L-type amino acid transporter 1-mediated L-leucine transport at the inner blood-retinal barrier

Masatoshi Tomi, Masahiko Mori, Masanori Tachikawa, Kazunori Katayama, Tetsuya Terasaki, Ken Ichi Hosoya

Research output: Contribution to journalArticle

67 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)2522-2530
Number of pages9
JournalInvestigative Ophthalmology and Visual Science
Volume46
Issue number7
DOIs
Publication statusPublished - 2005
Externally publishedYes

Fingerprint

Blood-Retinal Barrier
Amino Acid Transport Systems
Leucine
Retina
Endothelial Cells
Large Neutral Amino Acid-Transporter 1
Neurotransmitter Agents
Neutral Amino Acids
Retinal Vessels
Branched Chain Amino Acids
Aromatic Amino Acids
Glutamine
Phenylalanine
Alanine
Methionine
Real-Time Polymerase Chain Reaction
Permeability
Cell Line
Messenger RNA

ASJC Scopus subject areas

  • Ophthalmology

Cite this

L-type amino acid transporter 1-mediated L-leucine transport at the inner blood-retinal barrier. / Tomi, Masatoshi; Mori, Masahiko; Tachikawa, Masanori; Katayama, Kazunori; Terasaki, Tetsuya; Hosoya, Ken Ichi.

In: Investigative Ophthalmology and Visual Science, Vol. 46, No. 7, 2005, p. 2522-2530.

Research output: Contribution to journalArticle

Tomi, Masatoshi ; Mori, Masahiko ; Tachikawa, Masanori ; Katayama, Kazunori ; Terasaki, Tetsuya ; Hosoya, Ken Ichi. / L-type amino acid transporter 1-mediated L-leucine transport at the inner blood-retinal barrier. In: Investigative Ophthalmology and Visual Science. 2005 ; Vol. 46, No. 7. pp. 2522-2530.
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T1 - L-type amino acid transporter 1-mediated L-leucine transport at the inner blood-retinal barrier

AU - Tomi, Masatoshi

AU - Mori, Masahiko

AU - Tachikawa, Masanori

AU - Katayama, Kazunori

AU - Terasaki, Tetsuya

AU - Hosoya, Ken Ichi

PY - 2005

Y1 - 2005

N2 - 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.

AB - 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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