Wiechmann Allan F, Vrieze Melissa J, Dighe Radhika, Hu Ying
Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190, USA.
Invest Ophthalmol Vis Sci. 2003 Oct;44(10):4522-31. doi: 10.1167/iovs.03-0329.
Retinal circadian signals may have a role in maintaining the normal function and health of photoreceptors. Melatonin is an output of the retinal circadian oscillator and provides nocturnal signaling that is mediated through specific G-protein-coupled receptors. Melatonin receptors are expressed in retinal photoreceptor cells, and this study was undertaken to test the hypothesis that melatonin directly increases photoreceptor responses through melatonin receptors.
Transgenic Xenopus laevis frogs were generated using a DNA construct containing a Xenopus opsin promoter driving expression of a melatonin Mel(1c) receptor-green fluorescent protein (GFP) fusion protein (XOP-MEL(1c)-GFP). Electroretinogram (ERG) analysis on transgenic and normal tadpole eyes was performed in response to melatonin treatment, and the eyes were subsequently examined by confocal microscopy and GFP immunocytochemistry.
XOP-MEL(1c)-GFP transgenic frogs demonstrated GFP immunoreactivity in rod photoreceptor inner segments throughout the retina, indicating the rod-specific expression of the Mel(1c)-GFP fusion protein. ERG analysis of transgenic tadpole eyes showed that 1 to 100 nM melatonin increased the a- and b-wave amplitudes. Control transgenic (XOP-GFP) and normal frogs exhibited only modest ERG responses to 100-nM melatonin treatment. The effect of melatonin on a- and b-wave amplitudes in XOP-MEL(1c)-GFP transgenic frogs was dose dependent, with ERG responses occurring at physiological concentrations.
The results suggest that melatonin, acting through Mel(1c) receptors on rod photoreceptor membranes, directly stimulates the responsiveness of rod photoreceptors to light. This supports the hypothesis that melatonin acts both as an intracrine and paracrine circadian signal of darkness, and binds to specific receptors in photoreceptors and other retinal cells to increase visual sensitivity.
视网膜昼夜节律信号可能在维持光感受器的正常功能和健康方面发挥作用。褪黑素是视网膜昼夜节律振荡器的输出产物，并提供通过特定G蛋白偶联受体介导的夜间信号。褪黑素受体在视网膜光感受器细胞中表达，本研究旨在验证褪黑素通过褪黑素受体直接增强光感受器反应的假说。
使用包含非洲爪蟾视蛋白启动子的DNA构建体生成转基因非洲爪蟾蛙，该启动子驱动褪黑素Mel(1c)受体-绿色荧光蛋白（GFP）融合蛋白（XOP-MEL(1c)-GFP）的表达。对转基因和正常蝌蚪眼进行褪黑素处理后的视网膜电图（ERG）分析，随后通过共聚焦显微镜和GFP免疫细胞化学检查眼睛。
XOP-MEL(1c)-GFP转基因蛙在整个视网膜的视杆光感受器内节显示出GFP免疫反应性，表明Mel(1c)-GFP融合蛋白的视杆特异性表达。转基因蝌蚪眼的ERG分析表明，1至100 nM的褪黑素增加了a波和b波振幅。对照转基因（XOP-GFP）和正常蛙对100 nM褪黑素处理仅表现出适度的ERG反应。褪黑素对XOP-MEL(1c)-GFP转基因蛙a波和b波振幅的影响呈剂量依赖性，在生理浓度下出现ERG反应。
结果表明，褪黑素通过视杆光感受器膜上的Mel(1c)受体起作用，直接刺激视杆光感受器对光的反应性。这支持了褪黑素既作为黑暗的内分泌和旁分泌昼夜节律信号，又与光感受器和其他视网膜细胞中的特定受体结合以增加视觉敏感性的假说。
