Identification and Characterization of a Na+-Dependent Neutral Amino Acid Transporter, ASCT1, in Rabbit Corneal Epithelial Cell Culture and Rabbit Cornea

Abstract

Purpose: 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°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 K_m of 0.71 mM and a V_max value of 0.84 μ moles 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 (α -methylaminoisobutyric acid). Transport of L-Ala across rabbit cornea was also saturable (K_m 6.52 mM and V_max 1.09 × 10⁻² μ moles 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.