Mechanism of L-ascorbic acid uptake by rabbit corneal epithelial cells: evidence for the involvement of sodium-dependent vitamin C transporter 2.
OBJECTIVE
To investigate the mechanism of L-ascorbic acid uptake by rabbit corneal epithelial cells and to functionally characterize the specific transporter involved in this translocation process.


METHODS
Uptake studies were carried out with SIRC (Statens Seruminstitut Rabbit Cornea) and rPCEC (rabbit Primary corneal epithelial cell culture) in 12-well plates using [14C] Ascorbic acid (AA). Uptake was done in the presence of L-ascorbic acid and D-isoascorbic acid to delineate stereospecificity. Inhibition studies were performed in the presence of D-glucose a substrate for GLUT and also para amino hippuric acid (PAHA) a substrate for organic anion transporter. Effects of pH and sodium on the uptake of AA were characterized. Concentration dependency studies were performed. Energy dependence of AA uptake was investigated with ouabain and sodium azide in rPCEC. Reverse Transcription-polymerase chain reaction (RT-PCR) was also performed.


RESULTS
Uptake of AA was inhibited by about 90% and 50% respectively in the presence of L-ascorbic acid and D-isoascorbic acid in both SIRC and rPCEC. Uptake was unaltered by D-glucose and PAHA. The process was sodium dependent and saturable at higher concentrations. Ouabain and sodium azide significantly diminished the uptake process. It also decreased with a reduction in pH. The RT-PCR results showed the presence of SVCT2 but not SVCT1.


CONCLUSIONS
Uptake of AA across rabbit corneal epithelial cells appears to be a carrier mediated active process. A saturable, sodium dependent, and pH sensitive transporter with high specificity for L-ascorbic acid was functionally characterized and was identified as SVCT2.
Talluri
Ravi S
RS
School of Pharmacy, University of Missouri-Kansas City, Kansas City, Missouri 64110, USA.
Katragadda
Suresh
S
Pal
Dhananjay
D
Mitra
Ashim K
AK
D000818:Animals; D001205:Ascorbic Acid / Q000378:metabolism*; D001692:Biological Transport; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D019573:Epithelium, Corneal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005947:Glucose / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D029363:Organic Anion Transporters, Sodium-Dependent / Q000235:genetics / Q000378:metabolism*; D010042:Ouabain / Q000494:pharmacology; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019810:Sodium Azide / Q000494:pharmacology; D060347:Sodium-Coupled Vitamin C Transporters; D027981:Symporters / Q000235:genetics / Q000378:metabolism*; D010130:p-Aminohippuric Acid / Q000494:pharmacology
D029363:Organic Anion Transporters, Sodium-Dependent; D060347:Sodium-Coupled Vitamin C Transporters; D027981:Symporters; C005056:isoascorbic acid; D010042:Ouabain; D019810:Sodium Azide; D005947:Glucose; D001205:Ascorbic Acid; D010130:p-Aminohippuric Acid