Functional characterization of the aolute carrier LAT-1 (SLC7A5/SLC2A3) in human brain capillary endothelial cells with rapid UPLC-MS/MS quantification of intracellular isotopically labelled L-Leucine

• Verfasst von: Bay, Cindy [VerfasserIn]
• Verfasst von: Bajraktari-Sylejmani, Gzona [VerfasserIn]
• Verfasst von: Haefeli, Walter E. [VerfasserIn]
• Verfasst von: Burhenne, Jürgen [VerfasserIn]
• Verfasst von: Weiß, Johanna [VerfasserIn]
• Verfasst von: Sauter, Max [VerfasserIn]
• Titel: Functional characterization of the aolute carrier LAT-1 (SLC7A5/SLC2A3) in human brain capillary endothelial cells with rapid UPLC-MS/MS quantification of intracellular isotopically labelled L-Leucine
• Verf.angabe: Cindy Bay, Gzona Bajraktari-Sylejmani, Walter E. Haefeli, Jürgen Burhenne, Johanna Weiss and Max Sauter
• E-Jahr: 2022
• Jahr: 26 March 2022
• Umfang: 18 S.
• Fussnoten: Gesehen am 06.04.2022
• Titel Quelle: Enthalten in: International journal of molecular sciences
• Ort Quelle: Basel : Molecular Diversity Preservation International, 2000
• Jahr Quelle: 2022
• Band/Heft Quelle: 23(2022), 7, Artikel-ID 3637, Seite 1-18
• ISSN Quelle: 1422-0067
• ISSN Quelle: 1661-6596
• DOI: doi:10.3390/ijms23073637
• Datenträger: Online-Ressource
• Sprache: eng
• Bibliogr. Hinweis: Errata: Bay, Cindy: Correction
• Sach-SW: amino acid transport
• Sach-SW: blood-brain barrier
• Sach-SW: CNS
• Sach-SW: L-leucine
• Sach-SW: LAT-1
• Sach-SW: tandem mass spectrometry
• Sach-SW: UPLC
• K10plus-PPN: 1797784625
• K10plus-PPN:
  Universitätsbibliothek
• Lokale URL UB: Zum Volltext

Abstract

The solute carrier L-type amino acid transporter 1 (LAT-1/SLC7A5) is a viable target for drug delivery to the central nervous system (CNS) and tumors due to its high abundance at the blood-brain barrier and in tumor tissue. LAT-1 is only localized on the cell surface as a heterodimer with CD98, which is not required for transporter function. To support future CNS drug-delivery development based on LAT-1 targeting, we established an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) assay for stable isotopically labeled leucine ([13C6, 15N]-L-leucine), with a dynamic range of 0.1-1000 ng/mL that can be applied for the functional testing of LAT-1 activity when combined with specific inhibitors and, consequently, the LAT-1 inhibition capacity of new compounds. The assay was established in a 96-well format, facilitating high-throughput experiments, and, hence, can support the screening for novel inhibitors. Applicable recommendations of the US Food and Drug Administration and European Medicines Agency for bioanalytical method validation were followed to validate the assay. The assay was applied to investigate the IC50 of two well-known LAT-1 inhibitors on hCMEC/D3 cells: the highly specific LAT-1 inhibitor JPH203, which was also used to demonstrate LAT-1 specific uptake, and the general system L inhibitor BCH. In addition, the [13C6, 15N]-L-leucine uptake was determined on two human brain capillary endothelial cell lines (NKIM-6 and hCMEC/D3), which were characterized for their expressional differences of LAT-1 at the protein and mRNA level and the surface amount of CD98. The IC50 values of the inhibitors were in concordance with previously reported values. Furthermore, the [13C6, 15N]-L-leucine uptake was significantly higher in hCMEC/D3 cells compared to NKIM-6 cells, which correlated with higher expression of LAT-1 and a higher surface amount of CD98. Therefore, the UPLC-MS/MS quantification of ([13C6, 15N]-L-leucine is a feasible strategy for the functional characterization of LAT-1 activity in cells or tissue.