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 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 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 Ser1177 and Cav-1 Tyr14 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.

Thromboxane‐prostanoid receptor activation blocks ATP‐sensitive potassium channels in rat aortas

“…Our research group has evaluated the effects of NO donors and demonstrated that some of said NO donors can release NO in VSMCs and ECs. The NO donor [Ru(terpy)(bdq)NO] 3+ (Terpy) induces eNOS uncoupling and phosphorylation, to produce ROS 35 . We have also shown that in 2K‐1C rat aortas, K + channels are not sensitive to NO released from the NO donor 15‐ane 36 …”

Recruitment of TNF ligands to lipid rafts is mediated by their physical association with caveolin‐1

“…A more detailed study of this issue is required to reach a more convincing conclusion. Interestingly, all proteins interacting with both the N‐ and the C‐terminal domains of CAV1 are found as di‐, tri‐ and oligomers [27,42–46]. CAV1, as mentioned above, is capable of forming oligomers.…”

Endothelial modulation of a nitric oxide donor complex-induced relaxation in normotensive and spontaneously hypertensive rats

“…7D). Recently, by using human umbilical vein endothelial cells (HUVEC), we have demonstrated that TERPY promotes eNOS hyperactivation and eNOS phosphorylation at Ser 1177 [54]. In addition, TERPY increased eNOS expression only in mesenteric arteries of SHR (Fig.…”