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 analogues, 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.

Melatonin Receptors Trigger cAMP Production and Inhibit Chloride Movements in Nonpigmented Ciliary Epithelial Cells

“…This indirectly implies that the action of MT 1 /MT 2 may have an opposite effect, increasing the efflux of Cl 2 . Because MT 1 and MT 2 melatonin receptors are negatively coupled to adenylate cyclase and there is a concomitant reduction in the concentrations of cAMP, the blockade of these two receptors might involve PLC/PKC pathway as happens in some models (Bowler et al, 1996;Godson and Reppert, 1997;Dortch-Carnes and Tosini, 2013). Interestingly, new studies are appearing indicating that new second messenger systems can be coupled to melatonin receptors.…”

Melatonin and Its Analog 5-Methoxycarbonylamino-N-Acetyltryptamine Potentiate Adrenergic Receptor-Mediated Ocular Hypotensive Effects in Rabbits: Significance for Combination Therapy in Glaucoma

“…The presence of melatonin receptors in the ciliary body has been demonstrated in different species through the use of pharmacologic and molecular tools (Osborne and Chidlow, 1994;Pintor et al, 2001Pintor et al, , 2003. Indeed, melatonin receptors have been identified in the NPE cells of humans and frogs (Wiechmann and Wirsig-Wiechmann, 2001;Dortch-Carnes and Tosini, 2013).…”

Characterization of signaling pathways coupled to melatonin receptors in gastrointestinal smooth muscle

“…Then cells were centrifuged at 350 × g for 10 min to remove excess [ 3 H] inositol and resuspended in 10 ml of fresh medium. Lithium was added to a final concentration of 10 mM and the suspension was incubated for 10 min followed by melatonin (1 pM to 10 μM) or MT 2 receptor selective agonist IIK7 (100 nM) for 60 s. In some experiments cells were treated with melatonin in the presence and absence of a non-selective MT 1 /MT 2 receptor antagonist luzindole (100 mM), a selective MT 2 receptor antagonist 4P-PDOT, PLC β inhibitor U73122 (10 μM) or MLCK inhibitor ML-9 (1 μM) for 60 s [31-33]. Cultured smooth muscle cells were labeled with [ 3 H]myo-inositol (0.5 μCi/ml) for 24 h in inositol-free DMEM medium.…”