Hydroxyurea phosphorylated eNOS in HUVECs. (A) Left panel: hydroxyurea (at concentrations of 30 and 50 μM) induced phosphorylation of eNOS at Ser1177 after 30 seconds of treatment and the signal remained for more than 1 minute; bradykinin (1 μM) induced similar phosphorylation. Right panel: corresponding total eNOS protein levels in HUVECs during incubation with hydroxyurea and bradykinin. Results are representative of 4 independent experiments. (B) Densitometric analysis of panel A during 5 minutes of incubation of HUVECs with hydroxyurea and bradykinin. Results are represented as the ratio of pSer1177 to total eNOS bands intensity. (C) PI 3-kinase inhibitors: LY294002 (LY, 50 μM) and wortmannin (W, 100 nM) demonstrated incomplete inhibition of eNOS phosphorylation at Ser1177 in HUVECs after 1 minute of incubation with hydroxyurea (50 μM) (upper bands). PKA inhibitor H-89 (20 μM) induced complete inhibition of eNOS phosphorylation at Ser1177 in HUVECs after 1 minute of incubation with hydroxyurea (50 μM) (upper band). Lower bands show total eNOS protein levels. Results are representative of 4 independent experiments.