Identification and characterization of a Na+-dependent neutral amino acid transporter, ASCT1, in rabbit corneal epithelial cell culture and rabbit cornea.
OBJECTIVE
The aim of this study was to investigate the presence of a Na+-dependent neutral amino acid transporter, ASCT1, in rabbit primary corneal epithelial cell culture and rabbit cornea.


METHODS
Uptake studies were carried out on rabbit primary corneal epithelial culture (rPCEC) cells using 12-well plates. Transport studies were conducted with isolated rabbit corneas at 34 degrees C. Uptake and transport of L-alanine was determined at various concentrations. Inhibition studies were conducted in presence of various L- and D-amino acids, metabolic inhibitors like ouabain and sodium azide, and in the absence of sodium to delineate the functional characteristics of L-alanine uptake and transport. Reverse transcription-polymerase chain reaction (RT-PCR) was performed on total RNA harvested from rabbit cornea and rPCEC cells for identification of ASCT1.


RESULTS
Uptake of L-Ala was found to be saturable with a Km of 0.71 mM and a Vmax value of 0.84 micromoles min(-1) mg(-1) protein. Uptake was independent of pH and energy but depends on sodium. It was inhibited by serine, threonine, cysteine, and glutamine but did not respond to BCH (2-aminobicyclo [2,2,1] heptane-2-carboxylic acid) and MeAIB (alpha -methylaminoisobutyric acid). Transport of L-Ala across rabbit cornea was also saturable (Km 6.52 mM and Vmax 1.09 x 10(-2) micromoles min(-1) cm(-2)), energy independent, and subject to similar competitive inhibition. Presence of ASCT1 on rPCEC and on rabbit cornea was identified by RT-PCR.


CONCLUSIONS
L-Alanine, the chosen model substrate, was actively transported by Na+-dependent, neutral amino acid exchanger ASCT1, which was identified and functionally characterized on rPCEC cells and rabbit cornea.
Katragadda
Suresh
S
Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, Kansas City, Missouri 64110-2499, USA.
Talluri
Ravi Sankar
RS
Pal
Dhananjay
D
Mitra
Ashim K
AK
D000409:Alanine / Q000493:pharmacokinetics; D026921:Amino Acid Transport Systems, Neutral / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive; D001692:Biological Transport; D002478:Cells, Cultured; D019573:Epithelium, Corneal / Q000166:cytology / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007700:Kinetics; D008297:Male; D008353:Mannitol / Q000493:pharmacokinetics; D009994:Osmolar Concentration; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012964:Sodium / Q000378:metabolism
D026921:Amino Acid Transport Systems, Neutral; D007477:Ions; D008353:Mannitol; D012964:Sodium; D000409:Alanine