In adult respiratory distress syndrome (ARDS), platelets release thromboxane A₂ (TXA₂) which in turn promotes NADPH oxidase expression and superoxide (O₂^▪-) formation in the pulmonary vasculature (Muzaffar et al., 2004a). O₂^▪- then augments inflammation and hypertension associated with ARDS. NO inhibits NADPH oxidase expression through a cyclic GMP-mediated mechanism (Muzaffar et al., 2004b). Since sildenafil (SIL) prevents the hydrolysis of cyclic GMP via the inhibition of type V phosphodiesterase (type V PDE; Jeremy et al., 1997), SIL may be therapeutically effective in ARDS through an augmentation of this effect of NO. In order to test this proposal the effect of SIL and NO-donating SIL (SIL-NO) on O₂^▪- formation and gp91 ^phox expression was investigated in cultured porcine pulmonary artery endothelial cells (PAECs).

PAECs were incubated with 10 nM TXA₂ analogue, U46619 (± SIL or SIL-NO) for 16 hours and O₂^▪- formation measured spectrophometrically and gp91^phox protein using Western blotting (Muzaffar et al., 2004a). The role of NO and cyclic GMP in mediating effects was studied using m orpholinosydnonimine hydrochloride (SIN-1), the guanylyl cyclase inhibitor, 1H-{1,2,4}oxadiazolo{4,3-a}quinoxalin-1-one (ODQ) and the eNOS inhibitor, N⁵-(1-iminoethyl)-ornithine (L-NIL). Data below are expressed as mean ± SEM, n = 6. Statistics were carried out using ANOVA and post hoc unpaired students t-test with a Bonferonni adjustment.

Both SIL-NO and SIL elicited a concentration-dependent inhibition of O₂^▪- formation and gp91 phox expression, SIL-NO being 10-fold more potent than SIL (respective IC₅₀s; 480 pM and 5.8 nM). 100 µM ODQ completely reversed the inhibitory effect of both SIL-NO and SIL on O₂^▪- formation. U46619-stimulated O₂^▪- formation (15 ± 0.4 nmoles / hr) was inhibited by 1 µM SIN-1 (9.6 ± 0.7 nmoles / hr), an effect further augmented by 10 nM SIL (4.6 ± 0.5 nmoles / hr). The inhibitory effect of SIL (but not of SIL-NO) on U46619-stimulated O₂^▪- formation (6 ± 0.3 nmoles / hr) was partially reversed by 100 m M L-NIO (8.8 ± 0.2 nmoles / hr), indicating that SIL requires endogenous NO drive to exert an inhibitory effect.

SIL and SIL-NO are potent inhibitors of O₂^▪- formation in PAECs. This effect is mediated through the inhibition of type 5 PDE which in turn augments the inhibitory action of the NO-cGMP axis on gp91^phox expression. The action of SIL is mediated in part by endogenous NO-drive derived from eNOS. NO-SIL may be a more effective than SIL since this drug provides exogenous NO which would provide drive for guanylyl cyclase activation and NO formation is diminished by O₂^▪- in ARDS. SIL-NO may also be indicated for treating pulmonary hypertension due to its NO-donating capacity.

Muzaffar, S., et al. ( 2004a) Br. J. Pharmacol.141: 488-496
Muzaffar, S., et al. (2004b) Circulation: in press
Jeremy, J.Y., et al., (1997) Br. J. Urol. 79: 958-963