Excitatory amino acid transporter 1-mediated L-glutamate transport at the inner blood-retinal Barrier: Possible role in L-glutamate elimination from the retina

Tatsuhiko Sakurai, Shin Ichi Akanuma, Takuya Usui, Yoshiyuki Kubo, Masanori Tachikawa, Ken Ichi Hosoya

Research output: Contribution to journalArticlepeer-review

5 Citations (Scopus)

Abstract

The purpose of this study was to elucidate the transport mechanism(s) of L-glutamate (L-Glu), a neuroexcitatory neurotransmitter, in the inner blood-retinal barrier (BRB). The L-Glu transport was evaluated by an in vitro uptake study with a conditionally-immortalized rat retinal capillary endothelial cell line, TR-iBRB2 cells. L-Glu uptake by TR-iBRB2 exhibited time- and concentration-dependence, and was composed of high- and low-affinity processes with Michaelis-Menten constants (Km) of 19.3 μM and 275 μM, respectively. Under Na+-free conditions, L-Glu uptake by TR-iBRB2 involved one-saturable kinetics with a Km of 190 μM, which is similar to that of the low-affinity process of L-Glu uptake under normal conditions. Moreover, substrates/inhibitors of system Xc-, which is involved in blood-to-retina transport of compounds across the inner BRB, strongly inhibited the L-Glu uptake under Na+-free conditions, suggesting that Na+-independent low-affinity L-Glu transport at the inner BRB is carried out by system Xc-. Regarding the Na+-dependent high affinity process of L-Glu transport at the inner BRB, L-Glu uptake by TR-iBRB2 under normal conditions was significantly inhibited by substrates/inhibitors of excitatory amino acid transporter (EAAT) 1-5, but not alanine-serine-cysteine transporters. Reverse-transcription polymerase chain reaction (RT-PCR) analysis and immunoblot analysis demonstrated that mRNA and protein of EAAT1 are expressed in TR-iBRB2 cells, whereas mRNAs and/or proteins of EAAT2-5 are not. Immunohistochemical analysis revealed that EAAT1 protein is localized on the abluminal membrane of the retinal capillaries. In conclusion, EAAT1 most likely mediates Na+-dependent high-affinity L-Glu transport at the inner BRB and appears to take part in L-Glu elimination from the retina across the inner BRB.

Original languageEnglish
Pages (from-to)1087-1091
Number of pages5
JournalBiological and Pharmaceutical Bulletin
Volume38
Issue number7
DOIs
Publication statusPublished - 2015 Jul 1
Externally publishedYes

Keywords

  • Excitatory amino acid transporter 1
  • Inner blood-retinal barrier
  • L-glutamate

ASJC Scopus subject areas

  • Pharmacology
  • Pharmaceutical Science

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