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

Abstract

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.

Figure 1

Original tracings illustrating the effects of varying concentrations of LOE 908 and removal of extracellular Ca²⁺ on the increase in the intracellular free Ca²⁺ concentrations ([Ca²⁺]_i) in A7r5 cells induced by either a lower (1 nM) or higher (100 nM) concentration of endothelin-1 (ET-1). Fluo-3-loaded A7r5 cells were stimulated by either 1 nM (a and c) or 100 nM ET-1 (b and d) in the presence (a and b) or absence (c and d) of extracellular Ca²⁺. After the response to ET-1 reached a steady-state, varying concentrations of LOE 908 were added to the medium. In all experiments, nifedipine was added to the medium at the final concentration of 1 μM.

Figure 2

Original tracings illustrating the effects of SK&F 96365 and LOE 908 on the increase in [Ca²⁺]_i following addition of Ca²⁺ in A7r5 cells pretreated with thapsigarigin. A7r5 cells were loaded with fura-2 for SK&F 96365 (a) and with fluo-3 for LOE 908 (b). The A7r5 cells were preincubated in Ca²⁺-free Krebs-HEPES solution containing 0.1 μM thapsigargin for 10 min and subsequently for further 5 min in the same solution with or without either SK&F 96365 or LOE 908. The activities of Ca²⁺ influx were studied by addition of 2 mM Ca²⁺ to the bath solution.

Figure 3

Concentration-response curves for inhibitory effects of LOE 908 on Ca²⁺ influx through store-operated Ca²⁺ channel (SOCC) activated by either thapsigargin or ionomycin and through Ca²⁺-permeable nonselective cation channel (NSCC) activated by ET-1. For measurement of Ca²⁺ influx through SOCC, A7r5 cells loaded with fluo-3 were preincubated in Ca²⁺-free Krebs-HEPES solution containing 0.1 μM thapsigargin or 0.1 μM ionomycin for 10 min and subsequently for further 5 min in the same solution with or without varying concentrations of LOE 908. The activities of Ca²⁺ influx were studied by addition of 2 mM Ca²⁺ to the solution. For measurement of Ca²⁺ influx through NSCC, fluo-3-loaded A7r5 cells were preincubated in normal Krebs-HEPES with or without varying concentrations of LOE 908 for 5 min, and stimulated with 1 nM or 100 nM ET-1. The increases in [Ca²⁺]_i in the presence of LOE 908 were represented as percentages of those in its absence. Each point represents mean value±s.e.mean of six experiments. ^*P<0.01; significantly different from control values without pretreatment by LOE 908. §P<0.01; significantly different from the value with 100 nM ET-1.

Figure 4

Effects of LOE 908 or SK&F 96365 on whole-cell currents induced by a lower (1 nM) or higher (10 nM) concentration of ET-1 in A7r5 cells in the presence of 1 μM nifedipine. The cells were clamped at a holding potential of −60 mV with the whole-cell configuration, and ET-1 was added to the bath solution at the final concentration of 1 nM (a, c and e) or 10 nM (b, d and f). After the ET-1-induced currents reached a steady-state, SK&F 96365 (a and b) or LOE 908 (c and d) was added at 10 μM during the time intervals indicated by horizontal bars. At the time indicated by x, y and z in (c and d), voltage steps ranging from −100 to +80 mV in 20 mV increments were applied. (e and f) represent the current-voltage relationships obtained from the data in (c and d), respectively. The currents induced by ET-1 were obtained by subtracting currents at x from those at y, and the currents inhibited by LOE 908 were obtained by subtracting currents at z from those at y.

Figure 5

Summary of the effects of LOE 908 or SK&F 96365 on whole-cell currents induced by lower (1 nM) or higher (10 nM and 100 nM) concentrations of ET-1 in A7r5 cells in the presence of 1 μM nifedipine. Experimental protocols were the same as those in Figure 4, except that stimulation with 100 nM ET-1 was also performed. Each bar represents mean value±s.e.mean of the number of experiments shown in parentheses. ^*P<0.01, significantly different from control values without blockers. §rcub;P<0.01; significantly different from the value after pretreatment with SK&F 96365 alone.

Figure 6

Original tracings illustrating activation of whole-cell currents in A7r5 cells by thapsigargin. A7r5 cells were clamped at a holding potential of −60 mV with the whole-cell configuration: the pipette solution contained 10 mM (a) or 0.2 mM EGTA (b and c). Thapsigargin was added to the bath solution at the final concentration of 0.1 μM during the time indicated by horizontal bars. In the experiment (c) a blocker of the Cl⁻¹ channel, DIDS, was added to the bath solution at 1 mM.

Figure 7

Original tracings illustrating the effects of LOE 908, SK&F 96365 and their combination on the increase in [Ca²⁺]_i in A7r5 cells induced by either a lower (1 nM) or higher (100 nM) concentration of ET-1. A7r5 cells loaded with fura-2 (a) or fluo 3 (b and c) were stimulated by either 1 nM (a and b) or 100 nM ET-1 (c) in the presence or absence of 10 μM SK&F 96365, 10 μM LOE 908 or their combination. In all experiments, nifedipine was added to the medium at the final concentration of 1 μM.

Figure 8

Effects of LOE 908, SK&F 96365 and their combination on the increase in [Ca²⁺]_i in A7r5 cells induced by either a lower (1 nM) (a) or higher (100 nM) concentration of ET-1 (b). Experiments were performed as described in Figure 7. The ET-1-induced increases in [Ca²⁺]_i in the presence of channel blockers were represented as percentages of those in their absence. Each bar represents mean value±s.e.mean of five experiments. ^*P<0.01, significantly different from control values without blockers. §P<0.01; significantly different from the value after pretreatment with either LOE 908 or SK&F 96365 alone.