Quantitative analysis of ascorbic acid permeability of aquaporin 0 in the lens.
Aquaporin 0 (AQP0) is a lens-specific protein comprising more than 30% of lens membrane protein content and is a member of the aquaporin family. Water permeates through AQP0 much more slowly than other aquaporin family members, and other compounds, such as glycerol, also permeate AQP0. In the lens, ascorbic acid (AA) is found at high concentrations, protecting the lens from photochemical events such as photo-oxidation. The aim of the present study was to clarify the function of AQP0. Mouse fibroblast L-cells stably expressing AQP0 were established and incubated in medium containing AA, and intracellular AA levels were measured by high-performance liquid chromatography (HPLC) and 2,6-dichlorophenol-indophenol (DCPIP) analysis. Intracellular AA levels in AQP0-expressing cells quickly rose and reached saturation 10 min after incubation in medium containing 1000 μM AA. In contrast, AA levels in cells slowly decreased when AA was washed out from the medium. Cells overexpressing AQP0 increased the cellular uptake of AA in a time- and concentration-dependent manner. These data suggest that AA as well as water permeates AQP0. AQP0 expression on Xenopus oocyte membranes was achieved by the injection of AQP0 cRNA into oocytes that were incubated in medium containing AA. Intracellular AA levels were then measured by HPLC. AA uptake was demonstrated in the AQP0-expressing oocytes and was shown to quickly reach saturation. Intracellular AA concentration in oocytes increased in a time- and concentration-dependent manner. The data in the present study show that AA permeates AQP0, reveal the role of AQP0 in AA permeability ex vivo, and also indicate that there is a difference between the import and export of AA via AQP0. These findings suggest that AQP0 plays an important role in controlling lens AA content.
Department of Molecular Function and Physiology, Faculty of Pharmacy, Keio University, 1-5-30 Shibakoen, Minato-ku, Tokyo 105-8512, Japan.
Yosuke
Y
Nakazawa
Mikako
M
Oka
Akiko
A
Mitsuishi
Masayasu
M
Bando
Makoto
M
Takehana
D000818:Animals; D020346:Aquaporins / Q000378:metabolism*; D001205:Ascorbic Acid / Q000378:metabolism*; D002460:Cell Line; D005136:Eye Proteins / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D007908:Lens, Crystalline / Q000378:metabolism*; D008297:Male; D051379:Mice; D010539:Permeability; D051381:Rats; D017208:Rats, Wistar; D060347:Sodium-Coupled Vitamin C Transporters / Q000378:metabolism; D014867:Water / Q000378:metabolism*
D020346:Aquaporins; D005136:Eye Proteins; C555165:Slc23a2 protein, mouse; D060347:Sodium-Coupled Vitamin C Transporters; C030328:aquaporin 0; D014867:Water; D001205:Ascorbic Acid