Rapid Regulation of P-Glycoprotein at the Blood-Brain Barrier by Endothelin-1

Abstract

The ATP-driven xenobiotic transporter P-glycoprotein is a critical element of the blood-brain barrier. To study regulation of P-glycoprotein function, we measured specific transport [(3Ј-oxo-4-butenyl-4-methyl-threonine(1), (valine(2)) cyclosporin (PSC833)-sensitive] of the fluorescent cyclosporin A derivative [N-⑀(4-nitrobenzofurazan-7-yl)-D-Lys 8 ]-cyclosporin A (NBDL-CSA) into the lumens of isolated rat brain capillaries using confocal microscopy and quantitative image analysis. Luminal NBDL-CSA accumulation was rapidly and reversibly reduced in a concentration-dependent manner by 0.1 to 100 nM endothelin-1 (ET-1). In this concentration range, ET-1 did not affect junctional permeability. The ET B receptor agonist sarafotoxin 6c also reduced transport. An ET B receptor antagonist blocked effects of ET-1 and sarafotoxin 6c; an ET A receptor antagonist was without effect. Consistent with this, immunostaining and Western blotting showed expression of the ET B receptor in brain capillary membranes. NBDL-CSA transport was also reduced by sodium nitroprusside, a NO donor, and by phorbol ester, a protein kinase C (PKC) activator. Inhibition of NO synthase (NOS) or PKC abolished the ET-1 effects. Thus, ET-1, acting through an ET B receptor, NOS, and PKC rapidly and reversibly reduced transport mediated by P-glycoprotein at the blood-brain barrier.The structural basis of the blood-brain barrier is the nonfenestrated, brain capillary endothelium. Functionally, low passive permeability and the expression of selective, plasma membrane-bound transporters define this barrier tissue. These combine to both nourish the central nervous system (CNS) and protect it from potentially toxic chemicals. One critical selective component of the blood-brain barrier is Pglycoprotein, an ATP-driven, drug-efflux pump (Schinkel et al., 1994). This multispecific, primary active transporter, located at the luminal membrane of the brain capillary endothelium, is considered to be an important "gatekeeper" of the blood-brain barrier (Schinkel, 1999). As such, it protects the CNS from neurotoxicants and also limits brain penetration of a large number of drugs used to treat CNS disorders (Schinkel et al., 1996). In this regard, reduced P-glycoprotein function, whether the result of pump inhibition, genetic manipulation, or pathological change, substantially increases brain levels of administered drugs, including chemotherapeutics, human immunodeficiency virus protease inhibitors, anticonvulsants, antipsychotics, and glucocorticoids (Schinkel et al., 1996;Fellner et al., 2002;Goralski et al., 2003).For CNS therapy, it would clearly be advantageous to be able to modulate P-glycoprotein function over the short term while still retaining its protection over the longer term. One strategy that may accomplish this would be to transiently decrease specific efflux to the blood through rapid regulation. Unfortunately, we know little about the regulation of Pglycoprotein at the blood-brain barrier, and what we do know concerns mechanisms that take fro...

Activation of PKC Isoform βI at the Blood–Brain Barrier Rapidly Decreases P-Glycoprotein Activity and Enhances Drug Delivery to the Brain

“…Confocal images of capillaries were acquired using a Zeiss 510 meta-laser scanning confocal microscope (Zeiss, Jena, Germany) fitted with a  40 water-immersion objective (numerical aperture 1.2). Luminal fluorescence intensity was measured as described previously (Hartz et al, 2004;Miller et al, 2000) using NIH Image J software (WS Rasband, Image J, US National Institutes of Health, Bethesda, MD, USA, http://rsb.info.nih.gov/ij/, 1997 to 2008).…”

“…Brain capillaries were isolated from adult male retired breeder Sprague-Dawley rats as described previously (Hartz et al, 2004). Briefly, rats were killed by CO 2 inhalation, decapitated, and their brains were removed immediately to 41C phosphatebuffered saline (PBS) buffer (in mmol/L: KCl 2.7, KH 2 PO 4 1.5, NaCl 136.9, Na 2 HPO 4 4.3, CaCl 2 0.7, MgCl 2 0.5, D-glucose 5, and pyruvate 1, pH 7.4).…”

“…Tumor necrosis factor-a-induced loss of P-glycoprotein activity is signaled through TNF-a receptor 1, endothelin-1 (ET-1), ET B receptor, nitric oxide synthase, and protein kinase C (PKC). The effect of TNF-a or ET-1 is mimicked by treatment with 10 nmol/L PMA (phorbol-12-myristate-13-acetate), and is prevented by treatment with 10 nmol/L BIM-1 (bis-indolylmaleimide), indicating involvement of a classic PKC isoform (a/b I/II /g) (Hartz et al, 2004(Hartz et al, , 2006.…”

“…We previously suggested an alternative strategy for improving delivery of P-glycoprotein substrates to the brain: targeting signaling pathways that maintain transporter activity (Hartz et al, 2004(Hartz et al, , 2006. Studies from this laboratory have shown that exposing isolated rat brain capillaries to tumor necrosis factor (TNF)-a rapidly and reversibly reduces P-glycoprotein transport activity (Hartz et al, 2006).…”

Multidrug resistance gene and its relationship to ulcerative colitis and immune status of ulcerative colitis

“…ATP hydrolysis occurs at ATP-binding sites as an energy source, and the drug is then discharged into the extracellular region. As a result, P-gp reduces the oral bioavailability of substrates and MDR tumor cell toxicity, limiting the effects of the drugs (Hartz et al, 2004). Alternatively, this may also result from both internal drug resistance changes in UC and the development of inflammation (Salas et al, 2002).…”