Pharmacological properties of calcium entry channels in A7r5 cells activated by endothelin-1

Using whole-cell recordings of patch-clamp and monitoring of the intracellular free calcium (Ca2+) concentration ([Ca2+]i), we characterized Ca2+ entry channels in A7r5 cells activated by endothelin-1 (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 store-operated Ca2+ channel (SOCC). Furthermore, it was found that these channels can be discriminated pharmacologically using Ca2+ channel blockers such as 1-{beta-[3-(4-methoxyphenyl)propoxyl]-4-methoxyphenyl}-1-H-imidazoel hydrochloride (SK&F 96365) and (RS)-(3,4-dihydro-6,7-dimethoxyisoquinoline-1-gamma1)-2-phenyl-N,N-di-[2-(2,3,4-trimethoxyphenyl)ethyl] acetamide (LOE 908). NSCC-1 is resistant to SK&F 96365 but sensitive to LOE908, whereas NSCC-2 is sensitive to both drugs: SOCC is sensitive to SK&F 96365 but resistant to LOE 908. Using these channel blockers, we analyzed the Ca2+ entry channels involved in the ET-1-induced increase in [Ca2+]i of the cells. The increase induced by lower concentrations of ET-1 (less than or equal to 0.1 nM) was unaffected by SK&F 96365 but it was abolished by LOE 908. In contrast, the increase caused by higher concentrations of ET-1 (greater than or equal to 1 nM) was suppressed by SK&F 96365 or LOE 908 to about 35% of controls, and abolished by combined treatment with SK&F 96365 and LOE 908. These results show that the increase in [Ca2+] resulting from lower concentrations of ET-1 (less than or equal to0.1 nM) involves Ca2+ entry through only NSCC-1, whereas that resulting from higher concentrations of ET-1 involves Ca2+ entry through NSCC-1, NSCC-2 and SOCC, contributing 35%, 30% and 30%, respectively, to total Ca2+ entry.