P-glycoprotein expression in rat brain endothelial cells: evidence for regulation by transient oxidative stress

During ischaemia/reperfusion, cells of the blood-brain barrier are subjected to oxidative stress. This study uses primary cultured rat brain endothelial cells to examine the effect of such stresses on expression of multidrug transporters. H2O2 up to 500 muM applied to cell monolayers caused a concentration-dependent increase in expression of P-glycoprotein (Pgp) but not of multidrug resistance-associated protein (Mrp1). Concentrations >250 muM H2O2 decreased cell viability. Application of 100 muM H2O2 caused a significant increase after 48 h in Pgp functional activity, as assessed from [H-3]vincristine accumulation experiments. At this concentration, H2O2 produced a transient increase within 10 min followed by a sustained decrease in levels of intracellular reactive oxygen species (iROS), detectable by flow cytometry. Reoxygenation of cell monolayers after 6 h hypoxia gave rise to a similar transient increase in iROS and this also led to increased Pgp expression by 24 h. Increases were also observed within 4 h after both H2O2 and hypoxia/reoxygenation treatments in mdr1a and mdr1b mRNA. Evidence suggests this was due to enhanced transcription rather than mRNA stabilization. Therefore, oxidative stress, by changing Pgp expression, may affect movement of Pgp substrates in and out of the brain.