Sildenafil citrate and sildenafil nitrate (NCX 911) are potent inhibitors of superoxide formation and gp91phox expression in porcine pulmonary artery endothelial cells

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Abstract
Acute respiratory distress syndrome (ARDS) is associated with increased superoxide (O2•−) formation in the pulmonary vasculature and negation of the bioavailability of nitric oxide (NO). Since NO inhibits NADPH oxidase expression through a cyclic GMP‐mediated mechanism, sildenafil, a type V phosphodiesterase inhibitor, may be therapeutically effective in ARDS through an augmentation of NO‐mediated inhibition of NADPH oxidase. Therefore, the effect of sildenafil citrate and NO‐donating sildenafil (NCX 911) on O2•− formation and gp91phox (active catalytic subunit of NADPH oxidase) expression was investigated in cultured porcine pulmonary artery endothelial cells (PAECs). PAECs were incubated with 10 nM TXA2 analogue, 9,11‐dideoxy‐9α,11α‐methanoepoxy‐prostaglandin F2α (U46619) (±sildenafil or NCX 911), for 16 h and O2•− formation measured spectrophometrically and gp91phox using Western blotting. The role of the NO‐cGMP axis was studied using morpholinosydnonimine hydrochloride (SIN‐1), the diethylamine/NO complex (DETA‐NONOate), the guanylyl cyclase inhibitor, 1H‐{1,2,4}oxadiazolo{4,3‐a}quinoxalin‐1‐one (ODQ), and the protein kinase G inhibitor, 8‐bromoguanosine‐3′,5′‐cyclic monophosphorothioate, Rp‐isomer (Rp‐8‐Br‐cGMPS). NO release was studied using a fluorescence assay and O2•−–NO interactions by measuring nitrites. After a 16‐h incubation with 10 nM U46619, both NCX 911 and sildenafil elicited a concentration‐dependent inhibition of O2•− formation and gp91phox expression, NCX 911 being more potent (IC50; 0.26 nM) than sildenafil citrate (IC50; 1.85 nM). These inhibitory effects were reversed by 1 μM ODQ and 10 μM Rp‐8‐Br‐cGMPS. NCX 911 stimulated the formation of cGMP in PAECs and generated NO in a cell‐free system to a greater degree than sildenafil citrate. The inhibitory effect of sildenafil was augmented by 1 μM SIN‐1 and blocked partially by the eNOS inhibitor 10 μM N5‐(1‐iminoethyl)‐ornithine (L‐NIO). Acutely, sildenafil and NCX 911 also inhibited O2•− formation, again blocked by 1 μM ODQ. NCX 911 reacted with O2•− generated by xanthine oxidase, an effect that was inhibited by superoxide dismutase (500 U ml−1). Since O2•− formation plays contributory role in ARDS, both sildenafil citrate and NCX 911 may be indicated for treating ARDS through suppression of NADPH oxidase expression and therefore of O2•− formation and preservation of NO bioavailability. British Journal of Pharmacology (2005) 146, 109–117. doi:10.1038/sj.bjp.0706305

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