Figure 4.

DEP activation of NADPH oxidase is followed by TNF-α release and TNF-α signaling through TNF-R1 to up-regulate P-glycoprotein. A) TIMP-3, an inhibitor of TNF-α converting enzyme, blocks the DEP-mediated increase in P-glycoprotein expression (Western blot) and transport function (specific luminal NBD-CSA fluorescence). B) An antibody against TNF-α blocks the effect of DEPs on P-glycoprotein; an IgG control antibody has no effect. C) DEPs induce TNF-α in brain capillaries (26 kDa: TNF-α precursor protein, 17 kDa: TNF-α mature protein). D) Blocking TNF-R1 with H398 abolishes DEP up-regulation of P-glycoprotein. E) In isolated capillaries of wild-type mice, DEPs increase P-glycoprotein expression (Western blot) and transport activity (specific luminal NBD-CSA fluorescence). The specific TNF-R1 blocker, H398, blocks the DEP effect. F) In capillaries isolated from TNF-R1-deficient mice, DEPs had no effect on P-glycoprotein expression levels or transport activity. Note that different band intensities between controls in the Western blots of wild-type and TNF-R1 knockout mice result from different exposure times of the blotting membrane. G) Western blot showing that the NADPH oxidase inhibitor DPI blocks DEP up-regulation of P-glycoprotein. H) ELISA showing that blocking NADPH oxidase with DPI brings TNF-α levels back to control levels. Specific luminal NBD-CSA fluorescence values are means ± se for 10 capillaries from a single preparation (pooled tissue from 10 rats or 15 mice); scale 0–255 au. ***P < 0.001.