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 three radiolabeled P-glycoprotein substrate (including one chemotherapeutic) 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 examined whether the Glucocorticoid receptor (GR), a ligand-activated nuclear receptor targeted by both natural and synthetic glucocorticoids, regulates P-gp at CNS barriers. Naturally occurring glucocorticoids, cortisol in the human and corticosterone in the rat, regulate a wide range of physiological effects including gluconeogenesis, homeostasis, and apoptosis. However, the role of these hormones in maintenance of CNS barriers remains unresolved. Furthermore, the effects of synthetic glucocorticoids, a broad class of widely prescribed anti-inflammatory drugs, on CNS barriers are also poorly understood. However, these potent anti-inflammatory synthetic glucocorticoids are a mainstay in the treatment of cerebral edema and spinal cord injury. We hypothesize that both natural and synthetic glucocorticoids alter the expression and activity of P-gp at CNS barriers, thereby modifying drug delivery to the CNS. We have confirmed the basal expression of GR in both CNS barriers by qPCR and immunoblotting and further found that the depletion of corticosterone via adrenalectomy significantly decreased the expression and activity of P-gp in both the BBB and the BSCB. In-vivo treatment of both intact and adrenalectomized (ADX) rats with the synthetic glucocorticoid dexamethasone significantly increased P-gp activity and protein expression in both CNS barriers. In-vitro treatment of BBB and BSCB capillaries with dexamethasone also increased P-gp expression and activity and co-treatment with the GR antagonist, RU-486, abolished these increases. These results demonstrate that the endogenous glucocorticoid, corticosterone, maintains P-gp expression and activity at both the BBB and BSCB while the synthetic glucocorticoid, dexamethasone, in the presence or absence of endogenous glucocorticoids, increases the activity and expression of P-gp in a GR-dependent manner. Thus, natural glucocorticoids have a protective role in maintaining CNS barriers, while synthetic glucocorticoids may hinder delivery of therapeutic drugs to the CNS. Blood-Spinal Cord Barrier: 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. In a second project, we have collabortated with a group at Thomas Jefferson University medical School to how that P-glycoprotein and BCRP expression and transport activity is upregulated in spinal cord capillaries from an animal model of ALS and in spinal cord samples fron ALS patients. These finding indicate that one consequence of the disease is resistance to drugs that are P-glycoprotein or BCRP substrates.
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