Sildenafil Effect in HUVECs treatment reversed insulin effect in the absence of sildenafil (p,0.05), as expected [31]. In the absence of insulin LY markedly blunted sildenafil-induced NO production (p,0.01). However, LY did not completely reverse the effect of sildenafil since NO levels were not reduced to the baseline levels. These data suggest that in our model sildenafil effects are not mediated only through the PI3K pathway (Figure 4B). In order to confirm whether NO generation induced by sildenafil was due to eNOS activation, NO production studies were repeated in the presence of L-NAME, a NOS inhibitor. As expected, L-NAME treatment completely reversed to baseline levels both insulin- (data not shown) and sildenafil-induced NO generation (1mM/5h). (Figure 4 C). These results were confirmed also in HAEC (data not shown). Figure 5. NO production in insulin-resistance conditions after sildenafil treatment. Sildenafil treatment induces NO production in insulin resistance condition (HG and Gluc-N) in HUVEC (panel A) and HAEC (panel B). Full bars represent conditions without sildenafil, dashed bars represent sildenafil treatment. Means 6 S.E. (n = 6). *p,0.05 and **p,0.01 vs Ctrl; { p,0.05 and {{ p,0.01 vs insulin; { p,0.05 vs HG; $ p,0.05 vs sildenafil; # p,0.05 vs sildenafil+HG; ‘ p,0.05 vs Gluc-N. doi:10.1371/journal.pone.0014542.g005 PLoS ONE | www.plosone.org 6 January 2011 | Volume 6 | Issue 1 | e14542