title: Functional characterization and molecular identification of vitamin C transporter (SVCT2) in human corneal epithelial (HCEC) and retinal pigment epithelial (D407) cells.
abstract: OBJECTIVE
  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 [(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).
  
  
  RESULTS
  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 μM 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City , Kansas City, MO , USA .
authors.forename: Varun
authors.initials: V
authors.lastname: Khurana
authors.forename: Aswani Dutt
authors.initials: AD
authors.lastname: Vadlapudi
authors.forename: Ramya Krishna
authors.initials: RK
authors.lastname: Vadlapatla
authors.forename: Dhananjay
authors.initials: D
authors.lastname: Pal
authors.forename: Ashim K
authors.initials: AK
authors.lastname: Mitra
mesh_terms: D002460:Cell Line; D019573:Epithelium, Corneal / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D012313:RNA / Q000235:genetics*; D055213:Retinal Pigment Epithelium / Q000166:cytology / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D060347:Sodium-Coupled Vitamin C Transporters / Q000235:genetics / Q000378:metabolism*
chemical_list: D060347:Sodium-Coupled Vitamin C Transporters; D012313:RNA
keywords: Ascorbic acid kinetics; carrier-mediated transport and nutrient transporter; in vitro models; targeted drug delivery