The primary focus of this project has been 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, and 4) Western blotting to measure transporter expression. Using this approach, nine signaling pathways modulating p-glycoprotein transport activity have been identified. Results of these experiments are being used to construct detailed maps of each pathway to identify signaling targets that can be used to modify function of p-glycoprotein and other drug efflux transporters in the clinic. Since several pathways are triggered by signals associated with neurological disorders, e.g, inflammation, beta-amyloid, glutamate, reactive oxygen species, an important focus is on understanding how transporter function is altered in disease, an underappreciated issue for CNS pharmacotherapy.? ? Four signals rapidly (minutes) and reversibly reduce p-glycoprotein activity without changing transporter protein expression. 1) One pathway is triggered by lipopolysaccharide (LPS) or the proinflammatory cytokine, TNF-alpha. Even low levels of LPS or TNF-alpha abolish transporter activity without affecting tight junction integrity or activity of other luminal pumps. Regulation of activity is both rapidly reversible and cytoskeleton-dependent, suggesting removal of transporter to an internal compartment. Signaling is though endothelin release, ETB receptor, nitric oxide synthase (NOS) and protein kinase C isoform beta (PKC). 2) LPS activates a separate but minor NOS-dependent pathway. 3) Exposing brain capillaries to nanomolar concentrations of beta-amyloid protein rapidly reduces p-glycoprotein activity eventually causing targeting of transporter protein to the proteasome and reduced expression. These may be critical observations, since recent experiments show a role for blood-brain barrier p-glycoprotein in efflux of beta-amyloid from the brain and since autopsy samples show age is correlated positively with brain beta-amyloid levels and correlated negatively with p-glycoprotein. Our recent experiments with a transgenic mouse expressing human beta-amyloid protein show greatly depressed blood-brain barrier p-glycoprotein transport activity and expression. 4) Exposure to VEGF, a growth factor released following brain injury, causes rapid loss of transport activity with no change in transporter protein expression. Intraventricular injection of VEGF in rats and mice causes rapid loss of p-glycoprotein activity (with no change in tight junction permeability) as measured in vivo using brain perfusion. At a minimum, rapid, cytoskeletal-dependent trafficking of the transporter to an intracellular compartment appears to underlie all of these observations. The discovery of these rapid and reversible changes raises the possibility that in some instances targeted activation of signaling could provide a narrow window in time during which normally impermeant p-glycoprotein substrates, e.g., chemotherapeutics, would selectively enter the brain.? ? Five signaling pathways increase p-glycoprotein expression and transport function. 1) One is activated by endogenous metabolites and xenobiotics, e.g., steroid metabolites, chemotherapeutics, HIV protease inhibitors, glucocorticoids and anticonvulsives, that are ligands for the pregnane-X receptor. Using a transgenic mouse expressing human PXR we found that hPXR activation in vivo by the antibiotic rifampin (at plasma levels comparable to those measured in patients) increased blood-brain barrier p-glycoprotein activity, tightened the selective barrier and dramatically reduced the CNS efficacy of methadone, a p-glycoprotein substrate. 2) A second is activated by xenobiotics that are ligands for the constitutive androstane receptor (CAR). 3) Prolonged exposure to TNF-alpha activates a third pathway, suggesting a tightening of the selective blood-brain barrier with chronic inflammation. In this instance, signaling is through TNF-R1 and ET receptors, NOS, PKC and the transcription factor, NF-kB. 4) At autopsy, brains of people living in heavily polluted cities contain diesel exhaust particles (DEP). These particles are known to activate microglia to release reactive oxygen species. We found that low levels of DEP signaled through NADPH-oxidase, TNF-R1 and NOS, but not NF-kB to increase p-glycoprotein expression in brain capillaries. Our results suggest that DEP could target the blood-brain barrier, increasing p-glycoprotein expression and thus reducing CNS access of therapeutic drugs. 5) A fifth pathway is associated with upregulation of brain capillary p-glycoprotein following epileptic seizures, one explanation for drug-resistance in epilepsy. In brain capillaries, signaling was activated by micromolar glutamate acting through a NMDA-R, NOS, COX-2 and NF-kB. Glutamate was without effect in capillaries from COX-2 knockout mice. In an animal model of epilepsy, inhibiting COX-2 blocked focal upregulation of p-glycoprotein expression in the brain, suggesting a therapeutic approach to drug resistant epilepsy.? ? Initial studies now implicate all of these latter signaling pathways in the transcriptional regulation of BCRP and Mrp2 in rat and mouse brain capillaries. As with p-glycoprotein, transport assays show that specific transporter activity changes in step with transporter protein expression. Except for PXR and CAR dependent signaling, where expression of the three transporters increases with receptor activation, expression of p-glycoprotein, Mrp2 and BCRP does not necessarily change in parallel in response to a given signal. Together, these studies are beginning to define a complex signaling network that regulates expression and activity of key xenobiotic efflux transporters at the blood-brain barrier.
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