PURPOSE: The objective of this research was to investigate the presence of an Na(+)-independent, large neutral amino acid transporter, LAT1, on rabbit corneal epithelium and human cornea. METHODS: Freshly excised rabbit corneas were used for transport studies and SIRC (a rabbit corneal cell line) cells for uptake studies. Transport and uptake characteristics of [(3)H]-L-phenylalanine were determined at various concentrations and pH. Inhibition studies were conducted in the presence of other L- and D-amino acids and metabolic inhibitors, such as ouabain and sodium azide, and in the absence of sodium to delineate the mechanism of uptake and transport. Reverse transcription-polymerase chain reaction (RT-PCR) for large neutral amino acid transporter-1 (LAT1) was performed on total RNA from rabbit cornea, SIRC cells, and human cornea. RESULTS: SIRC uptake of L-Phe was found to be saturable, with K(m) of 73 +/- 9 microM, V(max) of 2.0 +/- 0.1 nanomoles/min per milligram protein, and K(d) of 0.44 +/- 0.6 microL/min per milligram protein. Uptake was independent of pH, energy, and Na(+); inhibited by D-Leu, D-Phe, and an L-system-specific inhibitor 2-aminobicyclo [2,2,1] heptane-2-carboxylic acid (BCH), but not inhibited by L-Ala and charged amino acids. Transport of L-Phe across rabbit cornea was also saturable (K(m) = 33 +/- 8 microM and V(max) = 0.26 +/- 0.03 nanomoles/min per square centimeter), energy independent, and subject to similar competitive inhibition. LAT1 was identified by RT-PCR in rabbit corneal, SIRC, and human corneal RNA. CONCLUSIONS: A Na(+)-independent, facilitative transport system, LAT1, was identified and functionally characterized on rabbit cornea. LAT1 was also identified on human cornea.
PURPOSE: The objective of this research was to investigate the presence of an Na(+)-independent, large neutral amino acid transporter, LAT1, on rabbit corneal epithelium and human cornea. METHODS: Freshly excised rabbit corneas were used for transport studies and SIRC (a rabbit corneal cell line) cells for uptake studies. Transport and uptake characteristics of [(3)H]-L-phenylalanine were determined at various concentrations and pH. Inhibition studies were conducted in the presence of other L- and D-amino acids and metabolic inhibitors, such as ouabain and sodium azide, and in the absence of sodium to delineate the mechanism of uptake and transport. Reverse transcription-polymerase chain reaction (RT-PCR) for large neutral amino acid transporter-1 (LAT1) was performed on total RNA from rabbit cornea, SIRC cells, and human cornea. RESULTS: SIRC uptake of L-Phe was found to be saturable, with K(m) of 73 +/- 9 microM, V(max) of 2.0 +/- 0.1 nanomoles/min per milligram protein, and K(d) of 0.44 +/- 0.6 microL/min per milligram protein. Uptake was independent of pH, energy, and Na(+); inhibited by D-Leu, D-Phe, and an L-system-specific inhibitor 2-aminobicyclo [2,2,1] heptane-2-carboxylic acid (BCH), but not inhibited by L-Ala and charged amino acids. Transport of L-Phe across rabbit cornea was also saturable (K(m) = 33 +/- 8 microM and V(max) = 0.26 +/- 0.03 nanomoles/min per square centimeter), energy independent, and subject to similar competitive inhibition. LAT1 was identified by RT-PCR in rabbit corneal, SIRC, and human corneal RNA. CONCLUSIONS: A Na(+)-independent, facilitative transport system, LAT1, was identified and functionally characterized on rabbit cornea. LAT1 was also identified on human cornea.