Abstract
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 K m of 73 ± 9 μM, V max of 2.0 ± 0.1 nanomoles/min per milligram protein, and K d 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 (K m = 33 ± 8 μM 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.
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, b
0,+, B
0,+ 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.
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.
Concentration Dependence.
Competitive Inhibition Studies.
Energy Dependence.
Competitive Inhibition Studies.
Energy Dependence.
Concentration Dependence.
Substrate Specificity.
Competitive Inhibition Studies.
Energy Dependence.
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
K m 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
K m 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
K m (∼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 K m for transportable substrates. In contrast, LAT2 is a relatively low-affinity transporter with K m for transportable substrates being several times higher than that observed in case of LAT1. Our K ms 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 α-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.
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;
[email protected].
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.
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