Flow- and bradykinin-induced nitric oxide production by endothelial cells is independent of membrane potential

1996 ◽  
Vol 270 (2) ◽  
pp. C546-C551 ◽  
Author(s):  
K. J. Gooch ◽  
J. A. Frangos
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Membrane Potential ◽  
Umbilical Vein ◽  
Fold Increase ◽  
No Production ◽  
Control Medium ◽  
Rich Medium ◽  
Membrane Hyperpolarization ◽  
Umbilical Vein Endothelial Cells

The objective of this study was to evaluate the role transmembrane potential plays in flow-induced nitric oxide (NO) production in endothelial cells (EC). NO production was monitored by measuring intracellular guanosine 3',5'-cyclic monophosphate (cGMP) and extracellular nitrite plus nitrate (NOx). Primary human umbilical vein endothelial cells (HUVEC) were exposed to laminar flow (22 dyn/cm2) of medium with 5.4 mM KCl (control medium) with or without 3 mM tetraethylammonium chloride (TEA) or 90 mM KCl (K(+)-rich medium). Bradykinin (BK) was added to time-matched stationary cultures to give a final concentration of 5 nM. With control medium, 30 s, 2 min, and 3 h of treatment with flow or 2 min of treatment with BK resulted in an approximately threefold increase in cGMP over stationary cultures. Depolarization with KCl or TEA did not influence cGMP production in flow-treated or stationary cultures. Flow of either control or potassium-rich medium resulted in an approximately 10-fold increase in average NOx production rate over 3 h compared with stationary cultures. Taken together these data indicate that neither membrane hyperpolarization nor normal membrane potential is necessary for flow- or BK-induced NO production by HUVEC.

Small- and intermediate-conductance Ca2+-activated K+ channels directly control agonist-evoked nitric oxide synthesis in human vascular endothelial cells

2007 ◽  
Vol 293 (1) ◽  
pp. C458-C467 ◽  
Author(s):  
Jian-Zhong Sheng ◽  
Andrew P. Braun
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Vascular Endothelial Cells ◽  
Umbilical Vein ◽  
Early Event ◽  
No Production ◽  
Primary Role ◽  
Membrane Hyperpolarization ◽  
Intracellular Ca ◽  
Umbilical Vein Endothelial Cells

The contribution of small-conductance (SKCa) and intermediate-conductance Ca2+-activated K+ (IKCa) channels to the generation of nitric oxide (NO) by Ca2+-mobilizing stimuli was investigated in human umbilical vein endothelial cells (HUVECs) by combining single-cell microfluorimetry with perforated patch-clamp recordings to monitor agonist-evoked NO synthesis, cytosolic Ca2+ transients, and membrane hyperpolarization in real time. ATP or histamine evoked reproducible elevations in NO synthesis and cytosolic Ca2+, as judged by 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM) and fluo-3 fluorescence, respectively, that were tightly associated with membrane hyperpolarizations. Whereas evoked NO synthesis was unaffected by either tetraethylammonium (10 mmol/l) or BaCl2 (50 μmol/l) + ouabain (100 μmol/l), depleting intracellular Ca2+ stores by thapsigargin or removing external Ca2+ inhibited NO production, as did exposure to high (80 mmol/l) external KCl. Importantly, apamin and charybdotoxin (ChTx)/ triarylmethane (TRAM)-34, selective blockers SKCa and IKCa channels, respectively, abolished both stimulated NO synthesis and membrane hyperpolarization and decreased evoked Ca2+ transients. Apamin and TRAM-34 also inhibited an agonist-induced outwardly rectifying current characteristic of SKCa and IKCa channels. Under voltage-clamp control, we further observed that the magnitude of agonist-induced NO production varied directly with the degree of membrane hyperpolarization. Mechanistically, our data indicate that SKCa and IKCa channel-mediated hyperpolarization represents a critical early event in agonist-evoked NO production by regulating the influx of Ca2+ responsible for endothelial NO synthase activation. Moreover, it appears that the primary role of agonist-induced release of intracellular Ca2+ stores is to trigger the opening of both KCa channels along with Ca2+ entry channels at the plasma membrane. Finally, the observed inhibition of stimulated NO synthesis by apamin and ChTx/TRAM-34 demonstrates that SKCa and IKCa channels are essential for NO-mediated vasorelaxation.

scholarly journals Cilostazol Induces eNOS and TM Expression via Activation with Sirtuin 1/Krüppel-like Factor 2 Pathway in Endothelial Cells

2021 ◽  
Vol 22 (19) ◽  
pp. 10287
Author(s):  
Chih-Hsien Wu ◽  
Yi-Lin Chiu ◽  
Chung-Yueh Hsieh ◽  
Guo-Shiang Tsung ◽  
Lian-Shan Wu ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Endothelial Nitric Oxide Synthase ◽  
Umbilical Vein ◽  
Sirtuin 1 ◽  
No Production ◽  
Beneficial Effects ◽  
Umbilical Vein Endothelial Cells ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Cilostazol was suggested to be beneficial to retard in-stent atherosclerosis and prevent stent thrombosis. However, the mechanisms responsible for the beneficial effects of cilostazol are not fully understood. In this study, we attempted to verify the mechanism of the antithrombotic effect of cilostazol. Human umbilical vein endothelial cells (HUVECs) were cultured with various concentrations of cilostazol to verify its impact on endothelial cells. KLF2, silent information regulator transcript-1 (SIRT1), endothelial nitric oxide synthase (eNOS), and endothelial thrombomodulin (TM) expression levels were examined. We found cilostazol significantly activated KLF2 expression and KLF2-related endothelial function, including eNOS activation, Nitric oxide (NO) production, and TM secretion. The activation was regulated by SIRT1, which was also stimulated by cilostazol. These findings suggest that cilostazol may be capable of an antithrombotic and vasculoprotective effect in endothelial cells.

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 Hydrogen-Rich Medium Ameliorates Lipopolysaccharides-Induced Mitochondrial Fission and Dysfunction in Human Umbilical Vein Endothelial Cells (Huvecs) via Up-Regulating HO-1 Expression

2021 ◽  
Author(s):  
Keliang Xie ◽  
Xing Mao ◽  
Naqi Lian ◽  
Yanyan Wang ◽  
Yuzun Wang ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Umbilical Vein ◽  
Mitochondrial Fission ◽  
Protective Effects ◽  
Atp Content ◽  
Rich Medium ◽  
Umbilical Vein Endothelial Cells

Abstract Background Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection. It has been showed that the change of mitochondrial dynamics has been proved to be one of the main causes of death in patients with severe sepsis. And hydrogen has been proved to exert its protective effects against sepsis via heme oxygenase-1 (HO-1). This study was designed to demonstrate that whether the benefit effects of hydrogen can maintain the dynamic process of mitochondrial fusion/fission to mitigate human umbilical vein endothelial cells (HUVECs) injury exposed to endotoxin through HO-1. Methods HUVECs cells cultured with medium which contained Lipopolysaccharides (LPS), Saline, hydrogen, Mdivi-1 (a dynamin-related protein 1 [Drp1] inhibitor) or zinc protoporphyrin Ⅸ (Znpp) (a HO-1 inhibitor) were also used in the research. Cell death and apoptosis were assessed using FITC annexin V and PI. Mitochondria were stained with Mitotracker orange and observed by confocal microscope. Oxygen consumption rate was assessed by seahorse xf24 extracellular analyzer. Mitochondrial membrane potential monitored by JC-1 dye. The expressions of Drp1 and HO-1 were tested by Western blot. The co-localization of Drp1 and mitochondria was determined by immunofluorescence. Results LPS caused a decrease in ATP content, mitochondrial membrane potential, and maximal respiration rate. At the same time, increased expression of Drp1 were observed in LPS-stimulated HUVECs, concomitantly with excessive mitochondrial fission. We found that hydrogen-rich medium can increase ATP content, mitochondrial membrane potential and maximal respiration rate, and decrease the expression of Drp1 in LPS-treated HUVECs. Meanwhile, hydrogen can ameliorate excessive mitochondrial fission caused by LPS. Furthermore, hydrogen-rich medium had a similar effect to Mdivi-1, a mitochondrial fission blocker. Both of them rescued the up-regulation of Drp1 and mitochondrial fission induced by LPS, then normalized mitochondrial shape after LPS stimulation. But after Znpp pretreatment, HO-1 expression was inhibited and the protective effects of hydrogen were abrogated. Conclusions Hydrogen-rich medium can alleviate the LPS-induced mitochondrial fusion/fission and dysfunction in HUVECs via HO-1 up-regulation.

Abstract P198: Differential Regulation of Endothelial Nitric Oxide Synthase Phosphorylation by Protease-Activated Receptors in Adult Human Endothelial Cells

Hypertension ◽  
2015 ◽  
Vol 66 (suppl_1) ◽  
Author(s):  
Lakeisha C Tillery ◽  
Evangeline D Motley-Johnson
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Endothelial Nitric Oxide Synthase ◽  
Umbilical Vein ◽  
Differential Regulation ◽  
No Production ◽  
Protease Activated Receptors ◽  
Umbilical Vein Endothelial Cells ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Protease-activated receptors (PARs) have been shown to regulate endothelial nitric oxide synthase (eNOS) through the activation of specific sites on the enzyme. It has been established that phosphorylation of eNOS-Ser-1177 leads to the production of the potent vasodilator nitric oxide (NO), and is associated with PAR-2 activation; while phosphorylation of eNOS-Thr-495 decreases NO production, and is coupled to PAR-1 activation. In this study, we demonstrate a differential regulation of the eNOS/NO pathway by the PARs using primary adult human coronary artery endothelial cells (HCAEC). Thrombin and the PAR-1 activating peptide, TFLLR, which are known to phosphorylate eNOS-Thr-495 in bovine and human umbilical vein endothelial cells, phosphorylated eNOS-Ser-1177 in HCAECs, and increased NO production. The PAR-1 responses were blocked using SCH-79797, a PAR-1 inhibitor, and L-NAME was used to inhibit NO production. A PAR-2 specific ligand, SLIGRL, which has been shown to phosphorylate eNOS-Ser-1177 in bovine and human umbilical vein endothelial cells, primarily regulated eNOS-Thr-495 phosphorylation and suppressed NO production in the HCAECs. PAR-3, known for its non-signaling potential, was activated by TFRGAP, a PAR-3 mimicking peptide, and only induced phosphorylation of eNOS-Thr-495 with no effect on NO production. In addition, we confirmed that PAR-mediated eNOS-Ser-1177 phosphorylation was calcium-dependent using the calcium chelator, BAPTA, and eNOS-Thr-495 phosphorylation was mediated via Rho kinase using the ROCK inhibitor, Y-27632. These data suggest a vascular bed specific differential coupling of PARs to the signaling pathways that regulate eNOS and NO production that may be responsible for the modulation of endothelial function associated with cardiovascular disease.

scholarly journals Anti-Vascular Inflammatory Effect of Ethanol Extract from Securinega suffruticosa in Human Umbilical Vein Endothelial Cells

Nutrients ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 3448
Author(s):  
Byung Hyuk Han ◽  
Chun Ho Song ◽  
Jung Joo Yoon ◽  
Hye Yoom Kim ◽  
Chang Seob Seo ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Cell Adhesion ◽  
Protective Effect ◽  
Adhesion Molecule ◽  
Umbilical Vein ◽  
Vascular Inflammation ◽  
No Production ◽  
Tnf Α ◽  
Umbilical Vein Endothelial Cells

Securiniga suffruticosa is known as a drug that has the effect of improving the blood circulation and relaxing muscles and tendons, thereby protects and strengthen kidney and spleen. Therefore, in this study, treatment of Securiniga suffruticosa showed protective effect of inhibiting the vascular inflammation in human umbilical vein endothelial cells (HUVECs) by inducing nitric oxide (NO) production and endothelial nitric oxide synthase (eNOS) coupling pathway. In this study, Securiniga suffruticosa suppressed TNF-α (Tumor necrosis factor–α) induced protein and mRNA levels of cell adhesion molecules such as intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and Interleukin-6 (IL-6). Pretreatment of HUVEC with Securiniga suffruticosa decreased the adhesion of HL-60 cells to Ox-LDL (Oxidized Low-Density-Lipoprotein)-induced HUVEC. Moreover, Securiniga suffruticosa inhibited TNF-α induced intracellular reactive oxygen species (ROS) production. Securiniga suffruticosa also inhibited phosphorylation of IκB-α in cytoplasm and translocation of NF-κB (Nuclear factor-kappa B) p65 to the nucleus. Securiniga suffruticosa increased NO production, as well increased the phosphorylation of eNOS and Akt (protein kinase B) which are related with NO production. In addition, Securiniga suffruticosa increased the protein expression of GTPCH (Guanosine triphosphate cyclohydrolase Ⅰ) and the production of BH4 in HUVEC which are related with eNOS coupling pathway. In conclusion, Securiniga suffruticosa has a protective effect against vascular inflammation and can be a potential therapeutic agent for early atherosclerosis.

scholarly journals Glossogyne tenuifolia Extract Increases Nitric Oxide Production in Human Umbilical Vein Endothelial Cells

2021 ◽  
Vol 14 (6) ◽  
pp. 577
Author(s):  
Chin-Feng Hsuan ◽  
Thung-Lip Lee ◽  
Wei-Kung Tseng ◽  
Chau-Chung Wu ◽  
Chi-Chang Chang ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Western Blot ◽  
Umbilical Vein ◽  
No Production ◽  
Enos Expression ◽  
Human Umbilical Vein ◽  
Whole Plant ◽  
Umbilical Vein Endothelial Cells ◽  
Glossogyne Tenuifolia

The vascular nitric oxide (NO) system has a protective effect in atherosclerosis. NO is generated from the conversion of L-arginine to L-citrulline by the enzymatic action of endothelial NO synthase (eNOS). Compounds with the effect of enhancing eNOS expression are considered to be candidates for the prevention of atherosclerosis. In this study, extracts from the aerial, root, and whole plant of Glossogyne tenuifolia (GT) were obtained with ethanol, n-hexane, ethyl acetate (EA), and methanol extraction, respectively. The effects of these GT extracts on the synthesis of NO and the expression of eNOS in human umbilical vein endothelial cells (HUVECs) were investigated. NO production was determined as nitrite by colorimetry, following the Griess reaction. The treatment of HUVECs with EA extract from the root of GT and n-hexane, methanol, and ethanol extract from the aerial, root, and whole plant of GT increased NO production in a dose-dependent manner. When at a dose of 160 μg/mL, NO production increased from 0.9 to 18.4-fold. Among these extracts, the methanol extract from the root of GT (R/M GTE) exhibited the most potent effect on NO production (increased by 18.4-fold). Furthermore, using Western blot and RT–PCR analysis, treatment of HUVECs with the R/M GTE increased both eNOS protein and mRNA expression. In addition, Western blot analysis revealed that the R/M GTE increased eNOS phosphorylation at serine1177 as early as 15 min after treatment. The chemical composition for the main ingredients was also performed by HPLC analysis. In conclusion, the present study demonstrated that GT extracts increased NO production in HUVECs and that the R/M GTE increased NO production via increasing eNOS expression and activation by phosphorylation of eNOS at serine1177.

scholarly journals Serum Containing Tao-Hong-Si-Wu Decoction Induces Human Endothelial Cell VEGF Production via PI3K/Akt-eNOS Signaling

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
DengKe Yin ◽  
ZhuQing Liu ◽  
DaiYin Peng ◽  
Ye Yang ◽  
XiangDong Gao ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Umbilical Vein ◽  
No Production ◽  
Hydroxysafflor Yellow A ◽  
Vegf Expression ◽  
Pi3k Inhibitor Ly294002 ◽  
Ischemic Disease ◽  
Umbilical Vein Endothelial Cells

Tao-Hong-Si-Wu decoction (TSD) is a famous traditional Chinese medicine (TCM) and widely used for ischemic disease in China. TSD medicated serum was prepared after oral administration of TSD (1.6 g/kg) twice a day for 3 days in rats. TSD medicated serum induced human umbilical vein endothelial cells (HUVECs) proliferation, VEGF secretion, and nitric oxide (NO) production. These promoted effects of TSD were partly inhibited by treatment with PI3K inhibitor (LY294002) or eNOS inhibitor (L-NAME), respectively, and completely inhibited by treatment with LY294002 and L-NAME simultaneously. Western blot analysis findings further indicated that TSD medicated serum upregulated p-Akt and p-eNOS expressions, which were significantly inhibited by LY294002 or L-NAME and completely inhibited by both LY294002 and L-NAME; these results indicated that TSD medicated serum induced HUVECs VEGF expression via PI3K/Akt-eNOS signaling. TSD medicated serum contains hydroxysafflor yellow A, ferulic acid, and ligustilide detected by UPLC with standards, so these effect of TSD medicated serum may be associated with these three active compounds absorbed in serum.

scholarly journals Endothelial adenosine kinase deficiency ameliorates diet-induced insulin resistance

2019 ◽  
Vol 242 (2) ◽  
pp. 159-172 ◽  
Author(s):  
Jiean Xu ◽  
Qiuhua Yang ◽  
Xiaoyu Zhang ◽  
Zhiping Liu ◽  
Yapeng Cao ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Insulin Resistance ◽  
Endothelial Cells ◽  
Adenosine Receptor ◽  
Metabolic Disorders ◽  
Umbilical Vein ◽  
Adenosine Kinase ◽  
No Production ◽  
Umbilical Vein Endothelial Cells ◽  
Endothelial Nitric Oxide

Insulin resistance-related disorders are associated with endothelial dysfunction. Accumulating evidence has suggested a role for adenosine signaling in the regulation of endothelial function. Here, we identified a crucial role of endothelial adenosine kinase (ADK) in the regulation of insulin resistance. Feeding mice with a high-fat diet (HFD) markedly enhanced the expression of endothelial Adk. Ablation of endothelial Adk in HFD-fed mice improved glucose tolerance and insulin sensitivity and decreased hepatic steatosis, adipose inflammation and adiposity, which were associated with improved arteriole vasodilation, decreased inflammation and increased adipose angiogenesis. Mechanistically, ADK inhibition or knockdown in human umbilical vein endothelial cells (HUVECs) elevated intracellular adenosine level and increased endothelial nitric oxide synthase (NOS3) activity, resulting in an increase in nitric oxide (NO) production. Antagonism of adenosine receptor A2b abolished ADK-knockdown-enhanced NOS3 expression in HUVECs. Additionally, increased phosphorylation of NOS3 in ADK-knockdown HUVECs was regulated by an adenosine receptor-independent mechanism. These data suggest that Adk-deficiency-elevated intracellular adenosine in endothelial cells ameliorates diet-induced insulin resistance and metabolic disorders, and this is associated with an enhancement of NO production caused by increased NOS3 expression and activation. Therefore, ADK is a potential target for the prevention and treatment of metabolic disorders associated with insulin resistance.

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