title: Excitatory Amino Acid Transporter 1-Mediated L-Glutamate Transport at the Inner Blood-Retinal Barrier: Possible Role in L-Glutamate Elimination from the Retina.
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.
authors.affiliation: Department of Pharmaceutics, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama.
authors.forename: Tatsuhiko
authors.initials: T
authors.lastname: Sakurai
authors.forename: Shin-ichi
authors.initials: S
authors.lastname: Akanuma
authors.forename: Takuya
authors.initials: T
authors.lastname: Usui
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Kubo
authors.forename: Masanori
authors.initials: M
authors.lastname: Tachikawa
authors.forename: Ken-ichi
authors.initials: K
authors.lastname: Hosoya
mesh_terms: D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D027341:Excitatory Amino Acid Transporter 1 / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000494:pharmacology*; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D012160:Retina / Q000378:metabolism*
chemical_list: D027341:Excitatory Amino Acid Transporter 1; D018698:Glutamic Acid