TY - JOUR
T1 - A functional in vitro model of rat blood-brain barrier for molecular analysis of efflux transporters
AU - Perrière, Nicolas
AU - Yousif, Salah
AU - Cazaubon, Sylvie
AU - Chaverot, Nathalie
AU - Bourasset, Fanchon
AU - Cisternino, Salvatore
AU - Declèves, Xavier
AU - Hori, Satoko
AU - Terasaki, Tetsuya
AU - Deli, Maria
AU - Scherrmann, Jean Michel
AU - Temsamani, Jamal
AU - Roux, Françoise
AU - Couraud, Pierre Olivier
N1 - Funding Information:
Grants: this work was supported by the Institut National de la Santé et de la Recherche Médicale, the Centre National de la Recherche Scientifique, the Université Paris-V and by a grant from the Association Nationale pour la Recherche et la Technologie to Nicolas Perrière, in collaboration with Synt:em.
Copyright:
Copyright 2009 Elsevier B.V., All rights reserved.
PY - 2007/8/31
Y1 - 2007/8/31
N2 - Physiological studies of the blood-brain barrier (BBB) are often performed in rats. We describe the functional characterization of a reproducible in vitro model of the rat BBB and its validation for investigating mechanisms involved in BBB regulation. Puromycin-purified primary cultures of brain endothelial cells, co-cultured with astrocytes in the presence of hydrocortisone (HC) and cAMP, presented low sucrose permeability (≤ 0.1 × 10- 3 cm/min) and high transendothelial electrical resistance (≥ 270 Ω cm2). Expression of specific BBB markers and their transcripts was detected by immunostaining and RT-PCR, respectively: tight junction proteins (claudin-3 and -5, ZO-1 and occludin) and transporters (P-gp, Bcrp and Oatp-2). RT-PCR experiments demonstrated a role of treatment by astrocytes, HC and cAMP in regulation of the transcript level of tight junction proteins (claudin-5 and ZO-1) as well as transporters (Mdr1a, Mrp3, Mrp4, Bcrp, Glut-1), while transcript level of Mdr1b was significantly decreased. The functionality of efflux pumps (P-gp, Mrps and Bcrp) was demonstrated in the presence of specific inhibitors (PSC833, MK571 or Ko143, respectively) by (i) assessing the uptake of the common substrates rhodamine 123 and daunorubicin and (ii) evaluating apical to basolateral and basolateral to apical polarized transport of daunorubicin. In addition, a good correlation (R = 0.94) was obtained between the permeability coefficients of a series of compounds of various lipophilicity and their corresponding in vivo rodent blood-brain transfer coefficients. Taken together, our results provide compelling evidence that puromycin-purified rat brain endothelial cells constitute a reliable model of the rat BBB for physiological and pharmacological characterization of BBB transporters.
AB - Physiological studies of the blood-brain barrier (BBB) are often performed in rats. We describe the functional characterization of a reproducible in vitro model of the rat BBB and its validation for investigating mechanisms involved in BBB regulation. Puromycin-purified primary cultures of brain endothelial cells, co-cultured with astrocytes in the presence of hydrocortisone (HC) and cAMP, presented low sucrose permeability (≤ 0.1 × 10- 3 cm/min) and high transendothelial electrical resistance (≥ 270 Ω cm2). Expression of specific BBB markers and their transcripts was detected by immunostaining and RT-PCR, respectively: tight junction proteins (claudin-3 and -5, ZO-1 and occludin) and transporters (P-gp, Bcrp and Oatp-2). RT-PCR experiments demonstrated a role of treatment by astrocytes, HC and cAMP in regulation of the transcript level of tight junction proteins (claudin-5 and ZO-1) as well as transporters (Mdr1a, Mrp3, Mrp4, Bcrp, Glut-1), while transcript level of Mdr1b was significantly decreased. The functionality of efflux pumps (P-gp, Mrps and Bcrp) was demonstrated in the presence of specific inhibitors (PSC833, MK571 or Ko143, respectively) by (i) assessing the uptake of the common substrates rhodamine 123 and daunorubicin and (ii) evaluating apical to basolateral and basolateral to apical polarized transport of daunorubicin. In addition, a good correlation (R = 0.94) was obtained between the permeability coefficients of a series of compounds of various lipophilicity and their corresponding in vivo rodent blood-brain transfer coefficients. Taken together, our results provide compelling evidence that puromycin-purified rat brain endothelial cells constitute a reliable model of the rat BBB for physiological and pharmacological characterization of BBB transporters.
KW - ABC transporters
KW - Blood-brain barrier
KW - Functional polarity
KW - In vitro model
KW - In vitro/in vivo correlation
KW - Tight junction
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U2 - 10.1016/j.brainres.2007.02.091
DO - 10.1016/j.brainres.2007.02.091
M3 - Article
C2 - 17434463
AN - SCOPUS:34248173562
SN - 0006-8993
VL - 1150
SP - 1
EP - 13
JO - Molecular Brain Research
JF - Molecular Brain Research
IS - 1
ER -