H2S-donating sildenafil (ACS6) inhibits superoxide formation and gp91phox expression in arterial endothelial cells: role of protein kinases A and G.
OBJECTIVE
Superoxide (O(2)(*-)), 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)(*-) formation and the expression of gp91(phox) (a catalytic subunit of NADPH oxidase) in porcine pulmonary arterial endothelial cells (PAECs) was investigated.


METHODS
PAECs were incubated with 10 ng mL(-1) tumour necrosis factor-alpha (TNFalpha) (+/-NaHS or ACS6), both of which released H(2)S, for 2 h or 16 h. O(2)(*-) 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.


RESULTS
After either 2- or 16-h incubations, O(2)(*-) 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 TNFalpha. 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.


CONCLUSIONS
H(2)S inhibited O(2)(*-) 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.
Department of Cardiac Surgery, Bristol Royal Infirmary, Bristol Heart Institute, University of Bristol, Bristol, UK. s.muzaffar@bristol.ac.uk
S
S
Muzaffar
J Y
JY
Jeremy
A
A
Sparatore
P
P
Del Soldato
G D
GD
Angelini
N
N
Shukla
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000187:drug effects / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000187:drug effects / Q000378:metabolism; D042783:Endothelial Cells / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects*; D006162:Guanylate Cyclase / Q000378:metabolism; D006862:Hydrogen Sulfide / Q000378:metabolism*; D020128:Inhibitory Concentration 50; D008297:Male; D019255:NADPH Oxidases / Q000187:drug effects / Q000378:metabolism; D058986:Phosphodiesterase 5 Inhibitors; D010879:Piperazines / Q000008:administration & dosage / Q000494:pharmacology*; D011651:Pulmonary Artery / Q000166:cytology; D012128:Respiratory Distress Syndrome / Q000188:drug therapy / Q000503:physiopathology; D013440:Sulfides / Q000008:administration & dosage / Q000494:pharmacology; D013450:Sulfones / Q000008:administration & dosage / Q000494:pharmacology*; D013481:Superoxides / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013552:Swine; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
C534144:1-piperazineacetic acid 4-((3-(4,7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo(4,3-d)pyrimidin-5-yl)-4-ethoxyphenyl)sulfonyl)-4-(3-thioxo-3H-1,2-dithiol-5-yl)phenyl ester; D058986:Phosphodiesterase 5 Inhibitors; D010879:Piperazines; D013440:Sulfides; D013450:Sulfones; D014409:Tumor Necrosis Factor-alpha; D013481:Superoxides; D000242:Cyclic AMP; D019255:NADPH Oxidases; D017868:Cyclic AMP-Dependent Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases; D000262:Adenylyl Cyclases; D006162:Guanylate Cyclase; C025451:sodium bisulfide; D006152:Cyclic GMP; D006862:Hydrogen Sulfide