Antioxidants transport via GLUT1 at the blood-retinal barrier

维生素C是体内的一种抗氧化剂，在保护视网膜免受氧化应激方面起着关键作用，与其他器官相比，它在视网膜中的浓度很高。本研究的目的是阐明维生素C在体内和体外通过血视网膜屏障（BRB）转运的机制。[^<14>C]采用大鼠体内整合图分析方法研究了脱氢抗坏血酸（DHAA）和[^<14>C]抗坏血酸（AA）在视网膜中的转运，并以条件永生化大鼠视网膜毛细血管内皮细胞系（r - ibrb）作为视网膜内BRB的体外模型来表征其转运机制。视网膜[^<14>C]DHAA的表观内流通透性清除率（CL）约为[^<14>C]AA的38倍，而心脏无明显差异。HPLC分析显示，大部分维生素C以AA形式在视网膜中积累。这些结果表明，维生素C主要以dha形式通过BRB运输，并以AA形式在大鼠视网膜中积累。[^<14>C] TR-iBRB细胞以Na^+不依赖和浓度依赖的方式摄取DHAA， Michaelis常数为93.4微米。这一过程被促性葡萄糖转运蛋白（GLUT）的底物和抑制剂所抑制。[^<14>C]葡萄糖以浓度依赖的方式抑制DHAA的摄取，50%的抑制浓度为5.56 mM。这些结果表明GLUTs参与了内部BRB的DHAA运输。利用大鼠视网膜血管内皮细胞磁分离法和实时定量PCR相结合的方法定量glts在BRB内的基因表达水平，结果表明，GLUT1在BRB内表达量最大，而GLUT3在BRB内未检测到。GLUT4的表达量比GLUT1少100倍，表明GLUT1在BRB内部负责DHAA的运输。链脲霉素诱导的糖尿病大鼠血中葡萄糖浓度高达20 mM时，视网膜[^<14 bb0 C]DHAA的CL被抑制31%。综上所述，维生素C主要通过BRB内部的GLUT1以dha的形式运输，并在视网膜中以AA的形式积累。高血糖会减少视网膜中维生素C的供给。在BRB内部，GLUT1的生理作用似乎涉及到从循环血液向视网膜供应维生素C和葡萄糖，以保护视网膜免受氧化应激。少

Vitamin C, which is an antioxidant in the body, plays a key role in protecting the retina from oxidative stress and is present in the retina at a high concentration compared with its presence in other organs. The purpose of study was to elucidate the mechanisms of vitamin C transport across the blood-retinal barrier (BRB) in vive and in vitro. [^<14>C]Dehydroascorbic acid (DHAA) and [^<14>C]ascorbic acid (AA) transport in the retina were examined using in vivo integration plot analysis in rats and the transport mechanism was characterized using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB) as an in vitro model of the inner BRB. The apparent influx permeability clearance (CL) of [^<14>C]DHAA of the retina was about 38-fold greater than that of [^<14>C]AA, whereas there was no major difference in the heart. HPLC analysis revealed that most of the vitamin C accumulates in AA form in the retina. These results suggest that vitamin C is mainly transported … More in DHAA form across the BRB and accumulates in AA form in the rat retina. [^<14>C]DHAA uptake by TR-iBRB cells took place in an Na^+-independent and concentration-dependent manner with a Michaelis constant of 93.4 microM. This process was inhibited by facilitative glucose transporter (GLUT)s' substrates and inhibitors. [^<14>C]DHAA uptake was inhibited by glucose in a concentration-dependent manner with a 50% inhibition concentration of 5.56 mM. These results suggest that GLUTs are involved in DHAA transport at the inner BRB. To quantify GLUTS' gene levels at the inner BRB using a combination of magnetic isolation method for rat retinal vascular endothelial cells and real-time quantitative PCR analysis, GLUT1 expression was the largest of three GLUTs, whereas GLUT3 was not detected at the inner BRB. The expression of GLUT4 was 100-fold less than that of GLUT1, suggesting that GLUT1 is responsible for DHAA transport at the inner BRB. When glucose concentration in the blood was high as 20 mM in streptozotocin-induced diabetic rats, CL of [^<14>C]DHAA of the retina was inhibited by 31%. In conclusion, vitamin C is predominantly transported as DHAA via GLUT1 at the inner BRB and accumulates as AA in the retina. Hyperglycemia reduces the supply of the vitamin C to the retina. The physiological role of GLUT1 at the inner BRB appears to involve the supply of vitamin C as well as glucose from the circulating blood to the retina to protect the retina against oxidative stress. Less