NO production in insulin-resistance conditions in presence of sildenafil As already established [7,8], in accordance with eNOS phosphorylation studies (figure 3), conditions simulating insulin resistance (HG and Gluc-N) markedly inhibited NO production induced by insulin in HUVECs (p,0.05). Sildenafil treatment (1 mM/72 h) significantly increased NO generation, both in the presence of HG (p,0.05) and Gluc-N (p,0.05). Moreover insulin blunted sildenafil-induced increase in NO generation under insulin resistance conditions. Such effect was significant in HG conditions (p,0.05), but did not reach statistical significance with Gluc-N (Figure 5A). Similar data were obtained in HAEC (Figure 5B). Discussion The present study confirms that HUVECs express PDE5 mRNA as previously reported [32] and shows for the first time that its expression is regulated by insulin. HUVECs acute treatment (5 h) with sildenafil induced a sharp increase in NO production independently of insulin stimulation. Furthermore, sildenafil was able to reverse the suppression in insulin-induced NO production, caused by the PI3-K inhibitor LY294002. Pre- treatment with L-NAME, a NOS inhibitor, completely blocks the burst of NO production induced by sildenafil, claiming for a NOS- dependent effect. Chronic treatment with sildenafil (72 h) increased NO production both in basal conditions as well as in insulin resistance conditions. It is well known that insulin promotes vasodilation and increases blood flow by modulating eNOS activity and expression through activation of the PI3-K/Akt-1 signalling [8,33]. As expected [9] Sildenafil Effect in HUVECs insulin stimulation induced NO production in HUVECs and inhibition of PI3K pathway by LY294002 reduced insulin- stimulated NO production. Our results show for the first time that in endothelial cells sildenafil enhances NO production by PDE5 inhibition and eNOS activation through a PI3K dependent pathway. We hypothesize that sildenafil, through specific inhibi- tion of PDE-5, could increase intracellular concentration of free Ca2+ by c-GMP. Moreover it is possible that sildenafil increases intracellular Ca2+ levels in a cGMP independent manner by activating K+ channels with large conductance (BKCa) [26]. It is known that the association of Ca2+/calmodulin complex with eNOS increases eNOS Ser1177 phosphorylation and decreases Thr495 dephosphorylation [34]. This mechanism could amplify NO production in response to the intracellular Ca2+ elevation. Our results show that sildenafil does not enhance Akt-1 phosphorylation in basal conditions, although Akt-1 Ser473pho- sphorylation has been shown to increase after sildenafil stimulation in striatal [35] and corpora cavernosa smooth muscle (20). Sildenafil differently, increases Akt-1 Ser473 phosphorylation in HUVECs when associated with insulin co-treatment in presence of higher oxidative stress levels (HG and Gluc-N), improving insulin action. These data are in accordance with previous studies demonstrating that sildenafil decreases ROS and nitrotyrosine cell production [36,37]. Interestingly, in basal conditions sildenafil increased NO production without modifying Akt-1 phosphoryla- tion (Fig. 2 and 3). Such effect was only partially reversed by PI3K inhibitor LY, without reaching baseline levels. This suggests a partial involvement of PI3K in the observed effect, whereas we can exclude the involvement of Akt-1 phosphorylation. It is possible that PI3K activates eNOS by modulating other kinases like Figure 6. Schematic summary of sildenafil action in endothelial cells. doi:10.1371/journal.pone.0014542.g006 PLoS ONE | www.plosone.org 7 January 2011 | Volume 6 | Issue 1 | e14542