Excitatory Amino Acid Transporter 1-Mediated L-Glutamate Transport at the Inner Blood–Retinal Barrier: Possible Role in L-Glutamate Elimination from the Retina

• Published in: Biological & pharmaceutical bulletin Vol. 38; no. 7; pp. 1087 - 1091
• Main Authors: Sakurai, Tatsuhiko, Akanuma, Shin-ichi, Usui, Takuya, Kubo, Yoshiyuki, Tachikawa, Masanori, Hosoya, Ken-ichi
• Format: Journal Article
• Language: English; Japanese
• Published: Japan The Pharmaceutical Society of Japan 01.07.2015; Pharmaceutical Society of Japan
• Subjects: Animals; Biological Transport; Cell Line; excitatory amino acid transporter 1; Excitatory Amino Acid Transporter 1 - genetics; Excitatory Amino Acid Transporter 1 - metabolism; Glutamic Acid - pharmacology; inner blood–retinal barrier; L-glutamate; Male; Rats; Rats, Wistar; Retina - metabolism
• ISSN: 0918-6158; 1347-5215
• DOI: 10.1248/bpb.b15-00226

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.