Acute inhibition of superoxide formation and Rac1 activation by nitric oxide and iloprost in human vascular smooth muscle cells in response to the thromboxane A2 analogue, U46619.
BACKGROUND
The over-production of superoxide (O(2)(-)) derived from NADPH oxidase (NOX) plays a central role in cardiovascular diseases. By contrast, nitric oxide (NO) and prostacyclin (PGI(2)) are vasculoprotective. The effect of the NO donor, NONOate and iloprost on O(2)(-) formation, p47(phox) and Rac(1) activation in human vascular smooth muscle cells (hVSMCs) was investigated.


METHODS
hVSMCs were incubated with 10nM thromboxane A(2) analogue, U46619 for 16h, and then with apocynin (a NOX inhibitor), NONOate or iloprost for 1h and O(2)(-) measured spectrophometrically. The role of cyclic AMP and cyclic GMP was examined by co-incubation of drugs with protein kinase (PK) A and G inhibitors listed above. Rac(1) was studied using pull-down assays.


RESULTS
NONOate and iloprost inhibited O(2)(-) formation, acutely, effects blocked by inhibition of PKG and PKA, respectively. Rac(1) and p47(phox) activation and translocation to the plasma membrane was completely inhibited by NONOate and iloprost, effects again reversed by co-incubation with PKG or PKA inhibitors.


CONCLUSIONS
NO and PGI(2) block the acute activity of NOX in hVSMCs via the cGMP-PKG axis (for NO) and by the cAMP-PKA axis (for iloprost) through inhibition of Rac(1) and p47(phox) translocation. These findings have implications in the pathophysiology and treatment of CVD.
Muzaffar
S
S
Bristol Heart Institute, University of Bristol, Bristol Royal Infirmary, Bristol, UK. S.Muzaffar@bris.ac.uk
Shukla
N
N
Bond
M
M
Sala-Newby
G
G
Angelini
G D
GD
Newby
A C
AC
Jeremy
J Y
JY
D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid / Q000494:pharmacology*; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D016285:Iloprost / Q000494:pharmacology*; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000378:metabolism*; D009569:Nitric Oxide / Q000494:pharmacology*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D013481:Superoxides / Q000378:metabolism*; D013928:Thromboxane A2 / Q000031:analogs & derivatives; D013997:Time Factors; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*
D047428:Protein Kinase Inhibitors; D013481:Superoxides; D009569:Nitric Oxide; D013928:Thromboxane A2; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D020830:rac1 GTP-Binding Protein; D016285:Iloprost