Miyamoto Y, Del Monte M A
School of Optometry, University of California-Berkeley 94720.
Invest Ophthalmol Vis Sci. 1994 Sep;35(10):3589-98.
To characterize glutamate uptake in the human retinal pigment epithelial (HRPE) cell line 165 and to determine the feasibility of using this cell line as an experimental model for glutamate transport studies.
Confluent monolayers of the HRPE cells cultured in 35-mm petri dishes were used to study glutamate uptake with regard to its ion dependence, substrate specificity, and kinetics. The radioactivity associated with the cells was measured by a liquid scintillation counter.
Glutamate uptake was noticeably stimulated by the presence of Na+ in uptake buffer. Acidic amino acids (100 microM; L-glutamate, L-aspartate, D-aspartate, and L-cysteate) and glutamate transporter inhibitors (100 microM; dihydrokainic acid, DL-threo-beta- hydroxyaspartic acid, and L-trans-pyrrolidine-2,4-dicarboxlic acid) interacted with uptake of radiolabeled glutamate. The activity of glutamate uptake depends on Na+ concentration. Glutamate uptake in the presence of Na+ does not have anion dependence. The uptake of glutamate was enhanced by external acidic environment and inhibited by 0.5 mM DIDS: Glutamate receptor antagonists (100 microM; [+/-]-2-amino-4-phosphonobutyric acid and 6-cyano-7- nitroquinoxaline-2,3-dione) did not inhibit glutamate uptake. Kinetic analysis shows that this transporter consists of at least two saturable systems.
The results of the present study demonstrate that a glutamate transporter is expressed in the HRPE cells. Its characteristics are similar to those of glutamate transporters observed in the RPE of laboratory animals. The human cell line 165 will be a useful tool for characterization of glutamate transport in the RPE. This study also provides clear evidence for the presence of a glutamate transporter in the human RPE.
表征人视网膜色素上皮（HRPE）细胞系165中的谷氨酸摄取，并确定使用该细胞系作为谷氨酸转运研究实验模型的可行性。
使用在35毫米培养皿中培养的汇合HRPE细胞单层，研究谷氨酸摄取的离子依赖性、底物特异性和动力学。通过液体闪烁计数器测量与细胞相关的放射性。
摄取缓冲液中Na+的存在显著刺激了谷氨酸摄取。酸性氨基酸（100微摩尔；L-谷氨酸、L-天冬氨酸、D-天冬氨酸和L-半胱氨酸）和谷氨酸转运体抑制剂（100微摩尔；二氢卡因酸、DL-苏式-β-羟基天冬氨酸和L-反式-吡咯烷-2,4-二羧酸）与放射性标记谷氨酸的摄取相互作用。谷氨酸摄取活性取决于Na+浓度。Na+存在时的谷氨酸摄取不具有阴离子依赖性。外部酸性环境增强了谷氨酸摄取，而0.5 mM DIDS抑制了谷氨酸摄取：谷氨酸受体拮抗剂（100微摩尔；[+/-]-2-氨基-4-膦酰丁酸和6-氰基-7-硝基喹喔啉-2,3-二酮）不抑制谷氨酸摄取。动力学分析表明，该转运体至少由两个可饱和系统组成。
本研究结果表明，HRPE细胞中表达了一种谷氨酸转运体。其特征与实验动物视网膜色素上皮中观察到的谷氨酸转运体相似。人细胞系165将是表征视网膜色素上皮中谷氨酸转运的有用工具。本研究还为人类视网膜色素上皮中存在谷氨酸转运体提供了明确证据。
