Tumor Necrosis Factor and Endothelin-1 Increase P-Glycoprotein Expression and Transport Activity at the Blood-Brain Barrier

The ATP-driven drug efflux pump, P-glycoprotein, is a critical and selective element of the blood-brain barrier and a primary impediment to pharmacotherapy of central nervous system (CNS) disorders. Thus, an understanding of how P-glycoprotein function is regulated has the potential to improve CNS therapy. We recently demonstrated rapid (minutes) and reversible inactivation of P-glycoprotein in rat brain capillaries signaled through tumor necrosis factor(TNF) and endothelin-1 (ET1), components of the brain’s innate immune response. In this study, we examined the longer-term consequences of continuous exposure of rat brain capillaries to low levels of TNFand ET-1. Exposing brain capillaries to TNFor ET-1 caused a rapid decrease in P-glycoprotein transport activity with no change in transporter protein expression. This was followed by a 2to 3-h plateau at the low activity level and then by a sharp increase in both transport activity and protein expression. After 6 h, transport activity and transporter protein expression was double that of control samples. TNFsignaled through TNFR1, which in turn caused ET release and action through ETA and ETB receptors, nitric-oxide synthase, protein kinase C and nuclear factorB (NFB) and finally increased P-glycoprotein expression and transport activity. Assuming similar effects occur in vivo, the present results imply a tightening of the selective blood-brain barrier with chronic inflammation and thus reduced efficacy of CNS-acting drugs that are P-glycoprotein substrates. Moreover, involvement of NFB raises the possibility that other effectors acting through this transcription factor may have similar effects on this key blood-brain barrier transporter. The blood-brain barrier, which resides within the brain capillary endothelium, is a formidable obstacle to the transfer of xenobiotics from blood to brain. Barrier function reflects the low paracellular permeability of the endothelium (tight junctions), a low rate of transcytosis, and high expression of certain multispecific, ATP-driven xenobiotic efflux pumps (Begley, 2004b). Luminal plasma membrane location, high expression level, transport potency, and affinity for a large number of therapeutics make one of these pumps, Pglycoprotein, a primary impediment to blood-brain barrier penetration of drugs and thus a major determinant of CNS efficacy (Schinkel et al., 1996; Begley, 2004a). Indeed, mice with disrupted P-glycoprotein genes exhibit substantially increased brain levels of administered P-glycoprotein substrates, including chemotherapeutic agents, HIV protease inhibitors, anticonvulsant agents, antipsychotic agents, and glucocorticoids (Schinkel et al., 1996; Goralski et al., 2003). Although the influence of blood-brain barrier P-glycoprotein on CNS pharmacotherapy is well documented, little is known about mechanisms that regulate its expression and function in that tissue. Such an understanding would be important in devising strategies to treat CNS disorders that involve altered barrier function [e.g., epilepsy (Loscher and Potschka, 2005)] or that require barrier modification for therapy [e.g., gliobastoma (Fellner et al., 2002)]. In this regard, we have been investigating mechanisms that regulate Pglycoprotein activity in intact rat brain capillaries and have recently linked the brain’s innate immune response to rapid functional inactivation of blood-brain barrier P-glycoprotein This research was supported by the Intramural Research Program of the National Institutes of Health, National Institute of Environmental Health Sciences. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.106.029512. ABBREVIATIONS: CNS, central nervous system; TNF, tumor necrosis factor; IL, interleukin; LPS, lipopolysaccharide; TNF-R1, TNF receptor 1; ET-1, endothelin-1; ETB, endothelin receptor B; NOS, nitric-oxide synthase; PKC, protein kinase C; TLR4, toll-like receptor 4; NFB, nuclear factorB; RES-701-1, cyclic (Gly1-Asp9) (Gly-Asn-Trp-His-Gly-Thr-Ala-Pro-Asp-Trp-Phe-Phe-Asn-Tyr-Tyr-Trp); JKC-301, D-aspartyl-propyl-D-isoleucyl-leucyl-Dtryptophan; SN50, H-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Val-Gln-Arg-Lys-Arg-Gln-Lys-Leu-Met-Pro-OH; SN50M, HAla-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Pro-Val-Glu-Arg-Asn-Gly-Gln-Lys-Leu-Met-Pro-OH; BIM, bisindolylmaleimide I; PMA, phorbol 12myristate 13-acetate; PBS, phosphate-buffered saline; BSA, bovine serum albumin; NBD-CSA, [N-(4-nitrobenzofurazan-7-yl)-D-Lys]cyclosporin A; PSC833, valspodar; MK571, 3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid; Mrp, multidrug-resistance associated protein; ECE, endothelin-converting enzyme; L-NMMA, N-monomethyl-L-arginine; SNP, sodium nitroprusside. 0026-895X/07/7103-667–675 MOLECULAR PHARMACOLOGY Vol. 71, No. 3 U.S. Government work not protected by U.S. copyright 29512/3177147 Mol Pharmacol 71:667–675, 2007 Printed in U.S.A. 667 at A PE T Jornals on M arch 1, 2017 m oharm .aspeurnals.org D ow nladed from (Hartz et al., 2004, 2006). Brain capillary endothelial cells, like endothelial cells throughout the body, express receptors for cytokines [e.g., tumor necrosis factor(TNF) (Nadeau and Rivest, 1999) and interleukin-1 (IL-1) (Konsman et al., 2004)] and inflammogens [e.g., lipopolysaccharide (LPS) (Chakravarty and Herkenham, 2005)]. They both respond to inflammatory stimuli and amplify inflammatory signals (Nguyen et al., 2002; Rivest, 2003). Thus, brain capillaries are both targets for and active participants in the innate immune response. Our experiments show that exposing isolated rat brain capillaries to the proinflammatory cytokine TNFcaused a rapid and reversible loss of P-glycoprotein transport activity (Hartz et al., 2004, 2006). This occurred through the following sequence of events: TNFacting through TNF receptor 1 (TNF-R1) released endothelin-1 (ET1), which signaled through an ETB receptor to activate nitricoxide synthase (NOS), and then protein kinase C (PKC); activation of PKC reduced P-glycoprotein transport activity. The inflammogen LPS, acting through toll-like receptor 4 (TLR4) activated this pathway and rapidly reduced P-glycoprotein activity (Hartz et al., 2006). In these short-term experiments, neither capillary tight junctional permeability nor P-glycoprotein expression (protein) was changed. The present report addresses the longer-term consequences of TNFexposure on blood-brain barrier P-glycoprotein expression and function. Available information on how extended exposure to TNFaffects blood-brain barrier P-glycoprotein is limited and inconsistent. For example, in mice, Shiga-like toxin II increases P-glycoprotein expression in whole brain by a TNF-dependent mechanism (Zhao et al., 2002). In rat brain capillary endothelial cell lines, TNFhas been found to increase P-glycoprotein mRNA and decrease transport function but not affect protein expression (Mandi et al., 1998; Theron et al., 2003). In this study, we showed in isolated, intact rat brain capillaries that continuous exposure to low levels of TNFand ET-1 affected P-glycoprotein transport activity and protein expression in a complex, time-dependent manner. In these experiments, we chose to measure expression as protein, because this correlates with transport function. The initial rapid reduction in transport activity first described by Hartz et al. (2004, 2006), was followed by a 2to 3-h plateau at the reduced activity level and then by a rapid increase. After 6 h, both transport activity and transporter protein expression was double that of control samples. This increase involved signaling through the TNF-R1 receptor, ETA and ETB receptors, NOS, PKC and the transcription factor nuclear factorB (NFB). These findings disclose a novel signaling pathway through which chronic inflammation can up-regulate P-glycoprotein expression and activity and thereby tighten the blood-brain barrier to CNS-acting drugs that are P-glycoprotein substrates. Materials and Methods Chemicals. ET-1, RES-701-1, JKC-301, NFB transcriptional activation inhibitor, and the NFB nuclear translocation inhibitor SN50 were purchased from Calbiochem-Novabiochem (LaJolla, CA). Monoclonal antibody to human TNF-R1 H398 was from AlexisAxxora (San Diego, CA), bisindolylmaleimide I (BIM) was from Invitrogen (Carlsbad, CA), and phosphoramidon and phorbol 12-myristate 13-acetate (PMA) were from A.G. Scientific (San Diego, CA). C219 antibody was purchased from Signet (Dedham, MA). [N-(4Nitrobenzofurazan-7-yl)-D-Lys]cyclosporin A (NBD-CSA) was custom-synthesized by R. Wenger (Basel, CH) (Schramm et al., 1995). PSC833 was a kind gift from Novartis (Basel, CH). All other chemicals were obtained from Sigma (St. Louis, MO). Rat Brain Capillary Isolation. Rat brain capillaries were isolated as described previously (Hartz et al., 2004, 2006). In brief, Sprague-Dawley male retired rats (Taconic, Germantown, NY) were euthanized by CO2 inhalation and decapitated. Brains were taken immediately and kept at 4°C in PBS buffer (2.7 mM KCl, 1.46 mM KH2PO4, 136.9 mM NaCl, and 8.1 mM Na2HPO4, supplemented with 5 mM D-glucose and 1 mM sodium pyruvate, pH 7.4). Rat brains were dissected and homogenized in PBS. After addition of Ficoll (final concentration, 15%; Sigma, St. Louis, MO), the homogenate was centrifuged at 5800g for 20 min at 4°C. The pellet was resuspended in PBS containing 1% BSA and passed over a glass bead column. Capillaries adhering to the glass beads were collected by gentle agitation in PBS (1% BSA) and washed three times in BSA-free PBS. Freshly isolated capillaries were used for transport experiments, immunostaining and plasma membrane isolation followed by West-