Constitutive androstane receptor-mediated up-regulation of ATP-driven xenobiotic efflux transporters at the blood-brain barrier

Abstract

ATP-driven efflux transporters at the blood-brain barrier both protect against neurotoxicants and limit drug delivery to the brain. In other barrier and excretory tissues, efflux transporter expression is regulated by certain ligand-activated nuclear receptors. Here we identified constitutive androstane receptor (CAR) as a positive regulator of P-glycoprotein, multidrug resistance-associated protein 2 (Mrp2), and breast cancer resistance protein (BCRP) expression in rat and mouse brain capillaries. Exposing rat brain capillaries to the CAR activator, phenobarbital (PB), increased the transport activity and protein expression (Western blots) of P-glycoprotein, Mrp2, and BCRP. Induction of transport was abolished by the protein phosphatase 2A inhibitor, OA. Similar effects on transporter activity and expression were found when mouse brain capillaries were exposed to the mouse-specific CAR ligand, 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP). In brain capillaries from CAR-null mice, TCPOBOP did not increase transporter activity. Finally, treating mice with 0.33 mg/kg TCPOBOP or rats with 80 mg/kg PB increased P-glycoprotein-, Mrp2-, and BCRP-mediated transport and protein expression in brain capillaries assayed ex vivo. Thus, CAR activation selectively tightens the blood-brain barrier by increasing transport activity and protein expression of three xenobiotic efflux pumps.

Fig. 1.

CAR expression in the rat and mouse brain capillaries. A, reverse transcriptase-polymerase chain reaction showed CAR expression in rat and mouse liver and brain capillaries. 1, rat liver (positive control); 2, rat brain capillaries; 3, mouse liver (positive control); 4, mouse brain capillaries; 5, negative control. B, Western blot showing CAR protein expression in rat and mouse brain capillaries and capillary membrane fractions. C, immunostaining for CAR protein in freshly isolated rat brain. Representative confocal image shows CAR was distributed throughout the cytoplasm, plasma membrane, and the nucleus of the endothelial cells. Left, CAR immunostaining image; right, transmitted light image. White scale bar, 10 μm.

Fig. 2.

Representative confocal images of rat brain capillaries showing luminal accumulation of fluorescent substrates specific for P-glycoprotein (A; 2 μM NBD-CSA), Mrp2 (B; 2 μM Texas Red) and BCRP (C; 2 μM BODIPY-prazosin). In each case, exposing capillaries to 1 mM PB for 3 h before incubation with transport substrate increased luminal substrate accumulation. Luminal accumulation was substantially reduced when specific transport inhibitors were added to the medium (5 μM PSC833 for P-glycoprotein, 0.3 μM LTC₄ for Mrp2, and 5 μM FTC for BCRP). White scale bar, 10 μm.

Fig. 3.

Increased transport activity of P-glycoprotein (A and D), Mrp2 (B and E), and BCRP (C and F) in isolated rat brain capillaries after exposure to 1 mM PB. A to C, time course of PB action. Capillaries were exposed to PB for the time indicated. During the last hour of incubation, 2 μM NBD-CSA (A), 2 μM Texas Red (B), or 2 μM BODIPY-prazosin (C) was present in the medium. D to F, effect of OA inhibition of PP2A with on PB-induced increases in transport (4-h exposure). In both sets of experiments, capillaries treated with PSC833 (P-glycoprotein), LTC₄ (Mrp2), FTC, or Ko143 (BCRP) indicate specific inhibition of transport. Shown are mean ± S.E.M. for 8 to 12 capillaries from a single preparation (pooled brains from 10 rats). **, p < 0.01, significantly higher than control; ***, p < 0.001, significantly higher than control.

Fig. 4.

Up-regulation of P-gp-, Mrp2-, and BCRP-mediated transport and expression in rat brain capillaries after exposure to 1 mM PB in vitro. A to C, increases of specific transport activity over multiple experiments (five preparations for P-glycoprotein, three preparations for Mrp2, and three preparations for BCRP). D, Western blots showing increased expression of all three ABC transporters in capillaries exposed to 1 mM PB for 5 h.

Fig. 5.

Inhibiting transcription (A, C, and E; 1 μM actinomycin D) or translation (B, D, and F; 100 μg/ml cycloheximide) blocked the effects of 1 mM PB on P-glycoprotein, Mrp2, and BCRP transport activity. Shown are mean ± S.E.M. for 8 to 12 capillaries from a single preparation (pooled brains from 10 rats). **, p < 0.01, significantly higher than control; ***, p < 0.001, significantly higher than control.

Fig. 6.

TCPOBOP increases transport activity in mouse brain capillaries in vitro. Freshly isolated mouse brain capillaries were exposed to TCPOBOP for 4 h; during the last hour, fluorescent substrates (NBD-CSA for P-glycoprotein, Texas Red for Mrp2, and BODIPY-prazosin for BCRP) and transport inhibitors were present in the medium. A, C, and E, concentration-dependent induction of transport with TCPOBOP. B, D, and F, induction was abolished when capillaries were pretreated for 30 min (before TCPOBOP exposure) with the PP2A inhibitor OA. Shown are mean ± S.E.M. for 8 to 12 capillaries from a single preparation (pooled brains from 10 rats). **. p < 0.01, significantly higher than control; ***, p < 0.001, significantly higher than control.

Fig. 7.

TCPOBOP does not increase transport activity in brain capillaries from CAR-null mice. Same protocol as described in Fig. 6. A, P-glycoprotein; B, Mrp2; C, BCRP. Shown are mean ± S.E.M. for 8 to 12 capillaries from a single preparation (pooled brains from 10 rats). *, p < 0.05, significantly higher than control; ***, p < 0.001, significantly higher than control.

Fig. 8.

Increased transport activity and expression of P-glycoprotein, Mrp2, and BCRP after exposure to CAR and PXR ligands in vivo. C3H mice were treated by intraperitoneal injection with 50 mg/kg PCN (PXR ligand) or 0.33 mg/kg TCPOBOP (CAR ligand) daily for 2 consecutive days. Brains and livers were collected on day 3. A to C, transport in freshly isolated mouse brain capillaries (1-h incubation with fluorescent substrate plus inhibitors). A, NBD-CSA; B, Texas Red; C, BODIPY-prazosin. Shown are mean ± S.E.M. for 8 to 12 capillaries from a single preparation (pooled brains from 10 mice). *, p < 0.05, significantly higher than control; ***, p < 0.001, significantly higher than control. D to F, increases in specific transport activity of P-glycoprotein, Mrp2, and BCRP over four to five TCPOBOP dosing experiments.

Fig. 9.

Increased protein expression of P-glycoprotein, Mrp2, and BCRP after exposure to CAR ligand TCPOBOP in vivo. Western blots of P-gp, Mrp2, and BCRP in membranes from brain capillaries and livers of vehicle-injected (control) mice and mice treated with the CAR ligand TCPOBOP.

Fig. 10.

Increased transport activity and expression of P-gp, Mrp2, and BCRP after exposure to the CAR activator PB in vivo. Rats were treated with intraperitoneal injection with 80 mg/kg PB daily for four consecutive days. Brains and livers were collected on day 5. A to C, transport in freshly isolated brain capillaries (1-h incubation with fluorescent substrate plus inhibitors). A, NBD-CSA; B, Texas Red; C, BODIPY-prazosin. Shown are mean ± S.E.M. for 8 to 12 capillaries from a single preparation (pooled brains from 10 rats). ***, p < 0.001, significantly higher than control. D, Western blots of P-glycoprotein, Mrp2, and BCRP in membranes from brain capillaries and livers of vehicle-injected (control) rats and rats treated with the CAR activator PB.