Characterization of Ca2+ channels and G proteins involved in arachidonic acid release by endothelin-1/endothelinA receptor

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 endothelinA receptors (CHO-ETAR). These channels can be distinguished by their sensitivity to Ca2+ channel blockers 1-(beta-[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-ETAR. ET-1 also induced AA release in CHO cells expressing ETAR truncated at the carboxyl terminal downstream of Cys385 (CHO-ETARDelta385) or an unpalmitoylated (Cys383 Cys385-388--> Ser383Ser385-388) ETAR (CHO-SerETAR), each of which is coupled with Gq or Gs/G12, respectively. In CHO-SerETAR, a dominant-negative mutant of G12 inhibited AA release. SK&F 96365 inhibited ET-1-induced AA release in CHO-ETARDelta385, whereas LOE 908 inhibited it in CHO-SerETAR. These results indicate the following: 1) ET-1-induced AA release depends on Ca2+ influx through NSCC-1, NSCC-2, and SOCC in CHO-ETAR; 2) Gq and G12 mediate AA release through ETAR in CHO cells; and 3) PI3K is involved in ET-1-induced AA release, which depends on NSCC-2 and SOCC.     

Involvements of voltage-independent Ca2+ channels and phosphoinositide 3-kinase in endothelin-1-induced PYK2 tyrosine phosphorylation

We demonstrated recently that endothelin-1 (ET-1) activates two types of Ca(2+)-permeable nonselective cation channels [designated nonselective cation channel (NSCC)-1 and NSCC-2] and a store-operated Ca(2+) channel (SOCC) in rabbit internal carotid artery vascular smooth muscle cells (ICA VSMCs). These channels can be distinguished by their sensitivity to Ca(2+) channel blockers 1-(beta-[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 LOE 908 and SK&F 96365, and SOCC is resistant to LOE 908 and sensitive to SK&F 96365. The purpose of the present study was to identify the Ca(2+) channels involved in the ET-1-induced, proline-rich tyrosine kinase 2 (PYK2) phosphorylation in ICA VSMCs. Based on sensitivity to nifedipine, an L-type voltage-operated Ca(2+) channel (VOCC) blocker, Ca(2+) influx through VOCC seems to play a minor role in the ET-1-induced PYK2 phosphorylation. In the presence of nifedipine, PYK2 phosphorylation was abolished by blocking Ca(2+) influx through NSCC-1, NSCC-2, and SOCC. The phosphoinositide 3-kinase (PI3K) inhibitors wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY 294002), inhibited ET-1-induced Ca(2+) influx through NSCC-2 and SOCC. In addition, these inhibitors blocked PYK2 phosphorylation that depends on Ca(2+) influx through NSCC-2 and SOCC. These results indicate that 1) Ca(2+) influx through NSCC-1, NSCC-2, and SOCC plays essential roles in ET-1-induced PYK2 phosphorylation, 2) NSCC-2 and SOCC are stimulated by ET-1 via a PI3K-dependent cascade, whereas NSCC-1 is stimulated via a PI3K-independent cascade, and 3) PI3K is involved in the PYK2 phosphorylation that depends on Ca(2+) influx through SOCC and NSCC-2.     

Extracellular Ca2+ influx and endothelin-1-induced intracellular mitogenic cascades in rabbit internal carotid artery vascular smooth muscle cells

Endothelin-1 (ET-1) has been proven to activate two types of Ca2+-permeable nonselective cation channels (designated NSCC-1 and NSCC-2) and a store-operated Ca2+ channel (SOCC) in rabbit internal carotid artery vascular smooth muscle cells (ICA VSMCs). Ca2+ influx through these channels plays an essential role for ET-1-induced mitogenesis in ICA VSMCs. The purpose of the current study was to investigate the effects of Ca2+ influx on intracellular pathways of ET-1-induced mitogenesis in ICA VSMCs using receptor-operated Ca2+ channel blockers, SK&F 96365 and LOE 908. We focused on extracellular-signal regulated kinase 1 and 2 (ERK1/2) in this context. PD 98059, an inhibitor of mitogen-activated protein kinase kinase, abolished the ET-1-induced increase in ERK1/2 activity, but only partially suppressed the mitogenesis. ERK1/2 activation by ET-1 was partially suppressed in the absence of extracellular Ca2+. Moreover, based on the sensitivity to SK&F 96365 and LOE 908, Ca2+ influx through NSCC-1, NSCC-2 and SOCC plays essential roles in the extracellular Ca2+-dependent component of ERK1/2 activity. In addition, Ca2+ influx through these channels was also involved in the PD 98059-resistant component of ET-1-induced mitogenesis. These results indicate that (1) the ET-1-induced mitogenesis involves both ERK1/2-dependent and -independent mechanisms in ICA VSMCs (2), ERK1/2 activation by ET-1 involves a Ca2+ influx-dependent cascade as well as a Ca2+ influx-independent cascade (3), Ca2+ influx through NSCC-1, NSCC-2 and SOCC has important roles in the Ca2+ influx-dependent component of ERK1/2-dependent mitogenesis, and (4) Ca2+ influx through these channels also plays important roles in mitogenic pathways downstream of ERK1/2.     

Characterization of voltage-independent Ca2+ channels activated by endothelin-1

To clarify Ca2+ entry channels involved in the endothelin-1 (ET-1)-induced increase in the intracellular concentration ([Ca2+]i), we performed whole-cell recordings of patch-clamp techniques and monitoring of [Ca2+]i with Ca2+ indicators fura-2 and fluo-3 in A7r5 cells (a cell line derived from rat thoracic aortic smooth muscle cells). With whole-cell recordings, lower concentrations (< or = 1 nM) of ET-1 activated a Ca(2+)-permeable nonselective cation channel (designated NSCC-1). In contrast, higher concentrations (> or = 1 nM) of ET-1 activated two types of Ca(2+)-permeable nonselective cation channel (designated NSCC-1 and NSCC-2) and store-operated Ca2+ channel (SOCC). Importantly, we found that these Ca2+ channels can be pharmacologically discriminated using blockers of the so-called receptor operated Ca2+ influx such as SK&F 96365 and LOE 908. That is, NSCC-1 is resistant to SK&F 96365 but sensitive to LOE 908; NSCC-2 is sensitive to both SK&F 96365 and LOE 908; SOCC is sensitive to SK&F 96365 but resistant to LOE 908. Using these blockers, we analyzed the ET-1-induced increase in [Ca2+]i. The increase in [Ca2+]i induced by lower concentrations of ET-1 was resistant to SK&F 96365 but sensitive to LOE 908. In contrast, the increase in [Ca2+]i induced by higher concentrations of ET-1 was partially suppressed to approximately 30% of controls by either SK&F 96365 or LOE 908 alone, and it was abolished by their combination. These results show that the increase in [Ca2+]i induced by lower concentrations (< or = 1 nM) of ET-1 results from Ca2+ influx through NSCC-1, whereas the increase in [Ca2+]i induced by higher concentrations (> or = 10 nM) of ET-1 results from Ca2+ influx through NSCC-1, NSCC-2 and SOCC.     

Effects of phosphoinositide 3-kinase on the endothelin-1-induced activation of voltage-independent Ca(2+) channels and mitogenesis in Chinese hamster ovary cells stably expressing endothelin(a) receptor

We recently demonstrated that endothelin-1 (ET-1) activates two types of Ca(2+)-permeable nonselective cation channel (designated NSCC-1 and NSCC-2) and a store-operated Ca(2+) channel (SOCC) in Chinese hamster ovary cells expressing endothelin(A) receptor (CHO-ET(A)R). In addition, these channels can be discriminated using Ca(2+) channel blockers (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) and 1-(beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole (SK&F 96365). LOE 908 is a blocker of NSCC-1 and NSCC-2, whereas SK&F 96365 is a blocker of SOCC and NSCC-2. In this study, we investigated the effects of phosphoinositide 3-kinase (PI3K) on the ET-1-induced activation of these channels and mitogenesis in CHO-ET(A)R using wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY 294002), inhibitors of phosphoinositide 3-kinase (PI3K). ET-1-induced Ca(2+) influx was partially inhibited in CHO-ET(A)R pretreated with wortmannin or LY 294002. In contrast, addition of wortmannin or LY 294002 after stimulation with ET-1 did not suppress Ca(2+) influx. The Ca(2+) channels activated by ET-1 in wortmannin or LY 294002-treated CHO-ET(A)R were sensitive to LOE 908 and resistant to SK&F 96365. Wortmannin also partially inhibited ET-1-induced mitogenesis. LOE 908, but not SK&F 96365, abolished the wortmannin-resistant part of mitogenesis. The IC(50) values (~30 nM) of wortmannin for the ET-1-induced Ca(2+) influx and mitogenesis were similar to those for the ET-1-induced PI3K activation. 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 seems to be required for the activation of the Ca(2+) entry, but not for its maintenance. In addition, PI3K is involved in the ET-1-induced mitogenesis that depends on the extracellular Ca(2+) influx through SOCC and NSCC-2.     

Ca(2+) influx through nonselective cation channels plays an essential role in endothelin-1-induced mitogenesis in C6 glioma cells

Ca(2+) channels activated by endothelin-1 (ET-1) in C6 glioma cells (C6 cells) were characterized using whole-cell patch-clamps and by monitoring the intracellular free Ca(2+) concentration ([Ca(2+)](i)), when administering Ca(2+) channel blockers such as LOE 908 and SK&F 96365. Using this methodology, the Ca(2+) channels involved in ET-1-induced mitogenesis were identified.The patch-clamp study and [Ca(2+)](i) monitoring showed that 10 nM ET-1 activated two types of Ca(2+)-permeable nonselective cation channels (NSCC); one was sensitive to LOE 908 but resistant to SK&F 96365 (NSCC-1) and the other was sensitive to both LOE 908 and SK&F 96365 (NSCC-2). Conversely, 0.1 nM ET-1 activated only NSCC-1.ET-1-induced mitogenesis in a concentration-dependent manner, with the maximum effect arising at concentrations > or =10 nM. LOE 908 completely suppressed the 10 nM ET-1-induced mitogenesis, whereas SK&F 96365 only partially suppressed it. The IC(50) values of these blockers for the ET-1-induced mitogenesis were similar to those for the 10 nM ET-1-induced increase in [Ca(2+)](i). In contrast, LOE 908 completely suppressed 0.1 nM ET-1-induced mitogenesis, whereas SK&F 96365 did not affect it.Collectively, these results demonstrate that the sustained increase in [Ca(2+)](i), via NSCC-1 and NSCC-2, may be essential for ET-1-induced mitogenesis in C6 cells. Moreover, the sensitivity of NSCC-1 to ET-1 is higher than that of NSCC-2 to ET-1.     

Ca2+ entry channels in rat thoracic aortic smooth muscle cells activated by endothelin-1.

The contraction of the rat aorta induced by endothelin-1 (ET-1) requires entry of extracellular Ca2+, but involvement of voltage-operated Ca2+ channel is minor. Using whole-cell recordings of patch-clamp and monitoring of the intracellular free Ca2+ concentration ([Ca2+]i), we characterized Ca2+ entry channels in A7r5 cells activated by ET-1. ET-1 activates three types of voltage-independent Ca2+ entry channels: two types of Ca2+-permeable nonselective cation channels (designated NSCC-1 and NSCC-2) and a store-operated Ca2+ channel (SOCC). Furthermore, it was found that these channels can be pharmacologically discriminated using Ca2+ channel blockers such as SK&F 96365 and LOE 908. NSCC-1 is resistant to SK&F 96365, but sensitive to LOE 908, whereas NSCC-2 is sensitive to both SK&F 96365 and LOE 908. SOCC is sensitive to SK&F 96365, but resistant to LOE 908. Using these channel blockers, we analyzed Ca2+ entry channels involved in the ET-1-induced contractions of rat thoracic aorta and increases in [Ca2+]i of single smooth muscle cells. The responses to lower concentrations of ET-1 (< or = 0.1 nM) were abolished by either SK&F 96365 or LOE 908 alone. In contrast, the responses to higher concentrations of ET-1 (> or = 1 nM) were suppressed by SK&F 96365 or LOE 908 to about 10% and 35% of controls, respectively, and abolished by combined treatment with SK&F 96365 and LOE 908. These results show that the responses of rat aorta to lower concentrations of ET-1 involve only one Ca2+ channel that is sensitive to SK&F 96365 and LOE 908 (NSCC-2), whereas those to higher concentrations of ET-1 involve NSCC-1, NSCC-2 and SOCC, contributing 10%, 55% and 35%, respectively, to total Ca2+ entry.     

Analysis of Ca2+ entry channels in endothelin-1-induced contraction of rat aorta using new blockers

1) We have recently shown that endothelin-1 (ET-1) activates two types of Ca(2+)-permeable nonselective cation channels (designated NSCC-1 and NSCC-2) and store-operated Ca2+ channel (SOCC). These channels can be pharmacologically discriminated using Ca2+ channel blockers such as SK&F 96365 and LOE 908. Here we characterized Ca2+ entry channels involved in ET-1-induced contractions of rat thoracic aortic rings and increases in the intracellular free Ca2+ concentration ([Ca2+]i) of single smooth muscle cells using these blockers. 2) LOE 908 or a blocker of voltage-operated Ca2+ channel nifedipine had no effect on the contractions and increases in [Ca2+]i induced by thapsigargin, whereas SK&F 96365 abolished them. 3) The contractions and increases in [Ca2+]i induced by ET-1 depended on extracellular Ca2+ but were resistant to nifedipine. The responses to lower concentrations (< or = 0.1 nM) of ET-1 were abolished by either SK&F 96365 or LOE 908. The responses to higher concentrations (> or = 1 nM) were abolished by SK&F 96365, but were partially resistant to LOE 908. 4) These results show that the contractions and increases in [Ca2+]i of rat aortic smooth muscles at lower concentrations of ET-1 involve only one Ca2+ entry channel which is sensitive to SK&F 96365 and LOE 908 (NSCC-2), whereas those at higher concentrations of ET-1 involve another Ca2+ entry channel which is sensitive to SK&F 96365 but resistant to LOE 908 (SOCC) in addition to the former channel.     

Pharmacological characterization of Ca2+ entry channels in endothelin-1-induced contraction of rat aorta using LOE 908 and SK&F 96365.

We have recently shown that endothelin-1 (ET-1) activates two types of Ca2+-permeable nonselective cation channels (designated NSCC-1 and NSCC-2) and store-operated Ca2+ channel (SOCC). These channels can be pharmacologically discriminated using Ca2+ channel blockers such as SK&F 96365 and LOE 908. Here we characterized Ca2+ entry channels involved in ET-1-induced contractions of rat thoracic aortic rings and increases in the intracellular free Ca2+ concentration ([Ca2+]i) of single smooth muscle cells using these blockers. LOE 908 or a blocker of voltage-operated Ca2+ channel nifedipine had no effect on the contractions and increases in [Ca2+]i induced by thapsigargin or ionomycin, whereas SK&F 96365 abolished them. The contractions and increases in [Ca2+]i induced by ET-1 depended on extracellular Ca2+ but were resistant to nifedipine. The responses to lower concentrations (< or =0.1 nM) of ET-1 were abolished by either SK&F 96365 or LOE 908. The responses to higher concentrations (> or = 1 nM) were abolished by SK&F 96365, but were partially resistant to LOE 908. SK&F 96365 inhibited the LOE 908-resistant contractions induced by higher concentrations of ET-1 with IC50 values similar to those for contractions induced by thapsigargin or ionomycin. These results show that the contractions and increases in [Ca2+]i of rat aortic smooth muscles at lower concentrations of ET-1 involve only one Ca2+ entry channel which is sensitive to SK&F 96365 and LOE 908 (NSCC-2), whereas those at higher concentrations of ET-1 involve another Ca2+ entry channel which is sensitive to SK&F 96365 but resistant to LOE 908 (SOCC) in addition to the former channel.     

Ca2+ entry channels involved in contractions of rat aorta induced by endothelin-1, noradrenaline,and vasopressin

Endothelin-1 (ET-1) has been shown to activate three types of Ca2+ channel, namely two Ca2+-permeable nonselective cation channels (designated NSCC-1 and NSCC-2) and a store-operated Ca2+ channel (SOCC), and that these channels can be discriminated by Ca2+ channel blockers such as LOE 908 (a blocker of NSCC-1 and NSCC-2) and SK&F 96365 (a blocker of NSCC-2 and SOCC). This study pharmacologically compared Ca2+ entry channels involved in contractions of rat thoracic aorta without endothelium induced by ET-1, noradrenaline (NA), or arginine-vasopressin (AVP). These agonists-induced contractions of aortic rings without endothelium and increases in the intracellular free Ca2+ concentration ([Ca2+]i) of cultured aortic smooth muscle cells were abolished by removal of extracellular Ca2+. A blocker of L-type voltage-operated Ca2+ channel (VOCC), nifedipine had no effect on the responses to ET-1, but it suppressed the responses to NA and AVP to 70% and 65% of control responses, respectively. LOE 908 partially suppressed the nifedipine-resistant responses to ET-1 and AVP, but not those to NA. SK&F 96365 also partially suppressed the nifedipine-resistant responses to ET-1 and AVP, whereas it abolished the responses to NA. LOE 908 in combination with SK&F 96365 abolished the nifedipine-resistant responses to either of the agonists. These results show that the contraction of rat aorta involves different Ca2+ entry channel depending on agonists: (a) NSCC-1, NSCC-2, and SOCC for ET-1; (b) VOCC and SOCC for NA; and (c) VOCC, NSCC-1, NSCC-2, and SOCC for AVP.     

Activation of three types of voltage-independent Ca2+ channel in A7r5 cells by endothelin-1 as revealed by a novel Ca2+ channel blocker LOE 908

1. We have shown that in addition to voltage-operated Ca2+ channel (VOC), endothelin-1 (ET-1) activates two types of Ca2+-permeable nonselective cation channel (NSCC) in A7r5 cells: its lower concentrations (< or = 1 nM; lower [ET-1]) activate only an SK&F 96365-resistant channel (NSCC-1), whereas its higher concentrations (> or = 10 nM; higher [ET-1]) activate an SK&F 96365-sensitive channel (NSCC-2) as well. 2. We now characterized the effects of a blocker of Ca2+ entry channel LOE 908 on NSCCs and store-operated Ca2+ channel (SOCC) in A7r5 cells, and using two drugs, clarified the involvement of these channels in the ET-1-induced increase in the intracellular free Ca2+ concentrations ([Ca2+]i). Whole-cell recordings and [Ca2+]i monitoring with fluo-3 were used. 3. LOE 908 up to 10 microM had no effect on increases in [Ca2+]i induced by thapsigargin or ionomycin, but SK&F 96365 abolished them. 4. In the cells clamped at -60 mV, both lower and higher [ET-1] induced inward currents with linear iv relationships and the reversal potentials of -15.0 mV. Thapsigargin induced no currents. 5. In the presence of nifedipine, lower [ET-1] induced a sustained increase in [Ca2+]i, whereas higher [ET-1] induced a transient peak and a sustained increase. The sustained increases by lower and higher [ET-1] were abolished by removal of extracellular Ca2+, and they were suppressed by LOE 908 to 0 and 35%, respectively, with the LOE 908-resistant part being abolished by SK&F 96365. 6. These results show that LOE 908 is a blocker of NSCCs without effect on SOCC, and that the increase in [Ca2+]i at lower [ET-1] results from Ca2+ entry through NSCC-1 in addition to VOC, whereas the increase at higher [ET-1] involves NSCC-1, NSCC-2 and SOCC in addition to VOC.     

Characterization of Ca(2+) channels involved in endothelin-1-induced mitogenic responses in vascular smooth muscle cells.

Ca(2+) channels involved in the endothelin-1-induced mitogenic response of cultured rat thoracic aorta smooth muscle cells, A7r5 cells, were characterized using the Ca(2+) channel blockers, LOE 908 and SK&F 96365. Stimulation of A7r5 cells with endothelin-1 induced a mitogenic response as well as a biphasic increase in the intracellular-free Ca(2+) concentration. Based on the sensitivity to nifedipine, a specific blocker of L-type voltage-operated Ca(2+) channel (VOCC), Ca(2+) influx through VOCC has a minor role in endothelin-1-induced mitogenic responses. On the other hand, Ca(2+) influx through voltage-independent Ca(2+) channels (VICCs) plays an important part in endothelin-1-induced mitogenesis. Moreover, based on their sensitivity to SK&F 96365 and LOE 908, VICCs consist of two types of Ca(2+)-permeable nonselective cation channels (designated NSCC-1 and NSCC-2) and a store-operated Ca(2+) channel (SOCC). Ca(2+) influx through NSCC-1, NSCC-2 and SOCC contributes to 35%, 30% and 35%, respectively, to the nifedipine-resistant component of the endothelin-1 mitogenic response.     

Effects of extracellular Ca2+ influx on endothelin-1-induced intracellular mitogenic cascades in C6 glioma cells

We have recently shown that endothelin-1 activates two types of Ca2+-permeable nonselective cation channels (NSCC-1 and NSCC-2) in C6 glioma cells. These channels can be distinguished by their sensitivity to blockers of the receptor-operated Ca2+ channel, 1-[b-(3-[4-methoxyphenyl]propoxy)-4-methoxyphenethyl]-1H-imidazole hydrochloride (SK&F 96365) and (R,S)-(3,4-dihydro-6,7-dimethoxy-isoquinoline-1-yl)-2-phenyl-N,N-di-[2-(2,3,4-trimethoxyphenyl)ethyl]-acetamide (LOE 908). 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. Moreover, extracellular Ca2+ influx through these channels plays an essential role in endothelin-1-induced mitogenesis in C6 glioma cells. The purpose of the present study was to investigate the effects of extracellular Ca2+ influx on intracellular pathways of endothelin-1-induced mitogenic responses in C6 glioma cells. We focused on extracellular signal-regulated kinase 1 and 2 (ERK1/2) in this context. An inhibitor of mitogen-activated protein kinase, 2-[2-amino-3-methoxyphenyl]-4H-1-benzopyran-4-one (PD 98059), abolished the endothelin-1-induced increase in ERK1/2 activity, but only partially suppressed the mitogenic response. ERK1/2 activation by endothelin-1 was partially suppressed in the absence of extracellular Ca2+. On the basis of the sensitivity to LOE 908 and SK&F 96365, Ca2+ influx through NSCC-1 and NSCC-2 plays an essential role in the extracellular Ca2+-dependent component of ERK1/2 activity. In contrast, Ca2+ influx through NSCC-2 is involved in the ERK1/2-independent component of endothelin-1-induced mitogenesis. These results indicate that (1) the endothelin-1-induced mitogenic response involves both ERK1/2-dependent and -independent mechanisms, (2) ERK1/2 activation by endothelin-1 involves an extracellular Ca2+ influx-dependent cascade as well as an extracellular Ca2+ influx-independent cascade, (3) because endothelin-1-induced mitogenesis is completely dependent on extracellular Ca2+ influx, extracellular Ca2+ influx also plays an important role in mitogenic pathways downstream of ERK1/2, (4) extracellular Ca2+ influx through NSCC-1 and NSCC-2 has an important role in the extracellular Ca2+ influx-dependent component of ERK1/2-dependent mitogenesis, (5) extracellular Ca2+ influx through NSCC-2 has an important role in ERK1/2-independent mitogenesis, and (6) Ca2+ influx through each Ca2+ channel may play a distinct role in intracellular mitogenic cascades.     

Characterization of Ca2+ channels involved in endothelin-1-induced contraction of rabbit basilar artery

This study attempted to characterize Ca2+ channels involved in endothelin-1-induced contraction of rabbit basilar artery using whole-cell patch-clamp and measurement of intracellular free Ca2+ concentration. Endothelin-1 activates two types of Ca2+-permeable nonselective cation channels (NSCC-1 and NSCC-2) and a store-operated Ca2+ channel (SOCC) in addition to the voltage-operated Ca2+ channel (VOCC). These channels can be discriminated using Ca2+ channel blockers, SK&F 96365 and LOE 908. Tension study was conducted to clarify the Ca2+ channels involved in endothelin-1-induced contraction of basilar artery. Endothelin-1-induced basilar artery contraction is fully dependent on extracellular Ca2+ influx. Based on sensitivity to nifedipine, an L-type VOCC blocker, VOCCs have a minor role in endothelin-1-induced contraction. Both LOE 908 and SK&F 96365 inhibit endothelin-1-induced contraction in a concentration-dependent manner, and their combination abolished it. The median inhibitory concentrations of these blockers for endothelin-1-induced contraction correlated well with those of the endothelin-1-induced [Ca2+]i responses. Thus, the inhibitory action of these blockers on endothelin-1-induced contraction may be mediated by blockade of NSCC-1, NSCC-2, and the SOCC. Extracellular Ca2+ influx through NSCC-1, NSCC-2, and SOCC may be essential for endothelin-1-induced basilar artery contraction.     

Pharmacological characterization of receptor-mediated Ca2+ entry in endothelin-1-induced catecholamine release from cultured bovine adrenal chromaffin cells

To clarify the mechanism for the endothelin-1 (ET-1)-induced release of catecholamines from the adrenal gland, we examined the effects of removal of extracellular Ca2+, blockers of L-, N-, P- and Q-types of voltage-operated Ca2+ channels (VOCC) such as nifedipine (L-type), omega-conotoxin GVIA (N-type), omega-agatoxin IVA (P-type) and omega-conotoxin MVIIC (Q-type) and blockers of voltage-independent Ca2+ entry channel such as SK&F 96365 and LOE 908 on release of catecholamines, the cytosolic free Ca2+ concentration ([Ca2+]i), and 45Ca2+ uptake in cultured bovine adrenal chromaffin cells. ET-1 but not ET-3 induced increases in release of catecholamines, [Ca2+]i, and 45Ca2+ uptake. The responses to ET-1 were abolished by the antagonist for ET(A) receptors, BQ-123, but not by the antagonist for ET(B) receptors, BQ-788, and they were abolished by removal of extracellular Ca2+. The increases were only partially inhibited (to about 65% of control) by nifedipine but unaffected by any of the omega-toxins. The nifedipine-resistant increase was inhibited by SK&F 96365 (to about 40%) and abolished by LOE 908 alone. These results indicate that ET-1 augments the release of catecholamines from adrenal chromaffin cells through ET(A) receptors, by activating two types of Ca2+ entry channels in addition to L-type VOCC: one (nonselective cation channel-1; NSCC-1) is sensitive to LOE 908 but resistant to SK&F 96365, whereas the other (NSCC-2) is sensitive to both LOE 908 and SK&F 96365.     

Failure of endothelin-1 to activate store-operated Ca2+ channels by lack of mobilization from intracellular Ca2+ stores in cultured bovine adrenal chromaffin cells

We have recently shown that in addition to L-type voltage-operated Ca2+ channel (VOC), endothelin-1 (ET-1) stimulation opens two types of Ca2+-permeable nonselective cation channels [designated nonselective cation channel-1 (NSCC-1) and NSCC-2]. However, in this Ca2+ entry, the involvement of store-operated Ca2+ channel (SOCC), which is suggested to exist in chromaffin cells, was unclear. Those NSCCs as well as SOCC can be pharmacologically discriminated using Ca2+ channel blockers such as SK&F 96365 and LOE 908. To clarify whether SOCC should actually exist and play a role in Ca2+ entry in chromaffin cells stimulated with ET-1, we examined the effects of removal of extracellular Ca2+, thapsigargin (TG, an inhibitor of endoplasmic reticulum Ca2+-ATPase), LOE 908 and SK&F 96365 on cytosolic free Ca2+ concentrations ([Ca2+]i) in cultured bovine adrenal chromaffin cells. After the cells were exposed to Ca2+-free medium followed by exposure to TG to deplete Ca2+ from the intracellular Ca2+ store, restoration of extracellular Ca2+ caused a gradual increase in [Ca2+]i (to about 200% of control). The increase was unaffected by LOE 908, but completely abolished by SK&F 96365. In the Ca2+-free medium, no increase in [Ca2+]i by ET-1 was observed, but the subsequent restoration of extracellular Ca2+ induced a rapid increase in [Ca2+]i (to the same level of [Ca2+]i as that evoked by ET-1 in the normal medium (1.0 mM Ca2+)). Since SK&F 96365 is also a blocker of SOCC, these results indicate that in bovine adrenal chromaffin cells, Ca2+ entry through SOCC (Ca2+ influx through the capacitative Ca2+ entry system) occurs but is comparably weak, and that it virtually does not work on the stimulation of ET-1.     

Effects of Ca(2+) influx through nonselective cation channel on noradrenaline-induced mitogenic responses

We have recently shown that noradrenaline induces extracellular Ca(2+) influx through nonselective cation channel (NSCC) in Chinese hamster ovary cells expressing alpha(1A)-adrenoceptors (CHO-alpha(1A)). Moreover, this NSCC is sensitive to (R,S)-(3,4-dihydro-6,7-dimethoxy-isoquinoline-1-yl)-2-phenyl-N,N-di-[2-(2,3,4-trimethoxyphenyl)ethyl]-acetamide (LOE 908) and resistant to 1-[b-(3-[4-Methoxyphenyl]propoxy)-4-methoxyphenethyl]-1H-imidazole hydrochloride (SK&F 96365). In the present study, we characterized the effects of extracellular Ca(2+) influx through NSCC on noradrenaline-induced mitogenic responses and activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) of CHO-alpha(1A) using LOE 908 and SK&F 96365. Noradrenaline induced a mitogenic response in CHO-alpha(1A). LOE 908 completely inhibited the noradrenaline-induced mitogenesis, whereas SK&F 96365 did not inhibit it. The IC(50) value of LOE 908 for noradrenaline-induced mitogenesis was similar to that for the noradrenaline-induced increase in intracellular free Ca(2+) concentration ([Ca(2+)](i)). Noradrenaline stimulated ERK1/2 activity. The magnitude of noradrenaline-induced ERK1/2 activity in the absence of extracellular Ca(2+) was 40% of that in the presence of extracellular Ca(2+). LOE 908 partially (60%) inhibited the noradrenaline-induced ERK1/2 activity, whereas SK&F 96365 did not inhibit it. The IC(50) value of LOE 908 for noradrenaline-induced ERK1/2 activity was similar to that for the noradrenaline-induced increase in [Ca(2+)](i). Collectively, these results demonstrate that extracellular Ca(2+) influx through LOE 908-sensitive and SK&F 96365-resistant NSCC may be essential for noradrenaline-induced mitogenesis in CHO-alpha(1A). Moreover, the noradrenaline-induced ERK1/2 activity involves two distinct pathways, one dependent on extracellular Ca(2+) influx through NSCC, whereas the other is independent of the influx.     

Molecular mechanisms for the activation of Ca2+-permeable nonselective cation channels by endothelin-1 in C6 glioma cells

We recently demonstrated that endothelin-1 (ET-1) activates two types of Ca(2+)-permeable nonselective cation channels (NSCC-1 and NSCC-2) in C6 glioma cells. It is possible to discriminate between these channels by using the Ca(2+) channel blockers SK&F 96365 (1-[beta-(3-[4-methoxyphenyl]propoxy)-4-methoxyphenethyl]-1H-imidazole hydrochloride) and LOE 908 [(R,S)-(3,4-dihydro-6,7-dimethoxy-isoquinoline-1-yl)-2-phenyl-N,N-di-[2-(2,3,4-trimethoxyphenyl)ethyl]-acetamide]. LOE 908 is a blocker for NSCC-1 and NSCC-2, whereas SK&F 96365 is an inhibitor for NSCC-2. The purpose of the present study was to identify the G-proteins that are involved in ET-1-activated Ca(2+) channels in C6 glioma cells. ET-1 activated only NSCC-1 in C6 glioma cells preincubated with U73122 (1-[6-[((17beta)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dione), a phospholipase C (PLC) inhibitor. Microinjection of the dominant negative mutant of G(12)/G(13) (G(12)G228A/G(13)G225A) abolished activation of NSCC-1 and NSCC-2. In contrast, pertussis toxin did not affect any of the Ca(2+) channels in the ET-1-stimulated C6 glioma cells. These results indicate that G(12)/G(13) may couple with endothelin receptors and play an important role in the activation of NSCCs in C6 glioma cells. Moreover, the activation mechanisms of NSCC-1 and NSCC-2 by ET-1 were different. NSCC-1 activation depended upon a G(12)/G(13)-dependent cascade, whereas NSCC-2 activation depended upon both G(q)/PLC- and G(12)/G(13)-dependent cascades.     

Effects of phosphoinositide 3-kinase on endothelin-1-induced activation of voltage-independent Ca2+ channels and vasoconstriction

We recently demonstrated that endothelin-1 (ET-1) activates two types of Ca(2+)-permeable nonselective cation channel (designated NSCC-1 and NSCC-2) and a store-operated Ca(2+) channel (SOCC) in rabbit basilar artery (BA) vascular smooth muscle cells (VSMCs). In this study, we investigated the effects of phosphoinositide 3-kinase (PI3K) on ET-1-induced activation of these channels and BA contraction by using PI3K inhibitors, wortmannin and LY 249002. To determine which Ca(2+) channels are activated via PI3K, monitoring of intracellular Ca(2+) concentration was performed. Role of PI3K in ET-1-induced vasoconstriction was examined by tension study using rabbit BA rings. Only NSCC-1 was activated by ET-1 in wortmannin- or LY 294002-pretreated VSMCs. In contrast, addition of these drugs after ET-1 stimulation did not suppress Ca(2+) influx. Wortmannin inhibited the ET-1-induced contraction of rabbit BA rings that depends on the Ca(2+) influx through NSCC-2 and SOCC. The IC(50) values of wortmannin for the ET-1-induced Ca(2+) influx and vasoconstriction were similar to those for the ET-1-induced PI3K activation. These results indicate that (1) NSCC-2 and SOCC are stimulated by ET-1 via PI3K-dependent cascade, whereas NSCC-1 is stimulated via PI3K-independent cascade; (2) PI3K is required for the activation of the Ca(2+) entry, but not for its maintenance; and (3) PI3K is involved in the ET-1-induced contraction of rabbit BA rings that depends on the extracellular Ca(2+) influx through SOCC and NSCC-2.     

Molecular mechanisms for the activation of voltage-independent Ca2+ channels by endothelin-1 in chinese hamster ovary cells stably expressing human endothelin(A) receptors

We demonstrated recently that in Chinese hamster ovary cells stably expressing human recombinant endothelin(A) receptors (CHO-ET(A)R), 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), which can be distinguished by Ca(2+) channel blockers such as 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenylethyl]-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). We also reported that CHO-ET(A)R couples with G12 in addition to G(q) and G(s). The purpose of the present study was to identify the G proteins involved in the activation of these Ca2+ channels by ET-1, using mutated ET(A)Rs with coupling to either G(q) or G(s)/G12 (designated ET(A)RDelta385 and SerET(A)R, respectively) and a dominant-negative mutant of G12 (G12G228A). ET(A)RDelta385 is truncated immediately downstream of Cys385 in the C terminus as palmitoylation sites, whereas SerET(A)R is unpalmitoylated because of substitution of all the cysteine residues to serine (Cys383Cys385-388 --> Ser383Ser385-388). In CHO-ET(A)RDelta385, stimulation with ET-1 activated only SOCC. In CHO-SerET(A)R or CHO-ET(A)R pretreated with U73122, an inhibitor of phospholipase C (PLC), ET-1 activated only NSCC-1. Dibutyryl cAMP alone did not activate any Ca2+ channels in the resting and ET-1-stimulated CHO-SerET(A)R. Microinjection of G12G228A abolished the activation of NSCC-1 and NSCC-2 in CHO-ET(A)R and that of NSCC-1 in CHO-SerET(A)R. These results indicate that ET(A)R activates three types of Ca2+ channels via different G protein-related pathways. NSCC-1 is activated via a G12-dependent pathway, NSCC-2 via G(q)/PLC- and G12-dependent pathways, and SOCC via a G(q)/PLC-dependent pathway.