Isolated Rat Brain Capillaries Research Articles

Similarities and differences in the localization, trafficking, and function of P-glycoprotein in MDR1-EGFP-transduced rat versus human brain capillary endothelial cell lines

BackgroundIn vitro models based on brain capillary endothelial cells (BCECs) are among the most versatile tools in blood–brain barrier research for testing drug penetration into the brain and how this is affected by efflux transporters such as P-glycoprotein (Pgp). However, compared to freshly isolated brain capillaries or primary BCECs, the expression of Pgp in immortalized BCEC lines is markedly lower, which prompted us previously to transduce the widely used human BCEC line hCMEC/D3 with a doxycycline-inducible MDR1-EGFP fusion plasmid. The EGFP-labeled Pgp in these cells allows studying the localization and trafficking of the transporter and how these processes are affected by drug exposure. Here we used this strategy for the rat BCEC line RBE4 and performed a face-to-face comparison of RBE4 and hCMEC/D3 wild-type (WT) and MDR1-EGFP transduced cells.MethodsMDR1-EGFP-transduced variants were derived from WT cells by lentiviral transduction, using an MDR1-linker-EGFP vector. Localization, trafficking, and function of Pgp were compared in WT and MDR1-EGFP transduced cell lines. Primary cultures of rat BCECs and freshly isolated rat brain capillaries were used for comparison.ResultsAll cells exhibited typical BCEC morphology. However, significant differences were observed in the localization of Pgp in that RBE4-MDR1-EGFP cells expressed Pgp primarily at the plasma membrane, whereas in hCMEC/D3 cells, the Pgp-EGFP fusion protein was visible both at the plasma membrane and in endolysosomal vesicles. Exposure to doxorubicin increased the number of Pgp-EGFP-positive endolysosomes, indicating a lysosomotropic effect. Furthermore, lysosomal trapping of doxorubicin was observed, likely contributing to the protection of the cell nucleus from damage. In cocultures of WT and MDR1-EGFP transduced cells, intercellular Pgp-EGFP trafficking was observed in RBE4 cells as previously reported for hCMEC/D3 cells. Compared to WT cells, the MDR1-EGFP transduced cells exhibited a significantly higher expression and function of Pgp. However, the junctional tightness of WT and MDR1-EGFP transduced RBE4 and hCMEC/D3 cells was markedly lower than that of primary BCECs, excluding the use of the cell lines for studying vectorial drug transport.ConclusionsThe present data indicate that MDR1-EGFP transduced RBE4 cells are an interesting tool to study the biogenesis of lysosomes and Pgp-mediated lysosomal drug trapping in response to chemotherapeutic agents and other compounds at the level of the blood–brain barrier.

Inflammation-Induced Attenuation of Prostaglandin D2 Elimination across Rat Blood-Brain Barrier: Involvement of the Downregulation of Organic Anion Transporter 3 and Multidrug Resistance-Associated Protein 4.

Prostaglandin (PG) D2 is a lipid mediator, and in the brain, overproduction of PGD2 is reportedly involved in the progression and exacerbation of neuroinflammation. The objective of this study was to elucidate PGD2 efflux transport, under normal and inflammatory conditions, across the blood-brain barrier (BBB), which is formed by brain capillaries. Elimination of [3H]PGD2 across the BBB of normal and lipopolysaccharide (LPS)-induced inflammatory rats was examined by the intracerebral microinjection technique. After intracerebral injection, the percentage of [3H]PGD2 remaining in the ipsilateral cerebrum decreased with time, with a half-life of 13 min. This [3H]PGD2 elimination across the BBB was significantly inhibited by the co-administration of unlabeled PGD2, which suggests carrier-mediated PGD2 efflux transport at the BBB. In isolated rat brain capillaries, mRNA expression of organic anion transporter (Oat) 3, organic anion-transporting polypeptide (Oatp) 1a4, and multidrug resistance-associated protein (Mrp) 4 was observed. In addition, co-administration of substrates/inhibitors for Oat3, Oatp1a4, and/or Mrp4, such as benzylpenicillin and cefmetazole, reduced [3H]PGD2 elimination across the BBB. Data suggest that Oat3 and Mrp4, but not Oatp1a4 are involved in PGD2 elimination across the BBB, as Oatp1a4-expressing Xenopus (X.) oocytes did not show the significant [3H]PGD2 uptake compared with water-injected X. oocytes. In LPS-treated rats, [3H]PGD2 elimination across the BBB and mRNA expression levels of Oat3 and Mrp4 were significantly decreased. Our data suggest that Oat3- and Mrp4-mediated PGD2 elimination across the BBB is attenuated under inflammatory conditions.

Effect of GenX on P-Glycoprotein, Breast Cancer Resistance Protein, and Multidrug Resistance-Associated Protein 2 at the Blood-Brain Barrier.

Background:Ammonium 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)propanoic acid (GenX) is a replacement for perfluorooctanoic acid in the production of fluoropolymers used in a variety of consumer products. GenX alters fetal development and antibody production and elicits toxic responses in the livers and kidneys of rodents. The GenX effect on the blood–brain barrier (BBB) is unknown. The BBB protects the brain from xenobiotic neurotoxicants and harmful endogenous metabolites.Objectives:We aimed to investigate the effects of GenX on the transport activity and expression of P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance–associated protein 2 (MRP2) at the BBB.Methods:Transporter activities were measured in isolated rat brain capillaries by a confocal microscopy–based method. ATPase (enzymatic hydrolysis of adenosine triphosphate to inorganic phosphate) levels were measured in vitro. Western blotting determined P-gp and BCRP protein levels. Cell survival after GenX exposure was determined for two human cell lines.Results:Nanomolar levels of GenX inhibited P-gp and BCRP but not MRP2 transport activities in male and female rat brain capillaries. P-gp transport activity returned to control levels after GenX removal. GenX did not reduce P-gp- or BCRP-associated ATPase activity in an in vitro transport assay system. Reductions of P-gp but not BCRP transport activity were blocked by a peroxisome proliferator–activated receptor () antagonist. GenX reduced P-gp and BCRP transport activity in human cells.Conclusion:In rats, GenX at rapidly (in 1–2 h) inhibited P-gp and BCRP transport activities at the BBB through different mechanisms. was required for the GenX effects on P-gp but not BCRP transport activity. https://doi.org/10.1289/EHP5884

Cytosolic phospholipase A2 is a key regulator of blood‐brain barrier function in epilepsy

Blood-brain barrier dysfunction in epilepsy contributes to seizures and resistance to antiseizure drugs. Reports show that seizures increase brain glutamate levels, leading to barrier dysfunction. One component of barrier dysfunction is overexpression of the drug efflux transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP). Based on our previous studies, we hypothesized that glutamate released during seizures activates cytosolic phospholipase A2 (cPLA2), resulting in P-gp and BCRP overexpression. We exposed isolated rat brain capillaries to glutamate ex vivo and used an in vivo-ex vivo approach of isolating brain capillaries from rats after status epilepticus (SE) and in chronic epileptic (CE) rats. Glutamate increased cPLA2, P-gp, and BCRP protein and activity levels in isolated brain capillaries. We confirmed the role of cPLA2 in the signaling pathway in brain capillaries from male and female mice lacking cPLA2. We also demonstrated, in vivo, that cPLA2 inhibition prevents overexpression of P-gp and BCRP at the blood-brain barrier in rats after status epilepticus and in CE rats. Our data support the hypothesis that glutamate signals cPLA2 activation, resulting in overexpression of blood-brain barrier P-gp and BCRP.-Hartz, A. M. S., Rempe, R. G., Soldner, E. L. B., Pekcec, A., Schlichtiger, J., Kryscio, R., Bauer, B. Cytosolic phospholipase A2 is a key regulator of blood-brain barrier function in epilepsy.

Impact of Zn2+ on ABC Transporter Function in Intact Isolated Rat Brain Microvessels, Human Brain Capillary Endothelial Cells, and in Rat in Vivo.

ABCtransporters act as efflux pumps, thereby influencing the pharmacokinetics and efficacy of many drugs. Zinc (Zn) is an essential trace element contributing to cellular growth and differentiation. It is increasingly recognized as an intracellular messenger. The present study aims at investigating the impact of Zn2+ on the function and regulation of ABC transporters at the blood-brain barrier (BBB). ABC transporter function was first studied in isolated rat brain capillaries. Zn2+ rapidly stimulated the activity of the multidrug resistance-related protein 2 (Mrp2), p-glycoprotein (P-gp), and breast cancer resistance protein (Bcrp). These short-term effects were independent of transporter de novo synthesis but based on Zn2+ triggering intracellular signaling to stimulate basal transport activity. Studies focused on Mrp2 and P-gp showed that Zn2+ induced signaling through an endothelin receptor type B (ETB)/nitric oxide synthase (NOS)/protein kinase C (PKC) pathway and caused, specifically, an activation of the isoform PKCα. Studies revealed signaling through the phosphatidylinositol 3-kinase (PI3K)/mechanistic target of rapamycin (mTOR) pathway, as well as induction of the downstream target serum- and glucocorticoid-inducible kinase 1 (SGK1). Short-term effects of Zn2+ were also demonstrated in human hCMEC/D3 cells. An initial in vivo study in rats suggested enhanced P-gp transport activity at the BBB due to elevated Zn2+ plasma levels. This work provides the first evidence for Zn2+ being a regulator of basal ABC transporter activity at the BBB, driving a rapid and nongenomic stimulation of transport function.

Matrix Metalloproteinase-Mediated Blood-Brain Barrier Dysfunction in Epilepsy.

The blood-brain barrier is dysfunctional in epilepsy, thereby contributing to seizure genesis and resistance to antiseizure drugs. Previously, several groups reported that seizures increase brain glutamate levels, which leads to barrier dysfunction. One critical component of barrier dysfunction is brain capillary leakage. Based on our preliminary data, we hypothesized that glutamate released during seizures mediates an increase in matrix-metalloproteinase (MMP) expression and activity levels, thereby contributing to barrier leakage. To test this hypothesis, we exposed isolated brain capillaries from male Sprague Dawley rats to glutamate ex vivo and used an in vivo/ex vivo approach of isolated brain capillaries from female Wistar rats that experienced status epilepticus as an acute seizure model. We found that exposing isolated rat brain capillaries to glutamate increased MMP-2 and MMP-9 protein and activity levels, and decreased tight junction protein levels, which resulted in barrier leakage. We confirmed these findings in vivo in rats after status epilepticus and in brain capillaries from male mice lacking cytosolic phospholipase A2 Together, our data support the hypothesis that glutamate released during seizures signals an increase in MMP-2 and MMP-9 protein expression and activity levels, resulting in blood-brain barrier leakage.SIGNIFICANCE STATEMENT The mechanism leading to seizure-mediated blood-brain barrier dysfunction in epilepsy is poorly understood. In the present study, we focused on defining this mechanism in the brain capillary endothelium. We demonstrate that seizures trigger a pathway that involves glutamate signaling through cytosolic phospholipase A2, which increases MMP levels and decreases tight junction protein expression levels, resulting in barrier leakage. These findings may provide potential therapeutic avenues within the blood-brain barrier to limit barrier dysfunction in epilepsy and decrease seizure burden.

P-gp Protein Expression and Transport Activity in Rodent Seizure Models and Human Epilepsy.

A cure for epilepsy is currently not available, and seizure genesis, seizure recurrence, and resistance to antiseizure drugs remain serious clinical problems. Studies show that the blood-brain barrier is altered in animal models of epilepsy and in epileptic patients. In this regard, seizures increase expression of blood-brain barrier efflux transporters such as P-glycoprotein (P-gp), which is thought to reduce brain uptake of antiseizure drugs, and thus, contribute to antiseizure drug resistance. The goal of the current study was to assess the viability of combining in vivo and ex vivo preparations of isolated brain capillaries from animal models of seizures and epilepsy as well as from patients with epilepsy to study P-gp at the blood-brain barrier. Exposing isolated rat brain capillaries to glutamate ex vivo upregulated P-gp expression to levels that were similar to those in capillaries isolated from rats that had status epilepticus or chronic epilepsy. Moreover, the fold-increase in P-gp protein expression seen in animal models is consistent with the fold-increase in P-gp observed in human brain capillaries isolated from patients with epilepsy compared to age-matched control individuals. Overall, the in vivo/ex vivo approach presented here allows detailed analysis of the mechanisms underlying seizure-induced changes of P-gp expression and transport activity at the blood-brain barrier. This approach can be extended to other blood-brain barrier proteins that might contribute to drug-resistant epilepsy or other CNS disorders as well.

PPAR-α, a lipid-sensing transcription factor, regulates blood–brain barrier efflux transporter expression

Lipid sensor peroxisome proliferator-activated receptor alpha (PPAR-α) is the master regulator of lipid metabolism. Dietary release of endogenous free fatty acids, fibrates, and certain persistent environmental pollutants, e.g. perfluoroalkyl fire-fighting foam components, are peroxisome proliferator-activated receptor alpha ligands. Here, we define a role for peroxisome proliferator-activated receptor alpha in regulating the expression of three ATP-driven drug efflux transporters at the rat and mouse blood–brain barriers: P-glycoprotein (Abcb1), breast cancer resistance protein (Bcrp/Abcg2), and multidrug resistance-associated protein 2 (Mrp2/Abcc2). Exposing isolated rat brain capillaries to linoleic acid, clofibrate, or PKAs increased the transport activity and protein expression of the three ABC transporters. These effects were blocked by the PPAR-α antagonist, GW6471. Dosing rats with 20 mg/kg or 200 mg/kg of clofibrate decreased the brain accumulation of the P-glycoprotein substrate, verapamil, by 50% (in situ brain perfusion; effects blocked by GW6471) and increased P-glycoprotein expression and activity in capillaries ex vivo. Fasting C57Bl/6 wild-type mice for 24 h increased both serum lipids and brain capillary P-glycoprotein transport activity. Fasting did not alter P-glycoprotein activity in PPAR-α knockout mice. These results indicate that hyperlipidemia, lipid-lowering fibrates and exposure to certain fire-fighting foam components activate blood–brain barrier peroxisome proliferator-activated receptor alpha, increase drug efflux transporter expression and reduce drug delivery to the brain.

Aβ40 Reduces P-Glycoprotein at the Blood–Brain Barrier through the Ubiquitin–Proteasome Pathway

Failure to clear amyloid-β (Aβ) from the brain is in part responsible for Aβ brain accumulation in Alzheimer's disease (AD). A critical protein for clearing Aβ across the blood-brain barrier is the efflux transporter P-glycoprotein (P-gp) in the luminal plasma membrane of the brain capillary endothelium. P-gp is reduced at the blood-brain barrier in AD, which has been shown to be associated with Aβ brain accumulation. However, the mechanism responsible for P-gp reduction in AD is not well understood. Here we focused on identifying critical mechanistic steps involved in reducing P-gp in AD. We exposed isolated rat brain capillaries to 100 nm Aβ40, Aβ40, aggregated Aβ40, and Aβ42. We observed that only Aβ40 triggered reduction of P-gp protein expression and transport activity levels; this occurred in a dose- and time-dependent manner. To identify the steps involved in Aβ-mediated P-gp reduction, we inhibited protein ubiquitination, protein trafficking, and the ubiquitin-proteasome system, and monitored P-gp protein expression, transport activity, and P-gp-ubiquitin levels. Thus, exposing brain capillaries to Aβ40 triggers ubiquitination, internalization, and proteasomal degradation of P-gp. These findings may provide potential therapeutic targets within the blood-brain barrier to limit P-gp degradation in AD and improve Aβ brain clearance. The mechanism reducing blood-brain barrier P-glycoprotein (P-gp) in Alzheimer's disease is poorly understood. In the present study, we focused on defining this mechanism. We demonstrate that Aβ40 drives P-gp ubiquitination, internalization, and proteasome-dependent degradation, reducing P-gp protein expression and transport activity in isolated brain capillaries. These findings may provide potential therapeutic avenues within the blood-brain barrier to limit P-gp degradation in Alzheimer's disease and improve Aβ brain clearance.

Reversible Dimers of the Atypical Antipsychotic Quetiapine Inhibit P-Glycoprotein-Mediated Efflux in Vitro with Increased Binding Affinity and in Situ at the Blood-Brain Barrier

The multidrug resistance transporter P-glycoprotein (P-gp) is highly expressed in the capillary endothelial cells of the blood-brain barrier (BBB) where it functions to limit the brain penetration of many drugs, including antipsychotic agents used to treat schizophrenia. Therefore, in an effort to inhibit the transporter, we designed dimers of the antipsychotic drug and P-gp substrate quetiapine (QT), linked by variable length tethers. In P-gp overexpressing cells and in human brain capillary endothelial hCMEC/D3 cells, the dimer with the shortest tether length (QT2C2) (1) was the most potent inhibitor showing >80-fold better inhibition of P-gp-mediated transport than monomeric QT. The dimers, which are linked via ester moieties, are designed to revert to the therapeutic monomer once inside the target cells. We demonstrated that the addition of two sterically blocking methyl groups to the linker (QT2C2Me2, 8) increased the half-life of the molecule in plasma 10-fold as compared to the dimer lacking methyl groups (QT2C2, 1), while retaining inhibitory potency for P-gp transport and sensitivity to cellular esterases. Experiments with purified P-gp demonstrated that QT2C2 (1) and QT2C2Me2 (8) interacted with both the H- and R-binding sites of the transporter with binding affinities 20- to 30-fold higher than that of monomeric QT. Using isolated rat brain capillaries, QT2C2Me2 (8) was a more potent inhibitor of P-gp transport than QT. Lastly, we showed that QT2C2Me2 (8) increased the accumulation of the P-gp substrate verapamil in rat brain in situ three times more than QT. Together, these results indicate that the QT dimer QT2C2Me2 (8) strongly inhibited P-gp transport activity in human brain capillary endothelial cells, in rat brain capillaries, and at the BBB in an animal model.

Acetaminophen Modulates P-Glycoprotein Functional Expression at the Blood-Brain Barrier by a Constitutive Androstane Receptor–Dependent Mechanism

Effective pharmacologic treatment of pain with opioids requires that these drugs attain efficacious concentrations in the central nervous system (CNS). A primary determinant of CNS drug permeation is P-glycoprotein (P-gp), an endogenous blood-brain barrier (BBB) efflux transporter that is involved in brain-to-blood transport of opioid analgesics (i.e., morphine). Recently, the nuclear receptor constitutive androstane receptor (CAR) has been identified as a regulator of P-gp functional expression at the BBB. This is critical to pharmacotherapy of pain/inflammation, as patients are often administered acetaminophen (APAP), a CAR-activating ligand, in conjunction with an opioid. Our objective was to investigate, in vivo, the role of CAR in regulation of P-gp at the BBB. Following APAP treatment, P-gp protein expression was increased up to 1.4-1.6-fold in a concentration-dependent manner. Additionally, APAP increased P-gp transport of BODIPY-verapamil in freshly isolated rat brain capillaries. This APAP-induced increase in P-gp expression and activity was attenuated in the presence of CAR pathway inhibitor okadaic acid or transcriptional inhibitor actinomycin D, suggesting P-gp regulation is CAR-dependent. Furthermore, morphine brain accumulation was enhanced by P-gp inhibitors in APAP-treated animals, suggesting P-gp-mediated transport. A warm-water (50°C) tail-flick assay revealed a significant decrease in morphine analgesia in animals treated with morphine 3 or 6 hours after APAP treatment, as compared with animals treated concurrently. Taken together, our data imply that inclusion of APAP in a pain treatment regimen activates CAR at the BBB and increases P-gp functional expression, a clinically significant drug-drug interaction that modulates opioid analgesic efficacy.

1α,25-Dihydroxyvitamin D3-liganded vitamin D receptor increases expression and transport activity of P-glycoprotein in isolated rat brain capillaries and human and rat brain microvessel endothelial cells.

Induction of the multidrug resistance protein 1 (MDR1)/P-glycoprotein (P-gp) by the vitamin D receptor (VDR) was investigated in isolated rat brain capillaries and rat (RBE4) and human (hCMEC/D3) brain microvessel endothelial cell lines. Incubation of isolated rat brain capillaries with 10 nM of the VDR ligand, 1α,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] for 4 h increased P-gp protein expression fourfold. Incubation with 1,25(OH)(2)D(3) for 4 or 24 h increased P-gp transport activity (specific luminal accumulation of NBD-CSA, the fluorescent P-gp substrate) by 25-30%. In RBE4 cells, Mdr1b mRNA was induced in a concentration-dependent manner by exposure to 1,25(OH)(2)D(3). Concomitantly, P-gp protein expression increased 2.5-fold and was accompanied by a 20-35% reduction in cellular accumulation of the P-gp substrates, rhodamine 6G (R6G), and HiLyte Fluor 488-labeled human amyloid beta 1-42 (hAβ(42)). In hCMEC/D3 cells, a 3 day exposure to 100 nM 1,25(OH)(2)D(3) increased MDR1 mRNA expression (40%) and P-gp protein (threefold); cellular accumulation of R6G and hAβ(42) was reduced by 30%. Thus, VDR activation up-regulates Mdr1/MDR1 and P-gp protein in isolated rat brain capillaries and rodent and human brain microvascular endothelia, implicating a role for VDR in increasing the brain clearance of P-gp substrates, including hAβ(42), a plaque-forming precursor in Alzheimer's disease.

Targeting blood-brain barrier sphingolipid signaling reduces basal P-glycoprotein activity and improves drug delivery to the brain

P-glycoprotein, an ATP-driven drug efflux pump, is a major obstacle to the delivery of small-molecule drugs across the blood-brain barrier and into the CNS. Here we test a unique signaling-based strategy to overcome this obstacle. We used a confocal microscopy-based assay with isolated rat brain capillaries to map a signaling pathway that within minutes abolishes P-glycoprotein transport activity without altering transporter protein expression or tight junction permeability. This pathway encompasses elements of proinflammatory- (TNF-α) and sphingolipid-based signaling. Critical to this pathway was signaling through sphingosine-1-phosphate receptor 1 (S1PR1). In brain capillaries, S1P acted through S1PR1 to rapidly and reversibly reduce P-glycoprotein transport activity. Sphingosine reduced transport by a sphingosine kinase-dependent mechanism. Importantly, fingolimod (FTY720), a S1P analog recently approved for treatment of multiple sclerosis, also rapidly reduced P-glycoprotein activity; similar effects were found with the active, phosphorylated metabolite (FTY720P). We validated these findings in vivo using in situ brain perfusion in rats. Administration of S1P, FTY720, or FTY729P increased brain uptake of three radiolabeled P-glycoprotein substrates, (3)H-verapamil (threefold increase), (3)H-loperamide (fivefold increase), and (3)H-paclitaxel (fivefold increase); blocking S1PR1 abolished this effect. Tight junctional permeability, measured as brain (14)C-sucrose accumulation, was not altered. Therefore, targeting signaling through S1PR1 at the blood-brain barrier with the sphingolipid-based drugs, FTY720 or FTY720P, can rapidly and reversibly reduce basal P-glycoprotein activity and thus improve delivery of small-molecule therapeutics to the brain.

Activating PKC-β1 at the blood—brain barrier reverses induction of P-glycoprotein activity by dioxin and restores drug delivery to the CNS

Upregulation of blood-brain barrier (BBB) P-glycoprotein expression causes central nervous system (CNS) pharmacoresistance. However, activation of BBB protein kinase C-β1 (PKC-β1) rapidly reduces basal P-glycoprotein transport activity. We tested whether PKC-β1 activation would reverse CNS drug resistance caused by dioxin acting through aryl hydrocarbon receptor. A selective PKC-β1 agonist abolished the increase in P-glycoprotein activity induced by dioxin in isolated rat brain capillaries and reversed the effect of dioxin on brain uptake of verapamil in dioxin-dosed rats. Thus, targeting BBB PKC-β1 may be an effective strategy to improve drug delivery to the brain, even in drug-resistant individuals.

Aryl hydrocarbon receptor‐mediated up‐regulation of ATP‐driven xenobiotic efflux transporters at the blood‐brain barrier

Many widespread and persistent organic pollutants, e.g., 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), activate the aryl hydrocarbon receptor (AhR), causing it to translocate to the cell nucleus, where it transactivates target genes. AhR's ability to target the blood-brain barrier is essentially unexplored. We show here that exposing isolated rat brain capillaries to 0.05-0.5 nM TCDD roughly doubled transport activity and protein expression of P-glycoprotein, an ATP-driven drug efflux pump and a critical determinant of drug entry into the CNS. These effects were abolished by actinomycin D or cycloheximide or by the AhR antagonists resveratrol and α-naphthoflavone. Brain capillaries from TCDD-dosed rats (1-5 μg/kg, i.p.) exhibited increased transport activity and protein expression of 3 xenobiotic efflux pumps, P-glycoprotein, multidrug resistance-associated protein 2, and breast cancer resistance polypeptide, as well as expression of Cyp1a1 and Cyp1b1, both AhR target genes. Consistent with increased P-glycoprotein expression in capillaries from TCDD-dosed rats, in situ brain perfusion indicated significantly reduced brain accumulation of verapamil, a P-glycoprotein substrate. These findings suggest a new paradigm for the field of environmental toxicology: toxicants acting through AhR to target xenobiotic efflux transporters at the blood-brain barrier and thus reduce brain accumulation of CNS-acting therapeutic drugs.

BCRP at the Blood−Brain Barrier: Genomic Regulation by 17β-Estradiol

At the blood-brain barrier (BBB), the ABC transporter breast cancer resistance protein (BCRP) actively extrudes a variety of therapeutic drugs, including cytostatics, and diminishes their pharmacological efficacy in the brain. Consequently, new strategies to circumvent BCRP-mediated multidrug resistance in the CNS are required. One major approach to increase brain drug levels is to manipulate signaling mechanisms that control transporter expression and function. In the present study, we investigated the long-term effect of 17β-estradiol on BCRP in an ex vivo model of isolated rat brain capillaries. BCRP function and protein expression were decreased after 6 h of incubation with nanomolar concentrations of 17β-estradiol in capillaries from male and female rats. Concomitantly, levels of BCRP mRNA were also reduced by 17β-estradiol suggesting that the transporter is down-regulated via a genomic pathway. Additionally, we identified the presence of both estrogen receptor (ER) subtypes α and β at the rat BBB. Experiments using selective ER agonists and antagonists revealed that ER subtype β is responsible for the hormone-induced reduction of BCRP function and protein expression. These findings were confirmed by the use of ERKO mice. Blocking the proteasome-dependent degradation by lactacystin reversed the 17β-estradiol-mediated decrease of BCRP supposing that transcriptional down-regulation of the efflux transporter is paralleled by protein degradation. This study demonstrates that 17β-estradiol induces the down-regulation of BCRP on transcriptional and translational levels via the activation of ERβ in rat brain capillaries after 6 h. These results could help to improve brain targeting of BCRP substrates in the treatment of CNS diseases such as brain tumors and also contribute to an enlarged understanding of BCRP-drug interactions at a chronic intake of phytoestrogens and oral contraceptives.

The aryl hydrocarbon receptor (AhR) upregulates P‐glycoprotein (P‐gp) at the blood‐brain barrier (BBB)

Widespread, persistent organic pollutants, e.g., polychlorinated biphenyls and 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD), activate AhR causing it to translocate to the cell nucleus where it induces transcription of target genes, e.g., cytochrome P450 1A1 and 1B1 and phase II drug metabolizing enzymes. AhR's ability to modulate expression of xenobiotic transport proteins is largely unexplored. We show here that exposing isolated rat brain capillaries to the AhR ligands, β‐naphthoflavone (βNF, 0.5–5μM) or TCDD (0.05–0.5nM), increased P‐gp‐mediated transport and P‐gp protein expression 2–3 fold. These effects were abolished by actinomycin D or cycloheximide or by the AhR antagonists, resveratrol and αNF. Dosing rats with TCDD (1–5μg/kg, ip) increased P‐gp mediated transport and protein expression in brain capillaries assayed ex vivo. This is the first evidence that AhR ligands upregulate P‐gp mediated transport in any tissue and that AhR can alter drug transport at the BBB. Thus, environmental toxicants that are AhR ligands selectively tighten the BBB, providing increased protection, but at the expense of reduced penetration of therapeutic drugs. Supported by the Intramural Research Program, NIH/NIEHS.

Activation of PKC Isoform βI at the Blood–Brain Barrier Rapidly Decreases P-Glycoprotein Activity and Enhances Drug Delivery to the Brain

P-glycoprotein is an ATP (adenosine triphosphate)-driven drug efflux transporter that is highly expressed at the blood-brain barrier (BBB) and is a major obstacle to the pharmacotherapy of central nervous system diseases, including brain tumors, neuro-AIDS, and epilepsy. Previous studies have shown that P-glycoprotein transport activity in rat brain capillaries is rapidly reduced by the proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha) acting through protein kinase C (PKC)-dependent signaling. In this study, we used isolated rat brain capillaries to show that the TNF-alpha-induced reduction of P-glycoprotein activity was prevented by a PKCbeta(I/II) inhibitor, LY333531, and mimicked by a PKCbeta(I/II) activator, 12-deoxyphorbol-13-phenylacetate-20-acetate (dPPA). Western blotting of brain capillary extracts with phospho-specific antibodies showed that dPPA activated PKCbeta(I), but not PKCbeta(II). Moreover, in intact rats, intracarotid infusion of dPPA potently increased brain accumulation of the P-glycoprotein substrate, [(3)H]-verapamil without compromising tight junction integrity. Thus, PKCbeta(I) activation selectively reduced P-glycoprotein activity both in vitro and in vivo. Targeting PKCbeta(I) at the BBB may prove to be an effective strategy for enhancing the delivery of small molecule therapeutics to the brain.

Rapid, reversible modulation of blood-brain barrier P-glycoprotein transport activity by vascular endothelial growth factor.

Increased brain expression of vascular endothelial growth factor (VEGF) is associated with neurological disease, brain injury, and blood-brain barrier (BBB) dysfunction. However, the specific effect of VEGF on the efflux transporter P-glycoprotein, a critical component of the BBB, is not known. Using isolated rat brain capillaries and in situ rat brain perfusion, we determined the effect of VEGF exposure on P-glycoprotein activity in vitro and in vivo. In isolated capillaries, VEGF acutely and reversibly decreased P-glycoprotein transport activity without decreasing transporter protein expression or opening tight junctions. This effect was blocked by inhibitors of the VEGF receptor flk-1 and Src kinase, but not by inhibitors of phosphatidylinositol-3-kinase or protein kinase C. VEGF also increased Tyr-14 phosphorylation of caveolin-1, and this was blocked by the Src inhibitor PP2. Pharmacological activation of Src kinase activity mimicked the effects of VEGF on P-glycoprotein activity and Tyr-14 phosphorylation of caveolin-1. In vivo, intracerebroventricular injection of VEGF increased brain distribution of P-glycoprotein substrates morphine and verapamil, but not the tight junction marker, sucrose; this effect was blocked by PP2. These findings indicate that VEGF decreases P-glycoprotein activity via activation of flk-1 and Src, and suggest Src-mediated phosphorylation of caveolin-1 may play a role in downregulation of P-glycoprotein activity. These findings also imply that P-glycoprotein activity is acutely diminished in pathological conditions associated with increased brain VEGF expression and that BBB VEGF/Src signaling could be targeted to acutely modulate P-glycoprotein activity and thus improve brain drug delivery.

Prevention of seizure-induced up-regulation of endothelial P-glycoprotein by COX-2 inhibition

In the epileptic brain, seizure activity induces expression of the blood–brain barrier efflux transporter, P-glycoprotein, thereby limiting brain penetration and therapeutic efficacy of antiepileptic drugs. We recently provided the first evidence that seizures drive P-glycoprotein induction through a pathway that involves glutamate-signaling through the NMDA receptor and cyclooxygenase-2 (COX-2). Based on these data, we hypothesized that selective inhibition of COX-2 could prevent seizure-induced P-glycoprotein up-regulation. In the present study, we found that the highly selective COX-2 inhibitors, NS-398 and indomethacin heptyl ester, blocked the glutamate-induced increase in P-glycoprotein expression and transport function in isolated rat brain capillaries. Importantly, consistent with this, the COX-2 inhibitor, celecoxib, blocked seizure-induced up-regulation of P-glycoprotein expression in brain capillaries of rats in vivo. To explore further the role of COX-2 in signaling P-glycoprotein induction, we analyzed COX-2 protein expression in capillary endothelial cells in brain sections from rats that had undergone pilocarpine-induced seizures and in isolated capillaries exposed to glutamate and found no change from control levels. However, in isolated rat brain capillaries, the COX-2 substrate, arachidonic acid, significantly increased P-glycoprotein transport activity and expression indicating that enhanced substrate flux to COX-2 rather than increased COX-2 expression drives P-glycoprotein up-regulation. Together, these results provide the first in vivo proof-of-principle that specific COX-2 inhibition may be used as a new therapeutic strategy to prevent seizure-induced P-glycoprotein up-regulation at the blood–brain barrier for improving pharmacotherapy of drug-resistant epilepsy.