Transport of Pregabalin Via L-Type Amino Acid Transporter 1 (SLC7A5) in Human Brain Capillary Endothelial Cell Line

Yu Takahashi, Tomohiro Nishimura, Kei Higuchi, Saki Noguchi, Yuma Tega, Toshiki Kurosawa, Yoshiharu Deguchi, Masatoshi Tomi

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

PURPOSE: The anti-epileptic drug pregabalin crosses the blood-brain barrier (BBB) in spite of its low lipophilicity. This study was performed to determine whether L-type amino acid transporters (LAT1/SLC7A5 and LAT2/SLC7A8) contribute to the uptake of pregabalin.

METHODS: Pregabalin uptake by LATs-transfected HEK293 cells or hCMEC/D3 cells, an in vitro human BBB model, was measured by LC-MS/MS analysis. Expression of LAT1 mRNA in hCMEC/D3 cells was determined by quantitative RT-PCR analysis.

RESULTS: Overexpression of LAT1, but not LAT2, in HEK293 cells significantly increased the cellular uptake of pregabalin, and the LAT1-mediated uptake was saturable with a Km of 0.288 mM. LAT1-mediated amino acid uptake was inhibited specifically and almost completely in the presence of 1 mM pregabalin. The uptake of pregabalin by hCMEC/D3 cells was sodium-independent, saturable (Km = 0.854 mM), and strongly inhibited by large amino acids at 1 mM, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid, a specific system L inhibitor, at 1 mM and by JPH203, a LAT1-selective inhibitor, at 10 μM. Pregabalin uptake in hCMEC/D3 cells was also decreased by 75% by the silencing of LAT1 gene using LAT1 siRNA.

CONCLUSIONS: Our results indicate that LAT1, but not LAT2, recognizes pregabalin as a substrate. It is suggested that LAT1 mediates pregabalin transport at the BBB.

Original languageEnglish
Number of pages1
JournalPharmaceutical Research
Volume35
Issue number12
DOIs
Publication statusPublished - 2018 Oct 29

Fingerprint

Large Neutral Amino Acid-Transporter 1
Amino Acid Transport Systems
Endothelial cells
Brain
Endothelial Cells
Cell Line
Blood-Brain Barrier
HEK293 Cells
Pregabalin
Amino Acids
Gene Silencing
Small Interfering RNA

Keywords

  • anti-epileptic drug
  • blood-brain barrier
  • L-type amino acid transporter
  • pregabalin

ASJC Scopus subject areas

  • Biotechnology
  • Molecular Medicine
  • Pharmacology
  • Pharmaceutical Science
  • Organic Chemistry
  • Pharmacology (medical)

Cite this

Transport of Pregabalin Via L-Type Amino Acid Transporter 1 (SLC7A5) in Human Brain Capillary Endothelial Cell Line. / Takahashi, Yu; Nishimura, Tomohiro; Higuchi, Kei; Noguchi, Saki; Tega, Yuma; Kurosawa, Toshiki; Deguchi, Yoshiharu; Tomi, Masatoshi.

In: Pharmaceutical Research, Vol. 35, No. 12, 29.10.2018.

Research output: Contribution to journalArticle

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abstract = "PURPOSE: The anti-epileptic drug pregabalin crosses the blood-brain barrier (BBB) in spite of its low lipophilicity. This study was performed to determine whether L-type amino acid transporters (LAT1/SLC7A5 and LAT2/SLC7A8) contribute to the uptake of pregabalin.METHODS: Pregabalin uptake by LATs-transfected HEK293 cells or hCMEC/D3 cells, an in vitro human BBB model, was measured by LC-MS/MS analysis. Expression of LAT1 mRNA in hCMEC/D3 cells was determined by quantitative RT-PCR analysis.RESULTS: Overexpression of LAT1, but not LAT2, in HEK293 cells significantly increased the cellular uptake of pregabalin, and the LAT1-mediated uptake was saturable with a Km of 0.288 mM. LAT1-mediated amino acid uptake was inhibited specifically and almost completely in the presence of 1 mM pregabalin. The uptake of pregabalin by hCMEC/D3 cells was sodium-independent, saturable (Km = 0.854 mM), and strongly inhibited by large amino acids at 1 mM, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid, a specific system L inhibitor, at 1 mM and by JPH203, a LAT1-selective inhibitor, at 10 μM. Pregabalin uptake in hCMEC/D3 cells was also decreased by 75{\%} by the silencing of LAT1 gene using LAT1 siRNA.CONCLUSIONS: Our results indicate that LAT1, but not LAT2, recognizes pregabalin as a substrate. It is suggested that LAT1 mediates pregabalin transport at the BBB.",
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T1 - Transport of Pregabalin Via L-Type Amino Acid Transporter 1 (SLC7A5) in Human Brain Capillary Endothelial Cell Line

AU - Takahashi, Yu

AU - Nishimura, Tomohiro

AU - Higuchi, Kei

AU - Noguchi, Saki

AU - Tega, Yuma

AU - Kurosawa, Toshiki

AU - Deguchi, Yoshiharu

AU - Tomi, Masatoshi

PY - 2018/10/29

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N2 - PURPOSE: The anti-epileptic drug pregabalin crosses the blood-brain barrier (BBB) in spite of its low lipophilicity. This study was performed to determine whether L-type amino acid transporters (LAT1/SLC7A5 and LAT2/SLC7A8) contribute to the uptake of pregabalin.METHODS: Pregabalin uptake by LATs-transfected HEK293 cells or hCMEC/D3 cells, an in vitro human BBB model, was measured by LC-MS/MS analysis. Expression of LAT1 mRNA in hCMEC/D3 cells was determined by quantitative RT-PCR analysis.RESULTS: Overexpression of LAT1, but not LAT2, in HEK293 cells significantly increased the cellular uptake of pregabalin, and the LAT1-mediated uptake was saturable with a Km of 0.288 mM. LAT1-mediated amino acid uptake was inhibited specifically and almost completely in the presence of 1 mM pregabalin. The uptake of pregabalin by hCMEC/D3 cells was sodium-independent, saturable (Km = 0.854 mM), and strongly inhibited by large amino acids at 1 mM, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid, a specific system L inhibitor, at 1 mM and by JPH203, a LAT1-selective inhibitor, at 10 μM. Pregabalin uptake in hCMEC/D3 cells was also decreased by 75% by the silencing of LAT1 gene using LAT1 siRNA.CONCLUSIONS: Our results indicate that LAT1, but not LAT2, recognizes pregabalin as a substrate. It is suggested that LAT1 mediates pregabalin transport at the BBB.

AB - PURPOSE: The anti-epileptic drug pregabalin crosses the blood-brain barrier (BBB) in spite of its low lipophilicity. This study was performed to determine whether L-type amino acid transporters (LAT1/SLC7A5 and LAT2/SLC7A8) contribute to the uptake of pregabalin.METHODS: Pregabalin uptake by LATs-transfected HEK293 cells or hCMEC/D3 cells, an in vitro human BBB model, was measured by LC-MS/MS analysis. Expression of LAT1 mRNA in hCMEC/D3 cells was determined by quantitative RT-PCR analysis.RESULTS: Overexpression of LAT1, but not LAT2, in HEK293 cells significantly increased the cellular uptake of pregabalin, and the LAT1-mediated uptake was saturable with a Km of 0.288 mM. LAT1-mediated amino acid uptake was inhibited specifically and almost completely in the presence of 1 mM pregabalin. The uptake of pregabalin by hCMEC/D3 cells was sodium-independent, saturable (Km = 0.854 mM), and strongly inhibited by large amino acids at 1 mM, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid, a specific system L inhibitor, at 1 mM and by JPH203, a LAT1-selective inhibitor, at 10 μM. Pregabalin uptake in hCMEC/D3 cells was also decreased by 75% by the silencing of LAT1 gene using LAT1 siRNA.CONCLUSIONS: Our results indicate that LAT1, but not LAT2, recognizes pregabalin as a substrate. It is suggested that LAT1 mediates pregabalin transport at the BBB.

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