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.

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Aims/hypothesis. Both diabetes mellitus and hyperhomocysteinaemia are risk factors for cardiovascular disease and are associated with impaired endothelial nitric oxide and with excess superoxide formation. To explore potential vasculopathic interactions between these risk factors, the effect of homocysteine on endothelium-dependent relaxation and cyclic GMP formation was investigated in aortae from diabetic rabbits. Methods. Rabbits were rendered diabetic by intravenous injection of alloxan. Six months later, the aortae were excised, cut into rings and mounted in an organ bath for isometric measurement of acetylcholineevoked relaxation in rings pre-contracted with phenylephrine. Cyclic GMP formation by aortic rings after stimulation with acetylcholine, calcium ionophore A23187 and sodium nitroprusside was assessed using radioimmunoassay. The effect of homocysteine on these parameters was then studied.Results. Ach-evoked relaxation and cyclic GMP formation induced with acetylcholine and calcium ionophore A23187 were impaired in aortae from diabetic rabbits compared with the control rabbits, effects that were reversed with superoxide dimutase (SOD) and augmented by 10-100 µmol/l homocysteine, an effect again reversed by SOD. Conclusion/interpretation. These data show that the bioavailability of nitric oxide is reduced in aortae from diabetic rabbits due to excess production of superoxide, an effect augmented by homocysteine. These results indicate that patients with diabetes mellitus could be susceptible to homocysteine-mediated angiopathy at lower concentrations than those that promote vasculopathy in non-diabetic patients. [Diabetologia (2002[Diabetologia ( ) 45:1325[Diabetologia ( -1331