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.
ACKNOWLEDGMENTS
The authors wish to dedicate this article to Dr. Cheng Yao. They also express their gratitude to Drs. B. W. Griffin, M. R. Hellberg, V. Sallee, and J. Veltman for helpful comments and suggestions during the initiation of the collaborative cell-provision and cell propagation procedures and the experimental work reported herein.
Declaration of interest: Authors are employees of Alcon Research, Ltd. No other commercial relationships exist. Furthermore, there is no conflict of interest.