Abstract P250: A High-throughput Nitric Oxide Measurement Assay Reveals That Angiotensin-(1-5) Is An AT2 Receptor Agonist

Hypertension ◽  
2021 ◽  
Vol 78 (Suppl_1) ◽  
Author(s):  
Igor M Souza Silva ◽  
Kenneth Kjærgaard ◽  
Christina Mortensen ◽  
Robson A Santos ◽  
Thiago Verano-Braga ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
High Throughput ◽  
Cho Cells ◽  
Receptor Agonist ◽  
No Production ◽  
At2 Receptor ◽  
Ang Ii ◽  
No Release ◽  
High Throughput Assay

The angiotensin AT2-receptor (AT2R) is a key component within the protective arm of the renin-angiotensin system (RAS), being involved in nitric oxide (NO) production and vasodilation. To this date, no quantitative high-throughput assay is available to identify AT2R agonists in vitro , which may be a reason for the low number of AT2R selective ligands in drug development programs. Objective: To design and validate a high-throughput method for detection of AT2R activation in vitro . Methods and Results: NO release was selected as readout for AT2R activation in AT2R transfected (CHO-AT2) and non-transfected (CHO-NT) CHO cells using DAF-FM (5x10-6 mol/L) as NO probe. Cells were seeded on 96-well plates and stimulated for 15 minutes with C21 or Ang II (established AT2R agonists, 10-6mol/L), Ang-(1-5) (molecule with unknown biological status, 10-6 mol/L) or Ang-(1-7) (Mas-receptor agonist, 10-7 mol/L). After fixation of cells, fluorescence signals were captured by fluorescence microscopy using an automated imaging system (ImageXpress Pico, Molecular Devices, San Jose, USA) and image analysis by ImageJ. In CHO-AT2, C21 (+34.78 ± 12.09%), Ang II (+28.76 ± 17.65%) and Ang-(1-5) (+78.00 ± 23.82%) increased NO release (one-way ANOVA, p < 0.05 vs control, at least 3 independent experiments), while Ang-(1-7) had no effect (+ 0.13 ± 4.74%). In CHO-NT, none of the compounds stimulated release of NO (C21 -4.91 ±10.24%; Ang II -4.79 ± 12.44%; Ang (1-5) -8.88 ± 18.16%; Ang-(1-7) -11.57 ± 19.95%) indicating that the responses in CHO-AT2 were AT2R specific. Conclusion: Measurement of NO release from AT2R transfected CHO cells by DAF-FM fluorescence in an automated way is suitable as high-throughput assay for the identification of AT2R-agonistic compounds in vitro , Application of the assay revealed that Ang-(1-5), which is commonly regarded as an inactive metabolite of Ang II, has AT2R agonistic properties.

scholarly journals Interaction of the endothelial nitric oxide synthase with the CAT-1 arginine transporter enhances NO release by a mechanism not involving arginine transport

2005 ◽  
Vol 386 (3) ◽  
pp. 567-574 ◽  
Author(s):  
Chunying LI ◽  
Wei HUANG ◽  
M. Brennan HARRIS ◽  
Jonathan M. GOOLSBY ◽  
Richard C. VENEMA
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Endothelial Nitric Oxide Synthase ◽  
Adaptor Protein ◽  
No Production ◽  
Arginine Transport ◽  
No Release ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

eNOS (endothelial nitric oxide synthase) catalyses the conversion of L-arginine into L-citrulline and NO. Evidence has been presented previously that eNOS is associated with the CAT (cationic amino acid transporter)-1 arginine transporter in endothelial caveolae, and it has been proposed that eNOS–CAT-1 association facilitates the delivery of extracellular L-arginine to eNOS. Definitive proof of a protein–protein interaction between eNOS and CAT-1 is lacking, however, and it is also unknown whether the two proteins interact directly or via an adaptor protein. In the present study, we raised a polyclonal antibody against CAT-1, and show using reciprocal co-immunoprecipitation protocols that eNOS and CAT-1 do indeed form a complex in BAECs (bovine aortic endothelial cells). In vitro binding assays with GST (glutathione S-transferase)–CAT-1 fusion proteins and eNOS show that the two proteins interact directly and that no single CAT-1 intracellular domain is sufficient to mediate the interaction. Overexpression of CAT-1 in BAECs by adenoviral-mediated gene transfer results in significant increases in both L-arginine uptake and NO production by the cells. However, whereas increased L-arginine transport is reversed completely by the CAT-1 inhibitor, L-lysine, increased NO release is unaltered, suggesting that NO production in this in vitro model is independent of CAT-1-mediated transport. Furthermore, eNOS enzymic activity is increased in lysates of CAT-1-overexpressing cells accompanied by increased phosphorylation of eNOS at Ser-1179 and Ser-635, and decreased association of eNOS with caveolin-1. Taken together, these data suggest that direct interaction of eNOS with CAT-1 enhances NO release by a mechanism not involving arginine transport.

Abstract 335: Atheroma-specific Delivery of Synthetic High-density Lipoprotein Containing Sphingosine-1-phosphate for Modulation of Vascular Inflammation.

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Emily E Morin ◽  
Yanhong Guo ◽  
Rui Kuai ◽  
Gergely Lautner ◽  
Mark E Meyerhoff ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Vascular Inflammation ◽  
The Body ◽  
Saline Control ◽  
No Production ◽  
No Release ◽  
Sphingosine 1 Phosphate ◽  
Anti Inflammatory

Introduction: Sphingosine-1-phosphate (S1P) is a potent anti-inflammatory signaling lipid carried in the body by circulating HDL. HDL has been shown to exhibit anti-inflammatory activities through activation of endothelial nitric oxide synthase (eNOS) and subsequent production and release of nitric oxide (NO) by endothelial cells. Objective: The aim of this study is to use synthetic HDL particles to selectively deliver S1P to the site of arterial plaques in order to exert anti-inflammatory activity and modulate the progression of atherosclerosis. Methods/Results: Synthetic HDL (sHDL) particles were prepared using the ApoA1 mimetic peptide 22A (PVLDLFRELLNELLEALKQKLK), dipalmitoylphosphatidylcholine (DPPC) and sphingomyelin. We also prepared sHDL containing either the hydrophobic dye, DiD, or S1P to assess the capability of sHDL to effectively reach atheroma site and induce nitric oxide (NO) release, respectively. The purity of all particles was determined to be > 97% and average particle size was 9.6 ± 0.4 nm for all preparations. To measure sHDL accumulation in the plaque, ApoE -/- mice were intravenously injected with 0.2 mg/kg HDL-DiD. Whole aortas were excised and analysed by IVUS imaging system, revealing significant accumulation of sHDL-DiD in the atherosclerotic lesions. We then tested the ability of sHDL to deliver S1P in vitro and induce NO production by treating human umbilical vein endothelial cells (HUVEC) with 1 mg/mL of 22A-DPPC-sHDL containing 0, 0.05, 0.5, or 5 nmol/mL of S1P using free 22A peptide (1 mg/mL) and saline as controls, and analyzing media by ozone chemiluminescence. Blank sHDL particles increased NO production two-fold over controls (0.27 ± 0.02 μM for 22A-DPPC-sHDLDL, 0.13 ± 0.01 μM PBS and 0.14 ± 0.02 μM for 22A peptide), while HDL-S1P further increased NO release: 0.35 ± 0.03, 0.44 ± 0.01, and 0.59 ± 0.01 μM for HDL with 0.05, 0.5, and 5 nmol/mL S1P, respectively. Conclusions: Our studies show that HDL is capable of delivering hydrophobic cargo to atherosclerotic plaques, making HDL a promising platform to deliver S1P for modulation vascular inflammation and atherosclerosis. In vitro studies have revealed that HDL-S1P is able to increase NO production 2 to 4-fold over saline control setting the basis for future in vivo studies.

Phosphorylation of endothelial nitric oxide synthase by atypical PKCζ contributes to angiopoietin-1–dependent inhibition of VEGF-induced endothelial permeability in vitro

Blood ◽  
2009 ◽  
Vol 114 (15) ◽  
pp. 3343-3351 ◽  
Author(s):  
Malika Oubaha ◽  
Jean-Philippe Gratton
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Endothelial Permeability ◽  
No Production ◽  
No Release ◽  
Dependent Inhibition ◽  
Transendothelial Permeability ◽  
Endothelial Nitric Oxide ◽  
Angiopoietin 1

Abstract Vascular endothelial growth factor (VEGF) is a potent angiogenic cytokine that also increases vascular permeability. Nitric oxide (NO) released from endothelial cells, after activation of endothelial NO synthase (eNOS), contributes to proangiogenic and permeability effects of VEGF. Angiopoietin-1 (Ang-1), via Tie2 receptors, shares many of the proangiogenic properties of VEGF on endothelial cells. However, in contrast to VEGF, Ang-1 protects blood vessels from increased plasma leakage, which contributes to their stabilization. Because eNOS-derived NO is central to increased permeability in response to VEGF, we investigated whether Ang-1 interferes with VEGF signaling to eNOS. We demonstrate that Ang-1 stimulation of endothelial cells inhibits VEGF-induced NO release and transendothelial permeability. In contrast to VEGF stimulation, Ang-1 causes a marked protein kinase C (PKC)–dependent increase in phosphorylation of eNOS on the inhibitory Thr497. Furthermore, using pharmacologic inhibitors, overexpression studies, and small interfering RNA-mediated gene silencing, we demonstrate that atypical PKCζ is responsible for phosphorylation of eNOS on Thr497 in response to Ang-1. In addition, PKCζ knockdown abrogates the capacity of Ang-1 to inhibit VEGF-induced NO release and endothelial permeability. Thus, inhibition of NO production by Ang-1, via phosphorylation of eNOS on Thr497 by PKCζ, is responsible, at least in part, for inhibition of VEGF-stimulated endothelial permeability by Ang-1.

Carbon monoxide induces vasodilation and nitric oxide release but suppresses endothelial NOS

1999 ◽  
Vol 277 (6) ◽  
pp. F882-F889 ◽  
Author(s):  
Christian Thorup ◽  
Caroline L. Jones ◽  
Steven S. Gross ◽  
Leon C. Moore ◽  
Michael S. Goligorsky
Keyword(s):  
Nitric Oxide ◽  
Carbon Monoxide ◽  
Feedback Regulation ◽  
No Production ◽  
Resistance Arteries ◽  
Vascular Effects ◽  
Nitric Oxide Release ◽  
In Vitro Measurements ◽  
No Release

The vascular effects of carbon monoxide (CO) resemble those of nitric oxide (NO), but it is unknown whether the two messengers converge or exhibit reciprocal feedback regulation. These questions were examined in microdissected perfused renal resistance arteries (RRA) studied using NO-sensitive microelectrodes. Perfusion of RRA with buffers containing increasing concentrations of CO resulted in a biphasic release of NO. The NO response peaked at 100 nM CO and then declined to virtually zero at 10 μM. When a series of 50-s pulses of 100 nM CO were applied repeatedly (150-s interval), the amplitude of consecutive NO responses was diminished. NO release from RRA showed dependence on l-arginine but notd-arginine, and the responses to CO were inhibited by pretreatment with NG-nitro-l-arginine methyl ester (l-NAME), an inhibitor of NO synthases (NOS). CO (100 nM) also suppressed NO release induced by 100 μM carbachol, a potent agonist for endothelial NOS (eNOS). RRA from rats in which endogenous CO production from inducible HO was elevated (cobalt chloride 12 h prior to study) also showed suppressed responses to carbachol. Furthermore, responses consistent with these findings were obtained in juxtamedullary afferent arterioles perfused in vitro, where the vasodilatory response to CO was biphasic and the response to acetylcholine was blunted. Collectively, these data suggest that the CO-induced NO release could be attributed to either stimulation of eNOS or to NO displacement from a cellular storage pool. To address this, direct in vitro measurements with an NO-selective electrode of NO production by recombinant eNOS revealed that CO dose-dependently inhibits NO synthesis. Together, the above data demonstrate that, whereas high levels of CO inhibit NOS activity and NO generation, lower concentrations of CO induce release of NO from a large intracellular pool and, therefore, may mimic the vascular effects of NO.

Neuregulin-1 Compensates for Endothelial Nitric Oxide Synthase Deficiency

2021 ◽  
Author(s):  
Hadis Shakeri ◽  
Jente R.A. Boen ◽  
Sofie De Moudt ◽  
Jhana O. Hendrickx ◽  
Arthur J.A. Leloup ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Cardiac Fibrosis ◽  
Separate Experiment ◽  
No Production ◽  
Ang Ii ◽  
Wild Type ◽  
Paracrine Factor ◽  
Renal Hypertrophy ◽  
Neuregulin 1 ◽  
Endothelial Nitric Oxide

Endothelial cells (ECs) secrete different paracrine signals that modulate the function of adjacent cells; two examples of these paracrine signals are nitric oxide (NO) and neuregulin-1 (NRG1), a cardioprotective growth factor. Currently, it is undetermined whether one paracrine factor can compensate for the loss of another. Herein, we hypothesized that NRG1 can compensate for endothelial NO synthase (eNOS) deficiency. Methods. We characterized eNOS null and wild type (WT) mice by cardiac ultrasound and histology and we determined circulating NRG1 levels. In a separate experiment, 8 groups of mice were divided into 4 groups of eNOS null mice and wild type (WT) mice; half of the mice received angiotensin II (Ang II) to induce a more severe phenotype. Mice were randomized to daily injections with NRG1 or vehicle for 28 days. Results. eNOS deficiency increased NRG1 plasma levels, indicating that ECs increase their NRG1 expression when NO production is deleted. eNOS deficiency also increased blood pressure, lowered heart rate, induced cardiac fibrosis, and affected diastolic function. In eNOS null mice, Ang II administration increased cardiac fibrosis, but also induced cardiac hypertrophy and renal fibrosis. NRG1 administration prevented the cardiac and renal hypertrophy and fibrosis caused by Ang II infusion and eNOS deficiency. Moreover, Nrg1 expression in the myocardium is shown to be regulated by miR-134. Conclusion. This study indicates that administration of endothelium-derived NRG1 can compensate for eNOS deficiency in the heart and kidneys.

scholarly journals Effects of chitosan on nitric oxide production and inducible nitric oxide synthase activity and mRNA expression in weaned piglets

2018 ◽  
Vol 60 (No. 8) ◽  
pp. 359-366
Author(s):  
J. Li ◽  
B. Shi ◽  
S. Yan ◽  
L. Jin ◽  
Y. Guo ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Mrna Expression ◽  
Inducible Nitric Oxide Synthase ◽  
No Production ◽  
Weaned Piglets ◽  
Inos Activity ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

The effects of chitosan on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) activity and gene expression in vivo or vitro were investigated in weaned piglets. In vivo, 180 weaned piglets were assigned to five dietary treatments with six replicates. The piglets were fed on a basal diet supplemented with 0 (control), 100, 500, 1000, and 2000 mg chitosan/kg feed, respectively. In vitro, the peripheral blood mononuclear cells (PBMCs) from a weaned piglet were cultured respectively with 0 (control), 40, 80, 160, and 320 &micro;g chitosan/ml medium. Results showed that serum NO concentrations on days 14 and 28 and iNOS activity on day 28 were quadratically improved with increasing chitosan dose (P &lt; 0.05). The iNOS mRNA expressions were linearly or quadratically enhanced in the duodenum on day 28, and were improved quadratically in the jejunum on days 14 and 28 and in the ileum on day 28 (P &lt; 0.01). In vitro, the NO concentrations, iNOS activity, and mRNA expression in unstimulated PBMCs were quadratically enhanced by chitosan, but the improvement of NO concentrations and iNOS activity by chitosan were markedly inhibited by N-(3-[aminomethyl] benzyl) acetamidine (1400w) (P&nbsp;&lt; 0.05). Moreover, the increase of NO concentrations, iNOS activity, and mRNA expression in PBMCs induced by lipopolysaccharide (LPS) were suppressed significantly by chitosan (P &lt; 0.05). The results indicated that the NO concentrations, iNOS activity, and mRNA expression in piglets were increased by feeding chitosan in a dose-dependent manner. In addition, chitosan improved the NO production in unstimulated PBMCs but inhibited its production in LPS-induced cells, which exerted bidirectional regulatory effects on the NO production via modulated iNOS activity and mRNA expression.

Decrease in ambient [Cl-] stimulates nitric oxide release from cultured rat mesangial cells

1994 ◽  
Vol 267 (1) ◽  
pp. F190-F195 ◽  
Author(s):  
H. Tsukahara ◽  
Y. Krivenko ◽  
L. C. Moore ◽  
M. S. Goligorsky
Keyword(s):  
Nitric Oxide ◽  
Mesangial Cells ◽  
Ionic Composition ◽  
Juxtaglomerular Apparatus ◽  
Cytosolic Calcium ◽  
No Synthase ◽  
Tubuloglomerular Feedback ◽  
No Production ◽  
Rapid Reduction ◽  
No Release

It has been hypothesized that fluctuations of the ionic composition in the interstitium of juxtaglomerular apparatus (JGA) modulate the function of extraglomerular mesangial cells (MC), thereby participating in tubuloglomerular feedback (TGF) signal transmission. We examined the effects of isosmotic reductions in ambient sodium concentration ([Na+]) and [Cl-] on cytosolic calcium concentration ([Ca2+]i) in cultured rat MC. Rapid reduction of [Na+] or [Cl-] in the bath induced a concentration-dependent rise in [Ca2+]i. MC are much more sensitive to decreases in ambient [Cl-] than to [Na+]; a decrease in [Cl-] as small as 14 mM was sufficient to elicit a detectable [Ca2]i response. These observations suggest that MC can be readily stimulated by modest perturbations of extracellular [Cl-]. Next, we examined whether activation of MC by lowered ambient [Cl-] influences cellular nitric oxide (NO) production. Using an amperometric NO sensor, we found that a 13 mM decrease in ambient [Cl-] caused a rapid, Ca2+/calmodulin-dependent rise in NO release from MC. This response was not inhibitable by dexamethasone, indicating the involvement of the constitutive rather than the inducible type of NO synthase in MC. In addition, the NO release was blunted by indomethacin pretreatment, suggesting that a metabolite(s) of cyclooxygenase regulates the activation of NO synthase in MC. Our findings that small perturbations in external [Cl-] stimulate MC to release NO, a highly diffusible and rapidly acting vasodilator, provide a possible mechanism to explain the transmission of the signal for the TGF response within the JGA.

scholarly journals New Cyclic Diarylheptanoids from Platycarya strobilacea

Molecules ◽  
2020 ◽  
Vol 25 (24) ◽  
pp. 6034
Author(s):  
Wen-bing Ding ◽  
Rui-yuan Zhao ◽  
Guan-hua Li ◽  
Bing-lei Liu ◽  
Hua-liang He ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Pc12 Cells ◽  
Stem Bark ◽  
No Production ◽  
Electronic Circular Dichroism ◽  
Pheochromocytoma Pc12 ◽  
Pheochromocytoma Pc12 Cells ◽  
Induced Apoptosis ◽  
Circular Dichroïsm

Five new cyclic diarylheptanoids (platycary A–E, compounds 1–5) and three previously identified analogues (i.e., phttyearynol (compound 6), myricatomentogenin (compound 7), and juglanin D (compound 8)) were isolated from the stem bark of Platycarya strobilacea. The structures of these compounds were determined using NMR, HRESIMS, and electronic circular dichroism (ECD) data. The cytotoxicity of compounds 1–5 and their ability to inhibit nitric oxide (NO) production, as well as protect against the corticosterone-induced apoptosis of Pheochromocytoma (PC12) cells, were evaluated in vitro using the appropriate bioassays. Compounds 1 and 2 significantly inhibited the corticosterone-induced apoptosis of PC12 cells at a concentration of 20 μΜ.

Tanshinone IIA Inhibits Angiotensin II-Induced Cell Proliferation in Rat Cardiac Fibroblasts

2011 ◽  
Vol 39 (02) ◽  
pp. 381-394 ◽  
Author(s):  
Paul Chan ◽  
Ju-Chi Liu ◽  
Li-Jen Lin ◽  
Po-Yuan Chen ◽  
Tzu-Hurng Cheng ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Cell Proliferation ◽  
Angiotensin Ii ◽  
Cardiac Fibroblasts ◽  
Tanshinone Iia ◽  
No Production ◽  
Ang Ii ◽  
Erk Phosphorylation ◽  
Enos Phosphorylation ◽  
Ros Formation

Tanshinone IIA extracted from Danshen, a popular medicinal herb used in traditional Chinese medicine, exhibits cardio-protective effects. However, the mechanism of its cardioprotective effect is not well established. The aims of this study were to examine whether tanshinone IIA may alter angiotensin II (Ang II)-induced cell proliferation and to identify the putative underlying signaling pathways in rat cardiac fibroblasts. Cultured rat cardiac fibroblasts were pre-treated with tanshinone IIA and stimulated with Ang II, cell proliferation and endothelin-1 (ET-1) expression were examined. The effect of tanshinone IIA on Ang II-induced reactive oxygen species (ROS) formation, and extracellular signal-regulated kinase (ERK) phosphorylation were also examined. In addition, the effect of tanshinone IIA on nitric oxide (NO) production, and endothelial nitric oxide synthase (eNOS) phosphorylation were tested to elucidate the intracellular mechanism. The increased cell proliferation and ET-1 expression by Ang II (100 nM) were partially inhibited by tanshinone IIA. Tanshinone IIA also inhibited Ang II-increased ROS formation, and ERK phosphorylation. In addition, tanshinone IIA was found to increase the NO generation, and eNOS phosphorylation. NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, and the short interfering RNA transfection for eNOS markedly attenuated the inhibitory effect of tanshinone IIA on Ang II-induced cell proliferation. The results suggest that tanshinone IIA prevents cardiac fibroblast proliferation by interfering with the generation of ROS and involves the activation of the eNOS-NO pathway.

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