Figure 5.

BI1029539 effect on glutamate-mediated up-regulation of P-gp and mPGES-1 levels in isolated brain capillaries. A) Isolated brain capillary from hmPGES-1 mice immunostained for hmPGES-1 (green); nuclei are counterstained with DAPI (blue). Scale bar, 5 μm. B) Negative control (no primary antibody) from left to right: transmitted light channel, green channel, blue channel, overlay of all 3 channels. Scale bar, 5 μm. C) Exposing isolated brain capillaries from hmPGES-1 mice to 100 μM glutamate increased P-gp protein expression levels. The glutamate effect on P-gp was blocked by 1 μM BI1029539, which by itself did not affect P-gp. Glutamate also slightly increased hmPGES1 protein levels in brain capillaries from hmPGES-1 mice; BI1029539 blocked this effect as well. BI1029539 itself did not affect hmPGES-1 protein levels. β-Actin was used as protein loading control. D) Representative images of brain capillaries from hmPGES-1 mice after addition of NBD-CSA to determine P-gp transport activity. Glutamate-treated capillaries show increased luminal NBD-CSA accumulation compared with controls. BI1029539 treatment maintained luminal NBD-CSA fluorescence at control levels; BI1029539 itself had no effect on luminal NBD-CSA fluorescence. Scale bar, 5 μm. E) Image analysis revealed that specific luminal NBD-CSA fluorescence was increased in glutamate-treated capillaries. BI1029539 blocked the glutamate effect and had no effect on P-gp transport activity by itself. For specific luminal NBD-CSA fluorescence, each data point represents means ± sem for 10 capillaries from 1 preparation of 50 hmPGES-1 mice. Units are arbitrary units (au; scale: 0–255). ***P < 0.001, significantly higher than controls.