The primary focus of this project is to understand regulation of the ATP-driven xenobiotic efflux pump, P-glycoprotein, at the blood-brain barrier. This focus is now expanded to include other blood-brain barrier efflux pumps, i.e., breast cancer resistance protein (BCRP) and multidrug resistance-associated protein 2 (Mrp2). To map the extracellular and intracellular signals that regulate these transporters, we use 1) pharmacological tools, 2) intact brain capillaries from rats and mice (including transgenics and knockouts), 2) fluorescent substrates, 3) confocal imaging to measure transport function, 4) Western blotting to measure transporter expression, and 5) brain perfusion in rats and mice in vivo to validate signaling-based changes in transporter function. Recent progress has been in three areas: identification of signals that rapidly reduce P-glycoprotein and BCRP activity without changing expression (non-genomic signaling), identification of ligand-activated nuclear receptors that upregulate transporter expression, and characterization of signals that alter P-glycoprotein expression in disease. Non-Genomic Signaling: Two distinct signaling pathways cause rapid but reversible loss of specific efflux transporter activity in isolated brain capillaries in vitro and in rats in vivo. In brain capillaries, activating VEGF signaling through src kinase and protein kinase C (PKC) isoform beta1 signaling separately reduce P-glycoprotein transport activity. In rats in vivo, both signals increase brain uptake of drugs that are P-glycoprotein substrates, but no increase in non-specific leakage through the blood-brain barrier. For both, loss of transport activity appears to arise from trafficking of the transporter from the plasma membrane to an intracellular vesicular compartment and we are developing techniques to actually measure loss of transporter protein from the luminal plasma membrane in vivo. With the identification of suitable agonists, VEGF and PKC provide enticing targets for clinically improving delivery of therapeutic drugs to the brain. In contrast, BCRP transport activity in rat and mouse brain capillaries is rapidly reduced by exposure to sub-nanomolar concentrations of estrogen. With time transporter expression decreases through increased degradation at the proteosome. This process is signaled primarily through estrogen receptor beta, PTEN, PI-3 kinase and Akt. Rapid loss of BCRP transport activity and delayed degradation also occurs in rats dose with estrogen. This provides a simple procedure to increase brain accumulation of drugs that are BCRP substrates. On the other hand, it suggests that environmental estrogens can target the blood-brain barrier to impair a transport-based protective mechanism. Nuclear Receptor Upregulation of Transporter Expression: We have found that therapeutic drugs, dietary constituents and environmental toxicants that specifically activate the nuclear receptors, Pregnane-X Receptor (PXR), Constitutive Androstane Receptor (CAR) or Aryl hydrocarbon Receptor (AhR), increase expression of blood-brain barrier P-glycoprotein, Mrp2 and BCRP in vitro (isolated brain capillaries) and in vivo (rats and mice). Such increased expression selectively tightens the barrier to a large number of therapeutic drugs, making CNS pharmacotherapy more difficult in exposed individuals. Moreover, this is one mechanism by which environment and dietary chemicals could alter responses to pharmacotherapy, making treatment of CNS diseases more difficult. On the other hand, our results also suggest that careful control of diet could be used to reduce expression of blood-brain barrier efflux transporters prior to CNS chemotherapy or to increase expression in those situations where there is a need for enhanced CNS protection. Transporter Expression in Disease: Our studies have focused on changes in expression of P-glycoprotein in epilepsy and Alzheimers disease. A substantial percentage of epileptics develop resistance to anti-epileptic drugs (AEDs). One basis for this is overexpression of drug efflux transporters at the blood-brain barrier. We have shown in an animal model of epilepsy that glutamate released during seizures signals through an NMDA receptor, PLA2, COX-2 and Nf-kB to upregulate P-glycoprotein expression. Such upregulation can be blocked in vitro and in vivo using inhibitors of COX-2, thereby increasing delivery of AEDs to the brain. In Alzheimers disease, we have shown that P-glycoprotein at the blood-brain barrier drives efflux of beta-amyloid from the brain. Moreover, we found substantially reduced blood-brain barrier expression and transport activity of P-glycoprotein in an mouse model of the disease (transgenic mouse expressing human APP). Treating these mice with a PXR ligand (above) for a week restored blood-brain barrier P-glycoprotein expression and transport activity and substantially reduced brain beta-amyloid levels. Together, these findings for animal models of epilepsy and Alzheimers disease show that targeting signals that regulate P-glycoprotein expression (up and down) has the potential to improve disease pharmacotherapy and possibly slow disease progression.
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