Melatonin receptor agonist-induced reduction of SNP-released nitric oxide and cGMP production in isolated human non-pigmented ciliary epithelial cells

Abstract

The present study was designed to determine the effects of melatonin and its receptor agonists on SNP-released nitric oxide (NO) and cGMP production in aqueous humor producing cells of the ciliary body because these effects may play a role in melatonin receptor-mediated regulation of intraocular pressure (IOP). NO release protocols were carried out using human non-pigmented ciliary epithelial (hNPCE) cells treated in dye free DMEM containing l-arginine (10−3 M). The cGMP experimental protocols were performed using dye free DMEM containing 3-isobutyl-1-methylxanthine (IBMX, 10−4 M). The effects of varying concentrations (10−13, 10−11, 10−9, 10−7, and 10−5 M) of melatonin, 5-MCA-NAT (putative MT3 agonist), N-butanoyl-2-(2-methoxy-6H-isoindolo[2, 1-a]indol-11-yl)ethanamine (IIK7; selective MT2 agonist) or S-27633-1 (selective MT1 agonist) on sodium nitroprusside (SNP)-released NO or cGMP production were determined in separate experiments. NO and cGMP levels were measured using a colorimetric assay or enzyme immunoassay (EIA), respectively. Melatonin receptor selectivity was evaluated using luzindole (LUZ; nonselective MT1/MT2 antagonist) or 4-phenyl-2-propionamidotetralin (4P-PDOT; selective MT2 antagonist). Melatonin, 5-MCA-NAT, and IIK7 all caused concentration-dependent reduction of SNP-released NO and cGMP production. The inhibitory actions of melatonin, 5-MCA-NAT and IIK7 were either completely blocked at 10−13, 10−11, and 10−9 M concentrations of the agonists or partially at 10−7 and 10−5 M in the presence of luzindole or 4P-PDOT. Results from this study suggest that melatonin and its analogs, 5-MCA-NAT and IIK7 inhibit SNP-released NO and cGMP production via activation of MT2 receptors in human NPCE cells. These actions may play a role in melatonin agonist-induced regulation of aqueous humor secretion and IOP.