Acute inhibition of superoxide formation and Rac 1 activation by nitric oxide and iloprost in human vascular smooth muscle cells in response to the thromboxane A 2 analogue, U46619

Abstract

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 10 nM thromboxane A 2 analogue, U46619 for 16 h, and then with apocynin (a NOX inhibitor), NONOate or iloprost for 1 h 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.