title: 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 ([Ca2+]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 [Ca2+]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 KD value of 108pM and Bmax of 12,400 sites/cell. ET-1, ET-2, ET-3 and big ET inhibited [125I]ET-1 binding to C6-BU-1 cells with KD values of 0.074, 0.167, 261 and 187 nM, respectively. All ETs produced a rapid increase in [Ca2+]i in C6-Bu-1 cells. EC50 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 KD values obtained from competition binding experiments and the EC50 values from [Ca2+]i response curves in C6-BU-1 cells (r = 0.996, p less than 0.004). Ten nM ET-1 produced about 85% of the maximal [Ca2+]i increase in C6-BU-1 cells which was reduced by 96% in the absence of extracellular calcium. Furthermore, diltiazem (10 microM) and nifedipine (1 microM) failed to block ET-induced [Ca2+]i mobilization. None of the ETs elevated [Ca2+]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 [Ca2+]i through dihydropyridine-insensitive, receptor-mediated calcium influx. Further, the ability of ETs to elevate [Ca2+]i in NG108-15 hybrid cells is due to the ET receptor inherent to the C6-BU-1 glioma parent line.
authors.affiliation: Department of Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406-0939.
authors.forename: M M
authors.initials: MM
authors.lastname: Gleason
authors.forename: H L
authors.initials: HL
authors.lastname: Wu
authors.forename: T L
authors.initials: TL
authors.lastname: Yue
authors.forename: G
authors.initials: G
authors.lastname: Feuerstein
authors.forename: P
authors.initials: P
authors.lastname: Nambi
mesh_terms: D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D016232:Endothelins / Q000378:metabolism* / Q000494:pharmacology; D016257:Fura-2; D005910:Glioma / Q000378:metabolism*; D006822:Hybrid Cells; D051379:Mice; D009447:Neuroblastoma / Q000378:metabolism*; D010972:Platelet Activating Factor / Q000494:pharmacology; D051381:Rats; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism*; D017466:Receptors, Endothelin; D013050:Spectrometry, Fluorescence; D014407:Tumor Cells, Cultured
chemical_list: D016232:Endothelins; D010972:Platelet Activating Factor; D011956:Receptors, Cell Surface; D017466:Receptors, Endothelin; D002118:Calcium; D016257:Fura-2