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Am J Physiol Cell Physiol 281: C1676-C1685, 2001;
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Vol. 281, Issue 5, C1676-C1685, November 2001

Ca2+ channels activated by endothelin-1 in CHO cells expressing endothelin-A or endothelin-B receptors

Yoshifumi Kawanabe1,2, Yasuo Okamoto1, Taijiro Enoki3, Nobuo Hashimoto2, and Tomoh Masaki1

Departments of 1 Pharmacology, 2 Neurosurgery, and 3 Anesthesiology, Kyoto University Faculty of Medicine, Kyoto 606-8507, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We compared the Ca2+ channels activated by endothelin-1 (ET-1) in Chinese hamster ovary (CHO) cells stably expressing endothelin type A (ETA) or endothelin type B (ETB) receptors using the Ca2+ channel blockers LOE-908 and SK&F-96365. In both CHO-ETA and CHO-ETB, ET-1 at 0.1 nM activated the Ca2+-permeable nonselective cation channel-1 (NSCC-1), which was sensitive to LOE-908 and resistant to SK&F-96365. ET-1 at 1 nM activated NSCC-2 in addition to NSCC-1; NSCC-2 was sensitive to both LOE-908 and SK&F-96365. ET-1 at 10 nM activated the same channels as 1 nM ET-1 in both cell types, but in CHO-ETA, it additionally activated the store-operated Ca2+ channel (SOCC), which was resistant to LOE-908 and sensitive to SK&F-96365. Up to 1 nM ET-1, the level of the formation of inositol phosphates (IPs) was low and similar in both cell types, but, at 10 nM ET-1, it was far greater in CHO-ETA than in CHO-ETB. These results show that, in CHO-ETA and CHO-ETB, ET-1 up to 10 nM activated the same Ca2+ entry channels: 0.1 nM ET-1 activated NSCC-1, and ET-1 >=  1 nM activated NSCC-1 and NSCC-2. Notably, in CHO-ETA, 10 nM ET-1 activated SOCCs because of the higher formation of IPs.

endothelin-1; endothelin receptor; calcium channel; calcium ion; Chinese hamster ovary


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ENDOTHELIN-1 (ET-1) is a 21-amino-acid peptide and is one of the most potent endogenous vasoconstricting agents discovered thus far (26). Subsequent studies have described its multiple, wide-ranging biological activities. The mitogenic activities of ET-1 indicate that it may play a role in the pathogenesis of certain clinical conditions, such as hyperlipoproteinemia and atherosclerosis (7, 12). ET-1 has also been identified as an autocrine/paracrine growth factor in some human cancer cell lines (21).

Recent reports have demonstrated that ET-1 induces contraction of rat aorta and cell proliferation by inducing extracellular Ca2+ influx (21, 27). The increase in intracellular free Ca2+ concentration ([Ca2+]i) induced by ET-1 usually consists of the following two phases: an initial transient increase and a subsequent sustained increase (4, 10, 11, 15). It is generally accepted that the initial transient increase results from mobilization of Ca2+ from the intracellular store, whereas the sustained increase results from entry of extracellular Ca2+ (6). We recently showed that the ET-1-induced sustained increase in [Ca2+]i in A7r5 cells (a cell line derived from rat thoracic aortic smooth muscle cells) mainly results from Ca2+ entry through several Ca2+ channels other than the voltage-operated Ca2+ channel (VOCC). These other Ca2+ channels include two types of Ca2+-permeable nonselective cation channels (designated NSCC-1 and NSCC-2) and the store-operated Ca2+ channel (SOCC; see Refs. 10 and 11). The NSCCs possess a permeability to Ca2+ that is approximately twofold higher than that to monovalent cations (11). The SOCC is highly specific for Ca2+ and is activated by depletion of the intracellular Ca2+ store (5). Of importance, it has been demonstrated that these channels can be distinguished using various Ca2+ channel blockers, such as SK&F-96365 and LOE-908 (3, 14). That is, 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 (11). In other words, LOE-908 is a blocker of NSCCs, whereas SK&F-96365 is a blocker of SOCC and NSCC-2. Moreover, the increase in [Ca2+]i via these channels plays a critical role in ET-1-induced vasoconstriction (27). Therefore, it is important to clarify the mechanisms through which ET-1 activates voltage-independent Ca2+ channels (VICCs). However, vascular smooth muscle cells (VSMCs) express both endothelin type A receptor (ETA) and endothelin type B receptor (ETB; see Refs. 1 and 2). Therefore, it is not known whether stimulation of ETA or ETB on VSMCs results in the activation of different types of Ca2+ channels. If different types of Ca2+ channels are activated, the mechanisms responsible for that difference are not known.

The transfection and functional expression of the cDNA clone for ETA or ETB into the same cell type provide a model system to study the precise signal transduction of a single receptor subtype without any ambiguity resulting from the presence of multiple receptor subtypes. Moreover, several mutant ETA and ETB clones (8, 18) may be useful for studying the mechanisms of the activation of Ca2+ channels by ET-1 in this system. We used Chinese hamster ovary (CHO) cells stably expressing ETA or ETB in the present study. The purpose of the present study was to identify and compare which Ca2+ channels are activated by ET-1 upon binding to ETA or ETB in CHO-ETA or CHO-ETB, respectively, using whole cell recordings of the patch-clamp technique and monitoring of [Ca2+]i, combined with the use of specific Ca2+ channel blockers such as LOE-908 and SK&F-96365. Next, we tried to find the cause of the differential activation of Ca2+ channels by ET-1 between CHO-ETA and CHO-ETB. The results of the present study clarified the mechanisms of the activation of VICCs by ET-1.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. CHO cells were maintained in Ham's F-12 medium supplemented with 10% FCS under a humidified 5% CO2-95% air atmosphere.

Stable expression of ETA or ETB in CHO cells. To obtain a cell line stably expressing ETA or ETB (CHO-ETA and CHO-ETB, respectively), we used a mammalian expression vector, pME18Sf, that carried a cDNA construct encoding human recombinant ETA receptor or ETB receptor. Construction and subcloning of receptor cDNAs were performed as described by Sakamoto et al. (19). Briefly, each expression vector was cotransfected with pSVbsrr plasmid into CHO cells by lipofection using Lipofectamine (Life Technologies, Tokyo, Japan) according to the manufacturer's instructions. Cell populations expressing the bsrr gene product were selected in Ham's F-12 medium supplemented with 10% FCS and 0.5 µg/ml blasticidin. From these selected populations, clonal cell lines were isolated by colony lifting and were maintained in the same selection medium.

Formation of inositol phosphates. The level of the formation of inositol phosphates (IPs) was determined as described previously (22). Briefly, CHO-ETA or CHO-ETB in 24-well plates were incubated with myo-[3H]inositol (final concentration, 5 µCi/ml) in 0.3 ml of Ham's F-12 medium supplemented with 10% FCS for 18 h. After being washed, the cells were incubated with or without various concentrations of ET-1 for 30 min, and the reaction was terminated by adding ice-cold perchloric acid. After neutralization with KOH and Tris, the samples were applied to small columns of AG1X8 (100-200 mesh, Cl- form; Bio-Rad, Hercules, CA) to separate the total IPs from the myo-[3H]inositol. The 3H-labeled IPs were eluted with 1 N HCl, and the radioactivity was counted with a liquid scintillation counter.

Monitoring of [Ca2+]i in CHO-ETA and CHO-ETB. The [Ca2+]i was monitored using the fluorescent probe fluo 3, as described previously (4). Briefly, CHO-ETA or CHO-ETB were loaded with fluo 3 by incubating the cells with 10 µM fluo 3-AM at 37°C under reduced light for 30 min. After being washed, the cells were suspended at a density of ~2 × 107 cells/ml, and 0.5-ml aliquots were used for measurement of fluorescence by a CAF 110 spectrophotometer (JASCO, Tokyo, Japan) with an excitation wavelength of 490 nm and an emission wavelength of 540 nm. At the end of the experiment, Triton X-100 and subsequently EGTA were added at a final concentration of 0.1% or 5 mM, respectively, to obtain the maximum fluorescence (Fmax) and the minimum fluorescence (Fmin). The [Ca2+]i was determined by the equilibrium equation [Ca2+]i = Kd(F - Fmin)/(Fmax - F), where F is the experimental value of fluorescence and the dissociation constant (Kd) was defined as 0.4 µM (16).

Electrophysiology. CHO-ETA or CHO-ETB were perfused with Krebs-HEPES solution and visualized with Nomarski optics (Zeiss, Tokyo, Japan). Whole cell recordings were made with thin-wall borosilicate glass patch pipettes (resistance, 3-5 MOmega ) as previously described (4). The Krebs-HEPES solution contained (in mM) 140 NaCl, 3 KCl, 2 CaCl2, 1 MgCl2, 11 glucose, and 10 HEPES (adjusted to pH 7.3 with NaOH). The pipettes were filled with cesium aspartate solution containing (in mM) 120 cesium aspartate, 20 CsCl, 2 MgCl2, 10 HEPES, and 10 EGTA (adjusted to pH 7.3 with CsOH). EGTA was added to the pipette solution at a final concentration of 10 mM; this concentration of EGTA had enough buffering capacity for Ca2+ to prevent a transient increase in [Ca2+]i (17), and the concentration of Ca2+ in the solution was maintained at 100 nM by adding the appropriate amount of CaCl2 as described previously (24). Tight-seal whole cell currents were recorded with a EPC7 patch-clamp amplifier (List, Darmstadt, Germany). The perfusion rate was maintained at 2.2 ~ 2.5 ml/min, and the bath volume was ~1.0 ml. All experiments were performed under voltage clamp at a holding potential of -60 mV at room temperature (22 ~ 24°C). To test the contribution of the Cl- current, the bath solution was switched from Krebs-HEPES to a solution with a low Cl- concentration that contained (in mM) 140 sodium gluconate, 3 KCl, 2 CaCl2, 1 MgCl2, 11 glucose, and 10 HEPES (pH 7.3). The permeability of Ca2+ through channels was measured in a Ca2+-N-methyl-D-glucamine (NMDG) solution containing (in mM) 30 CaCl2, 100 NMDG chloride, 11 glucose, and 10 HEPES (adjusted to pH 7.3 with Tris). Current-voltage relationships were obtained by applying voltage steps of 100-ms duration ranging from -100 to +80 mV in 20-mV increments, before and after application of ET-1 or channel blocker(s). The ET-1-induced current at each membrane potential was determined by subtracting the current before application of ET-1 from the current after its application. The drug-inhibited current was determined by subtracting the current after application of the drug from the current before its application.

Drugs. LOE-908 was kindly provided by Boehringer Ingelheim (Ingelheim, Germany). Other chemicals were obtained commercially from the following sources: ET-1 from Peptide Institute (Osaka, Japan); SK&F-96365 from Biomol (Plymouth Meeting, PA); fluo 3-AM from Dojindo Laboratories (Kumamoto, Japan); and 125I-labeled ET-1 and myo-[3H]inositol from Amersham Pharmacia Biotech (Buckinghamshire, UK).

Statistical analysis. All results were expressed as means ± SE.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Stable expression of ETA or ETB in CHO cells. By cotransfecting CHO cells with each expression plasmid and pSVbsrr and then selecting for resistance against blasticidin, we obtained eight individual clonal cell lines that stably expressed ETA and 10 individual clonal cell lines that stably expressed ETB. 125I-labeled ET-1 binding assays using membrane preparations from each clone gave Kd values of 30 ~ 150 pM and maximal binding (Bmax) values of 0.5 ~ 1.7 pmol/mg protein. Cell clones with a similar level of affinity for ET-1 and receptor density were used in the subsequent studies (the Kd and Bmax values of these cell clones were 52.8 ± 2.4 pM and 1.08 ± 0.16 pmol/mg protein, respectively, for CHO-ETA and 46.8 ± 5.3 pM and 0.97 ± 0.08 pmol/mg protein, respectively, for CHO-ETB).

Characterization of the currents induced by ET-1 in CHO-ETA and CHO-ETB with whole cell recordings of the patch clamp. To elucidate the ionic channels in CHO-ETA and CHO-ETB that were activated by ET-1, whole cell recordings of CHO-ETA and CHO-ETB were performed. Stimulation with various concentrations of ET-1 (0.1, 1, or 10 nM) induced an inward current in CHO-ETA and CHO-ETB held at -60 mV (Fig. 1, A-D). The currents induced by 0.1, 1, or 10 nM of ET-1 showed linear current-voltage relationships in both CHO-ETA and CHO-ETB with a reversal potential of -4.4 ± 1.5 mV (n = 15), -1.7 ± 2.7 mV (n = 15), or -0.8 ± 2.2 mV (n = 15), respectively, in CHO-ETA (Fig. 1E) and -5.2 ± 2.8 mV (n = 15), -1.0 ± 2.4 mV (n = 15), or -0.6 ± 3.3 mV (n = 15), respectively, in CHO-ETB (Fig. 1F).


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  		Fig. 1.   Whole cell recordings of the currents in Chinese hamster ovary (CHO) cells stably expressing endothelin type A or endothelin type B receptor (CHO-ETA or CHO-ETB, respectively) induced by incubation with various concentrations of endothelin-1 (ET-1). A-D: original tracings showing whole cell currents in CHO-ETA (A and B) and CHO-ETB (C and D) that were treated with ET-1. The cells were clamped at a holding potential of -60 mV with the whole cell configuration, and ET-1 (final concentration 0.1 or 10 nM) was added to the bath solution at the time indicated by the arrow. E and F: current-voltage relationships of the currents induced by 0.1, 1, or 10 nM ET-1 in CHO-ETA (E) and CHO-ETB (F). Cells were treated with 0.1, 1.0, or 10 nM ET-1. We applied a 100-ms voltage step at time x before ET-1 treatment and at time y after the cells had reached a steady state after ET-1 was added. The current was measured at time x and at time y, and the current at x was subtracted from the current at y. We applied voltage steps ranging from -100 to +80 mV in 20-mV increments. G and H: current-voltage relationships of the whole cell Ca2+ current in CHO-ETA (G) and CHO-ETB (H) that were induced by ET-1. Whole cell recordings of the patch clamp and application of voltage steps were performed using Ca2+-N-methyl-D-glucamine (NMDG) solution. open circle , 0.1 nM ET-1; , 1 nM ET-1; , 10 nM ET-1.

The current-voltage relationships of the currents induced by any of the three concentrations of ET-1 in either cell type were not affected by reducing the concentration of Cl- in the bath solution from 149 to 9 mM (data not shown). The reversal potential for 0.1, 1, or 10 nM ET-1 in a solution with a low Cl- concentration (9 mM) was -3.6 ± 2.2 mV (n = 6), -1.2 ± 1.4 mV (n = 6), and -0.5 ± 2.4 mV (n = 6), respectively, in CHO-ETA and -4.4 ± 1.8 mV (n = 6), -0.8 ± 2.3 mV (n = 6), and -0.4 ± 1.4 mV (n = 6), respectively, in CHO-ETB. These values did not significantly differ from those obtained in the cells incubated in Krebs-HEPES solution containing the normal concentration of Cl-.

To test whether the channels activated by ET-1 are permeable to Ca2+, all other cations in the bath solution were replaced with the nonpermeant cation NMDG while the concentration of Ca2+ was increased from 1 to 30 mM. Even under such conditions, ET-1 at 0.1, 1, or 10 nM induced an inward current in CHO-ETA and CHO-ETB held at -60 mV (Fig. 1, G and H). The reversal potential for 0.1, 1, or 10 nM ET-1 was -12.3 ± 1.6 mV (n = 6), -11.5 ± 1.2 mV (n = 6), and -10.8 ± 2.2 mV (n = 6), respectively, in CHO-ETA (Fig. 1G) and -12.1 ± 1.8 mV (n = 6), -11.5 ± 1.3 mV (n = 6), and -10.3 ± 1.4 mV (n = 6), respectively, in CHO-ETB (Fig. 1H).

Pharmacological properties of the whole cell currents induced by ET-1 in CHO-ETA and CHO-ETB. To determine the maximally effective concentration of various Ca2+ channel blockers, we first examined the effect of various concentrations (30 nM ~ 30 µM) of SK&F-96365 or LOE-908 on the whole cell currents in CHO-ETA and CHO-ETB induced by 0.1, 1, or 10 nM ET-1. SK&F-96365 and LOE-908 each inhibited ET-1-induced whole cell currents in a concentration-dependent manner, and the maximal effect was seen at concentrations >= 10 µM (data not shown). On the basis of these data, we decided to use 10 µM as the concentration of SK&F-96365 and LOE-908 in the following experiments.

In CHO-ETA, the current induced by 0.1 nM ET-1 was abolished by 10 µM LOE-908 (Fig. 2B), whereas it was not affected by 10 µM SK&F-96365 (Fig. 2A). The current inhibited by LOE-908 showed a linear current-voltage relationship and a reversal potential of -3.9 ± 3.0 mV (n = 6; Fig. 2E), indicating that LOE-908 suppressed the same current activated by ET-1. The currents induced by 1 and 10 nM ET-1 in both cell types were also abolished by 10 µM LOE-908 (10 nM; Fig. 2D); a major portion (~65%) of the current was suppressed by 10 µM SK&F-96365 (10 nM ET-1 in CHO-ETA, Fig. 2C). The characteristics of the currents inhibited by SK&F-96365 or LOE-908 were similar to those of the ET-1-induced currents in terms of the linear current-voltage relationship and the reversal potential of -2.6 ± 2.2 mV (n = 6) or -3.6 ± 2.3 mV (n = 6), respectively (Fig. 2F), indicating that both drugs suppressed the same currents activated by ET-1. The pharmacological properties of the whole cell currents induced by ET-1 in CHO-ETB were similar to those in CHO-ETA (Fig. 2, G and H). On the basis of these results, the current induced by 1 or 10 nM ET-1 was divided into two components. Namely, one component is sensitive to LOE-908 and resistant to SK&F-96365, and the second component is sensitive to both LOE-908 and SK&F-96365.


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  		Fig. 2.   Effect of SK&F-96365 and LOE-908 on the whole cell currents induced by various concentrations of ET-1 in CHO-ETA and CHO-ETB. A-D: original tracings showing the whole cell current in CHO-ETA that had been activated by 0.1 nM (A and B) or 10 nM (C and D) ET-1 and then treated with SK&F-96365 or LOE-908. The experimental protocol was the same as that described in Fig. 1. After the ET-1-induced current had reached a steady state, either SK&F-96365 (A and C) or LOE-908 (B and D) was added to the bath solution at a final concentration of 10 µM. E-H: current-voltage relationships of the currents induced by 0.1 or 10 nM ET-1 in CHO-ETA (E and F) and CHO-ETB (G and H). At the times indicated by x, y, and z, when the cells were in a stable condition, a voltage step of 100-ms duration was applied. Voltage steps ranging from -100 to +80 mV in 20-mV increments were applied. The ET-1-induced current at each membrane potential was obtained by subtracting the current at x from that at y or the current at z from that at y, and these were plotted against the membrane potential. E and G: open circle , current obtained by subtracting the current at x from that at y; , current obtained by subtracting the current at z from that at y. F and H: circles, subtracted currents in the presence of LOE-908; squares, subtracted currents in the presence of SK&F-96365; open circle , current obtained by subtracting the current at x from that at y; , current obtained by subtracting the current at z from that at y; , current obtained by subtracting the current at x from that at y; , current obtained by subtracting the current at z from that at y.

Pharmacological properties of the SOCC in CHO cells. Treatment of cells with thapsigargin (an inhibitor of Ca2+-pump ATPase on the membrane of the sarcoplasmic/endoplasmic reticulum) depletes the intracellular store of Ca2+ and thereby activates the Ca2+ channels on the plasma membrane called SOCCs or capacitative Ca2+ entry channels, causing a sustained increase in [Ca2+]i (23). Therefore, a sustained increase in [Ca2+]i is regarded as an index of the activity of SOCC. Our recent studies showed that the thapsigargin-induced increase in [Ca2+]i in A7r5 cells and in VSMCs in primary culture was abolished by SK&F-96365 but was not affected by nifedipine or LOE-908 (11, 27). We characterized the pharmacological properties of the SOCCs in CHO cells using thapsigargin.

The sustained increase in [Ca2+]i in wild-type CHO cells that had been induced by 0.1 µM thapsigargin was suppressed by SK&F-96365 in a concentration-dependent manner with an IC50 value of ~2 µM and was abolished at concentrations >= 10 µM (Fig. 3). However, the thapsigargin-induced increase in [Ca2+]i was not affected by LOE-908 (Fig. 3B) or nifedipine (data not shown) up to the concentration of 30 or 10 µM, respectively. These results demonstrate that the SOCCs in CHO cells have the same pharmacological properties as those in VSMCs in primary culture and A7r5 cells.


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  		Fig. 3.   Effects of various concentrations of SK&F-96365 or LOE-908 on the thapsigargin-induced increase in intracellular Ca2+ concentration ([Ca2+]i) in wild-type CHO cells as an index of the activity of store-operated Ca2+ channels. Wild-type CHO cells were loaded with fluo 3 and subjected to monitoring of [Ca2+]i. The cells were exposed to 0.1 µM thapsigargin during the period of time indicated by the horizontal hatched bar. After the [Ca2+]i reached a steady state, increasing concentrations of either SK&F-96365 or LOE-908 were added to the bath solution. A: original tracing showing the inhibitory effect of increasing concentrations of SK&F-96365 on the thapsigargin-induced increase in [Ca2+]i. B: concentration-response curves of the effect of SK&F-96365 () or LOE-908 (open circle ) on the thapsigargin-induced increase in [Ca2+]i. The level of inhibition of the increase by a drug was represented as the percentage of the level of [Ca2+]i after treatment with thapsigargin and just before the addition of the drug (control). Each point represents the mean ± SE of 5 experiments.

Basic properties of the ET-1-induced increase in [Ca2+]i in CHO-ETA and CHO-ETB. ET-1 at 0.1 nM induced a monophasic increase in [Ca2+]i in CHO-ETA and CHO-ETB, as monitored by a Ca2+ indicator, fluo 3 (Fig. 4, A and E). In contrast, higher concentrations (1 and 10 nM) of ET-1 induced a biphasic increase in [Ca2+]i consisting of an initial transient peak and a subsequent sustained increase (Fig. 4, B, C, F, and G). In experiments performed on cells incubated in a bath in which the extracellular Ca2+ had been removed, upon treatment with 1 or 10 nM ET-1, the transient peak was not affected, but the sustained increase induced by either concentration of ET-1 was abolished (data not shown), indicating that only the sustained increase in [Ca2+]i results from Ca2+ influx.


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  		Fig. 4.   A-C and E-G: original tracings showing the effect of treatment with various concentrations of ET-1 on the [Ca2+]i in CHO-ETA and CHO-ETB. CHO-ETA (A-C) and CHO-ETB (E-G) cells were loaded with a Ca2+ indicator, fluo 3, and subjected to monitoring of [Ca2+]i. ET-1 at 0.1, 1, or 10 nM was added to the bath solution at the time indicated by the arrow. D and H: concentration-response curves of the ET-1-induced increase in [Ca2+]i in CHO-ETA (D) and CHO-ETB (H). The transient increase (open circle ) and the sustained increase () of [Ca2+]i were plotted separately against the concentration of ET-1. Each point represents the mean ± SE of 5 experiments.

The magnitude of the transient peak and that of the sustained increase in [Ca2+]i depended on the concentration of ET-1 in both cell types (Fig. 4). However, the threshold concentration of ET-1 for induction of the transient peak or the sustained increase differed; among the tested concentrations of ET-1, the lowest concentration at which the transient increase in [Ca2+]i was seen was 1 nM ET-1, whereas the lowest concentration at which the sustained increase in [Ca2+]i was seen was 0.1 nM in both cell types (Fig. 4).

The concentration of ET-1 at which the magnitude of the increase in [Ca2+]i reached the maximum differed between CHO-ETA and CHO-ETB. In CHO-ETA, both the transient peak and the sustained increase reached a plateau at 10 nM ET-1 (Fig. 4D), whereas in CHO-ETB they reached a plateau at 1 nM ET-1 (Fig. 4H). Furthermore, the magnitude of the transient peak and that of the sustained increase in CHO-ETA were greater than those in CHO-ETB (Fig. 4).

Pharmacological analysis of the increase in [Ca2+]i induced by various concentrations of ET-1. In CHO-ETA, the sustained increase in [Ca2+]i upon treatment with ET-1 was suppressed by LOE-908 in a concentration-dependent manner with an IC50 value of ~3 µM, and maximal inhibition was observed at concentrations >= 10 µM (Figs. 5, B and D, and Fig. 7B). However, the extent of the maximal inhibition of LOE-908 differed depending on the concentration of ET-1; the inhibition amounted to 100% at 0.1 and 1 nM ET-1, whereas ~40% of the ET-1-induced increase in [Ca2+]i was left unsuppressed at 10 nM ET-1 (Fig. 7B). In CHO-ETB, the sustained increase in [Ca2+]i upon treatment with ET-1 was also suppressed by LOE-908 in a concentration-dependent manner with an IC50 value of ~3 µM, and maximal inhibition was observed at concentrations >= 10 µM (Figs. 6, B and D, and Fig. 7D). In this case, the inhibition by LOE-908 was virtually complete regardless of the concentration of ET-1 (Fig. 7D).


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  		Fig. 5.   Original tracings showing the effects of various concentrations of LOE-908 or SK&F-96365 on the [Ca2+]i in CHO-ETA that had been treated with 0.1 or 10 nM ET-1. Cells loaded with fluo 3 were stimulated with either 0.1 nM (A and B) or 10 nM (C and D) ET-1. After the [Ca2+]i reached a steady state, increasing concentrations of either SK&F-96365 (A and C) or LOE-908 (B and D) were added at the times indicated by the arrow.



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  		Fig. 6.   Original tracings showing the effect of various concentrations of SK&F-96365 (A and C) or LOE-908 (B and D) on the [Ca2+]i in CHO-ETB that had been treated with 0.1 nM (A and B) or 10 nM (C and D) ET-1. The experiments were performed as described in the legend for Fig. 5, using CHO-ETB instead of CHO-ETA.



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  		Fig. 7.   Concentration-response curves of the inhibition of the ET-1-induced increase in [Ca2+]i by SK&F-96365 or LOE-908 in CHO-ETA and CHO-ETB. CHO-ETA (A and B) and CHO-ETB (C and D) cells were loaded with a Ca2+ indicator, fluo 3, and subjected to monitoring of [Ca2+]i. The cells were stimulated with 0.1 nM (open circle ), 1 nM (), or 10 nM (triangle ) ET-1. After the [Ca2+]i reached a steady state, increasing concentrations of either SK&F-96365 (A and C) or LOE-908 (B and D) were added. The [Ca2+]i after stimulation with 0.1, 1, or 10 nM ET-1 was set at 100%, and the [Ca2+]i before stimulation with ET-1 was set at 0%. The [Ca2+]i after the addition of SK&F-96365 or LOE-908 was represented on this scale. Each point represents the mean ± SE of 5 experiments.

Different responses were obtained in the cells treated with SK&F-96365. That is, in CHO-ETA, the sustained increase in [Ca2+]i induced by 0.1 nM ET-1 was virtually resistant to SK&F-96365 (Figs. 5A and 7A). However, the increase induced by 1 or 10 nM ET-1 was suppressed by SK&F-96365 in a concentration-dependent manner, and maximal inhibition was observed at concentrations >= 10 µM (Figs. 5C and 7A); the extent of the inhibition by SK&F-96365 was larger at higher concentrations of ET-1 (the inhibition against 1 and 10 nM ET-1 amounted to 65 and 80%, respectively; Fig. 7A). In CHO-ETB, similar results were obtained except that the extent of the inhibition against 1 and 10 nM ET-1 did not differ (Fig. 6, A, and C, and Fig. 7C).

Summary of the inhibitory effects of the maximally effective concentration of LOE-908, SK&F-96365, and their combination on the ET-1-induced increase in [Ca2+]i. Table 1 summarizes the inhibitory effect of the maximally effective concentration (10 µM) of LOE-908, SK&F-96365, and their combination on the sustained increase in [Ca2+]i induced by ET-1.

                              
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  		Table 1.   Inhibitory effects of SK&F-96365, LOE-908, or their combination on the sustained increase in [Ca2+]i induced by various concentrations of ET-1 in CHO cells expressing ETA or ETB

In CHO-ETA and CHO-ETB, the sustained increase in [Ca2+]i induced by 0.1 nM ET-1 was not affected by SK&F-96365 but was abolished by LOE-908. In contrast, the sustained increase in [Ca2+]i induced by 1 nM ET-1 was partially suppressed by SK&F-96365, and it was abolished by LOE-908. The SK&F-96365-resistant part was abrogated by the combined treatment with LOE-908.

The increase in [Ca2+]i induced by 10 nM ET-1 in CHO-ETA and CHO-ETB showed different responses to these blockers. That is, in CHO-ETB, the responses to these blockers were the same as those that had been induced by 1 nM ET-1. However, in CHO-ETA the increase in [Ca2+]i was partially resistant to both SK&F-96365 and LOE-908, and the SK&F-96365-resistant part or LOE-908-resistant part was abrogated by the combined treatment with LOE-908 or SK&F-96365, respectively.

Formation of IPs in CHO-ETA and CHO-ETB after stimulation with ET-1. To gain insight into the mechanisms underlying the differential activation of Ca2+ channels by 10 nM ET-1 in CHO-ETA and CHO-ETB, we measured the level of the formation of IPs in CHO-ETA and CHO-ETB that had been treated with various concentrations of ET-1 (Fig. 8).


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  		Fig. 8.   Formation of total inositol phosphates (IPs) in CHO-ETA and CHO-ETB after stimulation with various concentrations of ET-1. Cells that had been incubated with myo-[3H]inositol for 18 h were stimulated by various concentrations of ET-1 for 30 min. The level of total IPs in the cell extract was determined as described in MATERIALS AND METHODS. , CHO-ETA; , CHO-ETB; open circle , wild-type CHO cells. Each point represents the mean ± SE of 5 experiments.

In both cell types, stimulation with ET-1 increased the formation of IPs in a concentration-dependent manner with an EC50 value of ~1 nM, and the maximal response was seen at a concentration of ET-1 of 10 nM. The level of formation of IPs in CHO-ETA was comparable to that in CHO-ETB up to 1 nM ET-1, but at concentrations of ET-1 >= 10 nM, it was approximately threefold higher than that in CHO-ETB (Fig. 8).

Effect of thapsigargin on the [Ca2+]i after stimulation with ET-1 in CHO-ETA and CHO-ETB. In CHO-ETA, thapsigargin induced a further increase in [Ca2+]i when it was added during the sustained increase in [Ca2+]i that had been induced by 1 nM ET-1 (Fig. 9A) or 0.1 nM ET-1 (data not shown), but it did not induce a further increase after stimulation with 10 nM ET-1 (Fig. 9B). In contrast, thapsigargin induced a further increase in [Ca2+]i in CHO-ETB regardless of whether the concentration of ET-1 was 1 or 10 nM (Fig. 9, C and D).


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  		Fig. 9.   Effect of thapsigargin on the [Ca2+]i in CHO-ETA and CHO-ETB that had been treated with 1 or 10 nM ET-1. CHO-ETA (A and B) and CHO-ETB (C and D) loaded with fluo 3 were stimulated with either 1 nM (A and C) or 10 nM (B and D) ET-1. After the [Ca2+]i reached a steady state, 0.1 µM thapsigargin was added at the time indicated by the arrow.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Characterization of the Ca2+ channels activated by ET-1 in CHO-ETA and CHO-ETB. In CHO-ETA and CHO-ETB, the whole cell currents induced by ET-1 at 0.1, 1, and 10 nM are considered to be conducted through NSCCs for the following reasons: 1) the current-voltage relationships are linear (Fig. 1); 2) the reversal potentials are close to 0 mV (Fig. 1); and 3) the reversal potentials were not affected by reducing the concentration of Cl- in the bath solution (see RESULTS). Furthermore, the channels are considered to be permeable to Ca2+ because the current could be induced by ET-1 even in a bath solution containing only Ca2+ as the movable cation; the permeability ratio of Ca2+ to Cs+ is ~1.6 (Fig. 1).

The current activated by 0.1 nM ET-1 in CHO-ETA and CHO-ETB consists of only one component in terms of its pharmacology. Judging from the findings that it was sensitive to LOE-908 and insensitive to SK&F-96365 (Fig. 2), the current is considered to be conducted through NSCC-1. In contrast, the current activated by 1 or 10 nM ET-1 can be divided into two components according to their sensitivity to the blockers. One component is sensitive to LOE-908 and resistant to SK&F-96365, whereas the second component is sensitive to both LOE-908 and SK&F-96365 (Fig. 2). The pharmacological criteria indicated that the former component is carried through NSCC-1, whereas the latter is carried through NSCC-2.

As reported previously (11), the Ca2+ current through SOCCs cannot be monitored under our conditions of whole cell recordings. This is probably because of latent activation of SOCCs under the basal condition, which results from the presence of a Ca2+ chelator, EGTA, in the pipette solution (to prevent Ca2+-activated currents). EGTA has been reported to deplete the intracellular Ca2+ store and activate SOCCs (9). In the present study, the activity of SOCCs was monitored by measuring the increase in [Ca2+]i after the addition of thapsigargin (Fig. 3). As reported for A7r5 cells and VSMCs in primary culture (11, 27), the SOCCs in CHO cells are sensitive to SK&F-96365 and resistant to LOE-908 (Fig. 3).

Taken together, these data show that the NSCC-1, NSCC-2, and SOCC in CHO cells possess the same pharmacology (i.e., sensitivity to LOE-908 and SK&F-96365) as the respective channel in A7r5 cells and VSMCs in primary culture (11, 27).

Ca2+ channels involved in the increase in [Ca2+]i induced by various concentrations of ET-1 in CHO-ETA and CHO-ETB. In CHO cells, VOCCs do not seem to be involved in the ET-1-induced increase in [Ca2+]i for the following reasons: 1) CHO cells are nonexcitable cells that usually lack VOCCs; 2) depolarization by high K+ stimulation did not elevate the [Ca2+]i (data not shown); and 3) the ET-1-induced increase in [Ca2+]i was resistant to specific blockers of L-type VOCC such as nifedipine (data not shown). Therefore, VICCs play a critical role in the ET-1-induced increase in [Ca2+]i.

In CHO-ETA and CHO-ETB, the increase in [Ca2+]i induced by the lowest effective concentration (0.1 nM) of ET-1 is considered to result from Ca2+ entry through only one type of Ca2+-permeable NSCC (Figs. 5-7). On the basis of its pharmacology (sensitive to LOE-908 and resistant to SK&F-96365; Fig. 7 and Table 1), this channel is regarded to be NSCC-1.

At 1 nM ET-1, the increase in [Ca2+]i in both cell types seemed to involve Ca2+ entry through two types of Ca2+-permeable NSCCs in terms of its sensitivity to channel blockers. That is, the major portion of the increase in [Ca2+]i (65%) was sensitive to both LOE-908 and SK&F-96365, whereas the remaining portion (35%) was sensitive to LOE-908 and resistant to SK&F-96365 (Fig. 7 and Table 1). From the pharmacological point of view, the former is mediated by Ca2+ entry through NSCC-2, whereas the latter is mediated by Ca2+ entry through NSCC-1.

Notably, different Ca2+ channels seemed to be activated by the saturating concentration (10 nM) of ET-1 in CHO-ETA and CHO-ETB. That is, the increase in [Ca2+]i in CHO-ETB induced by 10 nM ET-1 showed the same pharmacology as that induced by 1 nM ET-1 (Fig. 7, C and D, and Table 1), indicating that the increase involved Ca2+ entry through NSCC-1 and NSCC-2, which contributed ~35 and 65%, respectively, of the total Ca2+ entry. However, in CHO-ETA, the increase in [Ca2+]i consisted of three components in terms of the sensitivity to SK&F-96365 and LOE-908 (Fig. 7, A and C, and Table 1). One component, which contributed ~20% of the total increase in [Ca2+]i, was resistant to SK&F-96365 and sensitive to LOE-908, and the second component, which contributed ~40% of the total increase in [Ca2+]i, was resistant to LOE-908 and sensitive to SK&F-96365 (Table 1). According to the pharmacological criteria, the first component is NSCC-1, and the second component is SOCC. Because the LOE-908-sensitive portion (contributing ~60%) of the total increase in [Ca2+]i consisted of Ca2+ influx through NSCC-1 and NSCC-2, the contribution of Ca2+ influx through NSCC-2 was calculated to be 40%. Thus, in CHO-ETA treated with 10 nM ET-1, Ca2+ influx through NSCC-1, NSCC-2, and SOCC contributes ~20, 40, and 40%, respectively, of the total increase in [Ca2+]i. Moreover, on the basis of sensitivity to SK&F-96365 and LOE-908, the VICCs activated by ET-1 in CHO-ETA may be the same channels activated by ET-1 in A7r5 cells.

Mechanisms of the differential activation of SOCCs. The differential activation of SOCCs by 10 nM ET-1 seems to be the result of the larger amount of IP3 produced and hence depletion of the intracellular Ca2+ store in CHO-ETA for the following reasons: 1) when the level of the formation of IPs, which was used as an index of IP3 formation, was low and comparable between CHO-ETA and CHO-ETB (<= 1 nM ET-1), NSCCs but not SOCCs were activated in both cell types (Fig. 8 and Table 1); 2) when the level of formation of IPs was higher in CHO-ETA than in CHO-ETB (at 10 nM ET-1), SOCCs were activated only in CHO-ETA (Fig. 8 and Table 1); 3) thapsigargin, which specifically activates SOCCs by depleting the intracellular Ca2+ store, further enhanced the increase in [Ca2+]i induced by ET-1 only when the level of the formation of IPs was low (0.1 and 1 nM ET-1 for CHO-ETA; 0.1, 1, and 10 nM ET-1 for CHO-ETB; Fig. 9, A, C, and D). In contrast, thapsigargin did not further increase the [Ca2+]i in CHO-ETA that had been induced by 10 nM ET-1 (Fig. 9B).

To exclude the possibility that heterogeneity in the coupling of ETA to SOCC within CHO cells exists, we used different CHO-ETA and CHO-ETB clones to look for the absence or gain of SOCC coupling to endothelin receptors. Assays on several independent clones with various receptor densities suggested that, within the range of receptor densities that we could obtain, these differences were independent of the differences in receptor density. All clones expressing ETA or ETB gave essentially the same results (data not shown). Thus the difference in SOCC activation between ETA and ETB is the result of intrinsic differences between the two receptor subtypes.

Mechanisms of the activation of VICCs. The mechanisms underlying the activation of VICCs are presently not known. However, different mechanisms seem to be involved in the activation of these channels, because the concentration of ET-1 required for their activation differed. Several lines of evidence suggest that the alpha -subunit or beta gamma -subunit of G proteins is involved in the Ca2+ influx activated by stimulation of G protein-coupled receptors (13, 20, 25). The mechanisms underlying the activation of VICCs are now under investigation in our laboratory using CHO cells stably expressing mutant ETA and ETB with antisense oligonucleotides against G proteins and dominant-negative mutants of G proteins.


    ACKNOWLEDGEMENTS

We thank Boehringer Ingelheim (Ingelheim, Germany) for the kind donation of LOE-908.


    FOOTNOTES

Address for reprint requests and other correspondence: Y. Kawanabe, Dept. of Neurosurgery, Kyoto Univ. Faculty of Medicine, 54 Shougoin-Kawaharachou, Sakyo-ku, Kyoto 606-8507, Japan (E-mail: kawanabe{at}kuhp.kyoto-u.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 26 April 2001; accepted in final form 10 July 2001.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Cell Physiol 281(5):C1676-C1685
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