| Project/Area Number |
15390046
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Medical pharmacy
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| Research Institution | TOYAMA MEDICAL AND PHARMACEUTICAL UNIVERSITY |
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Principal Investigator |
HOSOYA Ken-ichi Toyama Medical and Pharmaceutical University, Faculty of Pharmaceutical Sciences, Professor, 薬学部, 教授 (70301033)
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| Co-Investigator(Kenkyū-buntansha) |
TOMI Masatoshi Toyama Medical and Pharmaceutical University, Faculty of Pharmaceutical Sciences, 薬学部, 助手 (30334717)
TERASAKI Tetsuya Tohoku University, New Industry Creation Hatchery Center, Professor, 未来科学技術共同研究センター, 教授 (60155463)
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| Project Period (FY) |
2003 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥13,200,000 (Direct Cost: ¥13,200,000)
Fiscal Year 2004: ¥7,500,000 (Direct Cost: ¥7,500,000)
Fiscal Year 2003: ¥5,700,000 (Direct Cost: ¥5,700,000)
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| Keywords | blood-retinal barrier / vitamin C / GLUT1 / oxidative stress / antioxidant / diabetes / TR-iBRB cells / transport mechanism / 担体輸送 / 条件的不死化細胞株 |
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| Research Abstract |
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
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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
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