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Nitric oxide donor [Ru(terpy)(bdq)NO] 3+ (TERPY) promotes eNOS activation
Author(s) -
Potje Simone Regina,
Oliveira Suellen D'Arc,
Chen Zhenlong,
Bonini Marcelo G,
Silva Roberto S.,
Bendhack Lusiane M,
Antoniali Cristina,
Minshall Richard D
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.957.1
Subject(s) - enos , hek 293 cells , chemistry , nitric oxide , umbilical vein , activator (genetics) , transfection , microbiology and biotechnology , medicine , endocrinology , nitric oxide synthase , biochemistry , in vitro , biology , receptor , gene
TERPY ([Ru(terpy)(bdq)NO] 3+ ) is a ruthenium complex nitric oxide (NO) donor that promotes relaxation of the mesenteric artery and aorta to a greater extent in hypertensive rats as compared to normotensive control. To better understand the mechanism of TERPY action, we sought to investigate whether it acts as both an NO donor and endothelial NO synthase (eNOS/NOS3) activator, as shown previously for nitroglycerin. Objective The aim of this study was to determine whether TERPY directly or indirectly (i.e., via NOS3 activation) increases the intracellular concentration of NO. Methods 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 time lengths (5–60 min). NOS3 expression and Ser 1179 phosphorylation were evaluated by Western Blot, and NO accumulation in HUVEC, HEK, and HEK‐eNOS cells was measured using a copper‐based fluorescent probe (1 μM). It was used to quantify the contribution of NO released from the donor source vs NO produced by eNOS (i.e, L‐NAME sensitive NO production). Results First, we demonstrated that in HEK, TERPY has the ability to release NO without additional stimulus, indicating that this compound is an NO donor. Moreover, in HEK‐eNOS cells, TERPY‐induced NO production was even greater with maximum values observed after 20 min. At this level of NO production, 26% could be blocked by co‐treatment with L‐NAME (non‐selective inhibitor of NOS, 0.1 mM). When cells were stimulated with TERPY for 20 min treatment, TERPY increased NOS3 Ser 1179 phosphorylation in HUVEC and HEK‐eNOS further suggesting that TERPY induces NOS3 activation. Consistent with the hypothesis that activated eNOS could be inactivated by Src‐phosphorylated caveolin‐1 as part of a negative feedback mechanism, we have observed caveolin‐1 Tyr14 phosphorylation after 20 min. Conclusion Taken together, these data suggest that TERPY acts as a long‐acting NO donor and also as a NOS3 activator, thereby increasing the NO bioavailability. The potential benefits and mechanisms of eNOS activation by TERPY, as compared to nitroglycerin, requires further characterization in cell cultures and rodent models of hypertension. Support or Funding Information Financial support: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) [Grant 232217/2014‐9], Fundação Lemann, and NIH HL‐60678.