Sildenafil Effect in HUVECs experimental conditions sildenafil co-treatment (1mM/72h) re- stored the ability of insulin to stimulate Akt-1 phosphorylation in presence of HG (+75% vs HG+insulin) or Gluc-N (+38% vs Gluc- N+insulin). Sildenafil improves eNOS Ser1177 phosphorylation in basal and in insulin-resistance conditions Since eNOS is a downstream effector of Akt-1, we studied eNOS Ser1177 phosphorylation in the same conditions shown in figure 2. Interestingly chronic (1 mM/72 h) and acute (1m M/5 h) sildenafil treatment enhanced eNOS Ser1177 phosphorylation by ,2 and 2.2 fold respectively (Fig. 3 and Fig. 4A) (p,0.05) under basal conditions. Moreover eNOS phosphorylation induced by acute sildenafil treatment is inhibited by LY294002 a PI3K inhibitor (Figure 4A). In accordance with previous data [30], HG reduced insulin induced eNOS Ser1177 phosphorylation by 25% (p,0.05). Sildenafil exposure (1 mM/72 h) markedly increased eNOS phosphorylation in HG conditions, both in the absence (+83% p,0.05) and in the presence (+74%, p,0.05) of insulin co- treatment (Figure 3), differently from what observed for Akt-1 Ser473 phosphorylation, where sildenafil was efficient only in the presence of insulin. As expected, eNOS phosphorylation was decreased by Gluc-N, in the presence of insulin (29%, p,0.05) and co-treatment with sildenafil was unable to improve eNOS phosphorylation, differently to what was observed for Akt-1 phosphorylation (Figure 3). Treatment with sildenafil induces NO production, not exclusively via PI3K pathway In order to confirm that the increase in eNOS Ser1177 phosphorylation induced by sildenafil displayed biological effects in term of NO production, we studied intracellular generation of NO by flow-cytometric analysis. Insulin (100 nM) stimulation for 1h induced a significant increase of NO production (p,0.05). Sildenafil treatment (1 mM/5 h) markedly enhanced NO produc- tion in HUVECs compared to control (p,0.01); such effect was ,1.5-fold higher than that obtained with insulin. Co-treatment with insulin blunted the effect of sildenafil, probably because of the insulin-induced increase in PDE-5 expression, previously de- scribed in fig. 1. In order to evaluate if sildenafil effects were mediated by PI3K pathway, the same experiments were performed in the presence of LY294002, a PI3K inhibitor. LY Figure 4. eNOS Ser1177 phosphorylation and NO production induced by acute treatment with sildenafil. A: Western Blot analysis of eNOS Ser1177 phosphorylation. Means 6 S.E. (n = 4). * p,0.05 vs Ctrl; u p,0.05 vs insulin; **p,0.01 vs sildenafil; uup,0.05 vs sildenafil+insulin. B: Sildenafil treatment (1 mM/5 h) induces NO production that is partially inhibited from LY. Means 6 S.E. (n = 6). *p,0.05 vs Ctrl; **p,0.01 vs Ctrl; { p,0.05 vs insulin; " p,0.05 vs LY; "" p,0.05 vs LY+insulin; # p,0.05 and ## p,0.01 vs sildenafil; I p,0.05 vs sildenafil+insulin. C: L-NAME effects on NO production induced by sildenafil treatment. L-NAME reduces NO production induced by acute treatment with sildenafil. Means 6 S.E. (n = 6). *p,0.05 vs Ctrl; # p,0.05 vs sildenafil. Full bar represent conditions without sildenafil, dashed bars represent sildenafil treatment. doi:10.1371/journal.pone.0014542.g004 PLoS ONE | www.plosone.org 5 January 2011 | Volume 6 | Issue 1 | e14542