Sildenafil citrate and sildenafil nitrate (NCX 911) are potent inhibitors of superoxide formation and gp91^phox expression in porcine pulmonary artery endothelial cells

Abstract

1 Acute respiratory distress syndrome (ARDS) is associated with increased superoxide (O 2 K À ) 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 K À 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-9a,11a-methanoepoxy-prostaglandin F 2a (U46619) (7sildenafil or NCX 911), for 16 h and O 2 K À formation measured spectrophometrically 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 0 ,5 0 -cyclic monophosphorothioate, Rp-isomer (Rp-8-BrcGMPS). NO release was studied using a fluorescence assay and O 2 K À -NO interactions by measuring nitrites.3 After a 16-h incubation with 10 nM U46619, both NCX 911 and sildenafil elicited a concentrationdependent inhibition of O 2 K À formation and gp91 phox expression, NCX 911 being more potent (IC 50 ; 0.26 nM) than sildenafil citrate (IC 50 ; 1.85 nM). These inhibitory effects were reversed by 1 mM ODQ and 10 mM 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 mM 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 K À formation, again blocked by 1 mM ODQ. NCX 911 reacted with O 2 K À generated by xanthine oxidase, an effect that was inhibited by superoxide dismutase (500 U ml À1 ). 4 Since O 2 K À 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 K À formation and preservation of NO bioavailability.

Novel soluble guanylyl cyclase stimulator BAY 41-2272 attenuates ischemia-reperfusion-induced lung injury

“…NO-cGMP signaling pathway has been shown to interfere with NADPH oxidase by inhibiting its assembly (10) and by inhibiting its expression (22). Indeed, we observed that BAY 41-2272 inhibited both the increase in ROS production and the Ppa increase induced by PMA, a stimulator of NADPH oxidase activity.…”

Reversal of Oxidative Stress-Induced Anxiety by Inhibition of Phosphodiesterase-2 in Mice

“…Furthermore, consistent with the effect of Bay 60-7550 on cGMP signaling and anxietyrelated behavior, it has been reported that mice deficient in cGMP-dependent protein kinase II, a mediator of the intracellular actions of cGMP, exhibit an anxiogenic-like behavioral phenotype (Werner et al, 2004). Moreover, PDE5, which is a cGMP-specific PDE, has been reported to inhibit NADPH oxidase via an increase in cGMP levels (Muzaffar et al, 2005).…”

Sildenafil Preserves Lung Endothelial Function and Prevents Pulmonary Vascular Remodeling in a Rat Model of Diastolic Heart Failure

“…42 Several mechanisms may account for the suppression of endothelial dysfunction by sildenafil, in that sildenafil can restore the expression of endothelial NO synthase, 43 promote its posttranslational activation by stimulating Akt-dependent endothelial NO synthase phosphorylation, 44 and endothelial Ca 2ϩ influx, 45 and inhibit Nox2 expression and superoxide production. 46 As lung endothelial dysfunction in the aortic banding model results primarily from increased ␤-actin expression and subsequent F-actin polymerization, cGMP-dependent activation of vasodilatorstimulated phosphoprotein and subsequent inhibition of actin filament formation may present a particularly intriguing mechanism by which sildenafil may restore endothelial function in this specific setting. 47 …”