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Research ArticleArticle

Quantitative Proteomics of Transporter Expression in Brain Capillary Endothelial Cells Isolated from P-Glycoprotein (P-gp), Breast Cancer Resistance Protein (Bcrp), and P-gp/Bcrp Knockout Mice

Sagar Agarwal, Yasuo Uchida, Rajendar K. Mittapalli, Ramola Sane, Tetsuya Terasaki and William F. Elmquist
Drug Metabolism and Disposition June 2012, 40 (6) 1164-1169; DOI: https://doi.org/10.1124/dmd.112.044719
Sagar Agarwal
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Yasuo Uchida
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Rajendar K. Mittapalli
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Ramola Sane
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Tetsuya Terasaki
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William F. Elmquist
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Abstract

The objective of this study was to quantitatively examine the protein expression of relevant transporters and other proteins in the brain capillary endothelial cells isolated from wild-type mice and P-glycoprotein (P-gp), breast cancer resistance protein (Bcrp), and P-gp/Bcrp knockout mice. After the isolation of brain capillary endothelial cells, a highly sensitive liquid chromatography-tandem mass spectrometry method with multiple reaction monitoring was used to determine the quantitative expression of membrane transporters at the blood-brain barrier (BBB) of the various mouse genotypes. Quantitative expression of 29 protein molecules, including 12 ATP-binding cassette transporters, 10 solute carrier transporters, five receptors, and two housekeeping proteins, was examined by quantitative proteomics in the four mouse genotypes. There was no significant difference in the expression of P-gp between the wild-type and Bcrp1(−/−) mice. Likewise, Bcrp expression was not significantly different between the wild-type and Mdr1a/b(−/−) mice. There was no significant difference in the expression of any of the measured proteins in the brain capillary endothelial cells across the genotypes, except for the lack of expression of the corresponding protein in the mice that had a genetic deletion of P-gp or Bcrp. In conclusion, using a quantitative proteomic approach, we have shown that there are no changes in the expression of several relevant transporters in brain capillary endothelial cells isolated from single and combination knockout mice. These data suggest that the mechanism behind the functional compensation between P-gp and Bcrp at the BBB is not related to compensatory changes in transporter expression.

Footnotes

  • This work was supported by the National Institutes of Health National Cancer Institute [Grant CA138437] (W.F.E.); and a grant from the Children's Cancer Research Fund at the University of Minnesota (to W.F.E.). S.A. was supported by the Doctoral Dissertation Fellowship from the University of Minnesota. R.S. was supported by the Ronald J. Sawchuk Fellowship. This study was also supported in part by a Grant-in-Aid for Scientific Research (S) [KAKENHI: 18109002] from the Japan Society for the Promotion of Science and by grants from the Research Seeds Quest Program and the Development of Creative Technology Seeds Supporting Program for Creating University Ventures from the Japan Science and Technology Agency.

  • Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

    http://dx.doi.org/10.1124/dmd.112.044719.

  • ↵Embedded Image The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material.

  • ABBREVIATIONS:

    BBB
    blood-brain-barrier
    P-gp
    P-glycoprotein
    Bcrp
    breast cancer resistance protein
    Mdr1
    gene encoding the murine P-glycoprotein
    Bcrp1
    gene encoding the murine breast cancer resistance protein
    ABC
    ATP-binding cassette
    FVB
    Friend leukemia virus strain B
    LC-MS/MS
    liquid chromatography-tandem mass spectrometry
    MRP
    multidrug resistance-associated protein
    MRM
    multiple reaction monitoring
    SLC
    solute carrier
    PBS
    phosphate-buffered saline
    HPLC
    high-performance liquid chromatography.

  • Received January 24, 2012.
  • Accepted March 8, 2012.
  • Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition: 40 (6)
Drug Metabolism and Disposition
Vol. 40, Issue 6
1 Jun 2012
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Research ArticleArticle

BBB TRANSPORTER EXPRESSION IN KNOCKOUT MICE

Sagar Agarwal, Yasuo Uchida, Rajendar K. Mittapalli, Ramola Sane, Tetsuya Terasaki and William F. Elmquist
Drug Metabolism and Disposition June 1, 2012, 40 (6) 1164-1169; DOI: https://doi.org/10.1124/dmd.112.044719

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Research ArticleArticle

BBB TRANSPORTER EXPRESSION IN KNOCKOUT MICE

Sagar Agarwal, Yasuo Uchida, Rajendar K. Mittapalli, Ramola Sane, Tetsuya Terasaki and William F. Elmquist
Drug Metabolism and Disposition June 1, 2012, 40 (6) 1164-1169; DOI: https://doi.org/10.1124/dmd.112.044719
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