The Endothelin/Sarafotoxin‐Induced Increase of the Proliferation of Undifferentiated and DMSO‐Differentiated GEM‐81 Goldfish Erythrophoroma Cells is Mediated by ET_B Receptors

Abstract

Endothelins (ETs) and sarafotoxins (SRTXs) have been reported to exert ET(B)-mediated effects on vertebrate pigment cells. GEM-81 cell line, a red pigment cell-derived cutaneous tumor of the teleost Carassius auratus, expresses ET(B) receptors and can be differentiated with 1.5% DMSO treatment, thus constituting an useful model to investigate ET and SRTX effects on cultured fish pigment cells. Our aim was to characterize the pharmacology and biological effects mediated by ET receptors in DMSO-differentiated and undifferentiated cells. ET subtype receptors and their respective Ki values in both cell types were determined by competitive binding assays using (125)I ET-1 and BQ-485 (an ET(A) antagonist) or BQ-788 (an ET(B) antagonist). BQ-788, but not BQ-485, significantly reduced (125)I-ET-1 binding in both cell types, with similar low (Ki > nM) affinities. To determine the proliferation effects of ETs/SRTXs, cells were treated for 72 h with the hormones, and counted in a hemocytometer. The proliferation assays were repeated for SRTX S6c in the presence or absence of BQ-788. The results demonstrated that, with the exception of ET-1 (biphasic effect) and ET-3 (no significant effect) in undifferentiated GEM-81 cells, all the tested hormones induced increases in the proliferation of both types of cells. The hormones were equipotent in DMSO-differentiated cells, which exhibited increased sensitivity to ETs, but not to SRTXs, as compared with undifferentiated cells. The BQ-788 antagonistic effect was also exerted on the proliferation responses to SRTX S6c. These results corroborate the long and important evolutionary history of the ET/SRTX receptor system in vertebrate pigment cells.

Melanopsin and Clock Genes: Regulation by Light and Endothelin in the Zebrafish ZEM-2S Cell Line

“…ETs are known to increase proliferation in several cell types (Kedzierski & Yanagisawa, 2001). The endothelins have been reported to play an important role in cardiovascular regulation and in the differentiation and regulation of neural crest derivatives, like pigment cells and craniofacial bone structures (Braasch et al, 2009;Filadelfi et al, 2004;Ramanzini et al, 2006). But most noteworthy is the demonstration that endothelin-1 induces circadian rhythmicity in Rat-1cells (Nakahata et al, 2006;Yagita et al, 2001) and mouse fibroblasts (Yagita et al, 2001).…”

Chromatic effects of endothelin family peptides in non-innervated fish,Synbranchus marmoratus, melanophores

“…In fact BQ-788, a selective antagonist for ET B , inhibited the aggregation induced by ET-1 in Z. temminck chemically desinnervated melanophores (Hayashi et al,'96), and the dispersion induced by ET-3 in O. latipes chemically desinnervated leucophores (Fujita and Fujii,'97). In addition, the GEM-81 cell line expresses ET B mRNA (Ono et al,'99) and this receptor mediates the ETs/SRTXs mitogenic effects in these cells (Filadelfi et al, 2004).…”

“…Endothelial cells apparently synthesize only ET-1, but in other tissues, like brain, kidneys, adrenal glands, intestine, etc., all three peptides may be produced (Inoue et al,'89;Masaki,'91;Sakurai et al,'92). ETs also stimulate cellular proliferation in different cell types, including normal human melanocytes (Kusuhara et al,'89;Yada et al,'91) and teleost erythrophoroma GEM-81 cells (Filadelfi et al, 2004); and have also other effects (Masaki,'91;Sakurai et al,'92;Rubanyi and Polokoff,'94;Schiffrin and Touyz,'98).…”

Modulation of rhodopsin gene expression and signaling mechanisms evoked by endothelins in goldfish and murine pigment cell lines

“…SRTXs and ETs act on the cardiovascular system of vertebrates through three ET receptor subtypes, ETA, ETB, and ETC, all belonging to the G-protein-coupled receptor superfamily (10)(11)(12). We have demonstrated that the erythrophoroma cell line of the teleost Carassius auratus, GEM-81, and B16 Mus musculus melanocytes express ETB (13) and ETA (14) receptors, respectively. ET receptors may activate a diversity of intracellular signaling pathways, including phospholipase C (PLC), phospholipase D (15) and the mitogen-activated protein kinase (MAPK) cascade (16).…”