Department of Pharmaceutics, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan.
Exp Eye Res. 2010 Apr;90(4):507-13. doi: 10.1016/j.exer.2010.01.003. Epub 2010 Jan 13.
The purpose of the present study was to elucidate the mechanisms of retina-to-blood transport of l-proline across the blood-retinal barrier (BRB) in vivo and in vitro, and to identify the responsible transporter(s). The vitreous humor/retina-to-blood transport of [(3)H]l-proline across the BRB was evaluated by microdialysis. Transport mechanisms of [(3)H]l-proline were investigated by cellular uptake using an in vitro model of the inner BRB (TR-iBRB2 cells). The mRNA level of system A was determined by quantitative real-time PCR analysis with specific primers. [(3)H]l-Proline and [(14)C]d-mannitol, which is a bulk flow marker, were bi-exponentially eliminated from the vitreous humor after vitreous bolus injection. The elimination rate constant of [(3)H]l-proline during the terminal phase was 1.6-fold greater than that of [(14)C]d-mannitol. The terminal elimination rate constant difference between [(3)H]l-proline and [(14)C]d-mannitol was reduced in the retinal presence of 3 mM l-proline and 5 mM alpha-methylaminoisobutyric acid, suggesting that l-proline is transported via a carrier-mediated retina-to-blood transport process across the BRB. [(3)H]l-Proline uptake by TR-iBRB2 cells appeared to be mediated through a saturable and Na(+)-dependent process. The corresponding Michaelis-Menten constant was 392 muM. This process was reduced by substrates for system A, suggesting that system A is involved in l-proline uptake. Of the isoforms of system A, ATA1, ATA2, and ATA3, ATA2 mRNA is predominantly expressed in TR-iBRB2 cells and isolated rat retinal endothelial cells. In conclusion, system A, most likely ATA2, is responsible for the retina-to-blood transport of l-proline across the inner BRB and may play a role in maintaining the concentration of small neutral amino acids in the retina.
本研究旨在阐明 l-脯氨酸在体内和体外经血视网膜屏障（BRB）向血液转运的机制，并鉴定负责转运的载体。通过微透析评估玻璃体液/视网膜中 [(3)H]l-脯氨酸向血液的转运。采用内 BRB 的体外模型（TR-iBRB2 细胞），通过细胞摄取研究 [(3)H]l-脯氨酸的转运机制。用定量实时 PCR 分析和特异性引物测定系统 A 的 mRNA 水平。[(3)H]l-脯氨酸和 [(14)C]d-甘露醇（一种大分子量示踪剂）经玻璃体腔注射后，呈双指数从玻璃体液中消除。终末相 [(3)H]l-脯氨酸的消除速率常数是 [(14)C]d-甘露醇的 1.6 倍。在视网膜存在 3mM l-脯氨酸和 5mM α-甲基氨基异丁酸的情况下，[(3)H]l-脯氨酸和 [(14)C]d-甘露醇之间的终末消除速率常数差异降低，提示 l-脯氨酸通过载体介导的视网膜向血液的转运过程穿过 BRB 进行转运。TR-iBRB2 细胞摄取 [(3)H]l-脯氨酸似乎是通过饱和和 Na(+)依赖的过程介导的。相应的米氏常数为 392μM。该过程被系统 A 的底物减少，提示系统 A 参与 l-脯氨酸摄取。在系统 A 的同工型 ATA1、ATA2 和 ATA3 中，ATA2mRNA 主要在 TR-iBRB2 细胞和分离的大鼠视网膜内皮细胞中表达。总之，系统 A，很可能是 ATA2，负责 l-脯氨酸穿过内 BRB 的向血液转运，可能在维持视网膜中小中性氨基酸的浓度中发挥作用。
