scholarly journals Superoxide-mediated inactivation of nitric oxide and peroxynitrite formation by tobacco smoke in vascular endothelium: studies in cultured cells and smokers

2009 ◽  
Vol 296 (6) ◽  
pp. H1781-H1792 ◽  
Author(s):  
Gonzalo Peluffo ◽  
Pablo Calcerrada ◽  
Lucia Piacenza ◽  
Nelson Pizzano ◽  
Rafael Radi
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Endothelial Dysfunction ◽  
Tobacco Smoke ◽  
Endothelial Nitric Oxide Synthase ◽  
Flow Mediated Dilation ◽  
Aortic Endothelial Cells ◽  
Bovine Aortic Endothelial Cells ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Tobacco smoke is known to cause nitric oxide (·NO) inactivation and endothelial dysfunction. In this work we evaluated the interplay between·NO and superoxide (O2·−) radicals and the consequent impact on·NO bioavailability and nitroxidative stress in bovine aortic endothelial cells exposed to cigarette smoke extract (CSE) and in smokers. Bovine aortic endothelial cells in the presence of CSE triggered O2·−production as indicated by spin-trapping electron paramagnetic resonance experiments. O2·−was produced both extracellulary (3.4 vs. 1.0 nmol·h−1·mg−1; CSE vs. control; cytochrome c3+reduction assay) and intracellularly (40% inhibition of cytosolic aconitase). CSE also led to the production of peroxynitrite as evaluated by dihydrorhodamine oxidation and protein tyrosine nitration on cells. O2·−and peroxynitrite formation were decreased by ascorbate and α-tocopherol. Additionally, CSE led to the oxidation of endothelial nitric oxide synthase increasing the monomeric inactive form of endothelial nitric oxide synthase. Smokers and age-matched healthy volunteers were supplemented orally with 500 mg ascorbate plus 400 IU all-rac-α-tocopherol every 12 h for 165 days. Smokers had endothelial dysfunction compared with control subjects (95% confidence interval: 2.5, 8.3 vs. 10.6, 14.2; P < 0.05) as assessed by flow-mediated dilation of the brachial artery, and plasma levels of protein 3-nitrotyrosine were 1.4-fold higher. The loss of flow-mediated dilation in smokers reverted after a long-term antioxidant supplementation (95% confidence interval: 13.9, 19.9; P < 0.05), reaching values comparable with the control population. Our data indicate that elements on tobacco smoke, most likely through redox cycling, divert·NO toward peroxynitrite by inducing O2·−production in vascular endothelial cells both in vitro and in vivo.

scholarly journals Potential pitfalls in analyzing structural uncoupling of eNOS: aging is not associated with increased enzyme monomerization

2019 ◽  
Vol 316 (1) ◽  
pp. H80-H88 ◽  
Author(s):  
Fumin Chang ◽  
Sheila Flavahan ◽  
Nicholas A. Flavahan
Keyword(s):  
Nitric Oxide ◽  
Endothelial Dysfunction ◽  
Nitric Oxide Synthase ◽  
Endothelial Nitric Oxide Synthase ◽  
Normal Activity ◽  
Aortic Endothelial Cells ◽  
Relative Expression ◽  
Fischer 344 ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Homodimer formation is essential for the normal activity of endothelial nitric oxide synthase (eNOS). Structural uncoupling of eNOS, with generation of enzyme monomers, is thought to contribute to endothelial dysfunction in several vascular disorders, including aging. However, low-temperature SDS-PAGE of healthy arteries has revealed considerable variation between studies in the relative expression of eNOS dimers and monomers. While assessing structural uncoupling of eNOS in aging arteries, we identified methodological pitfalls that might contribute to such variation. Therefore, using human cultured aortic endothelial cells and aortas from young and aged Fischer-344 rats, we investigated optimal approaches for analyzing the expression of eNOS monomers and dimers. The results demonstrated that published differences in treatment of cell lysates can significantly impact the relative expression of several eNOS species, including denatured monomers, partially folded monomers, dimers, and higher-order oligomers. In aortas, experiments initially confirmed a large increase in eNOS monomers in aging arteries, consistent with structural uncoupling. However, these monomers were actually endogenous IgG, which, under these conditions, has mobility similar to eNOS monomers. Increased IgG levels in aged aortas likely reflect the aging-induced disruption of endothelial junctions and increased arterial penetration of IgG. After removal of the IgG signal, there were low levels of eNOS monomers in young arteries, which were not significantly different in aged arteries. Therefore, structural uncoupling of eNOS is not a prominent feature in young healthy arteries, and the process is not increased by aging. The study also identifies optimal approaches to analyze eNOS dimers and monomers. NEW & NOTEWORTHY Structural uncoupling of endothelial nitric oxide synthase (eNOS) is considered central to endothelial dysfunction. However, reported levels of eNOS dimers and monomers vary widely, even in healthy arteries. We demonstrate that sample processing can alter relative levels of eNOS species. Moreover, endothelial dysfunction in aging aortas results in IgG accumulation, which, because of similar mobility to eNOS monomers, could be misinterpreted as structural uncoupling. Indeed, enzyme monomerization is not prominent in young or aging arteries.

scholarly journals Hepatocyte growth factor activates endothelial nitric oxide synthase by Ca2+- and phosphoinositide 3-kinase/Akt-dependent phosphorylation in aortic endothelial cells

2003 ◽  
Vol 374 (1) ◽  
pp. 63-69 ◽  
Author(s):  
Kennedy MAKONDO ◽  
Kazuhiro KIMURA ◽  
Naoki KITAMURA ◽  
Takanori KITAMURA ◽  
Daisuke YAMAJI ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Growth Factor ◽  
Endothelial Nitric Oxide Synthase ◽  
Aortic Endothelial Cells ◽  
Enos Activity ◽  
Phosphoinositide 3 Kinase ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide ◽  
Hepatocyte Growth

Hepatocyte growth factor (HGF) causes endothelium-dependent vasodilation, but its relation to endothelial nitric oxide synthase (eNOS) activity remains to be elucidated. Treatment of bovine aortic endothelial cells with HGF increased eNOS activity within minutes, accompanied by an increase of activity-related site-specific phosphorylation of eNOS. The phosphorylation was completely abolished by pretreatment of the cells with a phosphoinositide 3-kinase (PI3K) inhibitor (wortmannin) and by transfection of dominant-negative Akt, and the enzyme activity was inhibited by wortmannin. In addition, eNOS activity and phosphorylation were abolished by pretreatment of the cells with an intracellular Ca2+-chelator, bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid tetrakis(acetoxymethyl ester) (BAPTA/AM), with a suppression of Akt phosphorylation. These results suggest that HGF stimulates eNOS activity by a PI3K/Akt-dependent phosphorylation in a Ca2+-sensitive manner in vascular endothelial cells.

scholarly journals Mulberry Extract Attenuates Endothelial Dysfunction through the Regulation of Uncoupling Endothelial Nitric Oxide Synthase in High Fat Diet Rats

Nutrients ◽  
2019 ◽  
Vol 11 (5) ◽  
pp. 978 ◽  
Author(s):  
Geum-Hwa Lee ◽  
The-Hiep Hoang ◽  
Eun-Soo Jung ◽  
Su-Jin Jung ◽  
Soo-Wan Chae ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Endothelial Dysfunction ◽  
Nitric Oxide Synthase ◽  
High Fat Diet ◽  
Endothelial Nitric Oxide Synthase ◽  
High Fat ◽  
Enos Uncoupling ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Dyslipidemia is associated with endothelial dysfunction, which is linked to nitric oxide (NO) biology. The coupling of endothelial NO synthase with cofactors is a major step for NO release. This study is aimed to investigate the vascular pharmacology effects of mulberry in rat thoracic aorta and human vascular endothelial cells. In vitro, we investigated the protective effects of the mulberry extract and its main component cyanidin-3-rutinoside (C-3-R), against oxidized low-density lipoprotein (ox-LDL)-induced endothelial nitric oxide synthase (eNOS) uncoupling. Whereas ox-LDL significantly decreased NO levels in endothelial cells, mulberry extract, and C-3-R significantly recovered NO levels and phospho-eNOS Thr495 and Ser1177 expression. In vivo, mulberry was administered to 60% of high-fat diet (w/w)-fed Sprague Dawley (SD) rats for six weeks, in which endothelium-dependent relaxations were significantly improved in organ bath studies and isometric tension recordings. Consistently, aortic expressions of phospho-eNOS and nitrotyrosine were increased. Mulberry also raised serum NO levels, increased phosphorylation of eNOS, and reduced nitrotyrosine and intracellular reactive oxygen species (ROS) in aortas, showing that mulberry preserves endothelium-dependent relaxation in aortas from high-fat diet rats. We suggest that this effect is mediated through enhanced NO bioavailability, in which the regulation of ROS and its reduced eNOS uncoupling are involved.

scholarly journals Phytoestrogen Genistein Up-Regulates Endothelial Nitric Oxide Synthase Expression Via Activation of cAMP Response Element-Binding Protein in Human Aortic Endothelial Cells

Endocrinology ◽  
2012 ◽  
Vol 153 (7) ◽  
pp. 3190-3198 ◽  
Author(s):  
Hongwei Si ◽  
Jie Yu ◽  
Hongling Jiang ◽  
Hazel Lum ◽  
Dongmin Liu
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Endothelial Nitric Oxide Synthase ◽  
No Production ◽  
Enos Expression ◽  
Camp Response Element ◽  
Aortic Endothelial Cells ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide ◽  
Human Aortic Endothelial Cells

We previously reported that genistein, a phytoestrogen, up-regulates endothelial nitric oxide synthase (eNOS) and prevents hypertension in rats that are independent of estrogen signaling machinery. However, how genistein regulates eNOS expression is unknown. In the present study, we show that genistein enhanced eNOS expression and NO synthesis in primary human aortic endothelial cells. Inhibition of extracellular signal regulated kinase, phosphoinositol-3 kinase, or protein kinase C did not affect genistein-enhanced eNOS expression and NO synthesis. However, chemical inhibition of protein kinase A (PKA) or adenoviral transfer of the specific endogenous PKA inhibitor gene completely abolished PKA activity and genistein-stimulated eNOS expression and NO production. Accordingly, genistein induced PKA activity and subsequent phosphorylation of cAMP response element (CRE)-binding protein (CREB) at Ser133. Suppression of CREB by small interfering RNA transfection abolished genistein-enhanced eNOS expression and NO production. Consistently, deletion of the CRE site within human eNOS promoter eliminated genistein-stimulated eNOS promoter activity. These findings provide the first evidence to our knowledge that genistein may play a beneficial role in vascular function through targeting the PKA/CREB/eNOS/NO signaling pathway.

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