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 (less than or equal to 1 nM; lower [ET-1]) activate only an SK&F 96365-resistant channel (NSCC-1), whereas its higher concentrations (greater than or equal to 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 mu M 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] invokes NSCC-1, NSCC-2 and SOCC in addition to VOC.