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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.

The activity of NADPH oxidase (NOX) is blocked by nitric oxide (NO). Hydrogen sulfide (H₂S) is also produced by blood vessels. It is reasonable to suggest that H₂S may have similar actions to NO on NOX. In order to test this hypothesis, the effect of sodium hydrosulfide (NaHS) on O₂^– formation, the expression of NOX-1 (a catalytic subunit of NOX) and Rac₁ activity (essential for full NOX activity) in isolated vascular smooth muscle cells (hVSMCs) was investigated. hVSMCs were incubated with the thromboxane A₂ analogue U46619 ± NaHS for 1 or 16 h, and O₂^– formation, NOX-1 expression and Rac₁ activity were assessed. The possible interaction between H₂S and NO was also studied by using an NO synthase inhibitor, L-NAME, and an NO donor, DETA-NONOate. The role of K_ATP channels was studied by using glibenclamide. NaHS inhibited O₂^– formation following incubation of 1 h (IC₅₀, 30 nM) and 16 h (IC₅₀, 20 nM), blocked NOX-1 expression and inhibited Rac₁ activity. These inhibitory effects of NaHS were mediated by the cAMP-protein-kinase-A axis. Exogenous H₂S prevents NOX-driven intravascular oxidative stress through an a priori inhibition of Rac₁ and downregulation of NOX-1 protein expression, an effect mediated by activation of the adenylylcyclase-cAMP-protein-kinase-G system by H₂S.