Human Choroidal Melanocyte Signal Transduction Responses to Various Pharmacological Agents: Focus on Endothelin Receptors

Abstract

Purpose: The receptor-coupled signal transduction systems present in isolated human choroidal melanocytes (HCOMs) were investigated.Methods: [3H]-inositol phosphates ([3H]-IPs) generated in the cells were measured by ion-exchange chromatography. cAMP generated in the cells was quantified using an enzyme immunoassay.Results: Initially, HCOM cells were challenged with a relatively high concentration (e.g., 1 µM–1 mM) of a variety of pharmacological agents in order to determine which functional receptors were present in these cells. Full concentration-response pharmacological studies were subsequently conducted on endothelin receptors. While a number of prostaglandins (PGs) (e.g., PGD2, PGE2, PGF2α, cloprostenol, latanoprost acid, U-46619), histamine, carbachol, bombesin, and arginine-vasopressin were essentially inactive at stimulating the phosphoinositide (PI) hydrolysis response, endothelin-1 (ET-1) potently and efficaciously generated [3H]-IPs. Concentration-response studies yielded the following potency (EC50) and efficacy (Emax relative to ET-1) data: ET-1 EC50 = 3.4 ± 1.4 nM, Emax = 100%, n = 3; BQ-3020 (ETB receptor-selective agonist) EC50 = 13 ± 4 nM, Emax = 73 ± 2%, n = 3). The effects of ET-1 on [3H]-IPs production were blocked by the ETB receptor-selective antagonist, BQ-788 (IC50 = 10 ± 5 nM, n = 3), while the ETA receptor-selective antagonist (BQ-610) was essentially inactive. In the adenylyl cyclase (AC) assay, while isoproterenol (10 µM), ET-1 (1 µM) and PGE2 (10 µM) stimulated cAMP production, numerous other PGs (e.g., PGD2, PGF2α, PGI2, latanoprost, latanoprost acid, U-46619 and BW245C [all at > 10 µM]) were inactive.Conclusions: It is concluded that HCOMs express functionally active ETB receptors that mediate the production of [3H]-IPs. Additionally, HCOMs generate cAMP in response to ET-1, PGE2, and isoproterenol. These data may have relevance to the melanogenic activity of HCOM cells.