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) and the blood-spinal cord barrier. To map the extracellular and intracellular signals that regulate these transporters, we use 1) pharmacological tools, 2) intact brain and spinal cord 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 basal P-glycoprotein activity without changing expression (non-genomic signaling), identification of ligand-activated nuclear receptors that upregulate transporter expression, and characterization of the molecular basis for the blood-spinal cord barrier. Non-Genomic Signaling: P-glycoprotein is a major obstacle to the delivery of small molecule drugs across the blood-brain barrier and into the CNS. We have tested a novel, signaling-based strategy to overcome this obstacle. We mapped an extended, non-genomic signaling pathway that rapidly (minutes) and reversibly reduced P-glycoprotein transport activity without altering transporter protein expression;the defined pathway encompasses elements of proinflammatory, sphingolipid and protein kinase-based signaling. Central to this pathway is signaling through sphingosine-1-phosphate receptor 1 (S1PR1). S1P, the S1P analog, fingolimod (FTY720), currently in clinical trials for treatment of multiple sclerosis, and its active, phosphorylated metabolite (FTY720P) acted through S1PR1 to reduce P-glycoprotein transport activity. We validated these findings in vivo using in situ brain perfusion in rats. Administration of S1P, FTY720 or FTY729P increased brain uptake of a radiolabeled P-glycoprotein substrate without altering tight junctional permeability;blocking S1PR1 abolished this effect. Activation of this pathway also is effective in animals where P-glycoprotein expression/activity has increased due to exposure to the AhR ligand and persistent environmental pollutant, dioxin. Thus, targeting signaling through S1PR1 at the blood-brain barrier with a sphingolipid-based drug (FTY720) provides a means to rapidly and reversibly reduce basal P-glycoprotein activity and thus improve delivery of small molecule drugs to the brain. 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. Blood-Spinal Cord Barrier: This barrier lies within spinal cord capillaries. We have developed procedures to isolate these microvessels and study efflux transporter activity and expression. Initial experiments with capillaries from mice and rats indicate expression of three ABC transporters: P-glycoprotein, Mrp2 and Bcrp. As with the blood-brain barrier, expression of all three transporters is upregulated by ligands that activate the nuclear receptors, PXR, CAR and AhR;basal P-glycoprotein activity is rapidly reduced by TNF-alpha exposure, by activation of PKC-beta1 and by sphingolipid signaling. In all respects these results indicate that the molecular basis for xenobiotic transport and its regulation is similar for the two CNS barriers.
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