ET_BR (CHO-SerET_BR) and ET_BR truncated at the cytoplasmic tail downstream of Cys⁴⁰³ (CHO-ET_BRΔ403).

2. Based on the data obtained from actin stress fibre formation, CHO-ET_BR couple with G₁₃. Therefore, CHO-ET_BR couple with G_q, G_i and G₁₃. CHO-SerET_BR and CHO-ET_BRΔ403 couple with G₁₃ and G_q, respectively.

3. ET-1 activated NSCC-1 in CHO-ET_BR preincubated with phospholipase C (PLC) inhibitor, U73122, and in CHO-SerET_BR. On the other hand, ET-1 failed to activate Ca²⁺ channels in CHO-ET_BRΔ403. Microinjection of dominant negative mutants of G₁₃ (G₁₃G225A) abolished activation of NSCC-1 and NSCC-2 in CHO-ET_BR and that of NSCC-1 in CHO-SerET_BR.

4. Y-27632, a specific Rho-associated kinase (ROCK) inhibitor, did not affect the ET-1-induced transient and sustained increase in [Ca²⁺]_i in CHO-ET_BR.

5. These results indicate that (1) the cytoplasmic tail downstream of the palmitoylation sites of ET_BR, but not the palmitoylation site itself, is essential for coupling with G₁₃, (2) the activation mechanism of each Ca²⁺ channel by ET-1 is different in CHO-ET_BR. NSCC-1 activation depends on G₁₃-dependent cascade, and NSCC-2 activation depends on both G_q/PLC- and G₁₃-dependent cascades. Moreover, ROCK-dependent cascade is not involved in the activation of these channels.

Introduction

Endothelin-1 (ET-1) has a wide variety of biological effects on various tissues and cell types (Yanagisawa et al., 1988; Masaki, 1993) that are mediated by specific heterotrimetric guanine nucleotide-binding protein (G-protein)-coupled receptor (GPCR) subtypes, the endothelin_A receptor (ET_AR) and endothelin_B receptor (ET_BR) (Arai et al., 1990; Sakurai et al., 1990). When expressed in Chinese hamster ovary (CHO) cells, ET_AR couples with members of the G_q and G_s families and stimulates phospholipase C (PLC) and adenylyl cyclase, respectively. ET_BR couples with members of the G_q and G_i families and stimulates PLC and inhibits adenylyl cyclase, respectively (Aramori & Nakanishi, 1992; Takagi et al., 1995). Many GPCRs including ET_AR and ET_BR were shown to be palmitoylated at a cluster of cysteine residues located in the cytoplasmic tail (Horstmeyer et al., 1996; Okamoto et al., 1997). The functional role of palmitoylation and the cytoplasmic tail downstream of the palmitoylation site in coupling with G proteins has been studied for ET_AR and ET_BR in some detail. In the case of ET_BR, palmitoylation is essential for coupling of the receptor with both G_q and G_i, while the cytoplasmic tail downstream of the palmitoylation sites is also required for coupling with G_i (Okamoto et al., 1997). It is reported that both ET_AR and ET_BR can also couple with the G₁₂ subfamily, consisting of G₁₂ and G₁₃, in NIH3T3 cells (Mao et al., 1998). The G₁₂ subfamily has been shown to mediate important signaling pathways such as Rho/Rho-associated kinase (ROCK)-dependent formation of actin stress fibres (Buhl et al., 1995) and vascular smooth muscle cell contraction (Gohla et al., 2000). These reports suggested that the G₁₂ subfamily may play important roles in several ET-1-induced vascular disorders such as stroke or vasospasm. Thus, the control of G₁₂ subfamily activation may become a new treatment strategy for these conditions. Recently, it was shown that upon treatment with ET-1, ET_BR in fibroblast cell lines did not induce stress fiber formation (Gohla et al., 1999), whereas ET_BR in HEK 293 cells coupled to G₁₃ (Kitamura et al., 1999). These data imply that in some types of cells, stimulation of ET_BR can induce actin stress fibre formation via a member of G₁₂ family, although direct evidence is absent. Therefore, in the present study, we attempt to determine whether stimulation of ET_BR actually induces actin stress fibre formation in CHO cells expressing recombinant ET_BR (CHO-ET_BR), and if so, which subtypes of G proteins are involved in the formation. Furthermore, we analyse the domains of ET_BR that are necessary for coupling of the receptor with the G protein. For these purposes, we use dominant negative mutants of G₁₂ and G₁₃ as well as mutated ET_BRs that lose the ability to couple with G_q and/or G_i. Previous report demonstrated that CHO cells expressing ET_BR truncated at the cytoplasmic tail downstream of Cys⁴⁰³ (CHO-ET_BRΔ403) coupled with G_q but not with G_i, whereas CHO cells expressing the unpalmitoylated (Cys⁴⁰²Cys⁴⁰³Cys⁴⁰⁵→Ser⁴⁰²Ser⁴⁰³Ser⁴⁰⁵) ET_BR (CHO-SerET_BR) coupled with neither G_q nor G_i (Okamoto et al., 1997).

Recent reports demonstrated that ET-1-induced extracellular Ca²⁺ influx through voltage-independent Ca²⁺ channels (VICCs) plays a critical role for ET-1-induced vasoconstriction and cell proliferation (Zhang et al., 1999; Kawanabe et al., 2002). Thus, it is important to elucidate the activation mechanisms of VICCs by ET-1. We focused on investigating which G-protein subtypes were involved in activation of each Ca²⁺ channel by ET-1 in CHO-ET_BR. Transfection and functional expression of the cDNA clone for wild type or mutant ET_BRs into the same cell type provide a model system to study the precise characteristics of signal transduction by a single receptor subtype. We used CHO cells stably expressing wild-type or mutant ET_BRs in the present study. A recent report showed that ET-1 activates two types of Ca²⁺-permeable nonselective cation channels (designated NSCC-1 and NSCC-2) in CHO-ET_BR (Kawanabe et al., 2001). In particular, these channels can be distinguished using Ca²⁺ channel blockers, SK&F 96365 and LOE 908. Thus, 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 (Kawanabe et al., 2001). In the present study, we used a dominant negative mutant of G₁₃ and two types of mutated ET_BRs designated ET_BRΔ403 and SerET_BR to clarify the involvement of G_q, G_I and G₁₃ for Ca²⁺ channel activation by ET-1.

Methods

Mutagenesis

Wild-type G₁₂ and G₁₃ in pcDNA3(+) were kindly provided by Dr Manabu Negishi (Kyoto University, Japan). G₁₂G228A and G₁₃G225A, which were shown to be the dominant negative types (Gohla et al., 1999), were generated using the QuickChange™ site-directed mutagenesis kit (Stratagene, La Jolla, CA, U.S.A.). Mutations were verified by sequencing.

Cell culture

We used CHO-ET_BR, CHO-ET_BRΔ403 and CHO-SerET_BR, which were constructed as described previously (Okamoto et al., 1997). The K_D and B_max values of selected cell clones that were used in this study were 43±3 pM and 0.98±0.11 pmol (mg protein)⁻¹, respectively, for CHO-ET_BR; 122±10 pM and 1.66±0.12 pmol (mg protein)⁻¹, respectively, for CHO-ET_BRΔ403; and 38±3 pM and 1.29±0.10 pmol (mg protein)⁻¹, respectively, for CHO-SerET_BR. Cells were maintained in Ham's F-12 medium supplemented with 10% foetal calf serum (FCS) under a humidified 5% CO₂/95% air atmosphere.

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 incubated overnight in Ham's F-12 medium containing 1% FCS. Plasmids (100 ng μl⁻¹) encoding for G₁₂G228A and G₁₃G225A were microinjected into cell nuclei. As a control, expression plasmids without inserts were microinjected in an adjacent field on the same coverslip. Microinjection was performed using a manual microinjection system (Eppendorf, Hamburg, Germany) equipped with an Axiovert 100 inverted microscope (Carl-Zeiss, Frankfurt, Germany).

Stress fiber formation

After incubation of the cells with serum-free Ham's F-12 medium for 24 h, ET-1 was added at 37°C for 5 min. Cells were washed three times with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde in PBS at room temperature for 15 min. After being washed five times with PBS containing 0.1% Triton-X100 (PBS-Tx), the cells were incubated with fluorescein rhodamin-phalloidin (Molecular Probes, Eugene, OR, U.S.A.) in PBS-Tx (1 : 200) at room temperature for 10 min. After being washed five times with PBS-Tx, the labeled cells were mounted on glass slides and examined with an MRC 1024 laser-scanning confocal microscope (Bio-Rad, Hercules, CA, USA) equipped with an Axiovert 135 M inverted microscope (Carl-Zeiss, Frankfurt, Germany).

Measurement of intracellular free Ca²⁺ concentration ([Ca²⁺]_i)

[Ca²⁺]_i was measured using a fluorescent probe fluo-3. The measurement of fluorescence by a CAF 110 spectrophotometer (JASCO, Tokyo, Japan) was performed exactly as described in a previous report (Kawanabe et al., 2001).

Microfluorimetry of fluo-3 was done as described previously (Zhang et al., 1999). Briefly, the cells were seeded on 35-mm glass-bottomed plastic dishes (MatTek Corporation, Ashland MA, U.S.A.), which were marked with a cross to facilitate the localization of injected cells, were loaded with fluo-3 by incubating them with Ca²⁺-free Krebs-HEPES solution containing 10 μM fluo-3/AM for 30 min at 37°C under a reduced light. Ca²⁺-free Krebs-HEPES solution contained (in mM): NaCl 140, KCl 3, MgCl₂ 1, glucose 11 and HEPES 10 (pH 7.4, adjusted with NaOH). After washing with Krebs-HEPES solution (2.2 mM CaCl₂ was added to Ca²⁺-free Krebs-HEPES solution), they were kept in fresh Krebs-HEPES solution at 37°C for at least 30 min. Fluo-3 microfluorimetry was done at 25°C by an Attofluor Ratio- Vision real-time digital fluorescence analyser (Atto Instruments, Potomac, MD, U.S.A.) equipped with a Carl-Zeiss Axiovert-100 inverted epifluorescent microscope. A 100-W mercury burner served as the source of excitation. In measurement of [Ca²⁺]_i, fluo-3 was excited at 450–490 nm and fluorescence was detected at 515–565 nm. At the end of the experiment, ionomycin and subsequently EGTA were added at final concentrations of 10 μM and 10 mM, respectively, to obtain the fluorescence intensity maximum (F_max) and the fluorescence intensity minimum (F_min). [Ca²⁺]_i was determined from the equilibrium equation, [Ca²⁺]_i=K_d(F - F_min)/(F_max-F), where F was the experimental value of fluorescence and K_d was defined as 0.4 μM (Minta et al., 1989).

Drugs

Boehringer Ingelheim K.G. (Ingelheim, Germany) kindly provided LOE 908. (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide (Y-27632) was kindly provided by Welfide Corporation (Osaka, Japan). Chemicals were obtained from the following sources: ET-1 from the Peptide Institute (Osaka, Japan); rhodamin-phalloidin from Molecular Probes (Eugene, OR, U.S.A.); pertussis toxin and 1-(6-{[17β-3-methoxyestra-1,3,5(10)-trien-17-yl] amino}hexyl)-1H-pyrrole-2,5-dione (U73122) from Funakoshi Co. Ltd (Tokyo, Japan); SK&F 96365 from Biomol (Plymouth Meeting, PA, USA); and fluo-3/AM from Dojindo Laboratories (Kumamoto, Japan). All other chemicals were of reagent grade and were obtained commercially.

Statistical analysis

All results were expressed as mean±s.e.m.

Results

ET-1-induced actin stress fiber formation in CHO-ET_BR

We attempted to determine the structural basis for coupling of ET_BR with G₁₂/G₁₃ and subtypes of G proteins involved in ET-1-induced stress fibre formation. For these purposes, we examined the effects of inhibition of either one of the G protein-mediated signalling cascades by blockers and dominant negative mutants of G₁₂ or G₁₃ (G₁₂G228A or G₁₃G225A, respectively) on ET-1-induced actin stress fibre formation in CHO-ET_BRΔ403 and CHO-SerET_BR as well as CHO-ET_BR. As described previously (Gohla et al., 1999), G₁₂G228A inhibited ET-1-induced actin stress fibre formation in CHO cells expressing endothelin_A receptors (data not shown).

ET-1 induced actin stress fiber formation in CHO-ET_BR (Figure 1B). In contrast, ET-1 failed to induce stress fibre formation in CHO-ET_BR that had been preincubated with 10 μM Y-27632, a selective ROCK inhibitor (Figure 1C). Y-27632 was added 15 min before stimulation with ET-1. Stress fiber formation was not affected by pretreatment with 5 μM U73122, a PLC inhibitor, or microinjection of G₁₂G228A or G₁₃G225A in CHO-ET_BR (data not shown). When the cells were subjected to microinjection of G₁₃G225A followed by pretreatment with U73122, ET-1 failed to induce stress fiber formation (Figure 1D). In contrast, microinjection of G₁₂G228A in combination with pretreatment by U73122 had no effect on ET-1-induced stress fiber formation (data not shown). These results demonstrate that ET_BR couples to G₁₃ but not G₁₂ in CHO cells.

Figure 1.

Effects of Y-27632, U73122, G₁₂G228A and G₁₃G225A on the ET-1-induced actin stress fibre formation in CHO-ET_BR. Cells were stimulated with (B∼E) or without (A) 10 nM ET-1. The effects of preincubation with 10 μM Y-27632 (C), combination of G₁₃G225A microinjection and 5 μM U73122 preincubation (D) and combination of G₁₃G228A microinjection, PTX (50 ng ml⁻¹) preincubation and 5 μM U73122 preincubation (E) on ET-1-induced stress fibre formation are shown. Y-27632, U73122 and PTX were added 15 min before stimulation with ET-1. Expression plasmids encoding for G₁₂G228A and G₁₃G225A were microinjected into the cell nuclei 24 h before stimulation with ET-1. Actin stress fibers were visualized as described in Methods. Representative examples of stress fibres in individual cells are shown.

A previous report demonstrated that, in preadipocytes,α₂-adrenergic receptor activation of Rho was PTX sensitive (Betuing et al., 1998). In contrast, the other previous report showed that Rho-mediated effects on the cytoskeleton are PTX insensitive (Buhl et al., 1995). Therefore, we tried to clarify whether G_i played roles in ET-1-induced actin stress fibre formation in CHO-ET_BR using pertussis toxin (PTX; 50 ng ml⁻¹). It is generally accepted that PTX inhibits the effects of G_i (Takagi et al., 1995). The degree of ET-1-induced stress fiber formation in CHO-ET_BR that had been treated with combination of G₁₃G225A microinjection, PTX preincubation, and U73122 preincubation (Figure 1E) was similar to that in CHO-ET_BR that had been treated with combination of G₁₃G225A microinjection and U73122 preincubation (Figure 1D). These results indicate that G_i may not be involved in ET-1-induced actin stress fiber formation in CHO-ET_BR.

ET-1-induced actin stress fiber formation in CHO-SerET_BR and CHO-ET_BRΔ403

ET-1 induced stress fiber formation in CHO-SerET_BR (Figure 2B). Like CHO-ET_BR, ET-1 induced stress fiber formation was inhibited by preincubation of CHO-SerET_BR with Y-27632 (Figure 2C), but was not affected by microinjection of G₁₂G228A (Figure 2D). Notably, unlike CHO-ET_BR, it was inhibited by microinjection of G₁₃G225A (Figure 2E).

Figure 2.

Effects of Y27632, G₁₂G228A and G₁₃G225A on ET-1-induced actin stress fiber formation in CHO-SerET_BR. Cells were stimulated with (B∼E) or without (A) 10 nM ET-1. (C) Y-27632 at 10 μM was added 15 min before stimulation with ET-1. Expression plasmids encoding for G₁₂G228A (D) and G₁₃G225A (E) were microinjected into cell nuclei 24 h before stimulation with ET-1. Actin stress fibres were visualized as described in Methods. Representative examples of stress fibres in individual cells are shown.

ET-1 induced stress fiber formation in CHO-ET_BRΔ403, in which coupling of the receptor with G_q but not G_i was retained (Figure 3B). Like CHO-ET_BR, ET-1-induced stress fiber formation was inhibited by preincubation of CHO-ET_BRΔ403 with Y-27632 (Figure 3C). Notably, unlike CHO-ET_BR, it was inhibited by preincubation with U73122 (Figure 3D). In contrast, it was not affected by microinjection of G₁₂G228A or G₁₃G225A (Figure 3E,F).

Figure 3.

Effects of Y27632, U73122, G₁₂G228A and G₁₃G225A on ET-1-induced actin stress fiber formation in CHO-ET_BRΔ403. Cells were stimulated with (B∼F) or without (A) 10 nM ET-1. Y-27632 at 10 μM (C) and U73122 at 5 μM (D) were added 15 min before stimulation with ET-1. Expression plasmids encoding G₁₂G228A (E) and G₁₃G225A (F) were microinjected into the cell nuclei 24 h before stimulation with ET-1. Actin stress fibres were visualized as described in Methods. Representative examples of stress fibres in individual cells are shown.

These results indicate that SerET_BR, but not ET_BRΔ403, couples with G₁₃.

Basic properties of the ET-1-induced increase in [Ca²⁺]_i in CHO-ET_BR, CHO-ET_BRΔ403 and CHO-SerET_BR

ET-1 induced a biphasic increase in [Ca²⁺]_i in CHO-ET_BR, consisting of an initial transient phase and a subsequent sustained phase (Figure 4A). Both the transient and sustained increase in [Ca²⁺]_i were dependent on the concentrations of ET-1, and reached the maximal value at concentrations ⩾1 nM (Figure 4D,E).

Figure 4.

Original tracings illustrating the effects of ET-1 on the increase in [Ca²⁺]_i in CHO-ET_BR (A), CHO-ET_BRΔ403 (B) and CHO-SerET_BR (C). The cells were loaded with fluo-3 and stimulated with 10 nM ET-1 at the time indicated by arrows. 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-ET_BR, CHO-ET_BRΔ403 and CHO-SerET_BR. The values for CHO-ET_BR, CHO-ET_BRΔ403 and CHO-SerET_BR were represented by circles, squares and triangles, respectively. Each point represents the mean±s.e.m. of five experiments.

In CHO-ET_BRΔ403, which are coupled with G_q alone, stimulation with 1 nM ET-1 caused a transient peak but not sustained increase in [Ca²⁺]_i (Figure 4B). The magnitude of the transient increase in [Ca²⁺]_i in CHO-ET_BRΔ403 was essentially similar to that in CHO-ET_BR (Figure 4D).

In CHO-SerET_BR, which are coupled with G₁₃ (Figure 2), ET-1 failed to induce an initial transient increase in [Ca²⁺]_i, and it induced only a sustained increase in [Ca²⁺]_i (Figure 4C). The magnitude of the sustained increase in [Ca²⁺]_i in CHO-SerET_BR was lower than that in CHO-ET_BR (Figure 4E).

Pharmacological identification of Ca²⁺ channels activated by ET-1 in CHO-ET_BR and CHO-SerET_BR

As described previously (Kawanabe et al., 2001), in CHO-ET_BR, the ET-1-induced sustained increase in [Ca²⁺]_i was completely suppressed by the maximally effective concentration (10 μM) of LOE 908, whereas it was partially inhibited by the maximally effective concentration (10 μM) of SK&F 96365 (Figure 5A,B,E). PTX at 50 ng ml⁻¹ failed to affect the resting [Ca²⁺]_i and the ET-1-induced increase in [Ca²⁺]_i in CHO-ET_BR (data not shown). In CHO-SerET_BR, 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 (Figure 5C,D,F). These results demonstrate that ET-1 activates only NSCC-1 (LOE 908-sensitive and SK&F 96365 resistant) in CHO-SerET_BR.

Figure 5.

Original tracings illustrating the effects of various concentrations of LOE 908 and SK&F 96365 on the ET-1-induced sustained increase in [Ca²⁺]_i in CHO-ET_BR (A,B) and CHO-SerET_BR (C,D). The cells were loaded with fluo-3 and stimulated with 10 nM ET-1 at the time indicated by arrows. After [Ca²⁺]_i reached a steady-state, various concentrations of LOE 908 or SK&F 96365 was added as indicated by arrows. 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-ET_BR (E) and CHO-SerET_BR (F). The experimental protocols were described in Methods, and the values of [Ca²⁺]_i following 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.

Effects of inhibition of PLC on the species of ET-1-activated Ca²⁺ channels in CHO-ET_BR

In CHO-SerET_BR, 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_BR and confirm that PLC actually acts as an effector for activation of Ca²⁺ channels, we used U73122 in CHO-ET_BR stimulated by ET-1. ET-1 at 1 nM induced only the sustained increase in [Ca²⁺]_i in CHO-ET_BR treated with 5 μM U73122 (Figure 6A,B). The magnitude of the sustained increase in [Ca²⁺]_i was about 35% of that in the absence of U73122 (Figure 6C). 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 (Figure 6). These results indicate that ET-1 activates only NSCC-1 in CHO-ET_BR treated with U73122.

Figure 6.

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-ET_BR pretreated with U73122. The cells loaded with fluo-3 were incubated with 5 μM U73122 for 10 min before 10 nM ET-1 stimulation. 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. (C) Effects of maximally effective concentration of LOE 908 and SK&F 96365 on the ET-1-induced sustained increase in [Ca²⁺]_i in CHO-ET_BR pretreated with U73122. The experimental protocols were described in Methods, and the values of [Ca²⁺]_i following addition of 10 μM LOE 908 or 10 μM SK&F 96365 were determined. Each point represents the mean±s.e.m. of five experiments.

Effects of G₁₃ on the ET-1-induced sustained increase in [Ca²⁺]_i in CHO-ET_BR or CHO-SerET_BR

To assess the involvement of G₁₃ in the activation of Ca²⁺ channels, we investigated the effects of G₁₃G225A on the ET-1-induced increase in [Ca²⁺]_i in CHO-ET_BR and CHO-SerET_BR. In this experiment, G₁₃G225A was microinjected into CHO-ET_BR and CHO-SerET_BR, and the ET-1-induced increase in [Ca²⁺]_i in these cells was analysed using microfluorimetry.

ET-1 failed to induce sustained increase in [Ca²⁺]_i in CHO-ET_BR microinjected with G₁₃G225A (Figure 7A) and CHO-SerET_BR microinjected with G₁₃G225A (Figure 7B). These results indicate that G₁₃ plays critical roles in the activation of NSCC-1 and NSCC-2 caused by ET-1.

Figure 7.

Effects of G₁₃G225A on ET-1-induced sustained increase in [Ca²⁺]_i in CHO-SerET_BR (A) and CHO-ET_BR (B). The cells loaded with fluo-3 were stimulated by 10 nM ET-1. The sustained increase in [Ca²⁺]_i in the presence of G₁₃G225A is represented as a percentage of values in its absence. Data are presented as mean±s.e.m. of three experiments.

Effects of Y-27632 on the ET-1-induced sustained increase in [Ca²⁺]_i in CHO-ET_BR

It is generally accepted that Rho/Rho-kinase (ROCK) pathway is a downstream target of G₁₃ (Seasholtz et al., 1999). We examined the effects of ROCK on ET-1-induced increase in [Ca²⁺]_i in CHO-ET_BR. In this experiment, Y-27632 was used as a specific inhibitor of ROCK (Uehata et al., 1997). Y-27632 was added 15 min before stimulation with ET-1. Y-27632 at 10 μM did not affect the ET-1-induced transient and sustained increase in [Ca²⁺]_i (Figure 8).

Figure 8.

(A) Original tracing illustrating the effects of Y-27632 on the ET-1-induced increase in [Ca²⁺]_i in CHO-ET_BR. The cells loaded with fluo-3 were incubated with 10 μM Y-27632 for 15 min before 10 nM ET-1 stimulation. (B, C) Effects of 10 μM Y-27632 on 10 nM ET-1-induced transient (B) and sustained (C) increase in [Ca²⁺]_i in CHO-ET_BR. The experimental protocols were described in Methods. Data are presented as mean±s.e.m. of three experiments.

Discussion

As reported previously (Kawanabe et al., 2001), the ET-1-induced sustained increase in [Ca²⁺]_i in CHO-ET_BR results from extracellular Ca²⁺ influx through two types of Ca²⁺-permeable NSCC: NSCC-1 and NSCC-2 (Figures 4 & 5). The most important novelty of this study is that ET-1-induced NSCC-1 activation depends on G₁₃-dependent cascade, and NSCC-2 activation depends on both G_q/PLC- and G₁₃-dependent cascades in CHO-ET_BR.

To identify the G proteins involved in the activation of these Ca²⁺ channels by ET-1, we assessed whether G₁₂ and G₁₃ are coupled with CHO-ET_BR using actin stress fibre formation. A previous report demonstrated that G_q and G₁₂/G₁₃ play important roles in the majority of GPCR-induced, Rho-mediated effects on the cytoskeleton (Seasholtz et al., 1999). Based on sensitivity to U73122, G₁₂G228A and G₁₃G225A (Figure 1), we conclude that ET-1-induced stress fiber formation is mediated via two signalling pathways in CHO-ET_BR (i.e., the G_q/PLC- and G₁₃-dependent pathways), and also that only one of the two may be sufficient for actin stress fibre formation. Moreover, G_i may not be involved in ET-1-induced stress fiber formation, because treatment of the cells with sufficient concentrations of PTX did not affect ET-1-induced actin stress fiber formation in CHO-ET_BR (Figure 1E). This result is in agreement with the previous indication that Rho-mediated effects on the cytoskeleton are PTX insensitive (Buhl et al., 1995). On the other hand, based on sensitivity to Y-27632, the Rho/ROCK pathway plays important roles in ET-1-induced stress fiber formation in CHO-ET_BR (Figure 1C) as reported for a variety of cells (Mao et al., 1998; Gohla et al., 1999; Seasholtz et al., 1999). Therefore, Rho/ROCK pathway may be downstream of both the G_q/PLC- and G₁₃-dependent pathways. We deduced the structural determinant for coupling of ET_BR with G₁₃ based on data from experiments using mutated ET_BRs. That is, loss of coupling of ET_BRΔ403 with G₁₃ (Figure 3F) and retention of coupling of SerET_BR with G₁₃ (Figure 2E) clearly show that the cytoplasmic tail downstream of Cys⁴⁰³ but not the palmitoylation site of ET_BR is essential for coupling with G₁₃. Judging from these data, we conclude that the cytoplasmic tail downstream of the palmitoylation sites of ET_BR, but not the palmitoylation site itself, is essential for coupling with G₁₃.

Given that the subtypes of G proteins that are coupled with CHO-ET_BR has become clear, we analysed G proteins involved in the activation of NSCCs caused by ET-1. Judging from the data using U73122-treated CHO-ET_BR and CHO-ET_BRΔ403 (Figures 4 & 6), G_q/PLC-independent pathways as well as G_q/PLC-dependent pathways are involved in NSCC-2 activation, while only G_q/PLC-independent pathways are involved in NSCC-1 activation. To elucidate the mechanism of NSCC-1 activation, we examined the effects of G₁₃ using CHO-SerET_BR. ET-1-induced sustained increase in [Ca²⁺]_i is abrogated in CHO-SerET_BR microinjected with G₁₃G225A (Figure 7A). These results indicate that the G₁₃-dependent pathway seems to play an essential role for activation of NSCC-1 caused by ET-1. Moreover, these results support the indication that the cytoplasmic tail downstream of the palmitoylation sites of ET_BR, but not the palmitoylation site itself, is essential for coupling with G₁₃. Next, we examined the mechanisms of NSCC-2 activation using CHO-ET_BR microinjected with G₁₃G225A. ET-1 failed to induce sustained increase in [Ca²⁺]_i in these cells (Figure 7B). These results indicate that a G₁₃-dependent pathway plays an important role for activation of NSCC-2 as well as NSCC-1. In contrast, judging from the results using PTX, activation of NSCC-1 and NSCC-2 by ET-1 seems not to involve G_i-dependent pathway. In conclusion, NSCC-2 activation by ET-1 involves G₁₃-dependent pathway as well as G_q/PLC-dependent pathway. Recent reports demonstrated that G₁₃ regulates cell growth (Seasholtz et al., 1999). Moreover, extracellular Ca²⁺ influx through NSCCs is involved in ET-1-induced cell proliferation in a variety of cells (Kawanabe et al., 2001; 2002). Therefore, G₁₃ may be an effector for ET-1-induced cell proliferation by stimulating NSCCs. Collectively, among the subtypes of Gα proteins, G_q and G₁₃ play important roles for activation of NSCCs as follows; (1) NSCC-1 activation involves a G₁₃-dependent pathway. (2) NSCC-2 involves both G_q/PLC- and G₁₃-dependent pathways.

It is important to understand the mechanisms of ET-1 activation for each Ca²⁺ channel downstream of G_q and/or G₁₃. Because Rho/ROCK pathway is a downstream target of G₁₃ (Seasholtz et al., 1999), we investigated the effects of ROCK on the activation of Ca²⁺ channels by ET-1 using a selective ROCK inhibitor, Y-27632. However, Y-27632 did not affect ET-1-induced increase in [Ca²⁺]_i (Figure 8). This result indicates that G₁₃ activates NSCC-1 and NSCC-2 via ROCK-independent signaling pathways. Therefore, G₁₃ may have another intracellular signalling pathway for activating NSCCs. Further study is needed to identify the effectors downstream of G₁₃ for activation of NSCC-1 and NSCC-2.

Abbreviations

[Ca²⁺]_i

intracellular free Ca²⁺ concentration

CHO-ET_BR

Chinese hamster ovary cells that stably express human endothelin_B receptor

CHO-ET_BRΔ403

Chinese hamster ovary cells that express human endothelin_B receptor truncated at the carboxyl-terminal downstream of Cys403

CHO-SerET_BR

Chinese hamster ovary cells that express the unpalmitoylated (Cys⁴⁰²Cys⁴⁰³ Cys⁴⁰⁵ → Ser⁴⁰²Ser⁴⁰³ Ser⁴⁰⁵) human endothelin_B receptor

ET-1

endothelin-1

FCS

foetal calf serum

F_max

fluorescence intensity maximum

F_min

fluorescence intensity minimum

G₁₂G228A

dominant negative type of G₁₂

G₁₃G225A

dominant negative type of G₁₃

GPCR

heterotrimetric guanine nucleotide-binding protein (G-protein)-coupled receptor

NSCC

nonselective cation channel

IPs

inositol phosphates

PBS

phosphate-buffered saline

PBS-Tx

phosphate-buffered saline containing 0.1% Triton-X100

PLC

phospholipase C

PTX

pertussis toxin

ROCK

Rho-associated kinase

U73122

1-(6-{[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione

VICC

voltage-independent Ca²⁺ channel

Y-27632

(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide