B103 neuroblastoma cells predominantly express endothelin ETB receptor;: effects of extracellular Ca2+ influx on endothelin-1-induced mitogenesis

We sought to examine the effects of endothelin-1 on the intracellular free Ca2+ concentration ([Ca2+](i)) and mitogenic response in the neuroblastoma cell line, B103 (B103 cells). The results obtained from an [I-125]endothelin-1 binding assay demonstrated that B103 cells express the endothelin receptor. The B-max and K-d values for [I-125]endothelin-1 binding were 70 +/- 36 fmol/mg protein and 52 +/- 13 pM, respectively. Endothelin-1 failed to stimulate cAMP formation, but it did inhibit forskolin-induced cAMP formation. Endothelin-1 also stimulated the accumulation of [H-3]inositol phosphates. These results indicate that the endothelin receptor in B103 cells couples with G(i) and G(q) but not with G(s). Monitoring of [Ca2+](i) showed that endothelin-1 evoked a transient increase in [Ca2+](i); this remained even in the absence of extracellular Ca2+. However, no sustained, endothelin-1-induced increase in [Ca2+](i) due to extracellular Ca2+ influx was detected. The endothelin B receptor-selective antagonist, 2,6-Dimethylpiperidinecarbonyl-gamma -Methyl-Leu-N-in-[Methoxycarbonyl]-D-Trp-D-Nle (BQ 788), abolished the endothelin-1-induced increase in [Ca2+](i), while the endothelin ETA receptor-selective antagonist, cyclo-D-Asp-Pro-D-Val-Leu-D-Trp (BQ 123), failed to inhibit it. These results indicate that B103 cells express endothelin ETB receptor or an endothelin ETB-like receptor predominantly and have no Ca2+ channels activated by endothelin-1. Endothelin-1 activated mitogen-activated protein kinase in B103 cells. However, based on the data for 3-(4,5-dimethy-2-thiazolyl)-2,5-diphenyl tetrazolium. bromide, [H-3]thymidine incorporation, and apoptosis screening assays, endothelin-1 induces neither mitogenesis nor apoptosis. These results suggest that endothelin-1 has no role in the mitogenic response in B103 cells, and this is consistent with the notion that an endothelin-1-induced sustained increase in [Ca2+](i) plays a role in endothelin-1-induced cell proliferation. (C) 2001 Elsevier Science B.V. All rights reserved.