Localization and expression of the glutamate transporter, excitatory amino acid transporter 4, within astrocytes of the rat retina
Localization and expression of the glutamate transporter, excitatory amino acid transporter 4, within astrocytes of the rat retina
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大鼠视网膜星形胶质细胞内谷氨酸转运蛋白、兴奋性氨基酸转运蛋白 4 的定位和表达
DOI：
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发表时间：
2004
影响因子：
3.6
通讯作者：
E. Fletcher
中科院分区：
文献类型：
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作者：
Michelle M. Ward;A. Jobling;T. Puthussery;Lisa E. Foster;E. Fletcher
清除谷氨酸的机制对于维持视网膜的正常功能至关重要。迄今为止，已经从神经元组织中鉴定出五种兴奋性氨基酸转运蛋白，除兴奋性氨基酸转运蛋白4（EAAT 4）外，所有这些转运蛋白均在视网膜内表达。在这项研究中，我们研究了谷氨酸转运蛋白EAAT 4在大鼠视网膜的表达和定位，用RT-PCR和免疫细胞化学。使用大鼠EAAT 4特异性引物的RT-PCR显示在视网膜、皮质和小脑中有一个突出的296 bp产物。通过DNA测序确认EAAT 4片段的身份。我们使用实时PCR检测了EAAT 4在皮质、视网膜和小脑中的组织表达水平。在小脑中的表达水平最高。皮质中的表达约为小脑的3.1%，并且发现视网膜中的EAAT 4含量约为小脑总含量的0.8%。为了检查视网膜内表达EAAT 4的特定细胞类型，我们使用大鼠EAAT 4特异性抗血清进行免疫细胞化学。视网膜神经纤维层内的细胞突起被强烈标记为EAAT 4。用胶质细胞酸性蛋白（GFAP）双标记EAAT 4揭示了广泛的共定位，表明EAAT 4定位在视网膜内的星形胶质细胞内。EAAT 4和谷氨酸转运体EAAT 1（GLAST）的双标记揭示了广泛的共定位，表明视网膜中的星形胶质细胞表达至少两种类型的谷氨酸转运体。这些结果表明，视网膜内的星形胶质细胞处于良好的位置，以提供谷氨酸清除机制以及控制细胞的兴奋性。
Mechanisms for the removal of glutamate are vital for maintaining normal function of the retina. Five excitatory amino acid transporters have been characterized to date from neuronal tissue, all of which are expressed within the retina except excitatory amino acid transporter 4 (EAAT4). In this study we examined the expression and localization of the glutamate transporter EAAT4 in the rat retina using RT-PCR and immunocytochemistry. RT-PCR using rat EAAT4 specific primers revealed a prominent 296-bp product in the retina, cortex and cerebellum. The identity of the EAAT4 fragment was confirmed by DNA sequencing. We examined the tissue expression levels of EAAT4 in cortex, retina and cerebellum using real-time PCR. The highest expression level was found in the cerebellum. Expression in the cortex was approximately 3.1% that of the cerebellum and the retina was found to have approximately 0.8% the total cerebellar EAAT4 content. In order to examine the specific cell types within the retina that express EAAT4, we performed immunocytochemistry using a rat EAAT4 specific antiserum. Cellular processes within the nerve fibre layer of the retina were intensely labelled for EAAT4. Double labelling EAAT4 with glial fibrillary acidic protein (GFAP) revealed extensive colocalization indicating that EAAT4 is localized within astrocytes within the retina. Double labelling of EAAT4 and the glutamate transporter EAAT1 (GLAST) revealed extensive colocalization suggesting that astrocytes in the retina express at least two types of glutamate transporters. These results suggest that astrocytes within the retina are well placed to provide mechanisms for glutamate removal as well as controlling cellular excitability.