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assessed using Western blot analysis. The role of NAD[P]H oxidase and cGMP was further studied by using specific inhibitors of each. RESULTSSuperoxide formation was significantly greater in cells incubated with U46619 after 1 and 16 h incubation than in controls, an effect blocked by NADP(H) oxidase inhibitors. These effects of U46619 were inhibited by sildenafil (1 and 10 nmol/L), which in turn were negated by the guanylyl cyclase inhibitor, ODQ; 10 nmol/L sildenafil inhibited p47phox expression induced by U46619. CONCLUSIONSSildenafil is a potent inhibitor of superoxide formation in CVSMCs. This effect is mediated through the inhibition of PDE-5 which in turn augments the inhibitory action of the NOcGMP axis on NAD[P]H oxidase expression and activity. This mechanism constitutes a new pharmacological action of sildenafil, consolidates the potential role of superoxide in ED, and indicates that thromboxane A 2 may be an important mediator of intrapenile oxidative stress.

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Background and purpose: Superoxide (O 2 K À ), derived from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, is associated with acute respiratory distress syndrome (ARDS). NADPH oxidase activity and expression are blocked by nitric oxide (NO) and sildenafil. As another gas, hydrogen sulphide (H 2 S) is formed by blood vessels, the effect of sodium hydrosulphide (NaHS) and the H 2 S-donating derivative of sildenafil, ACS6, on O 2 KÀ formation and the expression of gp91 phox (a catalytic subunit of NADPH oxidase) in porcine pulmonary arterial endothelial cells (PAECs) was investigated. Experimental approach: PAECs were incubated with 10 ng mL À1 tumour necrosis factor-a (TNFa) ( ± NaHS or ACS6), both of which released H 2 S, for 2 h or 16 h. O 2 K À was measured. Expression of gp91 phox was measured by western blotting and the role of cyclic AMP (cAMP) and/or cyclic GMP was assessed using protein kinase inhibitors. Key results: After either 2-or 16-h incubations, O 2K À formation by PAECs was inhibited by NaHS or ACS6, with IC 50 values of about 10 nM and less than 1 nM, respectively. Both 100 nM NaHS and 1 nM ACS6 completely inhibited gp91 phox expression induced by TNFa. The effects of NaHS were blocked by the inhibition of protein kinase A (PKA), but not PKG, and not by the inhibition of guanylyl cyclase. Effects of ACS6 were blocked by inhibition of both PKA and PKG. Both NaHS and ACS6 augmented cAMP formation. Conclusion and implications: H 2 S inhibited O 2K À formation and upregulation of NADPH oxidase in PAECs through the adenylyl cyclase-PKA pathway. ACS6 may be effective in treating ARDS through both elevation of cAMP and inhibition of phosphodiesterase type 5 activity.