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