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, 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 [³H]-IPs. Concentration-response studies yielded the following potency (EC₅₀) and efficacy (E_max relative to ET-1) data: ET-1 EC₅₀ = 3.4 ± 1.4 nM, E_max = 100%, n = 3; BQ-3020 (ET_B receptor-selective agonist) EC₅₀ = 13 ± 4 nM, E_max = 73 ± 2%, n = 3). The effects of ET-1 on [³H]-IPs production were blocked by the ET_B receptor-selective antagonist, BQ-788 (IC₅₀ = 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 PGE₂ (10 µM) stimulated cAMP production, numerous other PGs (e.g., PGD₂, PGF_2α, PGI₂, latanoprost, latanoprost acid, U-46619 and BW245C [all at > 10 µM]) were inactive.

Conclusions: It is concluded that HCOMs express functionally active ET_B 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.

Keywords::

• Choroid
• Endothelin
• Melanocyte
• Pharmacology
• Signal transduction, prostaglandins

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.