Human choroidal melanocyte signal transduction responses to various pharmacological agents: focus on endothelin receptors.

The receptor-coupled signal transduction systems present in isolated human choroidal melanocytes (HCOMs) were investigated. [(3)H]-inositol phosphates ([(3)H]-IPs) generated in the cells were measured by ion-exchange chromatography. cAMP generated in the cells was quantified using an enzyme immunoassay. 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., PGD(2), PGE(2), PGF(2α), 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 [(3)H]-IPs. Concentration-response studies yielded the following potency (EC(50)) and efficacy (E(max) relative to ET-1) data: ET-1 EC(50) = 3.4 ± 1.4 nM, E(max) = 100%, n = 3; BQ-3020 (ET(B) receptor-selective agonist) EC(50) = 13 ± 4 nM, E(max) = 73 ± 2%, n = 3). The effects of ET-1 on [(3)H]-IPs production were blocked by the ET(B) receptor-selective antagonist, BQ-788 (IC(50) = 10 ± 5 nM, n = 3), while the ET(A) receptor-selective antagonist (BQ-610) was essentially inactive. In the adenylyl cyclase (AC) assay, while isoproterenol (10 µM), ET-1 (1 µM) and PGE(2) (10 µM) stimulated cAMP production, numerous other PGs (e.g., PGD(2), PGF(2α), PGI(2), latanoprost, latanoprost acid, U-46619 and BW245C [all at > 10 µM]) were inactive. It is concluded that HCOMs express functionally active ET(B) receptors that mediate the production of [(3)H]-IPs. Additionally, HCOMs generate cAMP in response to ET-1, PGE(2), and isoproterenol. These data may have relevance to the melanogenic activity of HCOM cells.