Comparison of endothelin binding and calcium mobilization in C6-BU-1 rat glioma and N18TG2 mouse neuroblastoma cells

Abstract

We have previously reported that endothelin (RT) receptor activation increases intracellular calcium concentrations ([Ca 2+] i) in NG108-15 cells, a hybrid of rat glioma C6-BU-1 and mouse neuroblastoma N18TG2 cells. This study was designed to further explore the origin of the ET receptor and [Ca 2+] i mobilization in the parent cell lines hybridized to form the NG108-15 cells. [ 125I]ET-1 bound to a single class of high affinity sites in C6-BU-1 cells with a K D value of 108pM and B max of 12400 sites/cell. ET-1, ET-2, ET-3 and big ET inhibited [ 125I]ET-1 binding to C6-BU-1 cells with K D values of 0.074, 0.167, 261 and 187 nM, respectively. All ETs produced a rapid increase in [Ca 2+] i in C6-Bu-1 cells. EC 50 values for ET-1, ET-2, ET-3 and big ET were 0.71, 1.14, 120 and 243 nM respectively. There was a significant correlation between the K D values obtained from competition binding experiments and the EC 50 values from [Ca 2+] i response curves in C6-BU-1 cells (r = 0.996, p < 0.004). Ten nM ET-1 produced about 85% of the maximal [Ca 2+] i increase in C6-BU-1 cells which was reduced by 96% in the absence of extracellular calcium. Furthermore, diltiazem (10 μM) and nifedipine (1 μM) failed to block ET-induced [Ca 2+] i mobilization. None of the ETs elevated [Ca 2+] i or displayed any specific [ 125I]ET-1 binding in N18TG2 cells. These data suggest that ET binds to a specific ET receptor in C6-BU-1 cells, and elevates [Ca 2+] i through dihydropyridine-insensitive, receptor-mediated calcium influx. Further, the ability of ETs to elevate [Ca 2+] i in NG108-15 hybrid cells is due to the ET receptor inherent to the C6-BU-1 glioma parent line.