Fig. 1.

Effects of H₂O₂ on cardiac myocyte NO synthesis and eNOS phosphorylation. (A) Adult mouse cardiac myocytes were loaded with the NO dye Cu₂(FL2E), and then treated either with PBS or H₂O₂ (10 μM). Fluorescence tracings are shown from a typical experiment, as well as representative differential interference contrast (DIC) and fluorescence images (1, 3, and 7 min). AU, arbitrary units. (B and C) Representative immunoblots from time course (B) or dose-response (C) experiments documenting the effects of H₂O₂ on eNOS phosphorylation at Ser1177 (peNOS¹¹⁷⁷) or Ser633 (peNOS⁶³³). (D) Cardiac myocytes were incubated with compound C (Comp C, 20 μM, 30 min) or vehicle, then treated with H₂O₂ and analyzed in immunoblots probed with antibodies as shown. (E) Immunoblot analyses from cardiac myocytes incubated with the PI3-kinase inhibitor wortmannin (1 μM, 30 min) or vehicle, then treated with H₂O₂. Below each representative immunoblot are shown the results of densitometric analyses from pooled data, documenting the changes in peNOS¹¹⁷⁷ and peNOS⁶³³ plotted relative to the signals present in unstimulated cells. Each data point represents the mean ± SE derived from at least three independent experiments; * indicates p < 0.05 and ** indicates p < 0.01.