Sildenafil citrate is a PDE5 inhibitor (PDE-5i) that belongs to the ‘‘new class’’ of PDE-5i. PDE-5 selectively degrades cyclic guanosine monophosphate (cGMP), hence its inhibition by sildenafil raises cGMP intracellular levels inducing vasodilatation. In recent years, several studies have shown that sildenafil initially used for the treatment of erectile dysfunction, may have other therapeutic applications [10–16]. Because of its potent vasodila- tory action, sildenafil has also been extensively studied for treating primary or hypoxia-induced pulmonary hypertension [11,12,14,15,17]. Endothelial dysfunction is an important defect that contributes to erectile dysfunction and vascular disease in diabetes and is associated with insulin resistance [18]. It has been reported that sildenafil can improve endothelial dysfunction after acute and chronic treatment [19]. In diabetic rats PDE-5i (SK-3530) can activate Akt-1 signaling and inhibit proapoptotic stimuli main- taining erectile function [20]. A recent report showed that chronic treatment with sildenafil improve glucose metabolism in a model of high-fat fed mice, without directly affecting Akt-1 phosphorylation [21]. The molecular mechanisms for these data still await demonstration; a possible involvement of cGMP pathway in insulin signal transduction could be hypothesized. Sildenafil Effect in HUVECs Since several reports have shown that endothelial dysfunction is associated with insulin resistance [22–24], the aim of the present study was to investigate the role of sildenafil in NO production and changes in insulin signaling in HUVECs in basal and insulin resistance conditions. Methods Materials Human umbilical endothelial cells (HUVECs), human arterial endothelial cells (HAEC) EBM-2 and EGM-2MV media were purchased from Lonza (Walkersville, MD, USA). Human fetal corpora cavernosa smooth muscle cells (hfCC) were a kind gift of Prof. Mario Maggi. Phospho-Akt-1 (Ser-473 and Thr-308), Akt-1, phospho eNOS (Ser-1177), eNOS and iNOS antibodies were obtained from Cell Signaling (Waltham, Mass). TRIZOL, M- MLV reverse transcriptase and Platinium High Fidelity Taq were purchased from Invitrogen (Paisley, UK). BSA, Arginine, Glucose and Glucosamine (Gluc-N) were obtained from Sigma (St Louis, Mo). LY 294002, nitro-L-arginine methyl ester (L-NAME) a non selective inhibitor of all NOS isoenzymes (26) and DAF-2DA were purchased from Calbiochem (San Diego, CA). Sildenafil powder was obtained from Viagra pills (Pfizer Inc); pills were ground into Figure 1. PDE5 and iNOS expression in human endothelial cells. A: PDE5 mRNA expression in HUVEC and hfCC cells: RT-PCR shows that HUVEC express detectable levels of PDE-5 mRNA. Actin was used as control. B: Western Blot analysis for PDE5 protein expression. C: Real time PCR shows that insulin (30 min) and/or sildenafil (5 h) treatments induce PDE5 and eNOS but not iNOS expression *p,0.05 vs Ctrl, **p,0.01 vs Ctrl; { p,0.05 vs sildenafil. D: A representative Western Blot shows that insulin (30 min) and/or sildenafil (5 h) treatments do not alter iNOS protein levels. doi:10.1371/journal.pone.0014542.g001 PLoS ONE | www.plosone.org 2 January 2011 | Volume 6 | Issue 1 | e14542