Sildenafil citrate (UK-92480 citrate) , a cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase type 5 (PDE5) Inhibitor, is used extensively for erectile dysfunction and less commonly for pulmonary hypertension.

PDE5:3.5 nM, PDE6:33 nM

在麻醉的狗体内,Sildenafil citrate增强盆骨神经刺激下阴茎的勃起功能.在Sprague-Dawley大鼠体内, 每天Sildenafil（20 mg/kg）以时间剂量依赖的方式改善勃起功能,同时Sildenafil明显减少细胞凋亡指数,并且增加akt 和eNOS的磷酸化作用.

在内皮细胞中,Sildenafil通过降低由高血糖引起的氧化应激和增加细胞内Ca2 +水平,改善胰岛素信号传导NO产生。Sildenafil citrate是一种强效的PDEⅤ型的可逆选择性抑制剂,其能够有效阻断cGMP的水解作用(Ki ～3 nM)。

COX enzyme assay in vitro: Expression of COX protein in insect cells is determined by assessing PG-synthetic capability in homogenates from cells incubated for 3 days with COX-1 or COX-2 baculovirus. Cells expressing COX-1 or COX-2 are homogenized and incubated with arachidonic acid (10 μM). COX activity is determined by monitoring PG production. No COX activity is detected in mock-infected Sf9 cells. Celecoxib are preincubated with crude 1% CHAPS homogenates (2-10 μg of protein) for 10 minutes before addition of arachidonic acid. PGE2 formed is detected by ELISA after 10 minute incubation.

Sildenafil is prepared in DMSO and stored, and then diluted with appropriate medium before use[2]. Cells at approximately 90% confluence are harvested with 0.1% trypsin/0.01% ethylene diamine tetraacetic acid (EDTA) solution and seeded into a 96-well plate at a density of 2×104 cells/well and grown in RPMI-1640 containing 10% FBS for three days, followed by serum starvation for three days. Cells are then incubated for different time with various concentration of serotonin or 1 μM Sildenafil followed by serotonin with or without U0126, as indicated. Control cells are treated in the same way except sterile PBS replaced the drug. After treatment, medium is changed to fresh medium, and cells are incubated with 5 g/L of MTT for four hours. MTT is then dissolved with 150 μL of 10% DMSO for 20 minutes. The optical densities (OD) in the 96-well plates are determined using a microplate reader at 570 nm[2].