Nongenomic ERα-AMPK Signaling Regulates Sex-Dependent Bcrp Transport Activity at the Blood-Brain Barrier

David B Banks; Sydney L Lierz; Ronald E Cannon; Kenneth S Korach

doi:10.1210/endocr/bqae081

. 2024 Jul 10;165(8):bqae081. doi: 10.1210/endocr/bqae081

Nongenomic ERα-AMPK Signaling Regulates Sex-Dependent Bcrp Transport Activity at the Blood-Brain Barrier

David B Banks ^1,², Sydney L Lierz ^3,⁴, Ronald E Cannon ⁵, Kenneth S Korach ^6,^✉

PMCID: PMC11272090 PMID: 38984714

Abstract

The blood-brain barrier (BBB) is an extensive capillary network that protects the brain from environmental and metabolic toxins while limiting drug delivery to the central nervous system (CNS). The ATP-binding cassette transporter breast cancer resistance protein (Bcrp) reduces drug delivery across the BBB by actively transporting its clinical substrates back into peripheral circulation before their entry into the CNS compartment. 17β-Estradiol (E2)-elicited changes in Bcrp transport activity and expression have been documented previously. We report a novel signaling mechanism by which E2 decreases Bcrp transport activity in mouse brain capillaries via rapid nongenomic signaling through estrogen receptor α. We extended this finding to investigate the effects of different endocrine-disrupting compounds (EDCs) and selective estrogen receptor modulators (SERMs) on Bcrp transport function. We also demonstrate sex-dependent expression of Bcrp and E2-sensitive Bcrp transport activity at the BBB ex vivo. This work establishes an explanted tissue-based model by which to interrogate EDCs and SERMs as modulators of nongenomic estrogenic signaling with implications for sex and hormonal regulation of therapeutic delivery into the CNS.

Keywords: 17β-estradiol, breast cancer resistance protein transporter, blood-brain barrier, estrogen receptor α, endocrine-disrupting compound

The blood-brain barrier (BBB) is a vascular capillary network of the central nervous system (CNS) that protects brain tissue from toxic solutes introduced into peripheral circulation by either endogenous metabolic processes or environmental exposure. Its neuroprotective function results from 2 key components. First, structural tight junctions that connect adjacent endothelial cells limit the passive permeability of larger molecular species. Second, ATP-binding cassette (ABC) transporters on both the circulation- and brain-facing sides of the capillary endothelium pump substrates between the CNS and blood compartments via active transport (1). The resulting barrier restricts the blood-to-brain delivery of CNS drugs handled as transporter substrates. The ABC transporter breast cancer resistance protein (Bcrp) contributes directly to this clinical challenge. The position of Bcrp at the blood-facing or luminal capillary membrane results in the net efflux of its clinical substrates including antiepileptic drugs and chemotherapeutics back into circulation (2, 3, 4, 5). Specifically targeting Bcrp-regulatory pathways at the capillary endothelium to decrease substrate efflux may augment CNS drug availability at target tissues without disrupting the inherent neuroprotection provided by the BBB (5).

Steroid hormone-initiated signaling by 17β-estradiol (E2) has been shown to drive both rapid and long-term changes in Bcrp transport activity and expression across tissues including the BBB and liver (6, 7, 8, 9, 10, 11). The mechanisms by which E2 exposure may cause rapid, reversible changes in Bcrp transport activity at the BBB are less well understood. E2 elicits its effects at the cellular level through canonical steroid hormone signaling, whereby ligand activation of estrogen receptor alpha or beta (ERα, ERβ) by E2 induces receptor dimerization within the nucleus where these receptors function as transcription factors to alter gene expression. Alternatively, E2 may activate discrete extranuclear signaling programs downstream of ERα, ERβ, or the G-protein coupled estrogen receptor (GPER). Rapid nongenomic estrogenic signaling through ERα and ERβ occurs independently of their transcription factor functions within the nucleus (12, 13). While extranuclear signaling through ERα has been shown to regulate endothelial cell function and vascular tone underlying cardiovascular homeostasis, its role at the capillary endothelium comprising the BBB remains unstudied (14, 15, 16).

Using freshly isolated mouse brain capillaries (MBCs) derived from male and ovary-intact female mice, we observed sex-dependent differences in Bcrp protein levels and E2-responsive Bcrp transport activity. The expression and transport differences observed were normalized by ovariectomy. Consistent with previous reports, we also observed a rapid reduction in specific Bcrp transport activity following exposure of MBCs to physiologically relevant concentrations of E2 (6, 7). For the first time, we report an E2-elicited reduction in specific Bcrp transport activity as a novel case of extranuclear, nongenomic signaling through ERα and AMP-activated protein kinase (AMPK) at the mouse BBB. We also investigated the activities of several endocrine-disrupting chemicals (EDCs) and selective estrogen receptor modulators (SERMs) in terms of Bcrp transport activity as a quantifiable result of estrogenic signaling. These findings could provide greater insight into sex-dependent differences in BBB and drug transport dynamics, as well as a new model for assessing rapid estrogenic activities of known and unknown EDCs and SERMs relevant to CNS drug delivery.

Materials and Methods

Materials

The Bcrp-specific inhibitor KO143, P-glycoprotein-specific inhibitor PSC833, Multidrug resistance-associated protein 2 (Mrp2)-specific inhibitor MK571, ERα agonist propyl pyrazole triol (PPT), ERβ agonist diarylpropionitrile (DPN), GPER agonist G1, Akt inhibitor GSK690693, PI3K inhibitor LY294002, and AMPK inhibitor dorsomorphin dihydrochloride were all purchased from Tocris Bioscience. Bisphenol A, bisphenol S, and endosulfan were obtained through the National Toxicology Program. The fluorescent breast cancer resistance protein (Bcrp) substrate BODIPY® FL prazosin was purchased from Invitrogen, the fluorescent P-glycoprotein substrate N-ε(4-nitrobenzofurazan-7-yl)-D-Lys8 cyclosporin A (NBD-CSA) was custom-synthesized, and the fluorescent Mrp2 substrate sulforhodamine 101 free acid (Texas Red) was purchased from Sigma-Aldrich. Rat monoclonal Bcrp antibody BXP-53 (Cat# MA1-35029, RRID:AB_2220311) was purchased from Enzo Life Sciences and rabbit polyclonal ERα antibody MC-20 (Cat# sc-542, RRID:AB_631470) was obtained from Santa Cruz Biotechnology. Rabbit polyclonal phospho-AMPKβ1 Ser108 and AMPKβ1 antibodies (Cat# 4181, RRID:AB_2169743; Cat# 4182, RRID:AB_2169740), as well as the nuclear stain DRAQ5, were obtained from Cell Signaling Technologies. Secondary goat anti-rat and anti-rabbit IgG (H + L) Alexa Fluor 488 antibodies (Cat# A-11006, RRID:AB_2534074; Cat# A-11008, RRID:AB_143165) and mouse monoclonal claudin-5 antibody (Cat# 35-2500, RRID:AB_2533200) were purchased from Invitrogen. Mouse monoclonal β-actin antibody (Cat# A5441, RRID:AB_476744), actinomycin D, cycloheximide, sodium cyanide (NaCN), Ficoll, and all other chemicals were all also purchased from Sigma-Aldrich.

Animals

All studies were approved by the National Institute of Environmental Health Sciences (NIEHS) Animal Care and Use Committee and conducted in agreement with the National Institutes of Health animal care and use guidelines. C57BL6J mice were obtained from Taconic. Estrogen receptor alpha knockout (ERαKO) and wild-type littermate mice were obtained from an NIEHS colony at Taconic (17). All animals were housed in temperature- and humidity-controlled space with a 12-hour light/dark cycle and ad libitum access to food and water. Animals were between the ages of 2 and 4 months at the time of each study, where female mice used for comparative studies were ovariectomized 2 weeks prior to sacrifice. Animals were killed by carbon dioxide and immediate decapitation. Brain capillaries were isolated and either immediately used for transport assay or immunohistochemistry experiments, or frozen for additional Western blotting analysis.

Capillary Isolation

Capillary isolation procedures were established and outlined previously (18, 19). Following carbon dioxide inhalation and decapitation, whole mouse brains were removed and transferred immediately to chilled phosphate-buffered saline (PBS) (in mM: 2.7 KCl, 1.5 KH₂PO4, 136.9 NaCl, 8.1 Na₂HPO4, 1 MgCl₂, 1 CaCl₂, 5 D-glucose, 1 sodium pyruvate, at pH 7.4). Cerebellum, midbrain, meninges and larger blood vessels, choroid plexus, white matter, and olfactory lobes were removed, and the remaining cerebral cortical tissue was homogenized. The resulting brain homogenate was combined with an equal volume of 30% Ficoll in PBS, after which capillaries were separated from remaining brain tissue by centrifugation (20 minutes, 5800g). Capillary pellets were washed with 1% bovine serum albumin in PBS and passed through a series of 30-μm cell strainers (pluriSelect), washed sequentially with PBS, and allowed to adhere to coverslip-bottomed imaging chamber slides for at least 15 minutes before treatment.

Transport Activity Assay

Transport assays with isolated brain capillaries using confocal microscopy- and imaging-based technologies were described previously (18, 19, 20, 21). All studies were conducted in imaging chamber slides filled with PBS at room temperature. BODIPY FL prazosin, NBD-CSA, or Texas Red, fluorescent substrates for Bcrp, P-glycoprotein, and Mrp2 respectively, were added with or without E2 or vehicle of equivalent volume and luminal fluorescence intensity was measured as an indicator of luminal substrate accumulation (22, 23, 24). In some studies, E2 was added with or without different signaling inhibitors. In every study, a specific ABC transporter inhibitor, KO143 for Bcrp, PSC833 for P-glycoprotein, and MK571 for Mrp2, was included as a treatment group in order to determine the transporter-specific component of luminal substrate accumulation. Capillaries were imaged through a 40× water-immersion objective (1.2 numeric aperture) with a Zeiss 510-inverted or 710-overhead confocal laser-scanning microscope, using a 488 nm laser line for BODIPY FL prazosin and NBD-CSA. A 543 nm laser line was used for Texas Red. Images were saved and the luminal florescence intensity was quantified using NIH Image J software as done previously (22, 23, 24).

Western Blotting

Capillary membrane and cytosolic fractions were isolated from control and E2-treated capillary pellet suspensions as characterized previously (25, 26, 27). Protein was assayed by the Bradford method to derive a standard curve. An aliquot of protein determined from the Bradford-derived standard curve was mixed with NuPAGE 4X sample buffer from Invitrogen. The mixture was loaded onto a 4% to 12% Bis-Tris NuPAGE gel, followed by electrophoresis and transfer to an Immobilon-FL membrane from Millipore. Odyssey Blocking Buffer from Li-Cor Biosciences was added to the membrane for 30 minutes at room temperature. The membrane was immunoblotted with primary antibodies against Bcrp (72 kDa, 1:50), claudin-5 (22-24 kDa, 1:100), AMPKβ1 (38 kDa, 1:1000), or phospho-AMPKβ1 (38 kDa, 1:1000) overnight at 4 °C. The membrane was then stained with the corresponding infrared dye-conjugated anti-mouse, anti-rat, or anti-rabbit secondary antibodies (1:1000, Li-Cor Biosciences) in PBS for 3 hours at room temperature and then washed in 0.05% Tween in PBS. The membrane was imaged using an Odyssey Infrared Imaging System from Li-Cor Biosciences. β-actin (42 kDa, 1:10000) was used as a loading control for all studies.

Immunostaining for Bcrp and ERα

After isolated capillaries adhered to the bottom of imaging chamber slides, they were fixed, permeabilized, and blocked in PBS as outlined previously (27). Capillaries in each treatment group were then incubated with primary antibodies against either Bcrp (1:50) or ERα (1:50) overnight at 4 °C, and then in secondary goat anti-rat or anti-rabbit IgG (H + L) Alexa Fluor 488 antibody (1:1000) with DRAQ5 (1:100) for 90 minutes at room temperature. Immunostained capillaries were imaged through a 40X water-immersion objective (1.2 numeric aperture) with a Zeiss 510-inverted confocal laser-scanning microscope using a 488 or 647 nm laser line. Images were saved and fluorescence intensity at each membrane and lumen was quantitated using NIH ImageJ software.

Statistical Analyses

Quantitative data are expressed as mean ± SEM. Statistical analyses of differences between experimental groups were performed by one-way analysis of variance (ANOVA) (Tukey multiple comparison test) or two-way ANOVA where appropriate using Prism software. Differences between experimental group means were considered significant when P < .05.

Results

Sex- and Gonad-Dependent Differences in Bcrp Substrate Transport and Bcrp and Claudin-5 Expression

We measured Bcrp transport activity in freshly isolated MBCs using a well-characterized confocal imaging-based assay to quantify luminal accumulation of the fluorescent Bcrp substrate BODIPY FL prazosin (6, 7, 23, 24). To determine any sex-based differences in Bcrp-mediated transport at the BBB ex vivo, we isolated MBCs from age-matched male, ovary-intact female, and ovariectomized (ovex) female mice. Brain capillaries from each of these 3 groups were allowed to reach a 30-minute steady state condition followed by incubation in media containing either 2 µM BODIPY FL prazosin alone to represent basal or control levels of Bcrp transport activity, or with the Bcrp inhibitor KO143 at a concentration of 10 µM. Figure 1A includes representative confocal micrographs of each of these conditions. Luminal BODIPY FL prazosin accumulation by fluorescence is noticeably reduced in capillaries derived from intact female mice relative to those from age-matched male and ovex female mice. KO143 exposure resulted in a visible and quantifiable decrease in luminal BODIPY FL prazosin accumulation in both male and ovex female MBCs to a level comparable to that in intact female MBCs at baseline (Fig. 1A and 1B). Specific Bcrp transport activity is quantified as the KO143-sensitive component of luminal BODIPY FL prazosin accumulation, or the component of substrate transport that is sensitive to treatment with an established pharmacological Bcrp transport inhibitor (6, 7, 23, 24).

Figure 1. — Gonadal status–dependent differences in luminal Bcrp substrate accumulation and Bcrp expression. (A) Representative confocal micrographs of mouse brain capillaries (MBCs) incubated for 30 minutes to steady state, followed by incubation in media containing BODIPY FL prazosin with or without KO143. Luminal fluorescence is visibly reduced in intact female MBCs compared to those isolated from male and ovex female mice. Exposure of male and ovex female MBCs to KO143 also results in a visible reduction in luminal fluorescence (scale bar, 10 µm). (B) Basal Bcrp transport activity is reduced in MBCs of intact female mice compared to those derived from age-matched male mice. This difference is normalized by ovariectomy. (C) Western blot demonstrating amount of monomeric Bcrp protein at MBC membranes differs by gonadal status. Each bar represents the mean value for 10 to 16 capillaries from a single isolation with tissue pooled from 10 mice. SEM bars depict variability and units are arbitrary fluorescence. Statistical comparisons: ***P < .001. Abbreviations: Bcrp, breast cancer resistance protein; ovex, ovariectomized.

To assess potential contributions of Bcrp expression to the observed sex-dependent differences in luminal substrate accumulation, we isolated brain capillary membranes from age-matched male, intact female, and ovex female mice. Western blot of isolate proteins indicated a reduced monomeric Bcrp protein amount at capillary membranes derived from intact female mice compared to those isolated from age-matched male and ovex female mice (Fig. 1C). These data indicate that differences in luminal BODIPY FL prazosin relative to sex and gonadal status correspond to those in Bcrp expression rather than functional Bcrp pump activity. Normalization with ovariectomy also indicates expression-level differences are mutable and likely depend on circulating steroid hormones, including E2, consistent with previous findings that sustained, longer-term E2 exposure downregulates Bcrp protein levels at the BBB (6, 7, 11).

E2 Rapidly and Reversibly Decreases Bcrp Transport Activity

We first determined whether an E2-elicited decrease in specific Bcrp transport activity occurred in MBCs. Figure 2A includes representative confocal micrographs of MBCs incubated for 30 minutes to steady state, followed by 30-minute incubation in media containing either 2 µM BODIPY FL prazosin alone or with 0.01 nM to 10 nM E2 to illustrate reductions in luminal BODIPY FL prazosin accumulation occurring with exposure to increasing concentrations of E2. Exposure of male MBCs to 0.01 nM to 1000 nM E2 resulted in a concentration-dependent decrease in specific Bcrp transport activity. Exposure to 0.01 nM to 100 nM E2 also resulted in a concentration-dependent decrease in specific Bcrp transport activity in ovex female MBCs, indicating that Bcrp sensitivity to E2 is comparable between the 2 groups (Fig. 2B). We utilized pooled brain capillaries isolated from male and ovex female mice, an E2 concentration of 1 nM, and exposure time of 30 minutes for all further experiments unless otherwise noted.

To determine the rate, duration, and potential reversibility of the E2-elicited decrease in specific Bcrp transport activity, we conducted a time course and reversibility assay (Fig. 2C) (22, 23, 24). Parallel samples of MBCs were incubated for 30 minutes to steady state before incubation in media containing 2 µM BODIPY FL prazosin and 1 nM E2 for 5, 15, and 30 minutes. After 30 minutes, E2-containing media was either (i) replaced with media containing only 2 µM BODIPY FL prazosin and incubated for an additional 5, 15, and 30 minutes; or (ii) maintained and imaged over the same time points for comparison relative to a sustained E2 exposure condition. Exposing MBCs to 1 nM E2 resulted in a significant decrease in specific Bcrp transport activity after 5 minutes with maximal reductions in transport observed in 30 minutes. Importantly, Bcrp transport activity increased significantly within 15 minutes of E2 removal relative to that in MBCs of the sustained exposure condition with a return to baseline levels within 30 minutes (Fig. 2C). These data indicate the E2 exposure results in a rapid and reversible decrease in specific Bcrp transport activity.

E2-Elicited Reduction in Bcrp Transport Activity Is Specific, Without Changes in Other Components of BBB Permeability

Bcrp expression differed between age-matched male, intact female, and ovex female MBCs. To determine if the reduction in specific Bcrp transport activity following 30-minute E2 exposure results from rapid degradation of Bcrp, we assessed its expression following E2 exposure by confocal imaging of immunostained MBCs and Western blotting. Using a Bcrp-specific antibody and nuclear stain DRAQ5 followed by confocal-based recognition of a fluorescent secondary antibody, we detected the presence of Bcrp in MBCs following 30-minute incubation in PBS alone or PBS containing 1 nM E2. E2 exposure caused no change in relative Bcrp abundance when normalized to DRAQ5 (Fig. 3A). Similarly, no qualitative or quantitative change in either Bcrp or claudin-5 protein expression was observed in membrane isolates of MBCs exposed to 1 nM E2 for 30 minutes relative to those incubated in control media by Western blot (Fig. 3B). These data indicate that the E2-elicited decrease in specific Bcrp transport activity does not involve alterations in Bcrp or claudin-5 expression.

Figure 3. — E2 does not affect Bcrp expression or other markers of MBC permeability. (A) Representative confocal micrographs of MBCs immunostained for Bcrp with and without nuclear stain DRAQ5 (scale bar, 10 µm). E2 exposure causes no change in relative Bcrp abundance when normalized to DRAQ5. (B) Western blot of MBC membranes showing no effect of 30-minute 1 nM E2 exposure on monomeric Bcrp protein or claudin-5 amount. (C) E2 exposure does not affect the activities of P-glycoprotein or Mrp2. (D) Representative confocal micrographs of MBCs incubated for 30 minutes to steady state, followed by incubation in media containing NBD-CSA or Texas Red with 1 nM E2 or 1 mM NaCN (scale bar, 10 µm). Each bar represents the mean value for 10 to 18 capillaries from a single isolation with tissue pooled from 5 mice. SEM bars depict variability and units are arbitrary fluorescence. Statistical comparisons: ***P < .001. Abbreviations: Bcrp, breast cancer resistance protein; E2, 17β-estradiol; NaCN, sodium cyanide.

To determine the potential of E2 exposure to decrease the activities of other ABC transporters present at the BBB, we examined the effect of E2 exposure on the luminal accumulation of the fluorescent P-glycoprotein substrate NBD-CSA and Mrp2 substrate Texas Red. MBCs were maintained to steady state as before and then incubated in media containing (i) 2 µM NBD-CSA or Texas Red alone; (ii) 2 µM NBD-CSA or Texas Red with 1 nM E2; or (iii) 2 µM NBD-CSA or Texas Red with 1 mM NaCN, which serves as a positive control for depleting intracellular adenosine triphosphate to inhibit active transport without transporter specificity (18). If E2 reduces Bcrp substrate transport in a nonspecific manner such as via ATP depletion or degradation of tight junctional complexes, E2 exposure would result in reduced luminal accumulation of off-target P-gp and Mrp2 substrates in addition to the Bcrp substrate BODIPY FL prazosin. However, exposing MBCs to 1 nM E2 had no effect on the luminal accumulation of either NBD-CSA or Texas Red while exposure to 10 mM NaCN resulted in a significant decrease in the luminal accumulation of both (Fig. 3C and 3D). Taken together, these data indicate that E2 modulates Bcrp transport function with relative specificity in that it does not alter the protein expression or membrane levels of Bcrp, specific activities of either P-glycoprotein or Mrp2, or other aspects of barrier permeability required for luminal fluorescent substrate accumulation and retention, including tight junction complex integrity and ATP availability (19, 24).

Extranuclear, Nongenomic Signaling Through ERα and AMPK Modulates Bcrp Transport Activity

The time course, reversibility, and specificity of the E2-elicited effect on Bcrp transport activity suggests a mechanism independent of new mRNA or protein synthesis. We first assessed the capacity of actinomycin D and cycloheximide, inhibitors of cellular transcriptional and translational machineries respectively, to block the decrease in specific Bcrp transport activity resulting from E2 exposure. Exposure of MBCs to either 1 µM actinomycin D or 200 µM cycloheximide did not attenuate the E2-elicited reduction in specific Bcrp transport activity, further supporting a nongenomic mode of action (Fig. 4A). Rapid, nongenomic E2-initiated signaling events have been shown to involve the classical nuclear receptors ERα and ERβ in addition to GPER in other systems (12, 13). We utilized the ERα-selective agonist PPT, ERβ-selective agonist DPN, and GPER-selective agonist G1 to investigate the sufficiency of activating each receptor to elicit a decrease in Bcrp transport activity. Exposing MBCs to 0.1 nM to 10 nM PPT resulted in a concentration-dependent decrease in specific Bcrp transport activity, whereas no effect was observed following exposure of MBCs to either 0.1 nM to 10 nM DPN or G1 (Fig. 4B). Therefore, selective agonism of ERα, but not of ERβ or GPER, elicits a reduction in Bcrp transport activity comparable to that observed with E2 exposure.

Figure 4. — Nongenomic, extranuclear signaling through ERα. (A) Inhibition of transcriptional and translational mechanisms does not block E2-elicited decrease in specific Bcrp transport activity. (B) The ERα-selective agonist PPT decreases specific Bcrp transport activity but DPN and G1 as agonists of ERβ and GPR30, respectively, do not. (C) Bcrp transport activity in MBCs from ERαKO mice is unaffected by exposure to 0.01 nM to 10 nM E2 compared to that of MBCs from their wild-type littermates. (D) Representative confocal micrographs of MBCs incubated for 30 minutes to steady state, followed by incubation in media containing BODIPY® FL prazosin with or without 1 nM E2. Luminal fluorescence is visibly reduced in E2-treated wild-type littermate MBCs but not in E2-treated ERαKO MBCs (scale bar, 10 µm). (E) Representative confocal micrographs of MBCs immunostained for ERα with and without nuclear stain DRAQ5 (scale bar, 10 µm). Each bar or point represents the mean value for 10 to 18 capillaries from a single isolation with tissue pooled from 5 mice. SEM bars depict variability and units are arbitrary fluorescence. Statistical comparisons: ***P < .001; **P < .01. Abbreviations: AMD, actinomycin D; Bcrp, breast cancer resistance protein; CHX, cycloheximide; E2, 17β-estradiol; ERα, estrogen receptor α; ERαKO, estrogenic receptor α knockout; WT, wild-type.

To determine a requirement of ERα for the decrease in specific Bcrp transport activity resulting from E2 exposure, we utilized the ERαKO mouse (17). In ERαKO MBCs, no significant change in Bcrp transport activity was observed following 30-minute exposure to 0.01 nM to 10 nM E2 whereas a reproducible dose-dependent decrease in Bcrp transport activity was observed in MBCs derived from their wild-type littermates. Further, exposure of MBCs from both ERαKO and wild-type littermate mice to 100 nM to 10000 nM E2 resulted in significant decreases in specific Bcrp transport activity (Fig. 4C). These data indicate that 0.01 nM to 10 nM E2 exposure reduces specific Bcrp transport activity in an ERα-dependent manner, but changes in luminal BODIPY FL prazosin accumulation in capillaries exposed to E2 concentrations at or above 100 nM reflect non-ERα-mediated processes. Figure 4D includes representative confocal micrographs of 1 nM E2-exposed ERαKO and wild-type littermate MBCs. We next investigated the expression of ERα in MBCs by confocal imaging of immunostained capillaries utilizing an ERα-specific antibody and nuclear stain DRAQ5 for relative localization. Figure 4E includes representative confocal micrographs of MBCs incubated for 30 minutes in PBS alone or containing 1 nM E2. In both conditions, ERα localized to the membranes and/or cytosol of MBCs by indirect immunofluorescence detection with minimal to no signal detected in capillary lumens or endothelial nuclei. Thus, modulation of Bcrp transport activity by E2 occurs by ERα-dependent signaling where persistent localization of the ERα at capillary membranes and/or cytosol without nuclear translocation is consistent with a rapid extranuclear ERα signaling program.

PI3K and Akt have been identified as downstream effectors of rapid ERα-mediated signaling in vascular endothelial cells (14, 28). If required for signal transduction here, pharmacologic blockade of their activity in MBCs should attenuate the decrease in specific Bcrp transport activity resulting from E2 exposure. Exposure of MBCs to either 25 μM LY294002 or 250 nM GSK690693, inhibitors of PI3K and Akt respectively, did not attenuate the E2-elicited reduction in specific Bcrp transport activity nor alter basal Bcrp transport activity. However, inhibition of AMPK with 500 nM dorsomorphin dihydrochloride completely blocked the E2-dependent decrease in specific Bcrp transport activity without affecting basal transport, indicating a requirement for AMPK activity but no apparent involvement of either PI3K or Akt (Fig. 5A and 5B). Further, Western blots of MBC cytosolic fractions showed increased phosphorylation of the AMPK β1 subunit with 30-minute 1 nM E2 exposure relative to control conditions and total AMPKβ1 (Fig. 5C). Taken together, these data indicate that E2 exposure decreases Bcrp transport activity via extranuclear, nongenomic ERα-dependent signaling requiring AMPK. We include an abbreviated working model in Fig. 5D.

Figure 5. — Signaling downstream of ERα involves AMPK. (A) Inhibition Akt and PI3K does not block E2-elicited decrease in specific Bcrp transport activity. (B) Treatment with an inhibitor of AMPK completely attenuates E2-elicited decrease in specific Bcrp transport activity. (C) Western blots of MBC cytosolic fractions showing increase in phosphorylated AMPKβ1 following exposure of MBCs to 1 nM E2 for 30 minutes. (D) Abbreviated working model. Each bar or point represents the mean value for 10 to 18 capillaries from a single isolation with tissue pooled from 5 mice. SEM bars depict variability and units are arbitrary fluorescence. Statistical comparisons: ***P < .001. Abbreviations: Bcrp, breast cancer resistance protein; DMD, dorsomorphin dihydrochloride; E2, 17β-estradiol; ERα, estrogen receptor α; ERαKO; GSK, GSK690693; LY, LY294002.

Effects of EDCs and Tamoxifen on Bcrp Transport Activity

Estrogenic signaling through ERα as a traditional nuclear receptor is well-documented and involves canonical nuclear hormone receptor dimerization and transcription factor activity to affect gene expression (12, 13). The agonist and antagonist properties of many established EDCs and SERMs have been characterized in terms of their transcriptional activities relative to endogenous E2 (29, 30, 31, 32, 33, 34). We tested the ability of 2 bisphenols found in manufactured plastics—bisphenol A (BPA) and bisphenol S (BPS)—as well as the chlorinated insecticide endosulfan and breast cancer drug tamoxifen to modulate specific Bcrp transport activity at the mouse BBB. Exposure of MBCs to 0.1 nM to 10000 nM BPA and BPS resulted in concentration-dependent decreases in specific Bcrp transport activity, while exposure to endosulfan over the same concentration range had no effect (Fig. 6A-6C). Interestingly, exposure of MBCs to 0.01 nM to 1 nM tamoxifen resulted in a significant increase in specific Bcrp transport activity, demonstrating inverse agonist activity relative to E2. Further, tamoxifen exposure had no effect on Bcrp transport at concentrations beyond 10 nM, the same range over which E2-dependent changes in Bcrp transport were no longer specific to or dependent on ERα (Fig. 6D).

Figure 6. — Bisphenols decrease whereas tamoxifen increases Bcrp transport activity. (A) Exposure of MBCs to 10 to 10000 nM (A) BPA and (B) BPS decrease Bcrp transport activity whereas (C) endosulfan has no effect over the same concentration range. (D) Exposure of MBCs to 0.01 nM to 1 nM tamoxifen increases Bcrp transport activity in MBCs. Each bar or point represents the mean value for 10 to 18 capillaries from a single isolation with tissue pooled from 5 mice. SEM bars depict variability and units are arbitrary fluorescence. Statistical comparisons: ***P < .001; **P < .01. Abbreviations: Bcrp, breast cancer resistance protein; BPA, bisphenol A; BPS, bisphenol S.

Discussion

We demonstrate that exposure of functional brain capillaries to nanomolar concentrations of the steroid hormone E2 results in rapid and specific reduction in Bcrp transport activity via extranuclear, nongenomic signaling through ERα and AMPK. This reduction was shown to occur independently of Akt-PI3K signaling, changes in Bcrp protein amount, P-glycoprotein and Mrp2 transport, tight junctional integrity, and ATP depletion. We then utilized this biological response to interrogate the rapid activities of known EDCs and SERMs at the mouse BBB. Rapid and long-term changes in Bcrp-mediated transport and expression downstream of sex-steroid treatment have been reported previously (6, 7, 11). Our results support previous findings regarding the concentration dependence, time course, reversibility, and specificity of the E2-elicited reduction in Bcrp transport activity. After confirming the estrogenic response in mice, we continued to investigate receptor modulation and the mechanism for required intracellular signaling. We report a novel case of extranuclear, nongenomic estrogenic signaling through ERα and downstream effector AMPK resulting in a rapid reduction in specific Bcrp transport activity. While previous studies indicated contributions by both ERα and ERβ, this discrepancy might result from differences in experimental design including species, strain, and hormonal status of animals used as well as time course (6).

AMPK activation during rapid ERα-mediated signaling has been reported previously via direct binding of ERα to AMPK or indirectly through LKB1 (35, 36). We determined involvement of AMPK here as well, demonstrating phosphorylation of the regulatory β1 subunit of AMPK following E2 exposure and attenuation of the E2-elicited decrease in Bcrp transport activity with blockade of AMPK activation using a selective inhibitor. The role of AMPK activation in protecting the BBB against oxidative stress has also been reported, and our results may suggest either the rapid involvement of or effect on Bcrp following oxidative stress at the BBB (37). Given the documented rapid actions of E2 in neurons and vascular endothelial cells, we reason that local production of E2 at sites of the cerebral cortex with relatively high aromatase expression may drive rapid and reversible changes in Bcrp-mediated transport at nearby capillaries of the BBB (38, 39, 40).

We utilized the E2-ERα-Bcrp relationship further to assay the rapid activities of EDCs and SERMs known to act as transcriptional ERα agonists and antagonists. We demonstrate the ability of BPA, BPS, and tamoxifen, but not endosulfan, to elicit rapid changes in Bcrp transport activity at the mouse BBB. Interestingly, the relative potencies of BPA and BPS shown are consistent with their relative activities as transcriptional ERα agonists by some reports (32, 33, 34). Additionally, the action of tamoxifen as an anti-estrogen through the activation function 2 (AF-2) domain of ERα at the transcription level may also translate here as exposure to tamoxifen increased Bcrp transport activity, reflecting inverse agonism activity. Unlike the bisphenols and tamoxifen, the transcriptional activity of endosulfan did not translate and may indicate a difference in its propensity for ERα agonism in the absence of nuclear translocation. Studies with different mouse models of mutated ERα function increase our understanding of extranuclear ERα-mediated signaling mechanisms and contributions of its different functional domains (41, 42). We reason for future studies to assay known and novel SERMs in terms of rapid Bcrp transport activity at the mouse BBB, and to utilize this model for further study of extranuclear ERα signaling at the receptor domain level.

Finally, we report a gonadal status–dependent difference in Bcrp-mediated transport at the mouse BBB with reduced expression of monomeric Bcrp protein at MBC membranes of intact female mice compared to those of male and ovex female mice. These findings are especially relevant to the documented role of ABC transporters in limiting therapeutic delivery to the brain. Successful therapeutic intervention often relies on the development of effective small-molecule drugs that target a disease mechanism at the molecular level. These agents must pass initial screening in murine models of disease before human clinical trials can commence. In some cases, observational differences in clinical outcomes that correlate strongly with patient demographics were not predicted by earlier-stage testing. Biological sex has been shown to drive differences in drug metabolism and clearance significant enough to require different dosing guidelines based on a patient's gonadal status, but only after the presentation of treatment disparity was observed clinically (43, 44). As a result, the importance of conducting initial studies in both male and female cohorts has gained greater attention over the past decade. Determining sex- and hormone- dependent differences in drug transport at the murine BBB may help predict disparities in patient outcome that occur following bench-to-clinic translation of CNS therapeutics. Differences in Bcrp expression by sex and gonadal status shown here and by other investigators were taken as a likely consequence of transcription regulation by E2. In this case, intact female mice would have reduced Bcrp expression at the BBB relative to male and ovex female mice primarily as a result of increased circulating estrogens synthesized by the ovaries. However, we show here comparable basal Bcrp transport activity in ERαKO MBCs compared to their wild-type littermates, consistent with previous reports demonstrating stable Bcrp expression in the absence of ERα and ERβ (6, 7). While our work focused on rapid estrogenic signaling involving Bcrp activity, we consider it worth noting that these findings support work demonstrating regulation of Bcrp expression by other ovarian sex steroid hormones including progesterone.

Acknowledgments

We thank Erica Scappini and C. Jeff Tucker of the NIEHS Fluorescence Microscopy and Imaging Center for confocal microscopy assistance. We thank members of the Receptor Biology Group for technical support and discussion during laboratory meetings: Yukitomo Arao, Laurel Coons, Katherine Hamilton, Sylvia Hewitt, and Tyler Ramsey. We also thank the staff at the NIEHS Animal Care Facilities for providing excellent care to the animals used in this study.

Abbreviations

ABC: ATP-binding cassette
AMPK: AMP-activated protein kinase
BBB: blood-brain barrier
Bcrp: breast cancer resistance protein
CNS: central nervous system
DPN: diarylpropionitrile
E2: 17β-estradiol
EDC: endocrine-disrupting compound
ERα/β: estrogen receptor alpha/beta
ERαKO: estrogen receptor alpha knockout
GPER: G-protein coupled estrogen receptor
MBC: mouse brain capillary
Mrp2: Multidrug resistance-associated protein 2
NIEHS: National Institute of Environmental Health Sciences
ovex: ovariectomized
PBS: phosphate-buffered saline
PPT: propyl pyrazole triol
SERM: selective estrogen receptor modulator

Contributor Information

David B Banks, Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA; The University of Chicago Pritzker School of Medicine, Chicago, IL 60637, USA.

Sydney L Lierz, Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA; North Carolina State University College of Veterinary Medicine, Raleigh, NC 27606, USA.

Ronald E Cannon, Laboratory of Toxicology and Toxicokinetics, National Cancer Institute, National Institutes of Health, Research Triangle Park, NC 27709, USA.

Kenneth S Korach, Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.

Funding

Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health to K.S.K., 1ZIAES070065

Author Contributions

Participated in research design: D.B.B., R.E.C, K.S.K.

Conducted experiments: D.B.B., S.L.L.

Performed data analysis: D.B.B., S.L.L.

Manuscript writing: D.B.B., R.E.C, K.S.K.

Disclosures

None of the authors have any disclosures.

Data Availability

Original data generated and analyzed during this study are included in this published article or in data repositories listed in References.

References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

Original data generated and analyzed during this study are included in this published article or in data repositories listed in References.

[bqae081-B1] 1. Abbott NJ, Patabendige AAK, Dolman DEM, Yusof SR, Begley DJ. Structure and function of the blood-brain barrier. Neurobiol Dis. 2010;37(1):13‐25. [DOI] [PubMed] [Google Scholar]

[bqae081-B2] 2. Abbott NJ. Blood-brain barrier structure and function and the challenges for CNS drug delivery. J Inherit Metab Dis. 2013;36(3):437‐449. [DOI] [PubMed] [Google Scholar]

[bqae081-B3] 3. Agarwal S, Hartz AMS, Elmquist WF, Bauer B. Breast cancer resistance protein and P-glycoprotein in brain cancer: two gatekeepers team up. Curr Pharm Des. 2011;17(26):2793‐2802. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bqae081-B4] 4. Römermann K, Helmer R, Löscher W. The antiepileptic drug lamotrigine is a substrate of mouse and human breast cancer resistance protein (ABCG2). Neuropharmacology. 2015;93:7‐14. [DOI] [PubMed] [Google Scholar]

[bqae081-B5] 5. Miller DS. Regulation of ABC transporters blood-brain barrier: the good, the bad, and the ugly. Adv Cancer Res. 2015;125:43‐70. [DOI] [PubMed] [Google Scholar]

[bqae081-B6] 6. Hartz AMS, Madole EK, Miller DS, Bauer B. Estrogen receptor beta signaling through phosphatase and tensin homolog/phosphoinositide 3-kinase/Akt/glycogen synthase kinase 3 down-regulates blood-brain barrier breast cancer resistance protein. J Pharmacol Exp Ther. 2010;334(2):467‐476. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bqae081-B7] 7. Hartz AMS, Mahringer A, Miller DS, Bauer B. 17-β-Estradiol: a powerful modulator of blood-brain barrier BCRP activity. J Cereb Blood Flow Metab. 2010;30(10):1742‐1755. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bqae081-B8] 8. Imai Y, Tsukahara S, Ishikawa E, Tsuruo T, Sugimoto Y. Estrone and 17beta-estradiol reverse breast cancer resistance protein-mediated multidrug resistance. Jpn J Cancer Res. 2002;93(3):231‐235. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bqae081-B9] 9. Sugimoto Y, Tsukahara S, Imai Y, Sugimoto Y, Ueda K, Tsuruo T. Reversal of breast cancer resistance protein-mediated drug resistance by estrogen antagonists and agonists. Mol Cancer Ther. 2003;2(1):105‐112. [PubMed] [Google Scholar]

[bqae081-B10] 10. Tanaka Y, Slitt AL, Leazer TM, Maher JM, Klaassen CD. Tissue distribution and hormonal regulation of the breast cancer resistance protein (Bcrp/Abcg2) in rats and mice. Biochem Biophys Res Commun. 2005;326(1):181‐187. [DOI] [PubMed] [Google Scholar]

[bqae081-B11] 11. Mahringer A, Fricker G. BCRP at the blood-brain barrier: genomic regulation by 17β-estradiol. Mol Pharm. 2010;7(5):1835‐1847. [DOI] [PubMed] [Google Scholar]

[bqae081-B12] 12. Hamilton KJ, Arao Y, Korach KS. Estrogen hormone physiology: reproductive findings from estrogen receptor mutant mice. Reprod Biol. 2014;14(1):3‐8. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bqae081-B13] 13. Hewitt SC, Winuthayanon W, Korach KS. What's new in estrogen receptor action in the female reproductive tract. J Mol Endocrinol. 2016;56(2):R55‐R71. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bqae081-B14] 14. Lu Q, Schnitzler GR, Ueda K, et al. ER alpha rapid signaling is required for estrogen induced proliferation and migration of vascular endothelial cells. PLoS One. 2016;11(4):e0152807. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bqae081-B15] 15. Kim SC, Boese AC, Moore MH, et al. Rapid estrogen receptor-α signaling mediated by ERK activation regulates vascular tone in male and ovary-intact female mice. Am J Physiol Heart Circ Physiol. 2018;314(2):H330‐H342. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bqae081-B16] 16. Niță A-R, Knock GA, Heads RJ. Signalling mechanisms in the cardiovascular protective effects of estrogen: with a focus on rapid/membrane signalling. Curr Res Physiol. 2021;4:103‐118. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bqae081-B17] 17. Hewitt SC, Kissling GE, Fieselman KE, Jayes FL, Gerrish KE, Korach KS. Biological and biochemical consequences of global deletion of exon 3 from the ER alpha gene. FASEB J. 2010;24(12):4660‐4667. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bqae081-B18] 18. Miller DS, Nobmann SN, Gutmann H, Toeroek M, Drewe J, Fricker G. Xenobiotic transport across isolated brain microvessels studied by confocal microscopy. Mol Pharmacol. 2000;58(6):1357‐1367. [DOI] [PubMed] [Google Scholar]

[bqae081-B19] 19. Hartz AMS, Bauer B, Fricker G, Miller DS. Rapid regulation of P-glycoprotein at the blood-brain barrier by endothelin-1. Mol Pharmacol. 2004;66(3):387‐394. [DOI] [PubMed] [Google Scholar]

[bqae081-B20] 20. Bauer B, Hartz AMS, Miller DS. Tumor necrosis factor alpha and endothelin-1 increase P-glycoprotein expression and transport activity at the blood-brain barrier. Mol Pharmacol. 2007;71(3):667‐675. [DOI] [PubMed] [Google Scholar]

[bqae081-B21] 21. Wang X, Campos CR, Peart JC, et al. Nrf2 upregulates ATP binding cassette transporter expression and activity at the blood-brain and blood-spinal cord barriers. J Neurosci. 2014;34(25):8585‐8593. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bqae081-B22] 22. Hawkins BT, Sykes DB, Miller DS. Rapid, reversible modulation of blood-brain barrier P-glycoprotein transport activity by vascular endothelial growth factor. J Neurosci. 2010;30(4):1417‐1425. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bqae081-B23] 23. Cannon RE, Peart JC, Hawkins BT, Campos CR, Miller DS. Targeting blood-brain barrier sphingolipid signaling reduces basal P-glycoprotein activity and improves drug delivery to the brain. Proc Natl Acad Sci U S A. 2012;109(39):15930‐15935. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bqae081-B24] 24. Banks DB, Chan GN, Evans RA, Miller DS, Cannon RE. Lysophosphatidic acid and amitriptyline signal through LPA1R to reduce P-glycoprotein transport at the blood-brain barrier. J Cereb Blood Flow Metab. 2018;38(5):857‐868. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bqae081-B25] 25. Bauer B, Hartz AMS, Pekcec A, Toellner K, Miller DS, Potschka H. Seizure-induced up-regulation of P-glycoprotein at the blood-brain barrier through glutamate and cyclooxygenase-2 signaling. Mol Pharmacol. 2008;73(5):1444‐1453. [DOI] [PubMed] [Google Scholar]

[bqae081-B26] 26. Hartz AMS, Bauer B, Block ML, Hong JS, Miller DS. Diesel exhaust particles induce oxidative stress, proinflammatory signaling, and P-glycoprotein up-regulation at the blood-brain barrier. FASEB J. 2008;22(8):2723‐2733. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bqae081-B27] 27. Wang X, Sykes DB, Miller DS. Constitutive androstane receptor-mediated up-regulation of ATP-driven xenobiotic efflux transporters at the blood-brain barrier. Mol Pharmacol. 2010;78(3):376‐383. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bqae081-B28] 28. Zhang T, Liang X, Shi L, et al. Estrogen receptor and PI3K/Akt signaling pathway involvement in S-(-)equol-induced activation of Nrf2/ARE in endothelial cells. PLoS One. 2013;8(11):e79075. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bqae081-B29] 29. Varayoud J, Monje L, Bernhardt T, Muñoz-de-Toro M, Luque EH, Ramos JG. Endosulfan modulates estrogen-dependent genes like a non-uterotrophic dose of 17beta-estradiol. Reprod Toxicol. 2008;26(2):138‐145. [DOI] [PubMed] [Google Scholar]

[bqae081-B30] 30. Li Y, Burns KA, Arao Y, Luh CJ, Korach KS. Differential estrogenic actions of endocrine-disrupting chemicals bisphenol A, bisphenol AF, and zearalenone through estrogen receptor α and β in vitro. Environ Health Perspect. 2012;120(7):1029‐1035. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bqae081-B31] 31. Li Y, Luh CJ, Burns KA, et al. Endocrine-disrupting chemicals (EDCs): in vitro mechanism of estrogenic activation and differential effects on ER target genes. Environ Health Perspect. 2013;121(4):459‐466. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bqae081-B32] 32. Kang JS, Choi J-S, Kim W-K, Lee Y-J, Park J-W. Estrogenic potency of bisphenol S, polyethersulfone and their metabolites generated by the rat liver S9 fractions on a MVLN cell using a luciferase reporter gene assay. Reprod Biol Endocrinol. 2014;12(1):102. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bqae081-B33] 33. Cano-Nicolau J, Vaillant C, Pellegrini E, Charlier TD, Kah O, Coumailleau P. Estrogenic effects of several BPA analogs in the developing zebrafish brain. Front Neurosci. 2016;10:112. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bqae081-B34] 34. Mesnage R, Phedonos A, Arno M, Balu S, Corton JC, Antoniou MN. Editor's highlight: transcriptome profiling reveals bisphenol A alternatives activate estrogen receptor alpha in human breast cancer cells. Toxicol Sci. 2017;158(2):431‐443. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bqae081-B35] 35. Lipovka Y, Chen H, Vagner J, Price TJ, Tsao T-S, Konhilas JP. Oestrogen receptors interact with the α-catalytic subunit of AMP-activated protein kinase. Biosci Rep. 2015;35(5):e00264. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bqae081-B36] 36. Kim SA, Lee KY, Kim J-R, Choi HC. Estrogenic compound attenuates angiotensin II-induced vascular smooth muscle cell proliferation through interaction between LKB1 and estrogen receptor α. J Pharmacol Sci. 2016;132(1):78‐85. [DOI] [PubMed] [Google Scholar]

[bqae081-B37] 37. Guo J-M, Shu H, Wang L, Xu J-J, Niu X-C, Zhang L. SIRT1-dependent AMPK pathway in the protection of estrogen against ischemic brain injury. CNS Neurosci Ther. 2017;23(4):360‐369. [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

Nongenomic ERα-AMPK Signaling Regulates Sex-Dependent Bcrp Transport Activity at the Blood-Brain Barrier

David B Banks

Sydney L Lierz

Ronald E Cannon

Kenneth S Korach

Abstract

Materials and Methods

Materials

Animals

Capillary Isolation

Transport Activity Assay

Western Blotting

Immunostaining for Bcrp and ERα

Statistical Analyses

Results

Sex- and Gonad-Dependent Differences in Bcrp Substrate Transport and Bcrp and Claudin-5 Expression

Figure 1.

E2 Rapidly and Reversibly Decreases Bcrp Transport Activity

Figure 2.

E2-Elicited Reduction in Bcrp Transport Activity Is Specific, Without Changes in Other Components of BBB Permeability

Figure 3.

Extranuclear, Nongenomic Signaling Through ERα and AMPK Modulates Bcrp Transport Activity

Figure 4.

Figure 5.

Effects of EDCs and Tamoxifen on Bcrp Transport Activity

Figure 6.

Discussion

Acknowledgments

Abbreviations

Contributor Information

Funding

Author Contributions

Disclosures

Data Availability

References

Associated Data

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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