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

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.

Breast Cancer Resistance Protein and P-Glycoprotein Influence In Vivo Disposition of 11C-Erlotinib

“…We addressed these questions by performing small-animal PET/MR experiments with 11 C-erlotinib without and with coinjection of a pharmacologic dose of unlabeled erlotinib (10 mg/kg) in wild-type and Abcb1a/b or Abcg2 knockout mice. Previous work has shown that Abcb1a/b (2/2) , Abcg2 (2/2) , and Abcb1a/b (2/2) Abcg2 (2/2) mice lack compensatory alterations in expression levels of other ABC and SLC transporters and are useful tools to study the effects of ABCB1 and ABCG2 on drug disposition (19). A potential limitation of using mice is their small blood volume, which prohibits arterial blood sampling, which is needed for quantitative analysis of PET data.…”

Diet-Induced Obesity Suppresses Expression of Many Proteins at the Blood–Brain Barrier

“…22 Proteomics analysis has identified secretory factors associated with adipogenesis in human adipose stromal vascular fraction cells and differentiated adipocytes. 23 Although proteomic methods have been used to characterize drug transporters and receptors at the BBB, 11,[24][25][26] the changes of BBB proteomes after DIO have not been shown previously. The proteins identified in our study are involved in a variety of cellular functions, cytoskeleton stability, stress response, vesicular transport, and energy metabolism ( Table 3).…”

Bcrp1;Mdr1a/b;Mrp2 Combination Knockout Mice: Altered Disposition of the Dietary Carcinogen PhIP (2-Amino-1-Methyl-6-Phenylimidazo[4,5-b]Pyridine) and Its Genotoxic Metabolites

“…3D). Since Mrp2 and Mrp3 are unlikely to play a role in the blood-brain barrier of FVB mice, where they are not detectably expressed (Soontornmalai et al, 2006;Agarwal et al, 2012), this indicates that the combined deficiency of Bcrp1 and Mdr1a/1b results in higher brain accumulation of PhIP than the single deficiency of Bcrp1. Thus, not only Bcrp1 (Enokizono et al, 2008), but also Mdr1a/1b contributes to protection of the brain from PhIP, consistent with the observed modest in vitro transport of PhIP by Mdr1a (van Herwaarden et al, 2003).…”