Nitric oxide donor [Ru( terpy)(bdq)NO](3+) induces uncoupling and phosphorylation of endothelial nitric oxide synthase promoting oxidant production

Abstract

Ru(terpy)(bdq)NO](3+) (TERPY) is a nitric oxide (NO) donor that promotes relaxation of the mesenteric artery and aorta in rats. We sought to investigate whether it acts as both an NO donor and endothelial NO synthase (eNOS) activator, as shown previously for nitroglycerin. Human umbilical vein endothelial cells (HUVECs) and human embryonic kidney 293 cells transfected with empty vector (HEK) or eNOS cDNA (HEK-eNOS) were treated with TERPY (1 mu M) for different lengths of time. eNOS expression, dimerization, and Ser(1177) phosphorylation, caveolin-1 (Cav-1) oligomerization, Cav-1 Tyr(14) phosphorylation were evaluated by Western blotting. Studies also assessed the production of reactive oxygen/nitrogen species (ROS/RNS) in HUVECs and HEK-eNOS cells. In HEK cells devoid of eNOS, TERPY released NO without additional stimulus indicating that is an NO donor. Moreover, in HEK-eNOS cells, TERPY-induced NO production that was blocked by L-NAME. In addition, TERPY increased ROS and ONOO- production which were blocked by more than 80% by BH4 (essential eNOS co-factor) and eNOS siRNA. These results suggest that TERPY-induced ROS and ONOO- production were originated from eNOS. HUVECs stimulated with TERPY showed increased eNOS Ser(1177) and Cav-1 Tyr(14) phosphorylation, and decreased eNOS dimerization, Cav-1 oligomerization, and Cav-1/eNOS interaction after 20 min. It suggests that TERPY induces eNOS hyperactivation and uncoupling by disrupting Cav-1/eNOS interaction and depleting BH4. Endothelium-dependent vasodilation in response to NO donor TERPY is associated with eNOS activation and uncoupling, and thereby appears to be mediated, at least in part, via eNOS-dependent ROS/RNS production.