Inhibition of ERK and JNK provides partial rescue against 2-ClHDA-induced barrier dysfunction.

<p>BMVEC were plated on gold microelectrodes and cultured to confluence. Barrier function of endothelial monolayers was continuously monitored by impedance sensing at 4 kHz. After stabilization, cells were challenged (arrow) with 2-ClHDA in the absence or presence of (A) 100 µM PD098059, (C) 25 µM SP600125, or (E) 25 µM SB203580. Results represent mean values ± SD from 4 independent experiments. 2-ClHDA concentrations were 5 (A) and 10 (C, E) µM. (B, D, and F) Statistical evaluation of relative barrier function at the indicated time periods post 2-ClHDA treatment in the absence or presence of the respective antagonist. Impedance was normalized to baseline and represent mean values ± SD of 4 independent experiments (**p<0.01; ***p<0.001; two-way ANOVA). (G) BMVEC were incubated with 2-ClHDA (25 µM) in the absence or presence of PD098059 (100 µM) for the times indicated. Cells were lysed, aliquots of protein lysates were subjected to SDS-PAGE and transferred to PVDF membranes. (Phospho)Specific polyclonal antibodies against ERK1/2, p38, or JNK1/2 were used as primary antibodies. Immunoreactive bands were visualized with peroxidase-conjugated secondary antibodies using the ECL-system. (H) Bar graphs represent the ratio of optical densities of immunoreactive phosphorylated proteins normalized to non-phosphorylated proteins.