Functional Expression of Ascorbic Acid Transporters in Lens

Abstract

Abstract: : Purpose: To determine:1. The presence of sodium-dependent vitamin C transporter 1 (SVCT1) and 2 (SVCT2) in mammalian lens and 2. The effects of glucose, H₂O₂ and agonists for PKA and PKC on ascorbic acid (ASA) transport in lens. Methods: RT-PCR and immunostaining were performed to identify lens SVCT1 and SVCT2. Regulation of ascorbic acid transporters was studied under hyperglycemic conditions. Glucose or ascorbic acid uptake by intact guinea pig and rabbit lenses was determined by incubation in ₃H labeled 2-deoxyglucose, and/or ₁₄C labeled ascorbic acid. PKA was activated using membrane permeant cAMP and PKC using PMA. Results: RT-PCR of lens total RNA and immunostaining of lens sections showed the presence of SVCT1 and SVCT2 in mouse and rabbit lens epithelium and fiber cells; however, expression of SVCT2 was higher than that of SVCT1. SVCT 1 and SVCT2 were localized more to the apical side of the lens epithelium. In fiber cells, the SVCTs showed uniform distribution. In lenses kept under hyperglycemic (50 mM) conditions for 48 h, ASA uptake was reduced as follows: guinea pig 42%, and rabbit 32%. Addition of the SVCT-specific inhibitors phloretin (100 µM) or phlorizin (100 µM) or 20 mM cold ASA reduced the uptake of ₁₄C labeled ascorbic acid by 73, 47 and 48%, respectively. Guinea pig lenses cultured for 2 days with 30 µM H₂O₂ in the culture medium showed an increase of 10-15% in ASA uptake; however, incubation for 20 days in 30 µM H₂O₂ inhibited ASA uptake by 36 ± 9%. ₃H labeled 2-deoxyglucose uptake was not altered in the presence of ASA in the medium. Guinea pig lens under hyperglycemic conditions, accumulated more glucose (28%) and less ascorbic acid (32%) than the controls. Incubation in 50 mM D-glucose for 24h inhibited ASA transport by 39% and 31%, for rabbit and guinea pig lenses respectively, even when the external glucose was near zero. PKA or PKC activation inhibited ASA transport. Conclusion: These data suggest that mammalian lenses contain Na-dependent vitamin C transporters SVCT1 and SVCT2. Long term exposure to high glucose or H₂O₂ decreased the uptake of ASA. Accumulation of more glucose and less ascorbic acid under hyperglycemic condition could cause oxidative damage to lens proteins. Internal rather than external glucose appears to regulate ASA transport in the lens. Alterations in the normal transport of the antioxidant ASA in lens cells could contribute to increased protein damage and could result in cataract under diabetic conditions.

Keywords: 387 diabetes • 417 gene/expression • 338 cataract