Functional Characterization of the Solute Carrier LAT-1 (SLC7A5/SLC3A2) in Human Brain Capillary Endothelial Cells with Rapid UPLC-MS/MS Quantification of Intracellular Isotopically Labelled L-Leucine
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, whic...
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2022-03-01
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| author | Cindy Bay Gzona Bajraktari-Sylejmani Walter E. Haefeli Jürgen Burhenne Johanna Weiss Max Sauter |
| author_facet | Cindy Bay Gzona Bajraktari-Sylejmani Walter E. Haefeli Jürgen Burhenne Johanna Weiss Max Sauter |
| author_sort | Cindy Bay |
| collection | DOAJ |
| description | 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 ([<sup>13</sup>C<sub>6</sub>, <sup>15</sup>N]-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 IC<sub>50</sub> 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 [<sup>13</sup>C<sub>6</sub>, <sup>15</sup>N]-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 IC<sub>50</sub> values of the inhibitors were in concordance with previously reported values. Furthermore, the [<sup>13</sup>C<sub>6</sub>, <sup>15</sup>N]-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 ([<sup>13</sup>C<sub>6</sub>, <sup>15</sup>N]-L-leucine is a feasible strategy for the functional characterization of LAT-1 activity in cells or tissue. |
| first_indexed | 2024-03-09T11:47:45Z |
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| spelling | doaj.art-a44f0b369a8e41ba9b21cec062ef77352023-11-30T23:19:51ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672022-03-01237363710.3390/ijms23073637Functional Characterization of the Solute Carrier LAT-1 (SLC7A5/SLC3A2) in Human Brain Capillary Endothelial Cells with Rapid UPLC-MS/MS Quantification of Intracellular Isotopically Labelled L-LeucineCindy Bay0Gzona Bajraktari-Sylejmani1Walter E. Haefeli2Jürgen Burhenne3Johanna Weiss4Max Sauter5Department of Clinical Pharmacology and Pharmacoepidemiology, Heidelberg University Hospital, Im Neuenheimer Feld 410, 69120 Heidelberg, GermanyDepartment of Clinical Pharmacology and Pharmacoepidemiology, Heidelberg University Hospital, Im Neuenheimer Feld 410, 69120 Heidelberg, GermanyDepartment of Clinical Pharmacology and Pharmacoepidemiology, Heidelberg University Hospital, Im Neuenheimer Feld 410, 69120 Heidelberg, GermanyDepartment of Clinical Pharmacology and Pharmacoepidemiology, Heidelberg University Hospital, Im Neuenheimer Feld 410, 69120 Heidelberg, GermanyDepartment of Clinical Pharmacology and Pharmacoepidemiology, Heidelberg University Hospital, Im Neuenheimer Feld 410, 69120 Heidelberg, GermanyDepartment of Clinical Pharmacology and Pharmacoepidemiology, Heidelberg University Hospital, Im Neuenheimer Feld 410, 69120 Heidelberg, GermanyThe 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 ([<sup>13</sup>C<sub>6</sub>, <sup>15</sup>N]-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 IC<sub>50</sub> 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 [<sup>13</sup>C<sub>6</sub>, <sup>15</sup>N]-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 IC<sub>50</sub> values of the inhibitors were in concordance with previously reported values. Furthermore, the [<sup>13</sup>C<sub>6</sub>, <sup>15</sup>N]-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 ([<sup>13</sup>C<sub>6</sub>, <sup>15</sup>N]-L-leucine is a feasible strategy for the functional characterization of LAT-1 activity in cells or tissue.https://www.mdpi.com/1422-0067/23/7/3637LAT-1amino acid transportCNSblood–brain barriertandem mass spectrometryUPLC |
| spellingShingle | Cindy Bay Gzona Bajraktari-Sylejmani Walter E. Haefeli Jürgen Burhenne Johanna Weiss Max Sauter Functional Characterization of the Solute Carrier LAT-1 (SLC7A5/SLC3A2) in Human Brain Capillary Endothelial Cells with Rapid UPLC-MS/MS Quantification of Intracellular Isotopically Labelled L-Leucine International Journal of Molecular Sciences LAT-1 amino acid transport CNS blood–brain barrier tandem mass spectrometry UPLC |
| title | Functional Characterization of the Solute Carrier LAT-1 (SLC7A5/SLC3A2) in Human Brain Capillary Endothelial Cells with Rapid UPLC-MS/MS Quantification of Intracellular Isotopically Labelled L-Leucine |
| title_full | Functional Characterization of the Solute Carrier LAT-1 (SLC7A5/SLC3A2) in Human Brain Capillary Endothelial Cells with Rapid UPLC-MS/MS Quantification of Intracellular Isotopically Labelled L-Leucine |
| title_fullStr | Functional Characterization of the Solute Carrier LAT-1 (SLC7A5/SLC3A2) in Human Brain Capillary Endothelial Cells with Rapid UPLC-MS/MS Quantification of Intracellular Isotopically Labelled L-Leucine |
| title_full_unstemmed | Functional Characterization of the Solute Carrier LAT-1 (SLC7A5/SLC3A2) in Human Brain Capillary Endothelial Cells with Rapid UPLC-MS/MS Quantification of Intracellular Isotopically Labelled L-Leucine |
| title_short | Functional Characterization of the Solute Carrier LAT-1 (SLC7A5/SLC3A2) in Human Brain Capillary Endothelial Cells with Rapid UPLC-MS/MS Quantification of Intracellular Isotopically Labelled L-Leucine |
| title_sort | functional characterization of the solute carrier lat 1 slc7a5 slc3a2 in human brain capillary endothelial cells with rapid uplc ms ms quantification of intracellular isotopically labelled l leucine |
| topic | LAT-1 amino acid transport CNS blood–brain barrier tandem mass spectrometry UPLC |
| url | https://www.mdpi.com/1422-0067/23/7/3637 |
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