title: B103 neuroblastoma cells predominantly express endothelin ET(B) receptor; effects of extracellular Ca(2+) influx on endothelin-1-induced mitogenesis.
abstract: We sought to examine the effects of endothelin-1 on the intracellular free Ca(2+) concentration ([Ca(2+)](i)) and mitogenic response in the neuroblastoma cell line, B103 (B103 cells). The results obtained from an [125I] endothelin-1 binding assay demonstrated that B103 cells express the endothelin receptor. The B(max) and K(d) values for [125I]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 [3H]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 [Ca(2+)](i) showed that endothelin-1 evoked a transient increase in [Ca(2+)](i); this remained even in the absence of extracellular Ca(2+). However, no sustained, endothelin-1-induced increase in [Ca(2+)](i) due to extracellular Ca(2+) 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 [Ca(2+)](i), while the endothelin ET(A) 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 ET(B) receptor or an endothelin ET(B)-like receptor predominantly and have no Ca(2+) 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, [3H]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 [Ca(2+)](i) plays a role in endothelin-1-induced cell proliferation.
authors.affiliation: Department of Neurosurgery, Kyoto University Faculty of Medicine, Kyoto, Sakyo, Japan. kawanabe@kuhp.kyoto-u.ac.jp
authors.forename: Y
authors.initials: Y
authors.lastname: Kawanabe
authors.forename: N
authors.initials: N
authors.lastname: Hashimoto
authors.forename: T
authors.initials: T
authors.lastname: Masaki
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D019332:Endothelin-1 / Q000378:metabolism / Q000494:pharmacology; D007295:Inositol Phosphates / Q000378:metabolism; D007457:Iodine Radioisotopes; D020928:Mitogen-Activated Protein Kinases / Q000187:drug effects / Q000378:metabolism; D009447:Neuroblastoma / Q000378:metabolism* / Q000473:pathology; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin / Q000096:biosynthesis*; D019284:Thapsigargin / Q000494:pharmacology; D014316:Tritium; D014407:Tumor Cells, Cultured
chemical_list: D019332:Endothelin-1; D007295:Inositol Phosphates; D007457:Iodine Radioisotopes; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin; D014316:Tritium; D019284:Thapsigargin; D000242:Cyclic AMP; D020928:Mitogen-Activated Protein Kinases; D002118:Calcium