Department of Pharmacology and Toxicology Morehouse School of Medicine, 720 Westview Drive, SW, Atlanta, GA 30310-1495, USA.
Exp Eye Res. 2013 Feb;107:1-10. doi: 10.1016/j.exer.2012.11.007. Epub 2012 Nov 29.
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 MT(3) agonist), N-butanoyl-2-(2-methoxy-6H-isoindolo[2, 1-a]indol-11-yl)ethanamine (IIK7; selective MT(2) agonist) or S-27633-1 (selective MT(1) 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 MT(1)/MT(2) antagonist) or 4-phenyl-2-propionamidotetralin (4P-PDOT; selective MT(2) 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 MT(2) receptors in human NPCE cells. These actions may play a role in melatonin agonist-induced regulation of aqueous humor secretion and IOP.
本研究旨在确定褪黑素及其受体激动剂对睫状体房水产生细胞中 SNP 释放的一氧化氮 (NO) 和环鸟苷酸 (cGMP) 产生的影响，因为这些影响可能在褪黑素受体介导的眼压 (IOP) 调节中发挥作用。NO 释放方案使用无色素睫状上皮 (hNPCE) 细胞在不含染料的 DMEM 中进行，其中含有 l-精氨酸 (10(-3) M)。cGMP 实验方案使用不含染料的 DMEM 进行，其中含有 3-异丁基-1-甲基黄嘌呤 (IBMX，10(-4) M)。在单独的实验中，确定了不同浓度（10(-13)、10(-11)、10(-9)、10(-7) 和 10(-5) M）的褪黑素、5-MCA-NAT（假定的 MT(3)激动剂）、N-丁酰基-2-（2-甲氧基-6H-异吲哚[2,1-a]吲哚-11-基）乙胺（IIK7；选择性 MT(2)激动剂）或 S-27633-1（选择性 MT(1)激动剂）对硝普钠 (SNP) 释放的 NO 或 cGMP 产生的影响。使用比色法或酶免疫测定 (EIA) 分别测量 NO 和 cGMP 水平。使用 luzindole (LUZ；非选择性 MT(1)/MT(2)拮抗剂) 或 4-苯基-2-丙酰胺四氢萘 (4P-PDOT；选择性 MT(2)拮抗剂) 评估褪黑素受体的选择性。褪黑素、5-MCA-NAT 和 IIK7 均导致 SNP 释放的 NO 和 cGMP 产生的浓度依赖性降低。在 10(-13)、10(-11) 和 10(-9) M 浓度的激动剂下，褪黑素、5-MCA-NAT 和 IIK7 的抑制作用完全被阻断，或在 10(-7) 和 10(-5) M 时被 luzindole 或 4P-PDOT 部分阻断。本研究结果表明，褪黑素及其类似物 5-MCA-NAT 和 IIK7 通过激活人 NPCE 细胞中的 MT(2)受体抑制 SNP 释放的 NO 和 cGMP 产生。这些作用可能在褪黑素激动剂诱导的房水分泌和 IOP 调节中发挥作用。
