Functional Characterization and Molecular Identification of Vitamin C Transporter (SVCT2) in Human Corneal Epithelial (HCEC) and Retinal Pigment Epithelial (D407) Cells

Purpose: The main goal of this study is to investigate the existence of sodium-dependent vitamin C transport system (SVCT2) and to define time-dependent uptake mechanism and intracellular regulation of ascorbic acid (AA) in human corneal epithelial (HCEC) and human retinal pigment epithelial (D407) cells. Methods: Uptake of [C-14] AA was studied in HCEC and D407 cells. Functional aspects of [C-14] AA uptake were studied in the presence of different concentrations of unlabeled AA, pH, temperature, metabolic inhibitors, substrates and structural analogs. Molecular identification of SVCT2 was examined with reverse transcription-polymerase chain reaction (RT-PCR). Results: Uptake of [C-14] AA was observed to be sodium, chloride, temperature, pH and energy-dependent in both cell lines. [C-14] AA uptake was found to be saturable, with K-m values of 46.14 +/- 6.03 and 47.26 +/- 3.24 mu M and V-max values of 17.34 +/- 0.58 and 31.86 +/- 0.56 pmol/min/mg protein, across HCEC and D407 cells, respectively. The process is inhibited by structural analogs (L-AA and D-Iso AA) but not by structurally unrelated substrates (glucose and PAHA). Ca++/calmodulin and protein kinase pathways play an important role in modulating uptake of AA. A 626 bp band corresponding to a vitamin C transporter (SVCT2) has been identified by RT-PCR analysis in both the cell lines. Conclusion: This research article reports regarding the ascorbic acid uptake mechanism, kinetics and regulation by sodium dependent vitamin C transporter (SVCT2) in HCEC and D407 cells. Also, SVCT2 can be utilized for targeted delivery in enhancing ocular permeation and bioavailability of highly potent ophthalmic drugs.