Effects of MAP kinase inhibitors on H2O2-promoted eNOS activation and phosphorylation.
Panel A shows the results of immunoblots analyzed in lysates prepared from adult murine cardiac myocytes treated with hydrogen peroxide (H2O2, 25 µM) for the indicated times. Cell lysates were analyzed in immunoblots probed using antibodies directed against phospho-MEK (Ser217/221), phospho-ERK1/2 (Thr202/Tyr204), total MEK, ERK, and GAPDH, as indicated. Below each immunoblot are the results of densitometric analyses from pooled data, showing the fold increase in protein phosphorylations (in arbitrary units) in cardiac myocytes treated with H2O2 at the indicated times plotted relative to the signals present in unstimulated cells. Each data point represents the mean ± SE derived from three independent experiments. The results are significant at the p<0.05 level. *indicates p<0.05 (ANOVA). Panel B adult mouse cardiac myocytes were loaded with the NO dye Cu2(FL2E), and then treated with PD98059 (37.4 µM), MEK inhibitor (1 µM) or vehicle followed by hydrogen peroxide (H2O2, 10 µM) treatment. Upper panel shows representative fluorescence images at 0, 2, 5, and 10 minutes followed treatments as indicated. Lower panel shows representative fluorescence tracings of a cell treated with PBS (green line), H2O2 (red line), H2O2 in the presence of MEK1/2 inhibitor (purple line), or H2O2 in the presence of PD98059 (blue line). The results shown are representative of three independent experiments that yielded equivalent results. In panel C, cardiac myocytes were incubated with PD98059 (50 µM, 30 min) or vehicle, then treated with H2O2 (25 µM, 15 min) and analyzed in immunoblots probed with antibodies as shown. Panel D shows immunoblot analyses from cardiac myocytes incubated with MEK inhibitor (1 µM, 30 min) or vehicle, then treated with H2O2 (25 µM, 15 min). Below each representative immunoblot are shown the results of densitometric analyses from pooled data, documenting the changes in phospho-eNOS (Ser1177) plotted relative to the signal present in unstimulated cells. Each data point represents the mean ± S.E. derived from at least three independent experiments; *indicates p<0.05 (ANOVA).