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

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 (HUVEC) and human embryonic kidney 293 cells transfected with empty vector (HEK) or eNOS cDNA (HEK-eNOS) were treated with TERPY (1 µM) for different lengths of time. eNOS expression, dimerization, and Ser1177 phosphorylation, caveolin-1 (Cav-1) oligomerization, Cav-1 Tyr14 phosphorylation were evaluated by Western blotting. Studies also assessed the production of reactive oxygen/nitrogen species (ROS/RNS) in HUVEC and HEK-eNOS cells. In HEK cells devoid of eNOS, TERPY released NO without additional stimulus indicating that it is an NO donor. Moreover, in HEK-eNOS cells, TERPY-induced NO production that could be 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, but not catalase, suggesting ROS and ONOO- production induced by TERPY originated from eNOS. HUVEC stimulated with TERPY showed increased eNOS Ser1177 and Cav-1 Tyr14 phosphorylation, and decreased eNOS dimerization, Cav-1 oligomerization, and Cav-1/eNOS interaction after 20 min suggesting 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.