Elsevier

Neuroscience

Volume 155, Issue 2, 13 August 2008, Pages 423-438
Neuroscience

Molecular neuroscience
Subcellular localization of transporters along the rat blood–brain barrier and blood–cerebral-spinal fluid barrier by in vivo biotinylation

https://doi.org/10.1016/j.neuroscience.2008.06.015Get rights and content

Abstract

Nutrient transporters and ABC efflux pumps at the blood–brain barrier are major determinants of drug penetration into the brain. Immunohistochemical analysis of transporter subcellular localization is challenging due to the close apposition of the luminal and abluminal microvessel plasma membranes. We employed in vivo perfusion of biotinylation reagent through rat brain microvessels to domain-specifically label proteins exposed on the microvessel luminal surface. Using this approach, we analyzed the luminal/abluminal localization of a number of blood–brain barrier transporters identified by quantitative PCR profiling as being highly expressed and enriched in rat brain endothelial cells compared with whole brain. We also examined the apical/basal–lateral distribution of transporters in the choroid plexus, a secondary site for transport of nutrients between the blood and CNS. We detected P-glycoprotein (Pgp) (Abcb1), ATP-binding cassette (Abc) g2, multidrug resistance protein (Mrp) 4 (Abcc4), glucose transporter 1 (Glut1) (Slc2a1), Lat1 (Slc7a5), and monocarboxylate transporter-1 (Mct1) (Slc16a1) on the luminal surface of rat cerebral microvessels by both immunofluorescence staining and Western blotting of in vivo biotinylated proteins. Mrp1 (Abcc1) appeared primarily abluminal by immunofluorescence staining, and was barely detectable in the biotinylated protein fraction. Organic anion transporter (Oat) 3 (Slc22a8), organic anion transporter polypeptide (Oatp) 2b1 (Slco2b1, Oatpb), and Mrp5 (Abcc5) were not detected on the luminal surface using either method, while Oatp1a4 (Slco1a4, Oatp2) appeared to partially localize to the microvessel lumen by immunofluorescence staining, but was not detected in the biotinylated protein fraction by Western blotting. Lat1, Mrp1 and Mrp4 were detected on the basal–lateral surface of lateral ventricle choroid plexus epithelial cells. Mrp5, Oct3 and Oatp2b1 (Oatpb) were detected in the ependymal cells lining the ventricle. We did not detect Pgp expression in choroid plexus by immunofluorescence staining. In vivo biotinylation provides a method for domain-specific labeling of luminal surface proteins within the capillaries of the blood–brain barrier, allowing for biochemical analysis of protein localization and facilitating optical discrimination of the luminal and abluminal endothelial surfaces.

Section snippets

Rat brain perfusion

All protocols involving animals were approved by the XenoPort Animal Use Committee in accordance with the recommendations outlined in the NIH Guide for the Care and Use of Laboratory Animals. Experiments were designed to minimize the number of animals used and to prevent animal suffering. Eight-week-old male Sprague–Dawley rats were anesthetized by i.m. injection of a mixture of 66.6 mg/kg ketamine and 3.3 mg/kg xylazine. Room temperature solutions were perfused using a programmable syringe

In vivo biotinylation of luminal proteins in rat brain microvessels

To domain-specifically label proteins expressed on the luminal surface of BMECs, the brain vasculature was perfused in vivo with the biotinylation reagent sulfo-NHS-SS-biotin. Sulfo-NHS-SS-biotin reacts with primary amines (epsilon amino groups of lysines and N-termini of proteins) at neutral pH and is membrane impermeable. The resulting disulfide-linked biotin tag can be released from the labeled protein using reducing agents such as DTT. Using perfusion conditions that maintain the integrity

Discussion

In this study, we have shown that in vivo biotinylation by perfusion with sulfo-NHS-SS-biotin can be used to domain-specifically label the luminal surface of cerebral microvessels allowing transporter localization by imaging and biochemical techniques. We have summarized the advantages and disadvantages of in vivo biotinylation compared with other methods for transporter localization at the BBB in Supplemental Table 2. The majority of studies have addressed the polarized localization of

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