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B Jain Vakkalagadda, S Dey, D Pal, AK Mitra; Identification and Functional Characterization of the Na+-Independent, Neutral Amino Acid Transporter, LAT1, on the Corneal Epithelium . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3256.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The objective of this research is to identify and functionally characterize the amino acid transporters on the corneal epithelium and to deliver amino acid prodrugs of ACV (glutamate-ACV) using such transport systems across the cornea. Methods: Freshly excised rabbit corneas (FERC) were used for transport studies and SIRC (rabbit corneal cell line) cells grown on 12-well plates for uptake studies. The transport of [14C]-mannitol and phenylalanine (Phe) containing 0.5 µCi/ml of radiolabelled Phe was studied across FERC. Uptake studies were carried out with [3H]-Phe at different concentrations, pH and temperature. Inhibition studies were carried out in presence of other L- and D- amino acids, glutamate-ACV and metabolic inhibitors like ouabain and sodium azide to delineate the mechanism of uptake and transport. Results: Uptake of L-Phe, was found to be saturable with increasing concentrations. The Km and Vmax for L-Phe on SIRC cells were found to be 51.86 (12.06) µM and 288.53 (24.78) ng/min/mg protein respectively. The amino acid transport system was found to be independent of pH and Na+ ions. Uptake was almost completely inhibited by a system L-specific inhibitor-BCH and also by D-leucine and D-Phe, while the small neutral amino acids (Ala, Pro) didn't have any significant effect, thus differentiating it from LAT2. Transport of L-Phe across FERC also showed a saturable profile at higher concentrations and inhibition with L-tyrosine. Also, transport of [14C] Gly was inhibited by glutamate-ACV. Conclusion: LAT1 amino acid transport system appears to be present on the rabbit cornea and SIRC. It is specific for aromatic amino acids and independent of pH unlike LAT2. Preliminary results indicate that glutamate-ACV is a substrate for the Gly transport system. Further studies with other ACV amino acid esters will help delineate the structural requirements for the transport systems and their relative affinities towards these substrates. RT-PCR will be done to confirm the presence of these transporters on the corneal epithelium.
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