Expression of drug transporters at the blood-brain barrier using an optimized isolated rat brain microvessel strategy.
Quantitative RT-PCR (qRT-PCR) and Western blotting studies on transporters at the blood-brain barrier (BBB) of isolated brain microvessels have produced conflicting data on their cellular distribution. A major problem is identifying cells expressing the genes of interest, since isolated brain microvessels are composed of several cell types and may be contaminated with mRNA or proteins from astrocytes and neurons. We isolated rat brain microvessels and examined microscopically samples at each step of isolation to evaluate microvessel purity. The expression of specific markers of endothelial cells (Glut-1, Flk-1), pericytes (Ng2), neurons (synaptophysin, Syn) and astrocytes (Gfap) was measured by qRT-PCR in order to select the protocol giving the least astrocyte and neuron mRNAs and the most endothelial mRNAs. We also evaluated the gene expression of drug transporters (Mdr1a, Mdr1b, Mrp1-5, Bcrp and Oatp-2) at each step to optimize their location in cells at the BBB. The Mdr1a, Mrp4, Bcrp and Oatp-2 gene profiles were similar to those of endothelium markers. The profiles of Mrp2 and Mrp3 closely resembled that of Ng2. Mrp5 and Mrp1 expression was not increased in the microvessel-enriched fraction, suggesting that they are ubiquitously expressed throughout the cortex parenchyma. We also evaluated by Western blotting the expression of P-gp, Mrp2, Gfap and Syn in the cortex and in the purest obtained microvessel fraction. Our results showed that P-gp expression strongly increased in microvessels whereas Mrp2 was not detected in any of the fraction. Surprisingly, Gfap expression increased in isolated microvessels whereas Syn was not detected. Our results showed that the strategy consisting of identifying gene expression at different steps of the protocol is useful to identify cells containing mRNA at the BBB and give overall similar results with protein expression.
Yousif
Salah
S
INSERM U705, CNRS, UMR 7157, University Paris 5, Neuropsychopharmacology of Addiction, Laboratory of Pharmacokinetics, Faculty of Pharmacy, Hôpital Fernand Widal, Paris, France.
Marie-Claire
Cynthia
C
Roux
Françoise
F
Scherrmann
Jean-Michel
JM
Declèves
Xavier
X
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics; D000818:Animals; D015415:Biomarkers / Q000032:analysis / Q000378:metabolism; D001812:Blood-Brain Barrier / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics*; D002536:Cerebral Arteries / Q000378:metabolism*; D042783:Endothelial Cells / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000235:genetics; D005904:Glial Fibrillary Acidic Protein / Q000235:genetics; D008297:Male; D026901:Membrane Transport Proteins / Q000235:genetics; D008833:Microcirculation / Q000378:metabolism*; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics; D009924:Organ Culture Techniques / Q000379:methods; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D015415:Biomarkers; D002352:Carrier Proteins; D005904:Glial Fibrillary Acidic Protein; D026901:Membrane Transport Proteins; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D012333:RNA, Messenger