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(Investigative Ophthalmology and Visual Science. 2005;46:2522-2530.)
© 2005 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.04-1175
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L-Type Amino Acid Transporter 1–Mediated L-Leucine Transport at the Inner Blood–Retinal Barrier

Masatoshi Tomi,1 Masahiko Mori,1 Masanori Tachikawa,2,3 Kazunori Katayama,1 Tetsuya Terasaki,2,4,5 and Ken-ichi Hosoya1

1From the Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Toyama, Japan; the 2Department of Molecular Biopharmacy and Genetics, Graduate School of Pharmaceutical Sciences, and the 4New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan; and 5Solution Oriented Research for Science and Technology of the Japan Science and Technology Agency (JST), Kawaguchi, Japan.

PURPOSE. L-type amino acid transporters (LATs) prefer branched-chain and aromatic amino acids, including neurotransmitter precursors. The objective of this study was to clarify the expression and function of LAT at the inner blood–retinal barrier (BRB).

METHODS. [3H]L-Leucine transport at the inner BRB was characterized by using in vivo integration plot analysis and a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2). The expression of the LAT1 was demonstrated by quantitative real-time RT-PCR, immunoblot, and immunohistochemical analyses.

RESULTS. The apparent influx permeability clearance of [3H]L-leucine in the rat retina was found to be 203 µL/(min · g retina), supporting a carrier-mediated influx transport of L-leucine at the BRB. [3H]L-Leucine uptake by TR-iBRB2 cells was an Na+-independent and concentration-dependent process with a Km of 14.1 µM. This process was more potently cis inhibited by substrates of LAT1, D-leucine, D-phenylalanine, and D-methionine, than those of LAT2, L-alanine, and L-glutamine. [3H]L-Leucine efflux from TR-iBRB2 cells was trans-stimulated by substrates of LAT1. The expression of LAT1 mRNA was 100- and 15-fold greater than that of LAT2 in TR-iBRB2 and magnetically isolated rat retinal vascular endothelial cells, respectively. The expression of LAT1 protein was observed in TR-iBRB2 and primary cultured human retinal endothelial cells and immunostaining of LAT1 was observed along the rat retinal capillaries.

CONCLUSIONS. LAT1 is expressed at the inner BRB and mediates blood-to-retina L-leucine transport. This transport system plays a key role in maintaining large neutral amino acids as well as neurotransmitters in the neural retina.






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