Functional characterization and molecular identification of vitamin C transporter (SVCT2) in human corneal epithelial (HCEC) and retinal pigment epithelial (D407) cells.

ABSTRACT: 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.Uptake of [(14)C] AA was studied in HCEC and D407 cells. Functional aspects of [(14)C] 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).Uptake of [(14)C] AA was observed to be sodium, chloride, temperature, pH and energy-dependent in both cell lines. [(14)C] AA uptake was found to be saturable, with Km values of 46.14?±?6.03 and 47.26?±?3.24??M and Vmax 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.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.

SUBMITTER: Khurana V 

PROVIDER: S-EPMC5527757 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature