title: 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.
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.
authors.affiliation: Department of Pharmaceutics, Brain Barriers Research Center, University of Minnesota, Minneapolis, MN 55455, USA.
authors.forename: Sagar
authors.initials: S
authors.lastname: Agarwal
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Uchida
authors.forename: Rajendar K
authors.initials: RK
authors.lastname: Mittapalli
authors.forename: Ramola
authors.initials: R
authors.lastname: Sane
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Terasaki
authors.forename: William F
authors.initials: WF
authors.lastname: Elmquist
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis* / Q000172:deficiency / Q000235:genetics; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000096:biosynthesis* / Q000172:deficiency / Q000235:genetics; D000818:Animals; D001812:Blood-Brain Barrier / Q000166:cytology / Q000378:metabolism; D002469:Cell Separation / Q000379:methods; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism*; D005786:Gene Expression Regulation; D051379:Mice; D018345:Mice, Knockout; D040901:Proteomics / Q000379:methods*
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; C501412:Abcg2 protein, mouse