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(Investigative Ophthalmology and Visual Science. 2003;44:2919-2927.)
© 2003 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.02-0907
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Identification and Functional Characterization of a Na+-Independent Large Neutral Amino Acid Transporter, LAT1, in Human and Rabbit Cornea

Blisse Jain-Vakkalagadda, Surajit Dey, Dhananjay Pal, and Ashim K. Mitra

From the Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, Kansas City, Missouri.


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
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 [3H]-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 Km of 73 ± 9 µM, Vmax of 2.0 ± 0.1 nanomoles/min per milligram protein, and Kd of 0.44 ± 0.6 µL/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 (Km = 33 ± 8 µM and Vmax = 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.

Cornea is the principal refractive element in the eye. Its optical qualities are imparted by its shape and transparency and its essential features maintained by the metabolic functions of the adjacent cell layers, the epithelium and the endothelium.1 The corneal epithelium itself is composed of five to six layers of columnar epithelial cells. Its barrier properties arise from the high electrical resistance of both the outermost cell membranes and the paracellular zonulae occludens, which restrict the paracellular movement of molecules across these layers. Delivery of hydrophilic compounds to the deeper corneal layers is thus a major challenge in ocular therapeutics.2 3 4 Solute transport through a transporter or a receptor is a mechanism of translocating hydrophilic compounds across lipid bilayers. Recently, a significant amount of work has been reported on membrane transporters and receptors in various tissues.5 6 7 8 9 10 11 12 13 Among the ocular tissues, conjunctiva has been reported to express a large number of transporter proteins including amino acid transporters, monocarboxylic acid transporter (MCT), and nucleoside transporter, which are believed to play a role in the absorption of drugs through the conjunctiva after topical administration. Nutrient transporters on the retina have also been identified.17 18 19 20 A few reports about the presence of carrier-mediated nutrient transport systems on the cornea are available, but most of them are believed to be present on the corneal endothelium. There is limited information on the mechanisms of amino acid transport across the corneal epithelium, which is the primary barrier for ocular drug absorption. A lactate-proton cotransporter and a glucose transporter (GLUT1) have been identified on the corneal epithelium.21 22 23 The oligopeptide transport system has been identified for the first time on the corneal epithelium in our laboratory.24 To enhance the corneal permeability of polar compounds a strategy can be adopted to use the transporters present on the corneal epithelium.

Numerous amino acid transport systems have been characterized at the molecular level including L, y+L, A, ASC, asc, b0,+, B0,+ and x-, Gly, n, and T.25 26 27 28 29 30 31 32 System L is a major amino acid transporter that transports large neutral amino acids in a Na+-independent manner.33 It was originally identified in Ehrlich ascites carcinoma cells.34 System L has been known to transport not only naturally occurring amino acids but also amino acid–related compounds such as L-dopa, a therapeutic drug for Parkinsonism; melphalan, an anticancer Phe mustard; triiodothyronine and thyroxine, two thyroid hormones; and gabapentin, an anticonvulsant.34 35 36 37 38 39 40 Two isoforms of the Na+-independent, large neutral amino acid transporter, LAT1 and LAT2 (L-type amino acid transporter 1 and 2 respectively), have recently been isolated, cloned and expressed in Xenopus oocytes.41 42 LAT2-mediated transport differs from that of LAT1 in pH dependence, substrate specificity, substrate affinity, tissue distribution, and interaction with D-amino acids.43

LAT1 preferentially transports large neutral amino acids, such as Leu, Ile, Val, Phe, Tyr, Trp, Met, and His. This transporter is highly upregulated and expressed in cultured cells and malignant tumors probably to support the high-level protein synthesis for continuous growth and proliferation.44 45 Ubiquitously expressed LAT2 transports not only large neutral amino acids but also small neutral amino acids.46 47 LAT1 has been cloned from rat, mouse, human and Xenopus tissues and its expression has been found to be restricted to certain tissues like brain, placenta, testis, and small intestine.48 There is no report, however, of the presence of this transport system on the corneal epithelium or any rabbit tissue.

In this study, Phe, a large neutral amino acid, was used as a model substrate to investigate the functional presence of a large neutral amino acid transport system on the corneal epithelium.

To minimize the use of animal tissues, we performed in vitro uptake studies using a rabbit corneal cell line SIRC (Statens Serum Institut rabbit cornea), which is a well-established cell culture model for corneal epithelium. This cell line has been used extensively for in vitro studies to assess corneal physiology, immunology, toxicology, and transport.48 49 50 51 52 53 54 55 The cell line forms five to six layers of epithelium in culture as characterized and reported previously from our laboratory,55 thus serving as a good in vitro model for the corneal epithelium. SIRC has not been investigated previously for the presence of possible membrane transporters and receptors. Herein, we report the uptake characteristics of Phe, using SIRC cells to identify a large neutral amino acid transporter on the corneal epithelium. These results were further confirmed by conducting transport studies across freshly excised rabbit cornea.


   Methods
Top
 Abstract
 Methods
Results
 Discussion
 References
 
Animals
New Zealand albino adult male rabbits weighing between 2.0 and 2.5 kg were obtained from Myrtle’s Rabbitry (Thompson Station, TN). Studies involving these rabbits were conducted according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Cell Culture
SIRC cells (passages 410-425; ATCC, Manassas, VA) were plated at a density of 500,000 cells/well on 12-well culture plates. The culture medium (minimum essential medium [Gibco-BRL-Invitrogen, Grand Island, NY]; 10% fetal bovine serum, [JRH Bio Sciences, Lenexa, KS]; lactalbumin; HEPES; sodium bicarbonate; penicillin [100 Units/mL], and streptomycin [100 µg/mL]) was replaced on alternate days. Cells were maintained at 37°C, in a humidified atmosphere of 5% CO2 and 90% relative humidity.

Uptake Experiments
Concentration Dependence.
Uptake studies were conducted based on the method of Surendran et al.56 with slight modifications. Briefly, at 10 to 12 days after seeding, the medium was removed, and cells were washed twice with Dulbecco’s phosphate-buffered saline (DPBS; pH 7.4), containing 130 mM NaCl, 2.5 mM KCl, 7.5 mM Na2HPO4, 1.5 mM KH2PO4, 1 mM CaCl2, 0.5 mM MgSO4, and 5 mM glucose. L-Phe solutions were prepared at different concentrations (0.01–2 mM) in DPBS, (pH 7.4), containing [3H]-L-Phe (0.5 µCi/mL; Amersham Pharmacia Biotech, Piscataway, NJ). Uptake was initiated by incubating the cells with 2 mL of each solution for a predetermined time at 37°C. Initial experiments revealed that uptake was linear for at least 25 minutes. All subsequent experiments were therefore performed with a 5-minute incubation period.

At the end of the incubation period, the solution was aspirated off, and cells were washed twice with 2 mL of ice-cold stop solution (210 mM KCl, 2 mM HEPES; pH 7.4), to arrest the cellular uptake. One milliliter of 0.3% NaOH containing 0.1% Triton-X solution was then added to each well to solubilize the cells overnight. A portion (500 µL) of that solution was then transferred to scintillation vials containing 5 mL of scintillation cocktail. Cellular radioactivity was quantified using a scintillation counter (Model LS-9000; Beckman Instruments Inc., Fullerton, CA). From each well, 20 µL of the solution was then taken to measure the protein content in each sample using the method of Bradford57 and bovine serum albumin as the standard (Bio-Rad protein assay kit, Hercules, CA). Nonspecific binding was subtracted from the overall uptake levels.

pH and Na+ Dependence.
When the effect of Na+ on amino acid uptake was studied, NaCl and Na2HPO4 in the buffer were replaced with equimolar quantities of choline chloride and K2HPO4, respectively. Buffer pH was varied from 5.5 to 8.5 for pH-dependence studies. In all cases, uptake of [3H]-L-Phe (0.5 µCi/mL) was conducted according to the procedure described previously.

Competitive Inhibition Studies.
To delineate the structural requirements for interaction with this amino acid carrier system, uptake experiments were performed using competitive inhibitors (various amino acids and amino acid type-drugs) based on the method of Hidalgo and Borchardt58 with slight modifications. Cells were incubated simultaneously with [3H]-L-Phe (0.5 µCi/mL) and 1 mM unlabeled amino acids as inhibitors, and uptake experiments were conducted as described earlier. Stereoselectivity of the carrier system was also determined, by using both L- and D-amino acids as inhibitors.

Energy Dependence.
These studies were performed by preincubating the cells for 30 minutes with buffer alone (control) or with 1-mM concentrations of either ouabain (an inhibitor of Na+K+-adenosine triphosphatase [ATPase]) or sodium azide (an inhibitor of oxidative phosphorylation). Uptake was then performed as described earlier with pH 7.4 buffer solution containing [3H]-L-Phe (0.5 µCi/mL).

Corneal Transport Studies
Transport of L-Phe across freshly excised rabbit cornea was performed according to the method of Tak et al.55 Briefly, New Zealand albino rabbits weighing 2.0 to 2.5 kg were killed by an overdose of pentobarbital through a marginal ear vein. Eyes were then carefully enucleated and washed with ice-cold DPBS (pH 7.4) to remove any traces of blood. Subsequently, a small incision was made in the sclera and the cornea was carefully excised, leaving some portion of the sclera attached to it for mounting on the diffusion apparatus. After separation of the lens and iris–ciliary–body, the cornea was washed with ice-cold DPBS (pH 7.4). It was then mounted on a diffusion apparatus (Side-bi-Side) maintained at 34°C (corneal temperature in vivo). Amino acid solutions (3 mL) were added on the epithelial side of the cornea (donor chamber). In the other half-chamber (receiver chamber), 3.2 mL of DPBS (pH 7.4) was added and solutions in both the chambers were stirred continuously using magnetic stirrer bars. Receiver chamber volume of DPBS was maintained slightly higher to generate hydrostatic pressure to maintain the curvature of the cornea throughout the experiment. Sink conditions prevailed during the entire experiment. One-hundred-microliter aliquots were removed from the receiver chamber at appropriate intervals and replaced with an equal volume of DPBS. Samples were transferred to scintillation vials containing 5 mL of scintillation cocktail, and radioactivity was measured with the help of a scintillation counter (Model LS-9000; Beckman Instruments, Inc.). [14C]-mannitol, a paracellular marker, was added to the donor solutions and its transport determined in a similar manner, to assess the integrity of the cornea for the duration of the experiment.

Saturation Kinetics of L-Phe Transport.
Transport of L-Phe across rabbit cornea was performed as described earlier, using L-Phe solutions prepared at different concentrations (0.5–50 µM) in DPBS (pH 7.4), containing [3H]-L-Phe (1 µCi/mL). Michaelis-Menten parameters Km and Vmax were determined by nonlinear regression.

Competitive Inhibition Studies.
Transport of [3H]-L-Phe (1 µCi/mL) was studied in the presence of various amino acids (1 mM) to determine the substrate specificity of the system responsible for the transport of L-Phe. Transport was also determined in the presence of 1 mM glycyl-sarcosine (a peptide transporter substrate) because of overlapping substrate specificity between large neutral amino acid and peptide transporter, as reported previously.56

Energy Dependence.
These studies were performed by preincubating the cornea with buffer alone (control), or 500 µM ouabain for 30 minutes. Transport of [3H]-L-Phe (1 µCi/mL) was then determined, as described earlier at pH 7.4 in DPBS.

RT-PCR and Sequencing
Reverse transcription-PCR was performed based on the method of Sugawara et al.,59 with slight modifications, using 1 µg of total RNA isolated from human cornea (kindly provided by Alcon Laboratories, Fort Worth, TX). The forward and reverse primers were 5'-TCT CAC TGC TTA ACG GCG TGT G-3', and 5'-TCC CTG GCC AAG TCT AAC AAT G-3', respectively. These primers correspond to the nucleotide positions 110-132 and 606-628 in hLAT1 cDNA, respectively. RT-PCR was performed with a commercial kit (GeneAmp; Applied Biosystems, Foster City, CA). The conditions for reverse transcription were as follows: denaturation of the template RNA for 10 minutes at 70°C and reverse transcription for 60 minutes at 42°C. The conditions for PCR amplification were as follows: denaturation for 1 minute at 94°C; annealing for 1 minute at 58°C, and extension for 1 minute at 72°C, 37 cycles; final extension for 10 minutes at 72°C. The resultant product (~520 bp) was subcloned in pGEM-T vector and sequenced from both T7 and SP6 directions with an automated DNA sequencer (377 Prism; Applied Biosystems) to establish its molecular identity. The sequence was analyzed on computer (GCG, ver. 10; Genetics Computer Group, Inc., Madison, WI). Under identical conditions, RT-PCR was also conducted on total RNA purified (acid guanidinium thiocyanate-phenol-chloroform extraction method) from freshly excised rabbit corneal epithelium as well as SIRC cells.60 The sense and antisense primers selected were 5'-GCC ATC ACC TTT GCC AAC TA-3' and 5'-AAT AGG CCA CAT TGG TCA AG-3', respectively. These primers correspond to the nucleotide positions 415-435 and 834-854 respectively in rat LAT1 cDNA and were selected from two highly conserved regions among rat LAT1, mouse LAT1, and human LAT1 sequences. The resultant product (~ 440 bp) was subcloned in pGEM-T vector and sequenced from both T7 and SP6 directions to establish its molecular identity.

Computer Analysis
Nucleotide sequence homology search was performed with basic local alignment tool (BLAST) through online connection to the National Center of Biotechnology Information (NCBI; Bethesda, MD) database (http://www.ncbi.nlm.nih.gov/BLAST/; provided in the public domain). Multiple nucleotide sequence comparisons were made with Clustal W (ver. 1.81) multiple sequence alignment tool from SwissProt, (http://www.ebi.ac.uk/clustalw/; provided in the public domain by the Swiss Institute of Bioinformatics, Geneva, Switzerland).

Data Treatment
Uptake data were fitted to a modified Michaelis-Menten equation which takes into account the carrier-mediated process (as described by the classic Michaelis-Menten equation) and the nonsaturable passive diffusion process

where V is the total rate of uptake, Vmax is the maximum uptake rate for the carrier-mediated process, Km (Michaelis-Menten constant) is the concentration at half the Vmax, and Kd is the rate constant for the nonsaturable diffusional component.

Data were fitted to the equation with a nonlinear least-squares regression analysis program (KaleidaGraph V3.09; Synergy Software, Reading, PA). In case of corneal transport of L-Phe, steady state flux values and permeability coefficients were calculated.55 The flux data were then fitted to the classic Michaelis-Menten equation.

Statistical Analysis
All experiments were conducted at least in triplicate, and results are expressed as the mean ± SD. Michaelis-Menten parameters Km and Vmax and the diffusional component Kd are expressed as the mean ± SE. Statistical analysis between two groups was performed with Student’s t-test. A difference between means was considered significant if P ≤ 0.05.


   Results
Top
 Abstract
 Methods
 Results
Discussion
 References
 
Uptake Studies
Concentration Dependence.
L-Phe rapidly accumulated within the cells. The uptake was linear initially, and the slope gradually decreased after 25 minutes. Therefore, all the subsequent studies and kinetic analyses were performed with data collected from initial 5-minute uptake studies.

Uptake of L-Phe consisted of a major (saturable) carrier-mediated process and a minor nonsaturable component evident at higher concentrations (Fig. 1) . Uptake data were fitted to the equation and kinetic parameters Km and Vmax calculated to be 73 ± 9 µM (mean ± SE) and 2.0 ± 0.1 nanomoles/min per milligram protein (mean ± SE) respectively. Kd had a value of 0.44 ± 0.6 µL/min per milligram protein (mean ± SE). Eadie-Hofstee transformation of the data pointed to the involvement of a single carrier in the uptake process (Fig. 1 , inset).



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  		FIGURE 1. Concentration-dependent L-Phe uptake in SIRC cells at 37°C, pH 7.4. Kd = 0.44 ± 0.6 µL/min per milligram protein (mean ± SE); Km = 73 ± 9 µM (mean ± SE), Vmax = 2.0 ± 0.1 nanomoles/min per milligram protein (mean ± SE). Inset: Eadie-Hofstee transformation of the data. Data points represent the mean ± SD (n = 3–4).

 
Substrate Specificity.
To investigate the substrate specificity of the carrier involved in the uptake of L-Phe, various amino acids and a structurally similar drug (L-dopa) were examined as potential inhibitors of uptake. Inhibition of [3H]-L-Phe uptake was observed preferentially with L-isomers of large neutral amino acids such as Phe, Tyr, Trp, and Leu (Fig. 2) . L-Dopa was also an effective inhibitor of uptake. Inhibition by small neutral (L-Ala, glycine), basic (L-lysine), and acidic (L-glutamate and L-aspartate) amino acids was negligible.



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  		FIGURE 2. Competitive inhibition of [3H]-L-Phe (0.5 µCi/mL) uptake in SIRC cells by specific inhibitors, L- and D-isomers of different amino acids and amino acid-type drugs (1 mM), indicating the substrate specificity and stereoselectivity of the transport system. Data are expressed as the mean ± SD (n = 3–4). *Significantly different from control (P ≤ 0.05).

 
The carrier exhibited stereoselectivity characteristic of LAT1 as D-isomers of Phe and Leu inhibited uptake to a large extent, whereas D-isomers of Val, Trp, and Tyr exhibited negligible inhibition (Fig. 2) .

We further investigated the possibility of involvement of multiple transport systems in the uptake process. Effects of 1 mM N-methylaminoisobutyric acid (NMAIB), a specific inhibitor of the Ala-preferring neutral amino acid transport system and BCH, a specific inhibitor of the Leu-preferring L-type amino acid transport system, were determined. Uptake of L-Phe was sensitive to the presence of BCH, whereas NMAIB did not have any significant effect on the uptake process (Fig. 2) . The data indicate that uptake is predominantly mediated by the Leu-preferring, system-L type, neutral amino acid transport system.

Effects of Metabolic Inhibitors, Na+ and pH.
The effects of metabolic inhibitors on the uptake of L-Phe were studied to determine whether the uptake requires energy expenditure. Sodium azide, a respiratory chain inhibitor and ouabain, an inhibitor of Na+K+-ATPase did not exhibit significant inhibition of uptake (Fig. 3) . Further, L-Phe uptake was not altered in a sodium-free buffer, indicating that the uptake was mediated by an Na+-independent system, characteristic of L-type large neutral amino acid transporter. Also, the kinetics did not change significantly with a change in buffer pH, confirming the involvement of LAT1-type large neutral amino acid transporter.43



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  		FIGURE 3. Uptake of [3H]-L-Phe (0.5 µCi/mL) in SIRC cells in absence and presence of metabolic inhibitors ouabain and sodium azide (1 mM) and in a sodium-free buffer. Uptake remained unaltered and therefore Na+ and energy independent. Data are expressed as the mean ± SD (n = 3–4).

 
Corneal Permeation Studies
Saturation Kinetics of L-Phe Transport.
Transport experiments were conducted for a period of 100 minutes with donor concentrations of L-Phe ranging from 0.5 to 50 µM. Flux and apparent permeability (Papp) values were calculated from a plot of cumulative amount of Phe transported across rabbit cornea as a function of time (Fig. 4) . Transport of Phe across the cornea was found to be concentration dependent and saturable at higher concentrations (Fig. 5) . The data were fitted to a classic Michaelis-Menten equation, and the kinetic parameters Km and Vmax calculated to be 33 ± 8 µM (mean ± SE) and 0.26 ± 0.03 nanomoles/min per square centimeter (mean ± SE), respectively. Eadie-Hofstee transformation of the data once again suggested the involvement of just a single carrier in the transport process (Fig. 5 inset). Corneal integrity was maintained for the entire duration of the experiment, because a constant 0.5% ± 0.08% mannitol was transported across rabbit cornea per hour for all the experiments.



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  		FIGURE 4. Time course of cumulative amount of 25 µM L-Phe transported across freshly excised rabbit cornea, 34°C, pH 7.4. Data points represent the mean ± SD (n = 3).

 


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  		FIGURE 5. Concentration dependent transport of L-Phe across freshly excised rabbit cornea at 34°C, pH 7.4. Michaelis-Menten parameters: Km = 33 ± 8 µM (mean ± SE), Vmax = 0.26 ± 0.03 nanomoles/min per square centimeter (mean ± SE). Inset: Eadie-Hofstee transformation of the data. Data points represent the mean ± SD (n = 3).

 
Competitive Inhibition Studies.
To investigate whether the carrier involved in the transport of L-Phe across rabbit cornea mimics the substrate specificity found in its uptake across SIRC cells, we performed inhibition studies using other amino acids and metabolic inhibitors. As depicted in Figure 6 , the transport of [3H]-L-Phe across rabbit cornea was inhibited markedly in the presence of L-Phe and L-Tyr, indicating the utilization of the same transport systems by the two aromatic amino acids. Basic (L-arginine) and acidic (L-glutamate) amino acids did not significantly alter the transport of [3H]-L-Phe, confirming our results obtained in uptake studies.



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  		FIGURE 6. Transport of [3H]-L-Phe (1 µCi/mL) across freshly excised rabbit cornea in the presence of different amino acids (1 mM), glycyl-sarcosine (1 mM), and ouabain (500 µM). L-Isomers of large neutral amino acids Phe and Tyr inhibited transport significantly, whereas small neutral amino acids, charged amino acids, dipeptides, and ouabain did not have any significant effect on transport. The substrate specificity and sodium independence suggest the involvement of a large neutral amino acid transport system on the rabbit cornea. Data are expressed as the mean ± SD (n = 3 to 6). *Significantly different from control (P ≤ 0.05).

 
Overlapping substrate specificities between large neutral amino acid transporter and peptide transporter have been reported, therefore the transport of L-Phe was determined in the presence of the dipeptide glycyl-sarcosine. The dipeptide did not cause any significant inhibition of L-Phe transport (Fig. 6) indicating that the peptide transporter is probably not involved in the transport of Phe across rabbit cornea.

Energy Dependence.
Uptake of L-Phe into SIRC cells was found to be energy and Na+ independent. Similarly, transport of L-Phe across rabbit cornea was found to be energy and Na+ independent, because no significant inhibition was seen in the presence of 500 µM ouabain (Fig. 6) .

RT-PCR and Sequencing
PCR products were analyzed by gel electrophoresis on 0.8% agarose. cDNA generated from total RNA isolated from rabbit corneal epithelium and SIRC cells was PCR amplified with the primers specific for rat LAT1 sequence (primers designed based on multiple sequence alignment between rat, mouse and human homologues of LAT1). A 440-bp product was obtained (Fig. 7) in both corneal epithelium and SIRC cells. The 440-bp fragment from rabbit corneal epithelial cDNA was subcloned and sequenced in both directions. The sequence obtained showed maximum homology to LAT1 sequence using the BLAST search program (NCBI) confirming the presence of LAT1 transporter on rabbit corneal epithelium and SIRC cells. A major ~520-bp band was obtained when PCR was performed with primers specific for human LAT1 sequence and cDNA generated from total RNA isolated from human cornea (Fig. 7) . This DNA fragment was directly subcloned into pGEM-T vector for amplification and sequenced. The sequence obtained from both the directions showed 100% homology to human LAT1 sequence when searched using the BLAST program, confirming the presence of LAT1 on human cornea. Thus, LAT1 was found to be present on both rabbit and human corneas.



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  		FIGURE 7. LAT1 cDNA was generated by reverse transcription-PCR amplification of total RNA from rabbit corneal epithelium (lane 2), SIRC (lane 3), and human cornea (lane 5). Aliquots of PCR products were analyzed by gel electrophoresis on 0.8% agarose. Ethidium bromide staining of the gel showed a major ~440-bp band corresponding to the amplified rabbit-LAT1 cDNA (lanes 2, 3) and ~520-bp band corresponding to the amplified human-LAT1 cDNA (lane 5). Lane 1: 1-kbp DNA ladder. The PCR products were confirmed to be LAT1 by subcloning and sequencing.

 

   Discussion
Top
 Abstract
 Methods
 Results
 Discussion
References
 
Amino acid transport across animal cell membranes is a highly complex process, primarily because of the existence of multiple transport systems with overlapping substrate specificities.61 System L is a major Na+-independent system for the transport of large neutral amino acids, including several essential amino acids. Imino acids, though zwitterionic in nature, are excluded by the system. LAT1 is reported in kidney, spleen, thymus, liver, small intestine, placenta, testis, brain, heart, lung, blood–brain barrier, and leukocytes.26 The presence of this transport system on the corneal epithelium has not been reported previously. Current knowledge of drug permeation across the cornea suggests passive diffusion to be the primary mechanism. Results from this study appear to indicate the presence of an amino acid transporter for Phe and other large neutral amino acids on the rabbit corneal epithelium.

Uptake of L-Phe by the SIRC cells was concentration dependent, with a Km of 73 µM and showed the involvement of a single carrier in the process (Fig. 1) . Transport of L-Phe across rabbit cornea was also found to be saturable with a Km of 33 µM. Neither the uptake into SIRC cells, nor the transport across rabbit cornea, was altered in the presence of any metabolic inhibitors, or in the presence of sodium-free buffer (Figs. 3 6) . These results suggest that large neutral amino acids cross the corneal epithelial cells through an energy- and sodium-independent facilitative transport system.

To delineate the structural requirements of this carrier and also to identify its substrate specificity, the effects of selected amino acids on [3H]-L-Phe uptake and transport were investigated. The uptake was inhibited significantly by large neutral amino acids such L-Phe, L-Tyr, L-Leu, and L-Ile and also by L-dopa (Fig. 2) . Inhibition of transport of [3H]-L-Phe was also seen in the presence of L-Phe and L-Tyr (Fig. 6) . A negligible effect of Ala and glycine on uptake of [3H]-L-Phe most likely indicates that this amino acid carrier system is specific for bulky amino acids (a suggestion supported by the effect of Leu). That it is a high-affinity transporter with a low Km (~15–50 µM)34 43 and neither anionic nor cationic amino acids affected Phe transport, further supports the hypothesis that the carrier system on the corneal epithelial cells belongs to a class of large neutral amino acid transporters. Among the amino acid transport systems, the A system is sodium-dependent and inhibited by NMAIB, the L system is sodium independent and inhibited by BCH, and the ASC system is sodium dependent but not inhibited by NMAIB.62 63 Based on the results presented in this report and the fact that the uptake of [3H]-L-Phe was inhibited almost completely by BCH but was nonresponsive to the absence of sodium and the presence of NMAIB (Fig. 2) , it appears that the primary carrier involved in Phe uptake and transport across corneal epithelium is the L system.

The functional characteristics of LAT1 cloned from rat and human tissues have been studied in detail.45 64 A detailed comparison of characteristics of LAT1 with those of LAT2 reveals important differences in substrate selectivity and affinity. LAT1 interacts preferentially with large neutral amino acids, whereas its affinity for short chain neutral amino acids is significantly lower. In contrast, LAT2 has broader substrate selectivity and interacts with short-chain as well as large neutral amino acids with comparable affinity.43 Competitive inhibition data from our studies suggest the presence of the LAT1 system on the corneal epithelium.

In terms of substrate affinity LAT1 is a high-affinity transporter with low Km for transportable substrates. In contrast, LAT2 is a relatively low-affinity transporter with Km for transportable substrates being several times higher than that observed in case of LAT1. Our Kms for Phe obtained (73 µM for uptake across SIRC and 33 µM for transport across rabbit cornea) lend further support to the fact that Phe utilizes the LAT1 system for its transport across the cornea.

We analyzed the pH dependence of the carrier involved and also its stereospecificity to confirm the involvement of LAT1. A unique biochemical property of LAT1 is its ability to interact with D-amino acids.45 D-Isomers of Leu, Phe, and Met are recognized as substrates by LAT1, whereas D-isomers of Val, His, Tyr, and Trp are not. In our uptake studies, D-isomers of Phe and Leu inhibited uptake of [3H] L-Phe to a large extent, whereas D-isomers of Val, Tyr, and Trp did not cause any significant inhibition (Fig. 2) suggesting the stereoselectivity specific to LAT1. Moreover, previous reports have shown that LAT1 activity is not influenced by pH in the range of 5.5 to 8.5, whereas transport activity of LAT2 is much higher at pH 6.5 than at pH 8.5.43 64 Studies using SIRC showed that the transport activity of the carrier involved is not affected within the pH range of 5.5 to 8.5 confirming the presence of LAT1 on the cornea.

Overlapping substrate specificity between the large neutral amino acid and the peptide transporter has been reported. Thus, we investigated whether the same holds true in the case of transport of L-Phe across rabbit cornea. However, no significant inhibition of L-Phe transport was seen in the presence of a known peptide substrate glycyl-sarcosine (Fig. 6) , indicating a high degree of substrate selectivity of the transporter for amino acids and minimal or no affinity for dipeptides.

Finally, the RT-PCR results confirmed the presence of LAT1 on SIRC, rabbit corneal epithelium, and human cornea. LAT1 from rat, mouse, Xenopus, and human tissues has previously been identified. LAT1 from rabbit DNA has not been cloned and sequenced. Thus, to identify the presence of LAT1 on rabbit corneal epithelium by PCR, we designed primers based on multiple sequence alignment between rat, mouse and human homologues of LAT1. A ~ 440-bp product was obtained, which when cloned and sequenced, showed maximum sequence homology to LAT1. This article reports for the first time the identification of LAT1 in a rabbit tissue. Tissue distribution of hLAT1 has been determined by Northern blot analysis. However, samples did not include any of the ocular tissues. We therefore performed RT-PCR on the RNA extracted from human cornea. A major ~520-bp band was obtained that was confirmed to be hLAT1 by subcloning and sequencing.

Results of studies aimed at delineating mechanisms of substrate recognition by LAT1 have shown that an {alpha}-amino group and a free carboxyl group at the C-terminal are required for affinity toward the transporter.65 In intestinal transport studies the amino acid transport system has not been found to be a very versatile and robust target compared to the peptide transporter. However the expression and tissue distribution of amino acid and peptide transport systems vary considerably. Thus, the rich amino acid transport systems located on the corneal epithelium may be targeted to significantly improve ocular drug absorption.66

In conclusion, this study demonstrates functional evidence of a high-affinity, Na+-independent Phe carrier system with characteristics similar to the LAT1 carrier on the SIRC cell line and on rabbit cornea. Biochemical evidence for the presence of this carrier has also been found on human cornea, SIRC cells, and rabbit corneal epithelium. The trends of inhibition observed in SIRC cells were consistent with the rabbit corneal studies. The SIRC cell line can thus be used as a valuable tool for in vitro screening to determine affinities of amino-acid–based drugs and prodrugs for LAT1. In the future, cloning and expression of LAT1 from rabbit cDNA will help us gain valuable insight into the characteristics of this corneal transporter and its species differentiation. Because corneal epithelium is the primary barrier to the absorption of drugs after topical administration, the presence of these transporters on the corneal epithelium may provide new opportunities for the design of transporter-targeted prodrugs with enhanced corneal permeability.


   Acknowledgements
 
The authors thank Alcon Laboratories, (Fort Worth, Texas), for their generous gift of human corneal RNA and Vadivel Ganapathy, Medical College of Georgia, Augusta, for guidance, training, and assistance with RT-PCR experiments.


   Footnotes
 
Supported by National Eye Institute Grants R01 EY09171-08 and R01 EY10659-07.

Submitted for publication September 5, 2002; revised January 31, 2003; accepted February 20, 2003.

Disclosure: B. Jain-Vakkalagadda, Alcon Laboratories (F); S. Dey, Alcon Laboratories (F); D. Pal, None; A.K. Mitra, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Ashim K. Mitra, Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, 5005 Rockhill Road, Kansas City, MO 64110-2499; mitraa{at}umkc.edu.


   References
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 Abstract
 Methods
 Results
 Discussion
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