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

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.

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

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

Multiple components of 2,4-dichlorophenoxyacetic acid uptake by rat choroid plexus.

Initial rates of uptake of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D; 20 microM) were measured in intact lateral choroid plexus from rat. Although inhibition of uptake by millimolar concentrations of estrone sulfate (ES) and unlabeled 2,4-D was maximal at 85%, inhibition by p-aminohippurate (PAH) saturated at about 50%. Inhibition by ES plus PAH was no greater than by ES or 2,4-D alone. Thus, inhibition studies indicated three distinct components of uptake; two mediated and one not. The sodium-dependent component of 2,4-D uptake coincided with the PAH-sensitive component, indicating uptake mediated by organic anion transporter subtype (Oat) 3. Consistent with this, efflux of 2,4-D from preloaded tissue was accelerated by all Oat3 substrates tested, and 2,4-D increased the efflux of the Oat3 substrate, PAH. Consistent with the inhibition data, kinetic analysis showed three components of 2,4-D uptake: a nonmediated component (linear kinetics), a high-affinity component, and a low-affinity component. The high-affinity component appeared to coincide with the PAH-sensitive and sodium-dependent component characterized in inhibition studies. The PAH-insensitive, low-affinity component was inhibited by ES, dehydroepiandrosterone sulfate, and taurocholate but not by 5-hydroxyindole acetic acid. Thus, the first step in transport of 2,4-D from cerebrospinal fluid to blood involves two transporters: Oat3 and a PAH-insensitive, sodium-independent transporter. Based on inhibitor profile, the latter may be Oatp3.

Fluorescein-methotrexate transport in rat choroid plexus analyzed using confocal microscopy.

One function of the vertebrate choroid plexus (CP) is removal of potentially toxic metabolites and xenobiotics from cerebrospinal fluid (CSF) to blood for subsequent excretion in urine and bile. We have used confocal microscopy and quantitative image analysis to follow transport of the large organic anion fluorescein-methotrexate (FL-MTX) from bath (CSF side) to blood vessels in intact rat CP and found concentrative transport from CSF to blood. With 2 microM FL-MTX in the bath, steady-state fluorescence in the subepithelium and vascular spaces exceeded bath levels by 5- to 10-fold, but fluorescence in epithelial cells was below bath levels. FL-MTX accumulation in subepithelium and vascular spaces was reduced by NaCN, Na removal, and by other organic anions, e.g., MTX, probenecid, and estrone sulfate. Increasing medium K 10-fold had no effect. None of these treatments affected cellular accumulation. However, two observations indicated that apical FL-MTX uptake was indeed mediated: first, cellular accumulation was a saturable function of medium substrate concentration; and second, digoxin and MK-571 reduced FL-MTX accumulation in the subepithelial/vascular spaces but also increased cellular accumulation severalfold. In the presence of digoxin and MK-571, cellular accumulation was concentrative, specific, and Na dependent. Thus transepithelial FL-MTX transport involved the following two mediated steps: Na-dependent uptake at the apical membrane and electroneutral efflux at the basolateral membrane, possibly on Oatp2 and Mrp1.

Organic anion transport in choroid plexus from wild-type and organic anion transporter 3 (Slc22a8)-null mice.

The choroid plexus actively transports endogenous, xenobiotic, and therapeutic compounds from cerebrospinal fluid to blood, thereby limiting their exposure to the central nervous system (CNS). Establishing the mechanisms responsible for this transport is critical to our understanding of basic choroid plexus physiology and will likely impact drug targeting to the CNS. We recently generated an organic anion transporter 3- (Oat3)-null mouse, which exhibited loss of PAH, estrone sulfate, and taurocholate transport in kidney and of fluorescein (FL) transport in choroid plexus. Here, we measured the uptake of four Oat3 substrates by choroid plexus from wild-type and Oat3-null mice to establish 1) the contribution of Oat3 to the apical uptake of each substrate and 2) the Na dependence of transport by Oat3 in the intact tissue. Mediated transport of PAH and FL was essentially abolished in tissue from Oat3-null mice. In contrast, only a 33% reduction in estrone sulfate uptake was observed in tissue from Oat3-null mice and, surprisingly, no reduction in taurocholate uptake could be detected. For PAH, FL, and estrone sulfate, all Oat3-mediated transport was Na dependent. However, estrone sulfate and taurocholate also exhibited additional mediated and Na-dependent components of uptake that were not attributed to Oat3, demonstrating the complexity of organic anion transport in this tissue and the need for further examination of expressed transporters and their energetics.

Mercuric chloride acting through Mg stimulates protein synthesis in Xenopus oocytes.

In Xenopus laevis oocytes, addition of HgCl2 (Hg) to the medium rapidly stimulated incorporation of [35S]methionine (MET) into protein, increasing incorporation up to five-fold over control values. The action of inorganic mercury persisted after removal of Hg. Microinjection of HgCl2 into oocytes maintained in buffer also increased MET incorporation. However, no such stimulation was found when Hg was microinjected into oocytes maintained under oil, suggesting that Hg action was dependent on a cell-medium interaction. Removing medium Mg2+ decreased insulin- and Hg-stimulated methionine incorporation. Increasing medium Mg2+ from 1 mM to 10 mM increased Hg-stimulated methionine incorporation twofold. Hypotonic swelling of oocytes stimulated hexose transport but inhibited protein synthesis. Together these data indicate that inorganic mercury activates translation in the oocyte through an Mg-dependent mechanism, possibly increased Mg2+ influx.

Confocal imaging of organic anion transport in intact rat choroid plexus.

We used confocal microscopy and quantitative image analysis to follow the movement of the fluorescent organic anion fluorescein (FL) from bath to cell and cell to blood vessel in intact rat lateral choroid plexus. FL accumulation in epithelial cells and underlying vessels was rapid, concentrative, and reduced by other organic anions. At steady state, cell fluorescence exceeded bath fluorescence by a factor of 3-5, and vessel fluorescence exceeded cell fluorescence by a factor of approximately 2. In cells, FL distributed between diffuse and punctate compartments. Cell and vessel accumulation of FL decreased when metabolism was inhibited by KCN, when bath Na(+) was reduced from 130 to 26 mM, and when the Na(+) gradient was collapsed with ouabain. Cell and vessel accumulation increased by >50% when 1-10 microM glutarate was added to the bath. Finally, transport of FL and carboxyfluorescein (generated intracellularly from carboxyfluorescein diacetate) from cell to blood vessel was greatly diminished when medium K(+) concentration ([K(+)]) was increased 10-fold. These results 1) validate a new approach to the study of choroid plexus function, and 2) indicate a two-step mechanism for transepithelial organic anion transport: indirect coupling of uptake to Na(+) at the apical membrane and electrical potential-driven efflux at the basolateral membrane.