scholarly journals In Endothelial Cells, the Activation or Stimulation of Soluble Guanylyl Cyclase Induces the Nitric Oxide Production by a Mechanism Dependent of Nitric Oxide Synthase Activation

2018 ◽  
Vol 21 ◽  
pp. 38-45 ◽  
Author(s):  
Ariane Migliato Martinelli ◽  
Carla Nascimento dos Santos Rodrigues ◽  
Thiago Francisco de Moraes ◽  
Gerson Jhonatan Rodrigues
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Nitric Oxide Synthase ◽  
Soluble Guanylate Cyclase ◽  
Soluble Guanylyl Cyclase ◽  
No Production ◽  
Selective Electrode ◽  
Nos Inhibitor ◽  
Oxide Synthase ◽  
Stimulation Of

Purpose. In endothelial cells, investigate if the soluble guanylate cyclase (sGC) activation or stimulation is able to potentiate the relaxation in vessels. Methods. Aortic and coronary rings with and without endothelium were placed in a myograph and cumulative concentration-effect curves for DETA-NO or ataciguat were performed. Nitric oxide (NO) were measured by fluorescence or by selective electrode in human umbilical endothelial cells (HUVECs) in response to some treatments, including ataciguat, 8-Br-cGMP and A23187. Results. The presence of the endothelium potentiated the relaxation induced by DETA-NO in aortic and coronary rings. In addition, in aortic rings the endothelium potentiated the relaxation induced by ataciguat. In the presence of nitric oxide synthase (NOS) inhibitor, the endothelium effect was abolished to DETA-NO or ataciguat, in both vessels. Ataciguat, 8-Br-cGMP and A23187 were able to induce NO production in HUVECs cells. In the presence of NOS inhibitor, the NO production induced by ataciguat and 8-Br-cGMP was abolished. Conclusions. Our results suggest that in aortic and coronary rings the endothelium potentiates the relaxation induced by activation or stimulation of sGC through a mechanism dependent of NOS activation. This article is open to POST-PUBLICATION REVIEW. Registered readers (see “For Readers”) may comment by clicking on ABSTRACT on the issue’s contents page.

TNF-alpha and IFN-gamma induce expression of nitric oxide synthase in cultured rat medullary interstitial cells

1995 ◽  
Vol 269 (2) ◽  
pp. F212-F217 ◽  
Author(s):  
K. S. Lau ◽  
O. Nakashima ◽  
G. R. Aalund ◽  
L. Hogarth ◽  
K. Ujiie ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Smooth Muscle ◽  
Nitric Oxide Synthase ◽  
Vascular Smooth Muscle ◽  
Guanylyl Cyclase ◽  
Soluble Guanylyl Cyclase ◽  
Tnf Alpha ◽  
No Production ◽  
Ifn Gamma ◽  
Oxide Synthase

Cytokines increase the expression of the inducible (type II) nitric oxide synthase (NOS) in macrophages, liver, and renal epithelial cells. Previously, we found that cultured rat medullary interstitial cells (RMIC) contain high levels of soluble guanylyl cyclase. To determine whether these cells can also produce NO, we studied the effects of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) on NO production, NOS II mRNA, and NOS II protein expression. Both TNF-alpha and IFN-gamma, in the presence of a low concentration of the other cytokine, caused dose-dependent increases in NO production. Exposure to TNF-alpha and IFN-gamma stimulated the production of NOS II mRNA, as determined by Northern blotting. Restriction mapping of reverse transcription-polymerase chain reaction products indicated that normal cells contained macrophage NOS II, whereas cytokine-stimulated cells contained primarily vascular smooth muscle NOS II and some macrophage NOS II. The appearance of NOS II protein was demonstrated by Western blotting. RMIC cell guanosine 3',5'-cyclic monophosphate accumulation increased 129-fold in response to the cytokines. NOS inhibitors decreased nitrite production. We conclude that 1) TNF-alpha and IFN-gamma induce the expression of vascular smooth muscle NOS II and production of NO in RMIC, and 2) NO acts as an autocrine activator of the soluble guanylyl cyclase in RMIC.

scholarly journals Endoplasmic Reticulum Ca2+ Release Modulates Endothelial Nitric-oxide Synthase via Extracellular Signal-regulated Kinase (ERK) 1/2-mediated Serine 635 Phosphorylation

2011 ◽  
Vol 286 (22) ◽  
pp. 20100-20108 ◽  
Author(s):  
Zhihong Xiao ◽  
Tingting Wang ◽  
Honghua Qin ◽  
Chao Huang ◽  
Youmei Feng ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Endoplasmic Reticulum ◽  
Protein Kinase ◽  
Nitric Oxide Synthase ◽  
Protein Phosphorylation ◽  
Endothelial Nitric Oxide Synthase ◽  
No Production ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Endothelial nitric-oxide synthase (eNOS) plays a central role in cardiovascular regulation. eNOS function is critically modulated by Ca2+ and protein phosphorylation, but the interrelationship between intracellular Ca2+ mobilization and eNOS phosphorylation is poorly understood. Here we show that endoplasmic reticulum (ER) Ca2+ release activates eNOS by selectively promoting its Ser-635/633 (bovine/human) phosphorylation. With bovine endothelial cells, thapsigargin-induced ER Ca2+ release caused a dose-dependent increase in eNOS Ser-635 phosphorylation, leading to elevated NO production. ER Ca2+ release also promoted eNOS Ser-633 phosphorylation in mouse vessels in vivo. This effect was independent of extracellular Ca2+ and selective to Ser-635 because the phosphorylation status of other eNOS sites, including Ser-1179 or Thr-497, was unaffected in thapsigargin-treated cells. Blocking ERK1/2 abolished ER Ca2+ release-induced eNOS Ser-635 phosphorylation, whereas inhibiting protein kinase A or Ca2+/calmodulin-dependent protein kinase II had no effect. Protein phosphorylation assay confirmed that ERK1/2 directly phosphorylated the eNOS Ser-635 residue in vitro. Further studies demonstrated that ER Ca2+ release-induced ERK1/2 activation mediated the enhancing action of purine or bradykinin receptor stimulation on eNOS Ser-635/633 phosphorylation in bovine/human endothelial cells. Mutating the Ser-635 to nonphosphorylatable alanine prevented ATP from activating eNOS in cells. Taken together, these studies reveal that ER Ca2+ release enhances eNOS Ser-635 phosphorylation and function via ERK1/2 activation. Because ER Ca2+ is commonly mobilized by agonists or physicochemical stimuli, the identified ER Ca2+-ERK1/2-eNOS Ser-635 phosphorylation pathway may have a broad role in the regulation of endothelial function.

VEGF upregulates ecNOS message, protein, and NO production in human endothelial cells

1998 ◽  
Vol 274 (3) ◽  
pp. H1054-H1058 ◽  
Author(s):  
John D. Hood ◽  
Cynthia J. Meininger ◽  
Marina Ziche ◽  
Harris J. Granger
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Umbilical Vein ◽  
Northern Blotting ◽  
No Production ◽  
Vascular Endothelial ◽  
Umbilical Vein Endothelial Cells ◽  
Oxide Synthase ◽  
Endothelial Growth Factor ◽  
Stimulation Of

Vascular endothelial growth factor (VEGF) is an endothelium-specific secreted protein that potently stimulates vasodilation, microvascular hyperpermeability, and angiogenesis. Nitric oxide (NO) is also reported to modulate vascular tone, permeability, and capillary growth. Therefore, we hypothesized that VEGF might regulate endothelial production of NO. The production of nitrogen oxides by human umbilical vein endothelial cells (HUVECs) was measured after 1, 12, 24, and 48 h of incubation with VEGF. VEGF treatment resulted in both an acute (1 h) and chronic (>24 h) stimulation of NO production. Furthermore, Western and Northern blotting revealed a VEGF-elicited, dose-dependent increase in the cellular content of endothelial cell nitric oxide synthase (ecNOS) message and protein that may account for the chronic upregulation of NO production elicited by VEGF. Finally, endothelial cells pretreated with VEGF for 24 h and subsequently exposed to A-23187 for 1 h produced NO at approximately twice the rate of cells that were not pretreated with VEGF. We conclude that VEGF upregulates ecNOS enzyme and elicits a biphasic stimulation of endothelial NO production.

scholarly journals Identification of Golgi-localized acyl transferases that palmitoylate and regulate endothelial nitric oxide synthase

2006 ◽  
Vol 174 (3) ◽  
pp. 369-377 ◽  
Author(s):  
Carlos Fernández-Hernando ◽  
Masaki Fukata ◽  
Pascal N. Bernatchez ◽  
Yuko Fukata ◽  
Michelle I. Lin ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Nitric Oxide Synthase ◽  
Endothelial Nitric Oxide Synthase ◽  
Cdna Clones ◽  
No Production ◽  
Human Endothelial Cells ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Lipid modifications mediate the subcellular localization and biological activity of many proteins, including endothelial nitric oxide synthase (eNOS). This enzyme resides on the cytoplasmic aspect of the Golgi apparatus and in caveolae and is dually acylated by both N-myristoylation and S-palmitoylation. Palmitoylation-deficient mutants of eNOS release less nitric oxide (NO). We identify enzymes that palmitoylate eNOS in vivo. Transfection of human embryonic kidney 293 cells with the complementary DNA (cDNA) for eNOS and 23 cDNA clones encoding the Asp-His-His-Cys motif (DHHC) palmitoyl transferase family members showed that five clones (2, 3, 7, 8, and 21) enhanced incorporation of [3H]-palmitate into eNOS. Human endothelial cells express all five of these enzymes, which colocalize with eNOS in the Golgi and plasma membrane and interact with eNOS. Importantly, inhibition of DHHC-21 palmitoyl transferase, but not DHHC-3, in human endothelial cells reduces eNOS palmitoylation, eNOS targeting, and stimulated NO production. Collectively, our data describe five new Golgi-targeted DHHC enzymes in human endothelial cells and suggest a regulatory role of DHHC-21 in governing eNOS localization and function.

scholarly journals Src Kinase Activates Endothelial Nitric-oxide Synthase by Phosphorylating Tyr-83

2005 ◽  
Vol 280 (43) ◽  
pp. 35943-35952 ◽  
Author(s):  
David Fulton ◽  
Jarrod E. Church ◽  
Ling Ruan ◽  
Chunying Li ◽  
Sarika G. Sood ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Nitric Oxide Synthase ◽  
Tyrosine Phosphorylation ◽  
Endothelial Nitric Oxide Synthase ◽  
Phosphorylation Site ◽  
Src Kinase ◽  
No Production ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

The endothelial nitric-oxide synthase (eNOS) is regulated in part by serine/threonine phosphorylation, but eNOS tyrosine phosphorylation is less well understood. In the present study we have examined the tyrosine phosphorylation of eNOS in bovine aortic endothelial cells (BAECs) exposed to oxidant stress. Hydrogen peroxide and pervanadate (PV) treatment stimulates eNOS tyrosine phosphorylation in BAECs. Phosphorylation is blocked by the Src kinase family inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Moreover, eNOS and c-Src can be coimmunoprecipitated from BAEC lysates by antibodies directed against either protein. Domain mapping and site-directed mutagenesis studies in COS-7 cells transfected with either eNOS alone and then treated with PV or cotransfected with eNOS and constitutively active v-Src identified Tyr-83 (bovine sequence) as the major eNOS tyrosine phosphorylation site. Tyr-83 phosphorylation is associated with a 3-fold increase in basal NO release from cotransfected cells. Furthermore, the Y83F eNOS mutation attenuated thapsigargin-stimulated NO production. Taken together, these data indicate that Src-mediated tyrosine phosphorylation of eNOS at Tyr-83 modulates eNOS activity in endothelial cells.

scholarly journals Nitric oxide synthase-mediated early nitric oxide-burst alleviates drought-induced oxidative damage in ammonium supplied-rice roots

2018 ◽  
Author(s):  
Cao Xiaochuang ◽  
Zhu Chunquan ◽  
Zhong Chu ◽  
Zhang Junhua ◽  
Zhu Lianfeng ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Oxidative Damage ◽  
Protective Role ◽  
Antioxidant Defenses ◽  
No Production ◽  
No Donor ◽  
Rice Roots ◽  
Nos Inhibitor ◽  
Oxide Synthase

AbstractAmmonium (NH4+) can enhance rice drought tolerance in comparison to nitrate (NO3-). The mechanism underpinning this relationship was investigated based on the time-dependent nitric oxide (NO) production and its protective role in oxidative stress of NH4+-/NO3--supplied rice under drought. An early burst of NO was induced by drought 3h after root NH4+ treatment but not after NO3- treatment. Root oxidative damage induced by drought was significantly higher in NO3- than in NH4+-treatment due to its reactive oxygen species accumulation. Inducing NO production by applying NO donor 3h after NO3- treatment alleviated the oxidative damage, while inhibiting the early NO burst increased root oxidative damage in NH4+ treatment. Application of nitric oxide synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) completely suppressed NO synthesis in roots 3h after NH4+ treatment and aggravated drought-induced oxidative damage, indicating the aggravation of oxidative damage might have resulted from changes in NOS-mediated early NO burst. Drought also increased root antioxidant enzymes activities, which were further induced by NO donor but repressed by NO scavenger and NOS inhibitor in NH4+-treated roots. Thus, the NOS-mediated early NO burst plays an important role in alleviating oxidative damage induced by drought by enhancing antioxidant defenses in NH4+-supplied rice roots.HighlightNOS-mediated early NO burst plays an important role in alleviating oxidative damage induced by water stress, by enhancing the antioxidant defenses in roots supplemented with NH4+

Abstract 3635: Inhibition of Inhibitor Kappa B Kinase Beta Augments Endothelial Nitric Oxide Synthase Activity and Nitric Oxide Production in Aortic Endothelial Cells

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Sumathy Mohan ◽  
Ryzard Konopinski ◽  
Mohan Natarajan
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Nitric Oxide Synthase ◽  
Endothelial Nitric Oxide Synthase ◽  
Vascular Endothelial Cells ◽  
No Production ◽  
Enos Activity ◽  
Hsp 90 ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

A decline in the bioavailability of nitric oxide (NO) that causes endothelial dysfunction is a hall-mark of diabetes. The availability of NO to the vasculature is regulated by endothelial nitric oxide synthase (eNOS) activity and the involvement of heat shock protein 90 (Hsp-90) in the regulation of eNOS activity has been demonstrated. Hsp-90 has been shown to interact with upstream kinases (inhibitor kappa B kinases α, β and γ) in non-vascular cells. In this study, we have investigated the interaction of Hsp-90-IKKβ in endothelial cells under conditions of high glucose (HG) as a possible mechanism that diminishes Hsp-90-eNOS interaction, which could contribute to reduced bioavailability of NO. We report for the first time that IKKβ interacts with Hsp-90 and this interaction is augmented by HG in vascular endothelial cells. HG also augments transcriptional (4.02 ± 0.81-folds) and translational (1.97 ± 0.17-fold) expression as well as the catalytic activity of IKKβ (2.04 ± 0.06-folds). Another important and novel finding is that both IKKβ and eNOS could be co-immunoprecipitated with Hsp-90 (Figures A & B ) thus indicating the possible existence of a complex of IKKβ and eNOS interacting with single pool of Hsp-90. Inhibition of Hsp-90 with geldanamycin (2μM) or Radicicol (20μM) mitigated (0.45 ± 0.04 -fold and 0.93 ± 0.16-fold, respectively) HG induced-IKKβ activity (2.5 ± 0.416-fold). Blocking of IKKβ expression by IKK inhibitor II (15μM wedelolactone) or siRNA improved Hsp-90-eNOS interaction and NO production under conditions of HG. These results illuminate a possible mechanism for the declining eNOS activity reported under conditions of HG.

Nebivolol-induced vasodilation of renal afferent arterioles involves β3-adrenergic receptor and nitric oxide synthase activation

2012 ◽  
Vol 303 (5) ◽  
pp. F775-F782 ◽  
Author(s):  
Ming-Guo Feng ◽  
Minolfa C. Prieto ◽  
L. Gabriel Navar
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Soluble Guanylate Cyclase ◽  
Adrenergic Blocker ◽  
Afferent Arterioles ◽  
Renal Microvasculature ◽  
Nos Inhibitor ◽  
Oxide Synthase ◽  
Arteriolar Vasodilation

Nebivolol is a β1-adrenergic blocker that also elicits renal vasodilation and increases the glomerular filtration rate (GFR). However, its direct actions on the renal microvasculature and vasodilator mechanism have not been established. We used the in vitro blood-perfused juxtamedullary nephron technique to determine the vasodilator effects of nebivolol and to test the hypothesis that nebivolol induces vasodilation of renal afferent arterioles via an nitric oxide synthase (NOS)/nitric oxide (NO)/soluble guanylate cyclase (sGC)/cGMP pathway and the afferent arteriolar vasodilation effect may be mediated through the release of NO by activation of NOS via a β3-adrenoceptor-dependent mechanism. Juxtamedullary nephrons were superfused with nebivolol either alone or combined with the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or the NOS inhibitor Nω-nitro-l-arginine (l-NNA) or the β-blockers metoprolol (β1), butoxamine (β2), and SR59230A (β3). Nebivolol (100 μmol/l) markedly increased afferent and efferent arteriolar diameters by 18.9 ± 3.0 and 15.8 ± 1.8%. Pretreatment with l-NNA (1,000 μmol/l) or ODQ (10 μmol/l) decreased afferent vasodilator diameters and prevented the vasodilator effects of nebivolol (2.0 ± 0.2 and 2.4 ± 0.6%). Metoprolol did not elicit significant changes in afferent vasodilator diameters and did not prevent the effects of nebivolol to vasodilate afferent arterioles. However, treatment with SR59230A, but not butoxamine, markedly attenuated the vasodilation responses to nebivolol. Using a monoclonal antibody to β3-receptors revealed predominant immunostaining on vascular and glomerular endothelial cells. These data indicate that nebivolol vasodilates both afferent and efferent arterioles and that the afferent vasodilator effect is via a mechanism that is independent of β1-receptors but is predominantly mediated via a NOS/NO/sGC/cGMP-dependent mechanisms initiated by activation of endothelial β3-receptors.

Roles of accumulated endogenous nitric oxide synthase inhibitors, enhanced arginase activity, and attenuated nitric oxide synthase activity in endothelial cells for pulmonary hypertension in rats

2007 ◽  
Vol 292 (6) ◽  
pp. L1480-L1487 ◽  
Author(s):  
Akihito Sasaki ◽  
Shouzaburoh Doi ◽  
Shuki Mizutani ◽  
Hiroshi Azuma
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Pulmonary Hypertension ◽  
Nitric Oxide Synthase ◽  
Pulmonary Artery ◽  
Protein Expression ◽  
No Production ◽  
Nos Inhibitors ◽  
Endogenous Nitric Oxide ◽  
Oxide Synthase

Nitric oxide (NO) has been suggested to play a key role in the pathogenesis of pulmonary hypertension (PH). To determine which mechanism exists to affect NO production, we examined the concentration of endogenous nitric oxide synthase (NOS) inhibitors and their catabolizing enzyme dimethylarginine dimethylaminohydrolase (DDAH) activity and protein expression (DDAH1 and DDAH2) in pulmonary artery endothelial cells (PAECs) of rats given monocrotaline (MCT). We also measured NOS and arginase activities and NOS protein expression. Twenty-four days after MCT administration, PH and right ventricle (RV) hypertrophy were established. Endothelium-dependent, but not endothelium-independent, relaxation and cGMP production were significantly impaired in pulmonary artery specimens of MCT group. The constitutive NOS activity and protein expression in PAECs were significantly reduced in MCT group, whereas the arginase, which shares l-arginine as a common substrate with NOS, activity was significantly enhanced in PAECs of MCT group. The contents of monomethylarginine (MMA) and asymmetric dimethylarginine (ADMA), but not symmetric dimethylarginine (SDMA), were increased in PAECs of MCT group. The DDAH activity and DDAH1, but not DDAH2, protein expression were significantly reduced in PAECs of MCT group. These results suggest that the impairment of cGMP production as a marker of NO production is possibly due to the blunted endothelial NOS activity resulting from the downregulation of endothelial NOS protein, accumulation of endogenous NOS inhibitors, and accelerated arginase activity in PAECs of PH rats. The decreased overall DDAH activity accompanied by the downregulation of DDAH1 would bring about the accumulation of endogenous NOS inhibitors.

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