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，维生素C在视网膜中的存在浓度较高。本研究的目的是阐明体内和体外维生素C跨血视网膜屏障(BRB)转运的机制。用大鼠视网膜毛细血管内皮细胞系(TR-iBRB)作为视网膜内膜的体外模型，采用体内积分图分析方法，研究了脱氢抗坏血酸(DHAA)和抗坏血酸(AA)在大鼠视网膜的转运情况。视网膜的表观内流通透性清除(CL)是[14&gt；C]AA的约38倍，而心脏则无明显差异。高效液相色谱分析表明，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主要是通过…运输的更多的以DHAA的形式存在于BRB中，并以AA的形式在大鼠视网膜中积累。Tr-iBRB细胞摄取DHAA具有浓度依赖性和安娜非依赖性，其米氏常数为93.4微米。这一过程被促进性葡萄糖转运体(GLUT)S的底物和抑制剂所抑制。葡萄糖对DHAA的摄取呈浓度依赖性抑制，半数抑制浓度为5.56 mM。这些结果表明，谷氨酸参与了DHAA在BRB内部的运输。采用大鼠视网膜血管内皮细胞磁分离法和实时定量聚合酶链式反应分析相结合的方法来定量检测BRB内侧的GLUT基因水平，GLUT1的表达是三种GLUT中最大的，而GLUT3在BRB内侧没有检测到。GLUT4的表达比GLUT1低100倍，提示GLUT1负责DHAA在BRB内侧的转运。在链脲佐菌素诱导的糖尿病大鼠，当血糖浓度高达20 mM时，视网膜DHAA的CL抑制31%。总而言之，维生素C主要以DHAA的形式通过GLUT1在BRB内侧运输，并以AA的形式在视网膜中积累。高血糖会减少维生素C对视网膜的供应。GLUT1在内部BRB的生理作用似乎涉及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的伤害。较少

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