Glutamate-Mediated Down-Regulation of the Multidrug-Resistance

Article pubs.acs.org/molecularpharmaceutics

Glutamate-Mediated Down-Regulation of the Multidrug-Resistance Protein BCRP/ABCG2 in Porcine and Human Brain Capillaries Josephine D. Salvamoser,† Janine Avemary,† Hiram Luna-Munguia,† Bettina Pascher,‡ Thekla Getzinger,‡ Tom Pieper,‡ Manfred Kudernatsch,§ Gerhard Kluger,‡,∥ and Heidrun Potschka*,† †

Institute of Pharmacology, Toxicology, and Pharmacy, Ludwig-Maximilians-University, Koeniginstrasse 16, D-80539 Munich, Germany ‡ Neuropediatric Clinic and Clinic for Neurorehabilitation, Epilepsy Center for Children and Adolescents and §Clinic for Neurosurgery and Epilepsy Surgery, Schön Klinik Vogtareuth, 83569 Vogtareuth, Germany ∥ Paracelsus Medical University, 5020 Salzburg, Austria ABSTRACT: Breast cancer resistance protein (BCRP) functions as a major molecular gatekeeper at the blood−brain barrier. Considering its impact on access to the brain by therapeutic drugs and harmful xenobiotics, it is of particular interest to elucidate the mechanisms of its regulation. Excessive glutamate concentrations have been reported during epileptic seizures or as a consequence of different brain insults including brain ischemia. Previously, we have demonstrated that glutamate can trigger an induction of the transporter Pglycoprotein. These findings raised the question whether other efflux transporters are affected in a comparable manner. Glutamate exposure proved to down-regulate BCRP transport function and expression in isolated porcine capillaries. The reduction was efficaciously prevented by coincubation with N-methyl-D-aspartate (NMDA) receptor antagonist MK-801. The involvement of the NMDA receptor in the down-regulation of BCRP was further confirmed by experiments showing an effect of NMDA exposure on brain capillary BCRP transport function and expression. Pharmacological targeting of cyclooxygenase-1 and -2 (COX-1 and -2) using the nonselective inhibitor indomethacin, COX-1 inhibitor SC-560, and COX-2 inhibitor celecoxib revealed a contribution of COX-2 activity to the NMDA receptor’s downstream signaling events affecting BCRP. Translational studies were performed using human capillaries isolated from surgical specimens of epilepsy patients. The findings confirmed a glutamate-induced downregulation of BCRP transport activity in human capillaries, which argued against major species differences. In conclusion, our data reveal a novel mechanism of BCRP down-regulation in porcine and human brain capillaries. Moreover, together with previous data sets for P-glycoprotein, the findings point to a contrasting impact of the signaling pathway on the regulation of BCRP and Pglycoprotein. The effect of glutamate and arachidonic acid signaling on BCRP function might have implications for brain drug delivery and for radiotracer brain access in epilepsy patients and patients with other brain insults. KEYWORDS: multidrug transporter, blood−brain barrier, NMDA receptor, drug resistance, glutamate, breast cancer resistance protein, ABCG2

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INTRODUCTION

considerable amount of attention. During not only epileptic seizures but also as a consequence of other brain insults such as brain ischemia, extracellular concentrations of glutamate can reach highly increased levels.8−10 Ex vivo and in vivo experiments revealed that glutamate induces expression of the BBB efflux transporter P-glycoprotein via an N-methyl- D -aspartate (NMDA) receptor/arachidonic acid signaling cascade.6,7,11 The elucidation of these signaling pathways suggested several targets for the control and modulation of P-glycoprotein expression, which were further confirmed by pharmacological studies.12−16

Breast cancer resistance protein (BCRP or ABCG2) is considered a major molecular gatekeeper at the blood−brain barrier (BBB).1−3 In line with a protective concept, the expression and function of BBB efflux transporters including BCRP is regulated in a highly dynamic manner allowing rapid adjustment to environmental and endogenous challenges.4,5 The mechanisms of multidrug transporter regulation at the BBB are of particular interest as up- and/or down-regulation might have multiple functional consequences. In particular, the exposure of brain tissue to harmful xenobiotics, the brain penetration and efficacy of central nervous system (CNS) therapeutic drugs, and the brain distribution of molecular biomarkers used for PET or SPECT imaging can be affected.4−7 Several signaling factors have been described as affecting the BBB expression and function of multidrug transporters. Among these factors, disease-triggered signaling events have attracted a © XXXX American Chemical Society

Received: December 17, 2014 Revised: March 5, 2015 Accepted: April 21, 2015

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DOI: 10.1021/mp500841w Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Article

Molecular Pharmaceutics P-glycoprotein and BCRP have been described as a cooperative team of gatekeepers, which team up to control brain exposure.1 Therefore, higher expression levels of BCRP in comparison with P-glycoprotein suggested a predominant role of this efflux transporter at the human BBB.17 Differences between P-glycoprotein and BCRP do not only seem to exist in the species-specific expression rates18 but also in their regulation.4,5,17,19 Thus, we were keen to compare the impact of glutamate exposure, NMDA receptor activation, and arachidonic signaling on expression and transport function of BCRP with that of P-glycoprotein (Figure 1).

(Frankfurt, Germany). NMDA and dizocilpine (MK-801) were from Abcam (Cambridge, UK) and Valspodar (PSC-833) from tebu-bio (Offenbach, Germany). The monoclonal antibody for BCRP (BXP-53) was obtained from ENZO (Lö r rach, Germany), and the polyclonal antibody for the glucose transporter 1 was purchased from Millipore (Schwalbach, Germany). Animals and Tissue. Male and female porcine brains were provided by the local Munich slaughterhouse from pigs 5−6 months old (Munich, Germany). Human brain tissue was obtained from surgical resection from drug-resistant patients with focal cortical dysplasia (Neuropediatric Department of the Schön Clinic, Vogtareuth, Germany). All patients had a history of chronic pharmacoresistant epilepsy. The mean age of all patients was 6.33 years. The parents of the patients gave informed consent, in accordance with the regulations set out by the ethics committee, respecting the Declaration of Helsinki. During the transport, pig brains and human brain specimens were kept in ice-cold buffer solution (referred to as isolation solution, consisting of 103 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 10 mM HEPES (N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid), 1.2 mM MgSO4·7H2O, and 2.5 mM CaCl2·2H2O, pH 7.4). Isolation of Porcine and Human Brain Capillaries. Brain capillaries were isolated using a method from earlier studies.11,12,22,23 Capillary isolation was started within 1 h after surgical dissection or sampling of porcine brain. After cleaning the brains from meninges, brains were dissected and then homogenized. Capillaries were separated from the homogenate by density centrifugation with 30% dextran (7500 g, 12 min, 4 °C). Separated capillaries were diluted in artificial cerebrospinal fluid (aCSF, containing isolation solution supplemented with 0.5% bovine serum albumin (BSA), 10 mM glucose, 25 mM NaHCO3, and 1 mM sodium pyruvate, pH 7.4) and filtered through a 210 μm nylon mesh. The filtrate was passed over a glass-bead column and was washed with aCSF. Capillaries adherent to glass-beads were collected by gentle agitation in isolation solution. Following a second centrifugation (7500 g, 12 min, 4 °C) capillaries were resolved in incubation solution (isolation solution supplemented with 10 mM glucose). According to the experimental design, freshly isolated capillaries were exposed to different compounds. The concentration of glutamate was chosen on the basis of previously used protocols,12,24−26 which have been designed to reflect glutamate release during seizures.9,27 The NMDA concentrations were chosen on the basis of earlier studies, in which we demonstrated that an NMDA concentration of 5 μM increased P-glycoprotein function and expression in rat brain capillaries and that an NMDA concentration of 3 and 5 μM increased MRP2 transport function in porcine brain capillaries.11,25 Moreover, we obtained evidence that concentrations higher than 5 μM exert toxic effects.11 The duration of glutamate and NMDA exposure was chosen on the basis of microdialysis studies assessing the course of the increase and normalization of extracellular glutamate concentrations in response to single seizures in patients and in a rat model.25,28 Glutamate (100 μM) or NMDA (1, 3, and 5 μM) were added for 30 min; then, the samples were transferred to glutamate-free medium and incubated for 5.5 h (in porcine capillaries) or 4.5 h (in human capillaries). Please note that the time points of investigations were chosen on the basis of a pilot experiment and previous experience regarding the viability of brain capillaries following glutamate exposure. In the pilot time course

Figure 1. Expression of BCRP in the luminal membrane and of the NMDA receptor in the abluminal membrane of brain capillary endothelial cells. Graphic also illustrates the hypothesis that glutamate signaling via the NMDA receptor and subsequent activation of arachidonic acid signaling involving the enzyme cyclooxygenase-2 affect BCRP function and expression.

The regulation of efflux transporters in disease conditions is of general interest because it can affect the exposure of brain tissue to harmful xenobiotics and can affect the brain penetration, efficacy, and the adverse effect potential of various therapeutics.20 Moreover, the antiepileptic drug lamotrigine has recently been identified as a substrate of BCRP. Thus, regulation of BCRP in response to seizure-associated glutamate release might also affect the efficacy of this antiepileptic drug.21 The impact of glutamate and NMDA exposure on BCRP transport function was studied in freshly isolated porcine capillaries as well as in human capillaries obtained from surgical specimens from patients with drugrefractory epilepsy. The role of the NMDA receptor was further confirmed by pharmacological antagonism with MK-801. Using porcine capillaries, we also demonstrated the involvement of cyclooxygenase-2 on the basis of pharmacological modulation with celecoxib. Furthermore, we analyzed the effect of the signaling factors on BCRP expression rates.

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EXPERIMENTAL SECTION Chemicals. Glutamate, MK-571, celecoxib, and indomethacin were purchased from Sigma-Aldrich (Taufkirchen, Germany) and fumitremorgin C (FTC), mouse monoclonal ß-actin antibody, and SC-560 from Sigma-Aldrich (Schnelldorf, Germany). BODIPY-Prazosin (BP) was provided by Invitrogen B


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Molecular Pharmaceutics

Figure 2. Immunohistochemical staining in porcine and human brain capillaries. Immunostaining of porcine brain capillaries of (A) breast cancer resistance protein (BCRP, green) and (B) the endothelial cell marker glucose transporter-1 (Glut-1, red) with (C) an overlay of BCRP and Glut-1 labeling. Immunostaining of human brain capillaries of (D) BCRP (green) and (E) the endothelial cell marker Glut-1 (red), with (F) an overlay of BCRP and Glut-1 labeling. Scale bar = 10 μm.

for 1 h at room temperature with 1.5 μM BP or BP plus 5 μM FTC,30−32 a specific inhibitor for BCRP. For each experiment, confocal images of up to 15 capillaries were acquired with a Zeiss LSM 510 microscope (Carl Zeiss GmbH, Jena, Germany) using a 40 × 1.2 water immersion objective for each exposure and incubation protocol. The 488 nm emission laser, a 488 nm dichroic mirror, and a 505 nm bandpass filter were used for the experiments with BP. Luminal BP fluorescence intensity was quantified using ImageJ 1.4.3.67 (Launcher Symmetry software, NIH, Bethesda, USA). The background fluorescence intensity was subtracted, and the average fluorescence intensity for each area was calculated. BCRP-mediated transport was defined as the difference between the total luminal BP accumulation and the total accumulation in the presence of FTC. Data are presented as BCRP-specific luminal BP fluorescence. Confocal images were not digitally modified except for a contrast enhancement using Irfanview 4.28 (http://www.irfanview.com/). Western Blot. Membrane fractions were isolated from porcine brain capillaries using a method described in earlier studies.32 Briefly, following the incubation period of 6 h, capillaries were washed in incubation buffer. CellLytic-MT Mammalian Tissue Lysis/Extraction Reagent and Complete

experiment in porcine capillaries, we also studied a 3.5 h time point after exposure to glutamate. This experiment did not reveal an effect of glutamate at this earlier time point. From 6 h on, the viability of the porcine capillaries is progressively compromised so that it is not possible to continue with experiments assessing active transport function. Because viability declines earlier in human capillaries, on the basis of previous experience,12,25 we had to use an earlier time point in experiments with human capillaries. MK-801 (0.1 μM) was added 5 min before glutamate addition; celecoxib (1 μM), indomethacin (5 μM), and SC-560 (50 nm) were added 15 min prior to glutamate exposure. Capillaries were then used for transport experiments, Western blotting, or immunohistochemical staining. All transport experiments and Western blots were performed in triplicate. Please note that samples of human tissue were limited in size because of surgical resection. Therefore, it was not possible to perform Western blot analysis. Transport Assay. The transport activity of BCRP in isolated brain capillaries was studied by adding the BCRP-specific fluorescent substrate BP.29 Capillaries were transferred to imaging chambers (Ibidi, Martinsried, Germany) and incubated C


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Figure 3. Transport assays in freshly isolated porcine brain capillaries with the fluorescent dye BODIPY-Prazosin (BP). Representative image showing BP accumulation under (A) control conditions, (B) following stimulation with glutamate (100 μM), and (C) following inhibition with the BCRP transport modulator fumitremorgin C (FTC, 5 μM); scale bars = 10 μm. (D) Quantification of BP accumulation (n = 3). Stimulation of capillaries with glutamate leads to a significant decrease in BP accumulation (p < 0.0001). Inhibition of BCRP with FTC reduced BP accumulation significantly (p < 0.0001). (E) BCRP-specific transport rate (data calculated from D). BCRP-specific transport rate is calculated by subtracting the accumulation of BP remaining after blockade of BCRP transport with the inhibitor FTC. (F) BCRP expression rates proved to be reduced in response to glutamate (n = 3). (G) Specificity of BP as a BCRP substrate. In contrast to FTC, the two modulators PSC-833 (for P-glycoprotein) and MK-571 (for multidrug-resistance associated proteins) did not exert an effect on accumulation of BP in the lumen of porcine capillaries (n = 3). Bars represent means ± SEM; *, p < 0.05, significant difference versus control.

temperature using Roti-Block (1:10) (Roth, Karlsruhe, Germany) and incubated overnight at 4 °C with adequate monoclonal antibody against BCRP (BXP-53, 1:500) and ßactin (1:2500). Following washing with TBS/T (3×), membranes were incubated for 1 h at room temperature with adequate secondary horseradish peroxidase-conjugated antibody (BXP-53 = 1:5000, ß-actin = 1:5000). Western blots were developed for visualization of the immunoreaction using enhanced chemoluminescence (ECL) detection and the Fusion-SL Advance 4.2 MP system (Peqlab, Erlangen, Germany). The quantification of BCRP band intensity was performed by densitometric analysis using the Bio1D.16.06 software (Vilber Lourmat, peqlab,

EDTA-free Protease Inhibitor (Sigma Fluka, Taufkirchen, Germany) were added (25:1). Capillaries were homogenized, left on ice for 1 h, and centrifuged at 10 000 g for 30 min at 4 °C. The supernatant was then centrifuged at 100 000 g for 1 h at 4 °C. Pellets were resuspended in lysis buffer containing complete EDTA-free protease inhibitor, and protein concentrations were determined by the Lowry method. The separation of the membrane fractions was done on a 10% sodium dodecyl sulfatepolyacrylamide gel, which was then blotted on polyvinylidene difluoride membrane (peqlab, Erlangen, Germany) using a transfer buffer. Membranes were blocked for 1 h at room D


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Figure 4. Effects of MK-801, and NMDA in freshly isolated porcine brain capillaries on transport activity and expression. Exposure to the noncompetitive NMDA receptor antagonist MK-801 prevented the effect of glutamate on (A) BCRP transport function (n = 3) and (B) expression (n = 3). Exposure of porcine brain capillaries to different concentrations of NMDA (1, 3, and 5 μM) corroborated a key role of the NMDA receptor with a significant down-regulation of (C) BCRP transport function (n = 3) (1 μM, p < 0.0001; 3 μM, p < 0.0001; 5 μM, p < 0.0001) and (D) expression rates (n = 3) (1 μM, p < 0,0065; 5 μM, p < 0.0106). Bars represent means ± SEM; *, p < 0.05, significant difference versus control.

Statistical Analysis. Data are presented as means ± standard error of the mean (SEM). For comparisons between control and treated groups, either Student’s t test or the Mann−Whitney U test was used. A value of p < 0.05 was considered statistically significant.

Erlangen, Germany). The protein levels were normalized against the expression level of β-actin as a loading control. Western blot images were not digitally modified except for a contrast enhancement using Irfanview 4.28 (http://www. irfanview.com/). Immunohistochemistry of Isolated Capillaries. Capillaries were fixed on microscope slides (HistoBond adhesion slides; Marienfeld, Lauda-Koenigshofen, Germany) with 4% paraformaldehyde in PBS for 15 min. Following washing with PBS, the capillary preparation was blocked for 2 h with PBS containing 0.5% Triton X-100 (AppliChem GmbH, Darmstadt, Germany) and 10% bovine serum albumin (Sigma-Aldrich, Taufkirchen, Germany). Capillaries were incubated overnight in a cover plate apparatus at 4 °C with monoclonal antibody against BCRP (BXP-53, 1:50) and the polyclonal antibody against glucose transporter 1 (anti-Glut-1, 1:500). Following washing with PBS, capillaries were incubated for 2 h with the fluorochrome-conjugated secondary antibody (1:200). Immunostainings were evaluated by using a confocal laser scanning microscope (Zeiss LSM 510 microscope, Carl Zeiss GmbH, Jena, Germany). Negative controls were performed with an identical procedure but without primary antibodies.

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RESULTS Immunohistochemical Analyses of BCRP and Glucose Transporter-1 in Porcine and Human Brain Capillaries. The immunohistochemical staining of porcine brain capillaries confirmed the colocalization of BCRP with the endothelial cell marker glucose transporter-1 (Figure 2A−C). BCRP expression proved to be prominent in the luminal membrane of brain capillary endothelial cells, whereas there was no evidence for a relevant expression in the abluminal membrane (Figure 2). Brain surgical tissue was obtained from one pediatric 8 year old patient with drug refractory epilepsy caused by focal cortical dysplasia (patient no. 4). This patient had exhibited uncontrolled epileptic seizures since 5.5 years. Capillaries were isolated from the surgical specimen and used to analyze the expression pattern of BCRP. The analysis revealed a comparable expression pattern of human BCRP colocalizing with glucose transporter-1 in the E


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Figure 5. Role of cyclooxygenase-2 in glutamate-associated regulation of BCRP. (A) Exposure of porcine brain capillaries to glutamate decreased the specific luminal BODIPY-Prazosin (BP) accumulation (p < 0.0001), demonstrating a reduction in BCRP transport activity. Co-incubation with the cyclooxygenase-2 inhibitor celecoxib kept BCRP transport activity at control level (n = 3). (B) In line with the data from the transport studies, celecoxib prevented the glutamate-induced decrease (p < 0.001) of BCRP expression rate (n = 3). (C−D) Effect of the cyclooxygenase-1/cyclooxygenase-2 inhibitor indomethacin on (C) transport activity (n = 3) and (D) protein expression rate (n = 3). Indomethacin prevented a significant glutamatemediated decrease of BCRP. Cyclooxygenase-1 inhibitor SC-560 did not affect the impact of glutamate on (E) transport activity (n = 3) and (F) protein expression rate (n = 3). Bars represent means ± SEM; *, p < 0.05, significant difference versus control.

according to the accumulation of the fluorescent BCRP substrate BP (1.5 μM, Figure 3A−C). Exposing brain capillaries to 100 μM glutamate decreased the luminal fluorescence (Figure 3B). This glutamate concentration was chosen to assess the impact of the neurotransmitter on BCRP function and expression, considering

luminal brain capillary endothelial cell membrane (Figure 2D− F). Glutamate Down-Regulates BCRP Transport Activity and Expression in Porcine Capillaries. The impact of glutamate on the transport activity of BCRP was assessed F


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Figure 6. Transport assays in freshly isolated human brain capillaries using the fluorescent dye BODIPY-Prazosin (BP) . Representative images showing BP accumulation in isolated human brain capillaries under (A) control conditions, (B) following stimulation with glutamate (100 μM), and (C) following BCRP inhibition with fumitremorgin C (FTC) (5 μM); scale bars = 10 μm. (D) Quantification of BP accumulation (n = 3). Stimulation of capillaries with glutamate leads to a significant decrease (p < 0.0001) in BP accumulation. Inhibition of BCRP with FTC reduced the BP accumulation significantly (p < 0.0001). (E) BCRP-specific transport rate (data calculated from graph D). The BCRP specific transport rate is calculated by subtracting the accumulation of BP remaining after blockade of BCRP transport with the inhibitor FTC. (F) Specificity of BP as a BCRP substrate. In contrast to FTC (p < 0.0001), the two modulators PSC-833 (for P-glycoprotein) and MK-571 (for multidrug-resistance associated proteins) did not exert an effect on accumulation of BP in the lumen of human capillaries (n = 3). Bars represent means ± SEM; *, p < 0.05, significant difference versus control.

were corrected to the BCRP-specific transport rate by subtracting the unspecific accumulation in the presence of FTC (Figure 3E). In line with this finding, BCRP expression rates proved to be down-regulated in response to glutamate (Figure 3F). Using modulators of other BBB efflux transporters, we assessed the specificity of BP as a BCRP substrate. Neither PSC-833 (5 μM), a P-glycoprotein modulator, nor MK-571 (20 μM), which inhibits transporters of the MRP (multidrugresistance-associated protein) family, exerted an effect on accumulation of BP in the lumen of porcine capillaries (Figure 3G). These data confirmed that BP is transported by the porcine

interstitial concentrations that glutamate reaches following its release from neurons during seizures8−10 and on the basis of earlier studies focusing on the regulation of P-glycoprotein in rodent, porcine, and human capillaries.11−13 As expected, exposing capillaries to FTC (5 μM), a modulator of BCRP, strongly reduced the effect on the BCRP-mediated luminal BP accumulation (Figure 3C). Exposure of porcine capillaries to glutamate for 30 min resulted in a 25% decrease of the luminal BP fluorescence intensity, reflecting reduced BCRP transport function (Figure 3D). FTC reduced the transport activity significantly by 49% in porcine brain capillaries (Figure 3D). As described in the Experimental Section, data from all experiments G


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Figure 7. Quantification of BODIPY-Prazosin (BP) accumulation in samples from three patients with epilepsy. (A) Data from transport assay experiments in human brain capillaries obtained from three patients (patient no. 1−3). Stimulation with glutamate (100 μM) decreased the specific luminal BP accumulation (p < 0.0001). (B−D) Transport data for individual patients. Glutamate-induced decrease in BCRP efflux function was evident in capillaries from all three patients. Bars represent means ± SEM; *, p < 0.05, significant difference versus control.

isoform of BCRP but not by P-glycoprotein or multidrugresistance-associated proteins. Role of the NMDA Receptor in the Regulation of BCRP Function and Expression in Porcine Capillaries. Considering that glutamate can exert its effect via different ionotropic and metabotropic membrane receptors, further experiments addressed the question of which receptor is involved in these effects. The experiments focused on a putative key role of the NMDA receptor because it proved to mediate glutamateassociated effects on P-glycoprotein in recent studies. Providing support for an involvement of this receptor, the noncompetitive NMDA receptor antagonist MK-801 efficaciously prevented the effect of glutamate on BCRP transport function (Figure 4A) and expression (Figure 4B). Therefore, MK-801 coincubation kept BCRP transport and expression at control levels (Figure 4A,B). Exposure of porcine capillaries to different concentrations of NMDA further corroborated a key role of the NMDA receptor with a significant down-regulation of BCRP transport function (1, 3, and 5 μM NMDA, Figure 4C) and expression rates (1 and 5 μM NMDA, Figure 4D). However, we did not observe a concentration-dependency in the impact of NMDA. Role of Cyclooxygenase-2 in the Regulation of BCRP Function and Expression in Porcine Capillaries. NMDA receptor activation can trigger the induction of arachidonic acid signaling involving the activity of cyclooxygenases. Previously, we have demonstrated that cyclooxygenase-2 is a key factor mediating effects of NMDA receptor activation on function and expression of the efflux-transporter P-glycoprotein.11,12 Here, we investigated the contribution of cyclooxygenase-2 on the glutamate-induced down-regulation of BCRP transport activity and expression rate using cyclooxygenase-2 inhibitor celecoxib. Figure 5A shows isolated porcine brain capillaries that were coincubated with celecoxib (1 μM). This coincubation kept BCRP transport function at control level during exposure to

glutamate so that a decrease in BP accumulation was prevented (Figure 5A). In line with this finding, the coincubation abolished the glutamate-mediated reduction in BCRP expression (Figure 5B). Considering the more pronounced clinical relevance of nonselective cyclooxygenase inhibitors, we addressed the question of whether glutamate’s effects can also be prevented using the cyclooxygenase-1/2 antagonist indomethacin (5 μM). Indomethacin prevented the glutamate-induced decrease of BCRP transport function and expression rate (Figure 5C,D). In a subsequent experiment, we ruled out that cyclooxygenase-1 significantly contributes to the impact of glutamate on BCRP transport. Therefore, we coincubated capillaries with highly selective cyclooxygenase-1 inhibitor SC-560 (Figure 5E,F). In these experiments the effect of glutamate proved to be unaffected by cyclooxygenase-1 inhibition. Glutamate Down-Regulates BCRP Transport Activity in Human Capillaries. Capillaries were obtained from three surgical specimens from pediatric patients suffering from drugresistant epilepsy caused by focal cortical dysplasia. Patients underwent surgery at the age of 3 years (female), 14 years (female), and 2 years (male), following 0.5, 13.2, and 1.4 year disease courses, respectively. In subsequent experiments, capillaries were either incubated under control conditions or exposed to glutamate with and without inhibitors of the glutamate signaling cascade. Figure 6A− C depicts human brain capillaries with intraluminal BP accumulation. Exposure of human capillaries to 100 μM glutamate (30 min) resulted in a 61% decrease of BP accumulation, reflecting reduced BCRP transport activity (Figure 6D,E). In line with the findings from porcine capillaries, neither PSC-833 nor MK-571 affected the luminal accumulation of BP. In apparent contrast, FTC efficaciously inhibited BP efflux, reducing its effect by 80% (Figure 6F). These data confirmed that BP is transported by the human isoform of BCRP but not by H


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Molecular Pharmaceutics human P-glycoprotein or multidrug-resistance-associated proteins. As described in the Experimental Section, data from all experiments were corrected to the BCRP-specific transport rate by subtracting the unspecific accumulation in the presence of FTC (as indicated in Figure 6E). When individual data were compared regarding the impact of glutamate on BCRP transport function (Figure 7A−D), the glutamate-induced decrease in BCRP efflux function was evident in capillaries from all three patients.

it needs to be kept in mind that human brain capillaries were from patients and that the molecular and cellular pathological consequences of the disease might also affect signaling pathways. However, the effects observed in the human capillaries proved to be in the same range as in the capillaries from healthy pigs. Although some controversies exist,38 functional expression of the NMDA receptor has been repeatedly described in brain capillary endothelial cells.11−13,16,39−42 Recently, we confirmed expression of the NR1 subunit and function of NMDA receptors in freshly isolated human brain capillaries.12 Expression of further ionotropic glutamate receptors, i.e., the AMPA and kainate receptors, has also been described in brain capillaries.39 Thus, it was of interest to determine the receptor mediating the effect of glutamate on BCRP function and expression. Our findings clearly indicate that the NMDA receptor plays a key role in down-regulating BCRP. This is supported by the fact that MK801 efficaciously prevented glutamate’s impact on BCRP, arguing against a critical role of other ionotropic or metabotropic glutamate receptors. NMDA receptor signaling has been linked with an activation of arachidonic acid signaling.43−45 This link has been attributed to a calcium-influx-triggered release of arachidonic acid from the outer membrane.46 In line with a downstream role of arachidonic acid signaling involving cyclooxygenase function, pharmacological inhibition of cyclooxygenase-2 enzyme activity using the inhibitor celecoxib efficaciously interfered with the impact of glutamate on BCRP function. The nonselective inhibition of both cyclooxygenase isoforms by indomethacin also proved to be efficacious. However, there was a trend toward a more limited effect on the impact of glutamate on BCRP expression. Taken together, the findings suggest that arachidonic signaling mediates the downstream effects of endothelial NMDA receptor activation on BCRP transport. Previously, we have reported that arachidonic acid signaling, resulting in enhanced generation of the prostanoid PGE2, can up-regulate expression and function of the efflux transporter P-glycoprotein in rodent, porcine, and human capillaries.11−13 Moreover, we have confirmed that this signaling pathway contributes to seizure-associated induction of P-glycoprotein using rodent models of status epilepticus and epilepsy.13,15,16 Taken together with the present findings, our data revealed a contrasting impact of arachidonic acid signaling on the BBB efflux transporters P-glycoprotein and BCRP. Recently, Yousif et al. have suggested that morphine induces Pglycoprotein and BCRP at the rat BBB through NMDA receptor and cyclooxygenase-2 activation.47 The conclusion was based on in vivo experiments, in which the impact of morphine on Pglycoprotein and BCRP expression was counteracted by NMDA receptor antagonist MK-801 and cyclooxygenase inhibitor meloxicam.47 In cultured immortalized hCMEC/D3, glutamate exposure was without effect on P-glycoprotein and BCRP mRNA, thus rather arguing against the main conclusions of the authors.47 In the same study, the prostaglandin E2 analogs and EP1 receptor agonists iloprost and misoprostol proved to increase mRNA expression of both transporters.47 Several factors might have been contributed to the discrepancy between the author’s conclusions and our present findings in isolated capillaries. These factors include involvement of other cell types in in vivo experiments, putative species and age differences, and limitations in the translation of RNA level alterations in protein alterations caused by posttranscriptional regulation as well as differences between immortalized cell lines and freshly isolated capillaries.

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DISCUSSION Central nervous system diseases and brain insults such as epilepsy and brain ischemia can be associated with excessive neuronal glutamate release.6,7 In a series of ex vivo and in vivo studies using isolated brain capillaries from rodents, pigs, and humans, we have demonstrated that glutamate can signal via the endothelial NMDA receptor, triggering arachidonic acid signaling, which results in transcriptional activation of the ABCB1 gene and enhanced functional expression of P-glycoprotein.11−13,16 These studies suggested that glutamate might be the key factor causing P-glycoprotein overexpression in the epileptic brain, which has been repeatedly described in rodent models and human epileptic tissue.11−13,16 The present findings indicate that an opposing trend is observed for the efflux transporter BCRP when brain capillaries are exposed to increased extracellular glutamate concentrations. Both transport activity and protein expression levels rapidly decreased in response to glutamate incubation. Considering that extracellular glutamate concentrations can reach excessive concentrations during epileptic seizures,9,10,26 this might imply that BCRP can be downregulated by seizure activity. So far, there are only a limited number of studies that analyzed BCRP expression in the epileptic brain. A qualitative and descriptive assessment of expression patterns, in one case with hippocampal sclerosis and two cases with focal cortical dysplasia, demonstrated colocalization of BCRP with P-glycoprotein and with major vault protein in microvascular endothelium from epileptogenic human brain tissue.33 Quantification of BCRP expression indicated that BCRP levels are not altered in tissue from patients with hippocampal sclerosis.34 Increased expression rates were limited to data describing BCRP expression in the microvasculature of epileptogenic brain tumors.34,35 Only one experimental study using a rodent temporal lobe epilepsy model has reported an overexpression of BCRP in blood vessels of the parahippocampal cortex.36 The fact that a down-regulation has not been observed in the epileptic rodent and human brain might be related to the complexity of signaling factors and cellular cross-talk activated during epileptic seizures. Respective signaling factors might affect transporter function and expression in a contrarious manner. Moreover, species differences might exist between the regulation in rodent capillaries on one hand and porcine or human capillaries on the other hand. Another factor that needs to be considered is age influencing the impact of regulatory mechanisms. Recently, Harati and colleagues have reported that the impact of endothelin on P-glycoprotein and BCRP expression differs in rats at postnatal days 21 and 84, corresponding to juvenile and adult stages of human brain maturation.37 In this context, it needs to be considered that the mean age of pigs at time of slaughter is 5−6 months. The age of the pediatric patients from which surgical tissue was sampled for the functional analyses in the present study were 2, 3, and 14 years. Thus, in the present study we can only conclude about regulation in brain capillaries from the juvenile brain. In addition, I


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the neurological adverse effects of a drug with peripheral targets or with CNS targets because of enhanced brain distribution. Our data reveal a novel mechanism of BCRP down-regulation in porcine and human brain capillaries. The impact of glutamate and archidonic acid signaling on BCRP function might have implications for brain drug delivery and for radiotracer brain access in epilepsy patients and patients with other brain insults associated with increased extracellular glutamate. In view of recent findings of the effect of age difference on the regulatory mechanisms of BCRP, future studies have to address the question whether the same regulatory patterns are observed in capillaries from the mature brain.

It is evident from the transport assays and the expression analysis that the impact of glutamate and NMDA on BCRP transport function is more pronounced than the impact on BCRP expression. In this context, it needs to be considered that specific changes in expression do not necessarily translate to comparable changes in transport activity measured during a specific period of time with accumulation of a substrate over time. Moreover, transporters can be rapidly redistributed to intracellular pools, resulting in an immediate reduction in transport activity.48 In contrast, expression rates might be affected in a delayed manner by enhanced intracellular degradation and/or reduced de novo synthesis. The analysis of downstream signaling gave first evidence that the same pathway that up-regulates P-glycoprotein11−13,16 can down-regulate BCRP. Various factors have been described that up- or downregulate P-glycoprotein and BCRP at the bloodbrain barrier.4−7,49 Both P-glycoprotein and BCRP can be positively regulated by xenobiotics, including therapeutic drugs and environmental toxic compounds, via ligand-activated receptors such as pregnane X receptor, constitutive androsterane receptor, and glucocorticoid receptors.4 Differential effects of signaling factors on P-glycoprotein versus BCRP have also been described. Whereas the inflammatory cytokine TNF-α upregulates P-glycoprotein, no such impact was observed for BCRP.50,51 In juvenile rats, endothelin-1 tended to decrease BCRP but did not affect P-glycoprotein.37 Thus, the glutamate/ NMDA receptor/arachidonic acid signaling pathway identified as a regulator of BCRP in the present study seems to add to the complex mechanisms that coordinate and orchestrate the function and expression of the major BBB gatekeepers Pglycoprotein and BCRP in response to endogenous and environmental challenges. The regulatory mechanisms of both transporters are of major relevance because they have been proposed to act as team players with overlapping substrate spectra.1 Their regulation can have major implications regarding brain exposure to toxic compounds, brain access and efficacy of CNS therapeutics, and molecular imaging biomarkers used for PET and SPECT. Considering the high extracellular concentrations of glutamate reached during epileptic seizure as well as during traumatic brain injury, brain ischemia, and stroke, this can have consequences for therapy and diagnostics in patients with epilepsy and stroke. Controversial findings have been reported regarding transport of antiepileptic drugs by BCRP.52,53 Taken together, the data rather argue against a major influence of BCRP on antiepileptic drug transport. However, recently the antiepileptic drug lamotrigine has been identified as a substrate of human BCRP.21 Glutamate-triggered down-regulation of BCRP may thus positively affect lamotrigine’s brain penetration and efficacy. In view of the fact that lamotrigine also serves as a substrate of human P-glycoprotein,54,55 the contrasting glutamate-associated regulation of both transporters might cancel out each other’s impact on lamotrigine brain penetration and efficacy. The regulation of BCRP must also be considered regarding other substrates of BCRP and the brain exposure to respective harmful xenobiotics and to various therapeutic drugs. The list of BCRP substrates includes highly toxic, mutagenic, and carcinogenic compounds as well as therapeutic drugs including antibiotic drugs, antiviral drugs, and cytostatic drugs used for tumor therapy.56,57 Downregulation of BCRP in disease states with excessive glutamate release might on one hand enhance efficacy of drugs targeting the CNS but on the other hand also increase

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AUTHOR INFORMATION

Corresponding Author

*Phone: +49-89-21802662. Fax: +49-89-218016556. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Olga Cabezas, Marion Fisch, Barbara Kohler, and Angela Vicidomini are acknowledged for their excellent technical assistance. Earlier studies on transporter regulation have been supported by DFG Po681/4-1.

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REFERENCES

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