Recent progress has been in 3 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 mapping of a signaling pathway by which DNA damage induces P-glycoprotein expression. Non-Genomic Signaling: P-glycoprotein is a major obstacle to the delivery of small molecule drugs across the blood-brain barrier (BBB) and into the CNS. We have tested a novel, signaling-based strategy to overcome this obstacle. We previously 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. Recent transport experiments with isolated brain capillaries have extended the signaling pathway downstream of S1PR1 to include PI-3K, Akt and mTOR. Western blots show rapid, transient phosphorylation (activation) of Akt following exposure to S1P. Consistent with a role for mTOR in signaling, micromolar concentrations of L-leucine rapidly reduce P-glycoprotein transport activity; these effects are blocked by the mTOR inhibitor, rapamycin. We used inhibitors of PTEN, a PI3/AKT repressor, to show that the PI3K/AKT pathway modulates the basal activity of P-glycoprotein at the BBB. Inhibitors of PTEN rapidly (minutes) increase P-glycoprotein transport activity in vitro. PTEN modulation of the S1P/AKT/mTOR signaling pathway will be validated in vivo by brain perfusion experiments. Lysophosphatidic acid (LPA) signaling at the blood-brain barrier: We identified a second non-genomic phospholipid signaling pathway that rapidly and reversibly reduces P-glycoprotein transport activity at low nM concentrations. Similar to S1P, LPA signals through a family of G-protein coupled receptors to fully inhibit P-glycoprotein activity in rat brain capillaries within 15 minutes. Removal of LPA causes a restoration of P-glycoprotein activity to control levels. Mrp-1 or Bcrp transport activity remained unchanged by LPA treatment indicating (1) the effect is specific for P-glycoprotein and (2) that leakage through the endothelial cell tight junctions is not responsible for the effect. Receptor antagonist for 1, 3 and 5 completely block the LPA repressive effect on P-glycoprotein transport. Ongoing work will focus on identifying the receptor and downstream signaling molecules responsible for the rapid and reversible inhibition of P-glycoprotein transport activity at the BBB. This project may lead to new strategies for drug delivery in patients where BBB P-glycoprotein limits pharmacotherapy. Nuclear Receptor Regulation of Transporter Expression: Progress in this area involved defining the role of the ligand-activated nuclear receptor PPAR-alpha in ABC transporter regulation. Peroxisome Proliferator Activated Receptor alpha, PPAR-alpha, is a master regulator of lipid metabolism. Endogenous free fatty acids act as natural PPAR-alpha ligands whereas anti-hyperlipidemic fibrate drugs are synthetic ligands. In addition, recent work has shown that environmental toxicants, like the flame retardant, perfluorooctanesulfonic acid (PFOS), can activate PPAR-alpha. PPAR-alpha regulates beta-oxidation of fatty acids. It also plays a role in regulation of drug metabolism by inducing expression of drug metabolizing enzymes and some ATP binding cassette (ABC) transporters in liver, kidney and skeletal muscle. We have found that clofibrate at uM concentrations and PFOS, at nM concentrations, increase transport activity and expression of P-glycoprotein, MRP2 and BCRP in isolated rat brain capillaries. All effects on transporter activity and expression were blocked by the PPAR-alpha antagonist, GW6471. The extent to which clofibrate and PFOS dosing as well as high fat diet alters blood-brain barrier transporter expression and drug delivery to the CNS remains to be determined. Increased Transporter expression in response to DNA damage: Involvement of p53 in the mechanism by which Nrf2 signals increased ABC transporter expression at the blood-brain barrier led us to investigate whether genotoxic stress had similar effects. Radiation-induced DNA damage activates a signaling pathway that can promote DNA repair and in the canonical pathway p53 activation occurs downstream of repair signaling in the nucleus. We have activated p53 signaling through low level (1-4 Gy) radiation-induced DNA damage in isolated rat and mouse brain capillaries. In irradiated capillaries, P-glycoprotein expression and transport activity increase in an ATM-, p53- and NF-kB-dependent manner. Alkaline comet assays show dose-dependent DNA damage in rat brain micro-capillaries exposed to 1-6 Gy radiation. Capillaries from Nrf2-null mice respond to ionizing radiation in the same manner as capillaries from wild-type mice. Thus DNA damage, not oxidative stress is the initiating event. Initial experiments with rats given 4 Gy to the head show increased expression and transport activity of P-glycoprotein in brain capillaries 24 hours later. Taken together, these results suggest that genomic stress through radiation-induced DNA damage initiates signaling that upregulates blood-brain barrier drug efflux transporter expression. These results have important implications for CNS therapy, since radiation is often combined pharmacotherapy to treat CNS tumors. Hyperglycemia alters transporter expression at the BBB: The cellular mechanism linking hyperglycemia to altered transporter function at the BBB is not known. Under hyperglycemic conditions we assessed P-gp transport activity in isolated rat brain capillaries by measuring luminal accumulation of a fluorescent cyclosporine A analog that is a specific P-gp substrate. Increasing medium glucose concentration from 5 mm (control) to 25 or 50 mM for 4 h reduced P-glycoprotein (P-gp) transport activity by 40 % and 70 %, respectively. In contrast, 4 h exposure to 50 mM glucose did not alter transport activity of two other efflux transporters, Bcrp and Mrp2, indicating that the BBB did not breakdown (no leakage). Neither galactose nor mannose at 50 mM affected P-gp transport activity, excluding the possibility of an osmotic effect. To determine whether such effects would be seen in vivo, we perfused rat brains for 30 min in situ with buffer containing 25 or 50 mM glucose, isolated brain capillaries and measured P-gp transport activity 3 h later. In vivo brain exposure to 25 or 50 mM glucose both reduced P-gp activity measured ex vivo by 50 % (compared to 10 mM glucose control). We used isolated brain capillaries to investigate the mechanism by which high glucose reduced P-gp activity. Inhibitors of Protein Kinase C (PKC) (BIM and LY333531), proteasomal function (MG115 and MG132), lysosomal fusion (Bafilomycin A1) and E2 ubiquitin ligase (Bay 11-7082) blocked the loss of P-gp activity caused by 50 mM glucose. Thus, PKC, proteasomal degradation and protein ubiquitination were involved. Surprisingly, western blots showed no decrease in P-gp protein expression following 4 h and 6 h exposure of capillaries to 50 mM glucose. We speculate that degradation of a P-gp-associated membrane protein, rather than the transporter itself, is responsible for the loss in transport activity.
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