Regulation of P-glycoprotein at the Blood-Brain Barrier

Over 1 billion people worldwide suffer from some type of central nervous system (CNS) disorder (WHO, The World Health Report, 2001). This includes depression, epilepsy, multiple sclerosis, brain tumors, Alzheimer’s and Parkinson’s disease, as well as infections of the brain such as meningitis and HIV encephalitis. Consequently, there is a strong demand for effective treatments. However, pharmacotherapy of CNS disorders is greatly impaired by poor blood-to-brain transport of a large number of therapeutic drugs. The structure responsible for the low CNS penetration of drugs is the blood-brain barrier. An important element of barrier function is the ATP-driven efflux transporter, P-glycoprotein (P-gp) that denies CNS entry to a myriad of therapeutics. Thus, a better understanding of the mechanisms regulating P-gp expression and transport function might provide new strategies to improve drug delivery to the brain and increase drug levels in the CNS. Therefore, the objective of this thesis was to study regulation of P-glycoprotein at the blood-brain barrier. P-glycoprotein transport function was assessed by measuring accumulation of the fluorescent, P-gp-specific substrate, NBD-cyclosporine A (NBD-CSA), in the lumens of isolated rat brain capillaries using confocal microscopy and quantitative image analysis. Exposing capillaries to the hormone endothelin-1 (ET-1) rapidly and reversibly reduced luminal NBD-CSA accumulation. Importantly, ET-1 did not affect tight junctions. Sarafotoxin, an ET-B receptor agonist, also reduced P-gp-mediated transport; the effects of ET-1 and sarafotoxin were blocked by an ET-B receptor antagonist, but not by an ET-A receptor antagonist. Immunostaining localized the ET-B receptor to the luminal and abluminal membranes of brain capillaries. NBD-CSA transport was also reduced by the NO donor, sodium nitroprusside (SNP), and by the protein kinase C (PKC) activator, PMA. Inhibition of NO synthase (NOS) or PKC blocked the effect of ET-1; PKC inhibition blocked the effects of SNP and PMA. Thus, P-glycoprotein function at the blood-brain barrier is regulated in the short-term by ET-1 acting through an ET-B receptor, NOS, NO and PKC (Hartz et al., 2004). Transcriptional regulation of P-glycoprotein by the ligand-activated transcription factor, pregnane X receptor (PXR), was also demonstrated. PXR was shown for the first time to be expressed in isolated rat brain capillaries by RT-PCR, Western blot analysis and immunostaining. Six-hour exposure of isolated capillaries to the PXR ligands, PCN or dexamethasone, significantly increased expression of P-gp in the plasma membrane. Consistent with this, P-gp immunostaining demonstrated significantly increased immunoreactivity at the luminal membrane of capillaries. Increased P-gp-mediated NBD-CSA transport into capillary lumens was also detected. No such increases in P-gp expression were found when capillaries were exposed to ligands that activate human PXR (hyperforin, paclitaxel), but do not activate rodent PXR. Importantly, an increase in P-glycoprotein expression and transport function was also found in capillaries isolated from rats injected with PCN and dexamethasone (Bauer et al., 2004)