Selective Impairment of Fluoro-Ascorbic Acid Uptake into Mouse 17EM15 Lens Epithelial Cells: A Comparative Study

Abstract

Abstract: : Purpose: Previous studies from our laboratory revealed that the human lens epithelial cell line HLE B-3 was surprisingly resistant toward glutathione and ascorbic acid depletion upon exposure to 50 mM D-galactose and D-glucose. In these studies, cells were probed with fluorinated ascorbic and dehydroascorbic acid, both of which are transported by the SVCT2 transporter. In order to find out if this property is unique to these cells, similar experiments were carried out with human choriocarcinoma JAR and mouse lens epithelial 17EM15 cells, a gift of Dr. John Reddan. Methods: Cells were grown to confluence and incubated with or without 50 mM D-glucose or D-galactose for 6 days, or treated for 24 hrs with 100 µM buthionine sulfoximine, a glutathione synthesis inhibitor. After washing, the cells were incubated with 1 mM F-ASA or F-DHA for 24 hrs, and lysed in cold 4% metaphosphoric acid. The supernatant was analyzed by 750 MHz NMR spectroscopy using 6-F-Glucose as internal standard. Results: When HLE B-3, JAR and 17EM15 cells were incubated with F-ASA, intracellular F-ASA concentration were ~ 400, 250 and 40 pmol/10⁶ cells respectively. When incubated with F-DHA, F-ASA concentrations were ~ 1200, 550, and 25 pmol/10⁶ cells, respectively. Presence of reducing sugars had little effect on F-ASA concentrations in HLE B-3 and JAR cells, but further depressed the already very low F-ASA levels in 17EM15 cells and increased its oxidation into the –212.4 ppm compound. Upon exposure to D-galactose, intracellular glutathione levels decreased by 0 and 50% in the HLE B-3 and 17EM15 cells, and by 25% and 50% upon exposure to glucose, respectively. In contrast, GSH concentration increased in hexose-treated JAR cells. BSO treatment suppressed GSH by 50% in 17EM15 and JAR cells, but by 100% in HLE B-3 cells. Conclusions: The discovery that mouse, in contrast to human, lens epithelial cells have a 10-20 fold decrease in fluoro-ascorbic acid uptake may explain why the lenticular rodent vitamin C concentration is close to 0 mM, while that of the human lens is > 1 mM, and suggests either presence of low SVCT2 concentration, or low affinity of the mouse transporter for ascorbic acid. Accordingly, the mouse cells were also more susceptible toward hexose mediated GSH depletion, though not by BSO. CR : None Support: NEI/NIH EY 07099 and P30EY11373

Keywords: cataract • oxidation/oxidative or free radical damage • protein modifications-post translational