Cellular Mechanisms Of Xenobiotic Transport

Environmental Health Sciences

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Abstract

The primary focus of this project is to understand the regulation of the ATP-driven xenobiotic efflux pump, p-glycoprotein, at the blood-brain barrier. To map the extracellular and intracellular signals that regulate this transporter, we use 1) pharmacological tools, 2) intact rat and mouse brain capillaries, 2) fluorescent p-glycoprotein substrates, 3) confocal imaging to measure transport function, and 4) Western blotting to measure transporter expression. With this approach, eight signaling pathways modulating p-glycoprotein transport activity have been identified. Current experiments are focused on constructing detailed maps of each pathway to identify targets that can be used to modify p-glycoprotein function in the clinic. Since several pathways are triggered by signals associated with neurological disorders, e.g, inflammation, beta-amyloid, glutamate, reactive oxygen species, a secondary focus is on understanding how transporter function is altered in disease, an underappreciated issue for CNS pharmacotherapy.[unreadable] [unreadable] 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. Signaling is though endothelin release, ETB receptor, nitric oxide synthase (NOS) and protein kinase C (PKC). 2) LPS activates a separate but minor NOS-dependent pathway. 3) p-Glycoprotein activity is rapidly reduced when capillaries were exposed to nanomolar concentrations of beta-amyloid. In a transgenic mouse expressing human beta-amyloid, blood-brain barrier p-glycoprotein transport activity and expression are dramatically reduced. 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. 4) Exposure to VEGF, a growth factor released following brain injury, causes rapid loss of transport activity with no change in transporter protein expression. 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.[unreadable] [unreadable] Four pathways increase p-glycoprotein function and protein expression. 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) Prolonged exposure to TNF-alpha activated a second pathway, suggesting a tightening of the selective blood-brain barrier with chronic inflammation. In this instance, signaling was through TNF-R1 and ET receptors, NOS, PKC and the transcription factor, NF-kB. 3) At autopsy, brains of people living in heavily polluted cities contain diesel exhaust particles (DEP). These particles activate microglia. 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. 4) A fourth pathway is associated with upregulation of brain capillary p-glycoprotein following epileptic seizures, one proposed explanation for drug-resistant epilepsy. In brain capillaries, signaling was activated by micromolar glutamate acting through a NMDA-R, NOS, COX-2 and NF-kB. 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.