MT2 Melatonin Receptor-Mediated Inhibition of Nitric Oxide Release and cGMP Production in Human Nonpigmented Ciliary Epithelial Cells

Abstract

Purpose: : It is well known that melatonin lowers intraocular pressure (IOP), but the mechanism has not been determined. Previous studies in our laboratory suggest that melatonin inhibits nitric oxide (NO) release and cGMP production in aqueous humor producing human nonpigmented ciliary epithelial (NPCE) cells via activation of MT₂ melatonin receptors. The present study was conducted in order to confirm the presence and functional activity of the MT₂ subtype in NPCE cells using selective agonists and antagonists of melatonin receptors.

Methods: : NO release protocols were carried out using NPCE cells treated in dye free DMEM containing protease inhibitor cocktail and L-arginine. Cyclic GMP experimental protocols were performed using dye free DMEM containing IBMX. The effects of S27633-1, IIK7 and 5-MCA-NAT, selective MT₁, MT₂ and putative MT₃ receptor agonists, respectively, on SNP-elevated NO and cGMP production were determined in separate experiments. NO and cGMP levels were measured using a colorimetric assay or enzyme immunoassay, respectively. Melatonin receptor agonist selectivity was evaluated using luzindole (nonselective MT₁/ MT₂ antagonist) or 4-PP-DOT (selective MT₂ antagonist).

Results: : IIK7 and 5-MCA-NAT (10^-11 - 10^-5 M) produced concentration-dependent reduction of NO release and cGMP production, whereas the MT₁ receptor agonist, S27633-1, had no effect on NO release. The effects of IIK7 and 5-MCA-NAT on NO release were significantly inhibited in the presence of luzindole and 4-PP-DOT. Further studies are being conducted to determine the effect of the melatonin receptor antagonists on IIK7 and 5-MCA-NAT-induced changes in cGMP production.

Conclusions: : Results from this study demonstrate that, similar to melatonin, 5-MCA-NAT-induced reduction of NO release was completely inhibited by luzindole, thus suggesting that this effect is mediated via activation of MT₁ or MT₂ receptors and not the MT₃ subtype. The effects of IIK7 on NO release are likely mediated by activation of MT₂ receptors. In summary, our findings provide evidence of the presence and functional activity of MT₂ receptors in our human NPCE cell line. These data suggest that the MT₂ receptor plays a role in melatonin receptor agonist-induced reduction of IOP.

Keywords: melatonin • second messengers: pharmacology/physiology • aqueous