氧化形式的维生素C跨大鼠血视网膜屏障的转运

Vitamin C transport in oxidized form across the rat blood-retinal barrier.

作者信息

Hosoya Ken-ichi, Minamizono Akito, Katayama Kazunori, Terasaki Tetsuya, Tomi Masatoshi

机构信息

Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Toyama, Japan.

出版信息

Invest Ophthalmol Vis Sci. 2004 Apr;45(4):1232-9. doi: 10.1167/iovs.03-0505.

DOI:10.1167/iovs.03-0505

PMID:15037592

Abstract

PURPOSE

To elucidate the mechanisms of vitamin C transport across the blood-retinal barrier (BRB) in vivo and in vitro. METHODS. [(14)C]Dehydroascorbic acid (DHA) and [(14)C]ascorbic acid (AA) transport in the retina across the BRB 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-iBRB2) as an in vitro model of the inner BRB.

RESULTS

The apparent influx permeability clearance (K(in)) per gram of retina of [(14)C]DHA and [(14)C]AA was found to be 2.44 x 10(3) microL/(min x g retina) and 65.4 microL/(min x g retina), respectively. In the retina and brain, the K(in) of [(14)C]DHA was approximately 38 times greater than that of [(14)C]AA, whereas there was no major difference in the heart. The K(in) of [(14)C]DHA in the retina was eight times greater than that in the brain. HPLC analysis revealed that most of the vitamin C accumulated in AA form in the retina. These results suggest that vitamin C is mainly transported in DHA form across the BRB and accumulates in AA form in the rat retina. In an in vitro uptake study in TR-iBRB2 cells, the initial uptake rate of [(14)C]DHA was 37 times greater than that of [(14)C]AA, which is in agreement with the results of the in vivo study. [(14)C]DHA uptake by TR-iBRB2 cells took place in an Na(+)-independent and concentration-dependent manner with a K(m) of 93.4 microM. This process was inhibited by substrates and inhibitors of glucose transporters. [(14)C]DHA uptake was inhibited by D-glucose in a concentration-dependent manner with a 50% inhibition concentration of 5.56 mM. Quantitative real-time PCR and immunostaining analyses revealed that expression of GLUT1 and -3 was greater than that of the Na(+)-dependent L-ascorbic acid transporter (SVCT)-2 in TR-iBRB2 cells.

CONCLUSIONS

Vitamin C is mainly transported across the BRB as DHA mediated through facilitative glucose transporters and accumulates as AA in the rat retina.

摘要

目的

阐明维生素C在体内和体外跨血视网膜屏障（BRB）转运的机制。方法：利用大鼠体内积分图分析检测[¹⁴C]脱氢抗坏血酸（DHA）和[¹⁴C]抗坏血酸（AA）在视网膜中跨BRB的转运情况，并以条件永生化大鼠视网膜毛细血管内皮细胞系（TR-iBRB2）作为内BRB的体外模型来表征转运机制。

结果

每克视网膜中[¹⁴C]DHA和[¹⁴C]AA的表观流入渗透清除率（Kₑₙ）分别为2.44×10³微升/（分钟·克视网膜）和65.4微升/（分钟·克视网膜）。在视网膜和脑中，[¹⁴C]DHA的Kₑₙ约为[¹⁴C]AA的38倍，而在心脏中无显著差异。视网膜中[¹⁴C]DHA的Kₑₙ比脑中高8倍。高效液相色谱分析显示，视网膜中大部分维生素C以AA形式积累。这些结果表明，维生素C主要以DHA形式跨BRB转运，并在大鼠视网膜中以AA形式积累。在TR-iBRB2细胞的体外摄取研究中，[¹⁴C]DHA的初始摄取率比[¹⁴C]AA高37倍，这与体内研究结果一致。TR-iBRB2细胞对[¹⁴C]DHA的摄取以不依赖Na⁺且浓度依赖的方式进行，Kₘ为93.4微摩尔。该过程受到葡萄糖转运体的底物和抑制剂的抑制。[¹⁴C]DHA摄取受到D-葡萄糖浓度依赖性抑制，50%抑制浓度为5.56毫摩尔。定量实时PCR和免疫染色分析显示，TR-iBRB2细胞中葡萄糖转运蛋白1和3的表达高于Na⁺依赖性L-抗坏血酸转运体（SVCT）-2。