L-type amino acid transporter 1-mediated L-leucine transport at the inner blood-retinal barrier.
OBJECTIVE
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 microL/(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 microM. 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.
Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Toyama, Japan.
Masatoshi
M
Tomi
Masahiko
M
Mori
Masanori
M
Tachikawa
Kazunori
K
Katayama
Tetsuya
T
Terasaki
Ken-ichi
K
Hosoya
D000818:Animals; D001692:Biological Transport; D001813:Blood-Retinal Barrier / Q000502:physiology*; D018929:Cell Culture Techniques; D004730:Endothelium, Vascular / Q000378:metabolism*; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D006168:Guinea Pigs; D015151:Immunoblotting; D027283:Large Neutral Amino Acid-Transporter 1 / Q000502:physiology*; D007930:Leucine / Q000378:metabolism*; D008297:Male; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D012171:Retinal Vessels / Q000166:cytology; D020133:Reverse Transcriptase Polymerase Chain Reaction
D027283:Large Neutral Amino Acid-Transporter 1; D012333:RNA, Messenger; D007930:Leucine