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

1 Acute respiratory distress syndrome ( ARDS) is associated with increased superoxide (O-2(center dot)-) 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 O-2(center dot)- formation and gp91(phox) (active catalytic subunit of NADPH oxidase) expression was investigated in cultured porcine pulmonary artery endothelial cells (PAECs). 2 PAECs were incubated with 10 nM TXA(2) analogue, 9,11-dideoxy-9 alpha,11 alpha-methanoepoxy-prosta-glandin F-2a (U46619) (+/- sildenafil or NCX 911), for 16 h and O-2(center dot)- formation measured spectro-phometrically and gp91(phox) 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 O-2(center dot)--NO interactions by measuring nitrites. 3 After a 16-h incubation with 10 nM U46619, both NCX 911 and sildenafil elicited a concentration-dependent inhibition of O-2(center dot)- formation and gp91(phox) expression, NCX 911 being more potent (IC50; 0.26 nM) than sildenafil citrate (IC50; 1.85 nM). These inhibitory effects were reversed by 1 mM ODQ and 10 mu M 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 mu M SIN-1 and blocked partially by the eNOS inhibitor 10 mM N-5-(1-iminoethyl)-ornithine (L-NIO). Acutely, sildenafil and NCX 911 also inhibited O-2(center dot)- formation, again blocked by 1 mM ODQ. NCX 911 reacted with O-2(center dot)- generated by xanthine oxidase, an effect that was inhibited by superoxide dismutase (500U ml(-1)). 4 Since O-2(center dot)- 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 O-2(center dot)- formation and preservation of NO bioavailability.