Lim Julie, Li Ling, Jacobs Marc D, Kistler Joerg, Donaldson Paul J
Department of Physiology, School of Medical Sciences, University of Auckland, Auckland, New Zealand.
Invest Ophthalmol Vis Sci. 2007 Nov;48(11):5142-51. doi: 10.1167/iovs.07-0649.
To correlate the distribution of glutathione (GSH) and its precursor amino acids (cysteine, glycine, and glutamate) with the expression of their respective amino acid transporters in the rat lens.
Whole rat lenses were fixed, cryoprotected, and cryosectioned in either an equatorial or axial orientation. Sections were double labeled with cystine, glycine, glutamate, GSH, GLYT1, or GLYT2 antibodies, and the membrane marker wheat germ agglutinin (WGA). Sections were imaged by confocal laser scanning microscopy. Cystine, glycine, glutamate, and GSH labeling were quantified by using image-analysis software and intensity profiles plotted as a function of distance from the lens periphery. Western blot analysis was used to verify regional differences in amino acid transporter expression.
Cystine and glycine labeling in equatorial sections was most intense in the outer cortex, was diminished in the inner cortex, but was increased again in the core relative to the inner cortex. Glutamate and GSH labeling was most intense in the outer cortex and was diminished in the inner cortex to a minimum that was sustained throughout the core. The distribution of cystine and glutamate levels correlated well with the expression patterns observed previously for the cystine/glutamate exchanger (Xc-) and the glutamate transporter (EAAT4/5), respectively. Although high levels of glycine labeling in the outer cortex correlated well with the expression of the glycine transporter GLYT1, the absence of GLYT1 in the core, despite an increase of glycine in this region, suggests an alternative glycine uptake system such as GLYT2 exists in the core. Equatorial sections labeled with GLYT2 antibodies, showed that labeling in the outer cortex was predominantly cytoplasmic, but progressively became more membranous with distance into the lens. In the inner cortex and core, GLYT2 labeling was localized around the entire membrane of fiber cells. Western blot analysis confirmed GLYT2 to be expressed in the outer cortex, inner cortex, and core of the lens. Axial sections labeled for glycine revealed a track of high-intensity glycine labeling that extended from the anterior pole through to the core that was associated with the sutures.
The mapping of GSH and its precursor amino acids has shown that an alternative glycine uptake pathway exists in mature fiber cells. Although GLYT1 and -2 are likely to mediate glycine uptake in cortical fiber cells, GLYT2 alone appears responsible for the accumulation of glycine in the center of the lens. Enhancing the delivery of glycine to the core via the sutures may represent a pathway to protect the lens against the protein modifications associated with age-related nuclear cataract.
将大鼠晶状体中谷胱甘肽（GSH）及其前体氨基酸（半胱氨酸、甘氨酸和谷氨酸）的分布与其各自氨基酸转运体的表达相关联。
将完整的大鼠晶状体固定、冷冻保护，并沿赤道或轴向进行冷冻切片。切片用胱氨酸、甘氨酸、谷氨酸、GSH、GLYT1或GLYT2抗体以及膜标记物麦胚凝集素（WGA）进行双重标记。通过共聚焦激光扫描显微镜对切片进行成像。使用图像分析软件对胱氨酸、甘氨酸、谷氨酸和GSH标记进行定量，并将强度分布图绘制为距晶状体周边距离的函数。采用蛋白质印迹分析来验证氨基酸转运体表达的区域差异。
赤道切片中胱氨酸和甘氨酸标记在外层皮质最强烈，在内层皮质减弱，但相对于内层皮质，在晶状体核中再次增加。谷氨酸和GSH标记在外层皮质最强烈，在内层皮质减弱至整个晶状体核中持续存在的最小值。胱氨酸和谷氨酸水平的分布分别与先前观察到的胱氨酸/谷氨酸交换体（Xc-）和谷氨酸转运体（EAAT4/5）的表达模式密切相关。尽管外层皮质中高水平的甘氨酸标记与甘氨酸转运体GLYT1的表达密切相关，但尽管该区域甘氨酸增加，晶状体核中却不存在GLYT1，这表明晶状体核中存在诸如GLYT2之类的替代甘氨酸摄取系统。用GLYT2抗体标记的赤道切片显示，外层皮质中的标记主要位于细胞质中，但随着向晶状体内深入，逐渐变得更靠近细胞膜。在内层皮质和晶状体核中，GLYT2标记位于纤维细胞的整个细胞膜周围。蛋白质印迹分析证实GLYT2在晶状体的外层皮质、内层皮质和晶状体核中均有表达。用甘氨酸标记的轴向切片显示，一条高强度甘氨酸标记轨迹从前极延伸至与缝线相关的晶状体核。
GSH及其前体氨基酸的图谱显示，成熟纤维细胞中存在替代的甘氨酸摄取途径。虽然GLYT1和GLYT2可能介导皮质纤维细胞中的甘氨酸摄取，但似乎只有GLYT2负责晶状体中心部位甘氨酸的积累。通过缝线增强甘氨酸向晶状体核的输送可能是一种保护晶状体免受与年龄相关性核性白内障相关的蛋白质修饰影响的途径。
