Dehydroascorbic acid uptake and intracellular ascorbic acid accumulation in cultured Müller glial cells (TR-MUL).
Vitamin C is mainly transported across the inner blood-retinal barrier (inner BRB) as dehydroascorbic acid (DHA) via a facilitative glucose transporter (GLUT) 1, and accumulates as ascorbic acid (AA) in the retina. Müller cells, huge glial cells, exhibit passive structural and metabolic functions for retinal neurons and the inner BRB. We characterized DHA transport and its corresponding transporter in a rat Müller cell line (TR-MUL5 cells). [(14)C]DHA uptake by TR-MUL5 cells took place in a time-dependent and Na(+)-independent manner. [(14)C]DHA uptake was inhibited by substrates and inhibitors of GLUTs, suggesting that Müller cells take up DHA via GLUTs. HPLC analysis revealed that most of the DHA taken up by TR-MUL5 cells was converted to AA and accumulated as AA in TR-MUL5 cells. [(14)C]DHA uptake by TR-MUL5 cells took place in a concentration-dependent manner with a Michaelis-Menten constant of 198 microM and was inhibited by cytochalasin B in a concentration-dependent manner with a 50% inhibition concentration of 0.283 microM. Although GLUT1, 3, and 4 mRNA are expressed in TR-MUL5 cells, quantitative real-time PCR revealed that GLUT1 mRNA expression was 5.85- and 116-fold greater than that of GLUT3 and 4, respectively. Western blot analysis supports the expression of GLUT1 protein with 45 kDa in TR-MUL5 cells. In conclusion, DHA is taken up by facilitative glucose transporters, most likely GLUT1, and converted to AA in TR-MUL5 cells.
Hosoya
Ken-ichi
K
Department of Pharmaceutics, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama, Japan. hosoyak@pha.u-toyama.ac.jp
Nakamura
Genki
G
Akanuma
Shin-ichi
S
Tomi
Masatoshi
M
Tachikawa
Masanori
M
D000818:Animals; D001205:Ascorbic Acid / Q000378:metabolism*; D001693:Biological Transport, Active / Q000187:drug effects; D001813:Blood-Retinal Barrier / Q000187:drug effects; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003627:Data Interpretation, Statistical; D003683:Dehydroascorbic Acid / Q000378:metabolism*; D051246:Glucose Transport Proteins, Facilitative / Q000378:metabolism; D008297:Male; D009457:Neuroglia / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction
D002352:Carrier Proteins; D051246:Glucose Transport Proteins, Facilitative; D012333:RNA, Messenger; D001205:Ascorbic Acid; D003683:Dehydroascorbic Acid