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

Ken Ichi Hosoya; Akito Minamizono; Kazunori Katayama; Tetsuya Terasaki; Masatoshi Tomi

doi:10.1167/iovs.03-0505

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

Ken Ichi Hosoya, Akito Minamizono, Kazunori Katayama, Tetsuya Terasaki, Masatoshi Tomi

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65 Citations (Scopus)

Abstract

PURPOSE. To elucidate the mechanisms of vitamin C transport across the blood-retinal barrier (BRB) in vivo and in vitro. METHODS. [¹⁴C] Dehydroascorbic acid (DHA) and [¹⁴C]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 [¹⁴C]DHA and [ ¹⁴C]AA was found to be 2.44 × 10³ μL/(min · g retina) and 65.4 μL/(min · g retina), respectively. In the retina and brain, the K_in of [¹⁴C]DHA was approximately 38 times greater than that of [¹⁴C]AA. whereas there was no major difference in the heart. The Kin of [¹⁴C]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 [¹⁴C]DHA was 37 times greater than that of [¹⁴C]AA, which is in agreement with the results of the in vivo study. [¹⁴C]DHA uptake by TR-iBRB2 cells took place in an Na ⁺-independent and concentration-dependent manner with a K_m of 93.4 μM. This process was inhibited by substrates and inhibitors of glucose transporters. [¹⁴C]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.

Original language	English
Pages (from-to)	1232-1239
Number of pages	8
Journal	Investigative Ophthalmology and Visual Science
Volume	45
Issue number	4
DOIs	https://doi.org/10.1167/iovs.03-0505
Publication status	Published - 2004 Apr 1
Externally published	Yes

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ASJC Scopus subject areas

Ophthalmology
Sensory Systems
Cellular and Molecular Neuroscience

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Hosoya, K. I., Minamizono, A., Katayama, K., Terasaki, T., & Tomi, M. (2004). Vitamin C transport in oxidized form across the rat blood-retinal barrier. Investigative Ophthalmology and Visual Science, 45(4), 1232-1239. https://doi.org/10.1167/iovs.03-0505

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title = "Vitamin C transport in oxidized form across the rat blood-retinal barrier",

abstract = "PURPOSE. To elucidate the mechanisms of vitamin C transport across the blood-retinal barrier (BRB) in vivo and in vitro. METHODS. [14C] Dehydroascorbic acid (DHA) and [14C]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 (Kin) per gram of retina of [14C]DHA and [ 14C]AA was found to be 2.44 × 103 μL/(min · g retina) and 65.4 μL/(min · g retina), respectively. In the retina and brain, the Kin of [14C]DHA was approximately 38 times greater than that of [14C]AA. whereas there was no major difference in the heart. The Kin of [14C]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 [14C]DHA was 37 times greater than that of [14C]AA, which is in agreement with the results of the in vivo study. [14C]DHA uptake by TR-iBRB2 cells took place in an Na +-independent and concentration-dependent manner with a Km of 93.4 μM. This process was inhibited by substrates and inhibitors of glucose transporters. [14C]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.",

author = "Hosoya, {Ken Ichi} and Akito Minamizono and Kazunori Katayama and Tetsuya Terasaki and Masatoshi Tomi",

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T1 - Vitamin C transport in oxidized form across the rat blood-retinal barrier

AU - Hosoya, Ken Ichi

AU - Minamizono, Akito

AU - Katayama, Kazunori

AU - Terasaki, Tetsuya

AU - Tomi, Masatoshi

PY - 2004/4/1

Y1 - 2004/4/1

N2 - PURPOSE. To elucidate the mechanisms of vitamin C transport across the blood-retinal barrier (BRB) in vivo and in vitro. METHODS. [14C] Dehydroascorbic acid (DHA) and [14C]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 (Kin) per gram of retina of [14C]DHA and [ 14C]AA was found to be 2.44 × 103 μL/(min · g retina) and 65.4 μL/(min · g retina), respectively. In the retina and brain, the Kin of [14C]DHA was approximately 38 times greater than that of [14C]AA. whereas there was no major difference in the heart. The Kin of [14C]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 [14C]DHA was 37 times greater than that of [14C]AA, which is in agreement with the results of the in vivo study. [14C]DHA uptake by TR-iBRB2 cells took place in an Na +-independent and concentration-dependent manner with a Km of 93.4 μM. This process was inhibited by substrates and inhibitors of glucose transporters. [14C]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.

AB - PURPOSE. To elucidate the mechanisms of vitamin C transport across the blood-retinal barrier (BRB) in vivo and in vitro. METHODS. [14C] Dehydroascorbic acid (DHA) and [14C]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 (Kin) per gram of retina of [14C]DHA and [ 14C]AA was found to be 2.44 × 103 μL/(min · g retina) and 65.4 μL/(min · g retina), respectively. In the retina and brain, the Kin of [14C]DHA was approximately 38 times greater than that of [14C]AA. whereas there was no major difference in the heart. The Kin of [14C]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 [14C]DHA was 37 times greater than that of [14C]AA, which is in agreement with the results of the in vivo study. [14C]DHA uptake by TR-iBRB2 cells took place in an Na +-independent and concentration-dependent manner with a Km of 93.4 μM. This process was inhibited by substrates and inhibitors of glucose transporters. [14C]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.

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