Induction of P-glycoprotein and Bcrp at the rat blood-brain barrier following a subchronic morphine treatment is mediated through NMDA/COX-2 activation.

Subchronic morphine treatment induces P-glycoprotein (P-gp) up-regulation at the blood-brain barrier. This study investigates the rate and extent to which P-gp and breast cancer-resistance protein (Bcrp) increase at the rat blood-brain barrier following subchronic morphine treatment. Rats were given increasing doses of morphine (10-40 mg/kg) or saline i.p. twice daily for 5 days. The brain cortex large vessels and microvessels were then mechanical isolated 6, 9, 12, 24, and 36 h after the last injection. The gene and protein expression of P-gp and Bcrp in morphine-treated and control rats were compared by qRT-PCR and western blotting. The levels of Mdr1a and Bcrp mRNAs were not significantly modified 6 h post morphine, but the Mdr1a mRNA increased 1.4-fold and Bcrp mRNA 2.4-fold at 24 h. P-gp and Bcrp protein expression in brain microvessels was unchanged 6 h post morphine and increased 1.5-fold at 24 h. This effect was more pronounced in large vessels than in microvessels. However, extracellular morphine concentrations of 0.01-10 µM did not modify the expressions of the MDR1 and BCRP genes in hCMEC/D3 human endothelial brain cells in vitro. MK-801 (NMDA antagonist) and meloxicam (cyclo-oxygenase-2 inhibitor) given after morphine treatment completely blocked P-gp and Bcrp up-regulation. Interestingly, misoprostol and iloprost, two well-known agonists of prostaglandin E2 receptors induced both MDR1 and BCRP mRNA levels in hCMEC/D3. Thus, morphine does not directly stimulate P-gp and Bcrp expression by the brain endothelium, but glutamate released during morphine withdrawal may do so by activating the NMDA/cyclo-oxygenase-2 cascade.

Transcriptomic and quantitative proteomic analysis of transporters and drug metabolizing enzymes in freshly isolated human brain microvessels.

We have investigated the transcriptomic and/or proteomic patterns of 71 solute carrier (SLC) and organic solute (OST) transporters, 34 ATP-binding cassette (ABC) transporters, and 51 metabolizing enzymes in human brain microvessels. We used quantitative RT-PCR and LC-MS/MS to examine isolated brain microvessels and cortex biopsies from 12 patients with epilepsia or glioma. SLC2A1/GLUT1, SLC1A3/EAAT1, and SLC1A2/EAAT2 were the main SLC proteins whereas ABCG2/BCRP, ABCB1/MDR1, ABCA2 and ABCA8 were the main ABC quantified in isolated brain microvessels; ABCG2/BCRP was 1.6-fold more expressed than ABCB1/MDR1, and ABCC4/MRP4 was 10 times less abundant than ABCB1/MDR1. CYP1B1 and CYP2U1 were the only quantifiable CYPs. Finally, GSTP1, COMT, GSTM3, GSTO1 and GSTM2 proteins were the main phase II enzymes quantified; UGTs and NATs were not detected. Our extensive investigation of gene and protein patterns of transporters and metabolizing enzymes provides new molecular information for understanding drug entry and metabolism in the human blood-brain barrier.

Effect of chronic exposure to morphine on the rat blood-brain barrier: focus on the P-glycoprotein.

Morphine may affect the properties of the blood-brain barrier (BBB) by modifying the expression of certain BBB markers. We have determined the effect of chronic morphine treatment on the expression and function of some BBB markers in the rat. The mRNAs of 19 selected genes encoding caveolins, endothelial transporters, receptors and tight junctions proteins in the total RNA of isolated cortex microvessels were assayed by quantitative RT-PCR (qRT-PCR). The expression of genes Mdr1a, Mrp1, Bcrp, Glut-1 and Occludin, was slightly increased, while that of Flk-1 was decreased in microvessels from morphine-treated rats. The expression of the Mrd1a and Mdr1b genes encoding the P-glycoprotein (P-gp) also increased in the whole hippocampus and cortex of morphine-treated rats. The Mdr1a gene induction (1.38-fold) observed by qRT-PCR was also confirmed using in situ hybridization technique (1.40-fold). Immunoblotting revealed an increase in P-gp expression in the hippocampus (1.8-fold) and cortex (1.36-fold) of morphine-treated rats, but no effect in isolated microvessels. In contrast, morphine treatment increased by 1.48-fold the expression of P-gp in a large vessel-enriched fraction. The integrity of the BBB, measured by in situ brain perfusion of [(14)C]-sucrose, and the activity of P-gp at the BBB, measured with the P-gp substrate [(3)H]-colchicine, were not modified by morphine. Immunohistofluorescence experiments revealed that P-gp expression is restricted to large vessels and microvessels in control rats and that morphine treatment did not induce the expression of P-gp in the brain parenchyma (astrocytes or neurons). Taken together, our results showed that chronic morphine treatment does not significantly alter BBB integrity or P-gp activity. The impact of morphine-mediated P-gp induction observed in large vessels remains to be determined in terms of brain disposition of drugs that are P-gp substrates.

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.

A functional in vitro model of rat blood-brain barrier for molecular analysis of efflux transporters.

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 (< or =0.1 x 10(-3) cm/min) and high transendothelial electrical resistance (> or =270 Omega cm(2)). 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.

Interplay of drug metabolizing CYP450 enzymes and ABC transporters in the blood-brain barrier.

The recent identification of drug-metabolizing enzymes cytochrome P450 (CYP) in the human blood-brain barrier (BBB) raises the question of whether these enzymes act in concert with ATP-binding cassette (ABC) transporters to limit the brain distributions of drugs. We recently demonstrated several CYP genes in freshly isolated human brain microvessels; the main isoforms expressed were CYP1B1 and CYP2U1. Many studies using different experimental approaches have revealed that P-glycoprotein (P-gp, ABCB1), breast cancer resistance protein (BCRP, ABCG2) and the multidrug resistance-associated protein 4 (MRP4, ABCC4) are the main ABC transporters in the human BBB. The first part of this review covers recent studies on the expression, regulation and function of CYP450 and ABC transporters in the rodent and human BBBs. The second part focuses on the possible interplay between some CYPs and certain ABC transporters at the BBB, which makes it a determining element of brain drug concentrations and thus of the effects of centrally acting drugs.

Aryl hydrocarbon receptor-dependent upregulation of Cyp1b1 by TCDD and diesel exhaust particles in rat brain microvessels.

BACKGROUND: AhR activates the transcription of several target genes including CYP1B1. Recently, we showed CYP1B1 as the major cytochrome P450 (CYP) enzyme expressed in human brain microvessels. Here, we studied the effect of AhR activation by environmental pollutants on the expression of Cyp1b1 in rat brain microvessels. METHODS: Expression of AhR and Cyp1b1 was detected in isolated rat brain microvessels. AhR was immunovisualised in brain microvessel endothelial cells. The effect of AhR ligands on Cyp1b1 expression was studied using isolated brain microvessels after ex vivo and/or in vivo exposure to TCDD, heavy hydrocarbons containing diesel exhaust particles (DEP) or Δ9-tetrahydrocannabinol (Δ9-THC). RESULTS: After ex vivo exposure to TCDD (a highly potent AhR ligand) for 3 h, Cyp1b1 expression was significantly increased by 2.3-fold in brain microvessels. A single i.p. dose of TCDD also increased Cyp1b1 transcripts (22-fold) and Cyp1b1 protein (2-fold) in rat brain microvessels at 72 h after TCDD. Likewise, DEP treatment (in vivo and ex vivo) strongly induced Cyp1b1 protein in brain microvessels. DEP-mediated Cyp1b1 induction was inhibited by actinomycin D, cycloheximide, or by an AhR antagonist. In contrast, a sub-chronic in vivo treatment with Δ9-THC once daily for 7 seven days had no effect on Cyp1b1 expression CONCLUSIONS: Our results show that TCDD and DEP strongly induced Cyp1b1 in rat brain microvessels, likely through AhR activation.

Influence of development, HIV infection, and antiretroviral therapies on the gene expression profiles of ABC transporters in human lymphocytes.

The efficacy of drugs acting on lymphocytes like anticancer, immunosuppressive, and antiretroviral drugs depends on their intracellular concentrations, which could be modulated by membrane efflux pumps belonging to the ABC transporter superfamily. The gene expression profiles of 6 main ABC transporters (MDR1, MRP1, MRP3, MRP4, MRP5, and BCRP) were established in lymphocytes from birth to adulthood using blood samples from 57 children and 15 adults (34 and 5 HIV-infected, respectively). Gene expression levels were quantified by quantitative RT-PCR. In adults, the MRP1 gene had the highest expression, followed by the MRP5 gene. BCRP and MRP4 genes were significantly higher expressed at birth than after 1 month of life. Neither HIV infection nor antiretroviral therapies modulated the gene expression profiles of ABC transporters. In conclusion, drugs that are substrates of BCRP and MRP4, like zidovudine, may have an altered efficacy in newborns.

ABC transporters and the accumulation of imatinib and its active metabolite CGP74588 in rat C6 glioma cells.

Imatinib (Glivec, Gleevec, STI571) is a small tyrosine kinase inhibitor that is currently in phase II clinical trials in patients with recurrent glioblastoma. Its therapeutic benefit is minimal, although it is greater in some patients when combined with hydroxyurea. Imatinib is transported by human and rodent ATP-binding cassette (ABC) transporters like P-glycoprotein (Pgp) and the breast cancer resistance protein (BCRP). We have investigated whether ABC transporters determine the pharmacokinetics of imatinib and its pharmacological active metabolite CGP74588 in rat C6 glioma cells. ABC transporter expressions were measured by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). C6 cells express high concentrations of the Pgp-encoding gene Mdr1b and a 10-fold smaller amount of the Pgp-encoding gene Mdr1a. The relative expression of ABC transporter genes are: Mdr1b>Mrp4>Mrp1>Mrp5>Mdr1a>Mrp3>Mrp2>Bcrp. The accumulation of imatinib into C6 cells increased linearly with the extracellular concentration of imatinib (0.5-50microM) and was not increased by zosuquidar (selective Pgp inhibitor) or elacridar (inhibitor of both Pgp and Bcrp). In contrast, there was less CGP74588 than imatinib in C6 cells and its concentration increased with the extracellular concentration in a sigmoid fashion. Lastly, 10microM valspodar (selective Pgp inhibitor), elacridar and zosuquidar all increased the accumulation of CGP74588 by 2.5-fold. Thus CGP74588 is readily transported by the Pgp in rat C6 gliomas cells, which raises the question of the role of Pgp in the resistance of recurrent glioblastomas to imatinib.