Effects of endothelins on signal transduction and proliferation in human melanocytes

We demonstrate here that human melanocytes could be regulated by endothelin (ET) derivatives, potent vasoconstrictive peptides synthesized by endothelial cells, to stimulate their proliferation and melanization via a receptor-mediated signal transduction pathway. Receptor-binding assay using [125I]ET indicated that unlabeled ET-1 or ET-2 competitively inhibited each binding of labeled ETs to melanocytes with a concentration for half-maximal inhibition (IC50) of 0.7 or 0.9 nM, respectively. The dissociation constant (Kd) and the number of sites of the specific bindings of ET-1 and those of ET-2 were almost the same (Kd: 1.81 nM, binding sites: 7.0-8.0 x 10(4) per cell). Upon incubation with cultured cells, the mass contents of inositol 1,4,5-trisphosphate and intracellular calcium level were substantially increased by 10 nM ET-1, ET-2, and ET-3, but not by big-ET with maximal response at 80-130-s postincubation. The addition of ET-1 and ET-2 at 1-50 nM concentrations caused human melanocytes to significantly stimulate DNA [( 3H]thymidine incorporation) and melanin synthesis (3H2O release and [14C] thiouracil incorporation). Furthermore, ETs exhibited an additive stimulatory effect on basic fibroblast growth factor-stimulated DNA synthesis. In a long-term serum-free culture system, the strongest stimulation of growth by 10 nM ET-1 or ET-2 was observed in the presence of 10 nM cholera toxin and 0.2% bovine pituitary extract, resulting in a 4.5-fold increase in cell number for 12 culture days. These findings strongly suggest involvement of ET in the mechanism regulating proliferation and melanization of human melanocytes.