H2S-donating sildenafil (ACS6) inhibits superoxide formation and gp91phox expression in arterial endothelial cells: role of protein kinases A and G

Background and purpose: Superoxide (O-2(center dot-)), 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 (H2S) is formed by blood vessels, the effect of sodium hydrosulphide (NaHS) and the H2S-donating derivative of sildenafil, ACS6, on O-2(center dot-) 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(-1) tumour necrosis factor-alpha (TNF alpha) (+/- NaHS or ACS6), both of which released H2S, for 2 h or 16 h. O-2(center dot-) 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-2(center dot-) formation by PAECs was inhibited by NaHS or ACS6, with IC50 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 TNF alpha. 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: H2S inhibited O-2(center dot-) 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.