Identification and functional expression of a carrier-mediated riboflavin transport system on rabbit corneal epithelium.
OBJECTIVE
To investigate the functional expression of a carrier-mediated transport mechanism for riboflavin (vitamin B2) across cultured rabbit primary corneal epithelial cells (rPCECs) and intact rabbit cornea. The secondary objective was to understand the physiological significance behind the presence of such a transport system for riboflavin on the apical side of the corneal epithelium.


METHODS
rPCECs and freshly excised rabbit corneas were selected as in vitro and ex vivo models, respectively. Transport and uptake characteristics of [3H]riboflavin were determined at various time points, concentrations, temperatures, and pH. Substrate specificity, energy, and ion dependence studies were carried out to characterize the translocation mechanism. Rabbit tear analysis was done with liquid chromatography/tandem mass spectrometry (LC-MS/MS) to understand the physiological relevance of this transporter.


RESULTS
The uptake process in rPCECs was found to be concentration dependent and saturable at higher concentrations. The process was also independent of pH, Na+, and Cl- but dependent on energy and temperature. Unlabeled riboflavin and its structural analogues caused significant inhibition, whereas unrelated vitamins did not interfere with the process. Transport of [3H]riboflavin across rabbit cornea was also saturable at higher concentration and energy dependent but Na+ independent. Substrate specificity studies across intact rabbit cornea produced results similar to the uptake studies in cultured rPCECs. LC-MS/MS analysis of rabbit tears showed the presence of riboflavin.


CONCLUSIONS
Results suggest the presence of a specialized, high-affinity transport mechanism for riboflavin that is expressed on the apical side of rabbit corneal epithelium and may in turn be responsible for influx of riboflavin from tears to cornea.
Hariharan
Sudharshan
S
Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, Kansas City, Missouri 64112-2499, USA.
Janoria
Kumar G
KG
Gunda
Sriram
S
Zhu
Xiaodong
X
Pal
Dhananjay
D
Mitra
Ashim K
AK
D019297:2,4-Dinitrophenol / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport; D002478:Cells, Cultured; D002853:Chromatography, Liquid; D003078:Colchicine / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019573:Epithelium, Corneal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D006863:Hydrogen-Ion Concentration; D008297:Male; D026901:Membrane Transport Proteins / Q000378:metabolism*; D011817:Rabbits; D012256:Riboflavin / Q000493:pharmacokinetics*; D013666:Tears / Q000378:metabolism; D013696:Temperature
D004791:Enzyme Inhibitors; D026901:Membrane Transport Proteins; C019079:riboflavin-binding protein; D019297:2,4-Dinitrophenol; D003078:Colchicine; D012256:Riboflavin