Identification and functional characterization of a Na+-independent large neutral amino acid transporter, LAT1, in human and rabbit 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.

Molecular evidence and functional expression of P-glycoprotein (MDR1) in human and rabbit cornea and corneal epithelial cell lines.

PURPOSE: Efflux pumps such as P-glycoprotein (P-gp; MDR1) are believed to be a major barrier to drug delivery. The purpose of this work was to determine whether cornea and corneal epithelial cells expresses the functionally active P-gp efflux pump. METHOD: Cultured rabbit primary corneal epithelial cells (rPCECs) and a corneal cell line (Statens Seruminstitut rabbit cornea [SIRC] cells) were selected as the model. Rhodamine-123 (Rho-123), a P-gp substrate, was used as a P-gp probe. To confirm gene expression, RT-PCR was performed with appropriate pairs of primers for rabbit and human MDR1. Subcloning, sequencing, and protein sequence determination were performed to confirm P-gp. RESULTS: Permeability of [(3)H] cyclosporin A (CsA) across SIRC cells was found at 1.74 x 10(-6) cm/s in the apical-to-basolateral and 5.1 x 10(-6) cm/s in the basolateral-to-apical directions. Uptake of Rho-123 across both SIRC cells and rPCECs was time and temperature dependent. Rho-123 uptake in SIRC cells was 14.4 picomoles/mg protein and in the presence of CsA (10 micro M) was 70.8 picomoles/mg protein at 3 hours. Uptake in rPCECs was the highest at 3 hours. Western blot analysis indicated a 170-kDa band confirming the presence of P-gp. Human cornea was also checked for the presence of P-gp. RT-PCR data indicated one single band, which was subcloned and sequenced to confirm the presence of P-gp. The protein sequence deduced from the fragment product indicated more than 89% homology with human MDR1. CONCLUSIONS: Functional and molecular characterization showed the existence of P-gp in human cornea, rabbit cornea, and a rabbit corneal cell line. This knowledge of the existence of P-gp will help in development of better ocular drug delivery strategies.

Carrier mediated uptake of L-tyrosine and its competitive inhibition by model tyrosine linked compounds in a rabbit corneal cell line (SIRC)--strategy for the design of transporter/receptor targeted prodrugs.

The objective of this study was to investigate the presence of amino acid transporters on the corneal epithelium and to enhance corneal drug absorption through prodrug modification targeted to the amino acid transporters. SIRC was used as a model cell line representing the corneal epithelium. Uptake studies were carried out using [3H] L-tyrosine at 37 degrees C. Temperature, energy and pH dependence studies were carried out. The uptake seems to be composed of a major saturable and minor non-saturable component (V(max) =2.9+/-0.62 nmoles/min/mg protein, K(m) =71+/-21 microM, K(d) =2.6+/-0.6 nl/min/mg protein). No significant inhibition of uptake was observed in the presence of metabolic inhibitors or in the absence of sodium. Competitive inhibition studies were performed in the presence of various amino acids and model tyrosine conjugates (p-nitro and p-chloro benzyl ether conjugate of L-tyrosine). Uptake was inhibited by neutral aromatic and large neutral aliphatic amino acids. L-Tyrosine uptake was inhibited by its ether conjugates in a concentration dependent manner suggesting that these compounds may be sharing the same transport mechanism. This study provides biochemical evidence of the presence of a large neutral amino acid transport system on the corneal epithelium, which may be utilized to enhance the corneal drug transport.

Identification of a Na+-dependent cationic and neutral amino acid transporter, B(0,+), in human and rabbit cornea.

The purpose of this study was to identify and functionally characterize an active transport system for L-arginine on rabbit corneal epithelium and human cornea and study its interaction with the amino acid ester prodrugs of acyclovir (Anand, B. S.; Mitra, A. K. Pharm. Res. 2002, 19, 1194-1202). Transport characteristics of [3H]-L-arginine across freshly excised rabbit corneas were determined at various concentrations, in the absence of sodium and chloride ions. Inhibition studies were conducted in the presence of other amino acids, ouabain, and amino acid ester prodrugs of acyclovir (glycine-ACV, phenylalanine-ACV and gamma-glutamate-ACV). Reverse transcription-polymerase chain reaction (RT-PCR) for amino acid transporter B(0,+) was carried out on total RNA isolated from rabbit cornea, rabbit corneal epithelium, and human cornea. Transport of L-Arg across rabbit cornea was saturable (Km = 306 +/- 72 microM and Vmax = 0.12 +/- 0.01 nmol min(-1) cm(-2)) and was Na+, Cl-, and energy dependent. Transport was inhibited by neutral and cationic amino acids and a B(0,+) system specific inhibitor, BCH (Sloan, J. L.; Mager, S. J. Biol. Chem. 1999, 274, 23740-23745), but not by anionic amino acids. Amino acid prodrugs of ACV (Glu-ACV and Phe-ACV) also inhibited transport of [3H]-L-Arg across rabbit cornea. Amino acid transporter B(0,+) was identified by RT-PCR and its identity confirmed by subcloning and sequencing in rabbit cornea, rabbit corneal epithelium, and human cornea. A Na+-, Cl(-)-, and energy-dependent carrier for L-Arg, B(0,+), was identified on rabbit corneal epithelium and human cornea. Glu-ACV and Phe-ACV appear to be substrates for this transporter. The presence of such transporters on the corneal epithelium may provide new opportunities for transporter-targeted prodrug design for enhanced corneal absorption.