Shear Stress Effect on the Production of Nitric Oxide in Cultured Rat Aorta Endothelial Cells

2002 ◽  
Author(s):  
Jianfeng Ye ◽  
Baoguo Chen ◽  
Lisa X. Xu
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Shear Stress ◽  
Protein Tyrosine Kinase ◽  
Vascular Endothelial Cells ◽  
Free Calcium ◽  
Enos Gene ◽  
No Production ◽  
Stress Effect ◽  
Dependent Manner

Atherosclerotic lesions tend to develop in regions where there are separations from unidirectional laminar blood flow, typically near branches, bifurcations, regions of arterial narrowing, and curvatures in the arteries (1, 2). Obviously, homodynamic forces play a key role in atherosclerosis. Studies also indicate that vascular endothelium function disturbance, especially impairment of endothelium dependent vasodilation, is involved (3). Shear stress affects endothelial cells in many ways, such as cytoskeletal rearrangement, decrease of intracellular pH, release of PGI2 and some growth factors (PDGF, FGF, ECGF, TGF-b, etc), activation of IP3 and mitogen-activated protein kinases, and the significant increase in the production of nitric oxide (1,2,4,5). As an important function factor of vascular endothelial cells, nitric oxide (NO) is closely related to the endothelial dysfunction and atherosclerosis (6). Endothelial derived nitric oxide involves in many events in the vasculature, including vasodilation, inhibition of platelet aggregation, adhesion molecule expression, and vascular smooth muscle proliferation, which are directly or indirectly related to atherosclerosis. Endothelial cells release NO more potently in response to increased shear stress than to agonists that raise intracellular free calcium concentration [Ca2+]i. Studies have indicated that NO production increases with a calcium/CaM dependent manner in the first few minutes after exposed to shear stress, followed by a sustained NO production that occurs more than 30min which is Ca2+ independent (7). The activation of eNOS by shear stress, which modulated by Ca/CaM, G protein, tyrosine kinase phosphorylation and eNOS gene expression, is responsible for the increase of NO production (8). However, the contribution of extracellular calcium to the production of NO is somewhat contradictory.

Adenosine enhances nitric oxide production by vascular endothelial cells

1995 ◽  
Vol 269 (2) ◽  
pp. C519-C523 ◽  
Author(s):  
J. M. Li ◽  
R. A. Fenton ◽  
B. S. Cutler ◽  
J. G. Dobson
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Vascular Tone ◽  
Vascular Endothelial Cells ◽  
Competitive Inhibitor ◽  
No Production ◽  
Dependent Manner ◽  
Bathing Medium ◽  
Adenosine Receptor Antagonist ◽  
Potent Vasodilator

Adenosine per se is a potent vasodilator of vascular smooth muscle. Endothelial cells modulate vascular tone via the release of nitric oxide (NO), which also elicits vasodilation. This study was undertaken to determine whether adenosine could directly stimulate endothelial cells to enhance NO production, which could subsequently reduce vascular tone. NO production was evaluated in porcine carotid artery endothelial cells (PCAEC) and human saphenous vein endothelial cells (HSVEC) seeded on multiwell plates, grown to confluence, and treated with adenosine for 1 h. The bathing medium was collected, and the NO production was determined as reflected by the formation of NO2- and NO3-. NO production by PCAEC was significantly increased by adenosine in a dose-dependent manner, whereas there was only an insignificant tendency for an increase by HSVEC. The addition of the NO synthase competitive inhibitor, NG-monomethyl-L-arginine (NMMA), or the adenosine receptor antagonist, theophylline, prevented the increase in NO production by adenosine. The results suggest that adenosine stimulates, by a receptor-mediated mechanism, the production of NO by arterial, but not by venous, endothelial cells.

Role of G proteins in shear stress-mediated nitric oxide production by endothelial cells

1994 ◽  
Vol 267 (3) ◽  
pp. C753-C758 ◽  
Author(s):  
M. J. Kuchan ◽  
H. Jo ◽  
J. A. Frangos
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Shear Stress ◽  
G Protein ◽  
G Proteins ◽  
No Production ◽  
Dependent Manner ◽  
Protein Activation ◽  
Synthase Inhibitor

Exposure of cultured endothelial cells to shear stress resulting from well-defined fluid flow stimulates the production of nitric oxide (NO). We have established that an initial burst in production is followed by sustained steady-state NO production. The signal transduction events leading to this stimulation are not well understood. In the present study, we examined the role of regulatory guanine nucleotide binding proteins (G proteins) in shear stress-mediated NO production. In endothelial cells not exposed to shear stress, AIF4-, a general activator of G proteins, markedly elevated the production of guanosine 3',5'-cyclic monophosphate (cGMP). Pretreatment with NO synthase inhibitor N omega-nitro-L-arginine completely blocked this stimulation. Incubation with guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), a general G protein inhibitor, blocked the flow-mediated burst in cGMP production in a dose-dependent manner. Likewise, GDP beta S inhibited NOx (NO2 + NO3) production for the 1st h. However, inhibition was not detectable between 1 and 3 h. Pertussis toxin (PTx) had no effect on the shear response at any time point. The burst in NO production caused by a change in shear stress appears to be dependent on a PTx-refractory G protein. Sustained shear-mediated production is independent of G protein activation.

Role of cytoskeleton in shear stress-induced endothelial nitric oxide production

1997 ◽  
Vol 273 (1) ◽  
pp. H347-H355 ◽  
Author(s):  
H. L. Knudsen ◽  
J. A. Frangos
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Shear Stress ◽  
Plasma Membrane ◽  
Cytochalasin D ◽  
Induced Response ◽  
No Production ◽  
Dependent Manner ◽  
Mechanochemical Transduction

To study the role of the cytoskeleton in mechanochemical transduction, human umbilical vein endothelial cells were exposed to cytoskeleton-disrupting or -stabilizing agents, and the flow-induced production of nitric oxide (NO) as monitored by intracellular levels of guanosine 3',5'-cyclic monophosphate (cGMP) was examined. A shear stress of 20 dyn/cm2 elevated cGMP levels approximately twofold relative to basal (stationary) levels (1.9 +/- 0.1 pmol cGMP in stationary controls; P < 0.01). Treatment with 1 microM phalloidin and 0.5 microM cytochalasin D did not significantly affect the flow-induced response (1.77 +/- 0.23 and 2.89 +/- 0.18 pmol cGMP in stationary controls, respectively), whereas disruption of microtubules with 0.5 microM colchicine significantly elevated the response (3.64 +/- 0.18 pmol cGMP in stationary controls; P < 0.01). The NO synthase inhibitor NG-amino-L-arginine abrogated all flow-induced elevations of cGMP, indicating that increased cGMP levels were mediated by NO. Cytoskeletal disruption with 0.2 microM cytochalasin D or 0.5 microM colchicine did not alter cGMP levels in response to 10 nM bradykinin. The role of the plasma membrane in mechanochemical transduction was examined by treatment with cholesteryl hemisuccinate, which attenuated the flow-induced response in a dose-dependent manner. In conclusion, the pathways of flow- and bradykinin-mediated NO production in endothelial cells did not require actin filament turnover or intact actin or microtubule cytoskeletons, and cholesterol, possibly by stiffening the plasma membrane, attenuated the flow response.

Laminar Flow on Endothelial Cells Suppresses eNOS O-GlcNAcylation to Promote eNOS Activity

2021 ◽  
Author(s):  
Sarah Basehore ◽  
Samantha Bohlman ◽  
Callie Weber ◽  
Swathi Swaminathan ◽  
Yuji Zhang ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Shear Stress ◽  
Laminar Flow ◽  
No Production ◽  
Disturbed Flow ◽  
Enos Activity ◽  
Enos Phosphorylation ◽  
Steady Laminar ◽  
In Cells

Rationale: In diabetic animals as well as high glucose cell culture conditions, endothelial nitric oxide synthase (eNOS) is heavily O-GlcNAcylated, which inhibits its phosphorylation and nitric oxide (NO) production. It is unknown, however, whether varied blood flow conditions, which affect eNOS phosphorylation, modulate eNOS activity via O-GlcNAcylation-dependent mechanisms. Objective: The goal of this study was to test if steady laminar flow, but not oscillating disturbed flow, decreases eNOS O-GlcNAcylation, thereby elevating eNOS phosphorylation and NO production. Methods and Results: Human umbilical vein endothelial cells (HUVEC) were exposed to either laminar flow (20 dynes/cm2 shear stress) or oscillating disturbed flow (4{plus minus}6 dynes/cm2 shear stress) for 24 hours in a cone-and-plate device. eNOS O-GlcNAcylation was almost completely abolished in cells exposed to steady laminar but not oscillating disturbed flow. Interestingly, there was no change in protein level or activity of key O-GlcNAcylation enzymes (OGT, OGA, or GFAT). Instead, metabolomics data suggest that steady laminar flow decreases glycolysis and hexosamine biosynthetic pathway (HBP) activity, thereby reducing UDP-GlcNAc pool size and consequent O-GlcNAcylation. Inhibition of glycolysis via 2-deoxy-2-glucose (2-DG) in cells exposed to disturbed flow efficiently decreased eNOS O-GlcNAcylation, thereby increasing eNOS phosphorylation and NO production. Finally, we detected significantly higher O-GlcNAcylated proteins in endothelium of the inner aortic arch in mice, suggesting that disturbed flow increases protein O-GlcNAcylation in vivo. Conclusions: Our data demonstrate that steady laminar but not oscillating disturbed flow decreases eNOS O-GlcNAcylation by limiting glycolysis and UDP-GlcNAc substrate availability, thus enhancing eNOS phosphorylation and NO production. This research shows for the first time that O-GlcNAcylation is regulated by mechanical stimuli, relates flow-induced glycolytic reductions to macrovascular disease, and highlights targeting HBP metabolic enzymes in endothelial cells as a novel therapeutic strategy to restore eNOS activity and prevent EC dysfunction in cardiovascular disease.

Abstract 153: Bile Acid Receptor TGR5 Agonism Represents Anti-inflammatory Effects via Stimulating Nitric Oxide Production in Vascular Endothelial Cells

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Taiki Kida ◽  
Yoshiki Tsubosaka ◽  
Masatoshi Hori ◽  
Hiroshi Ozaki ◽  
Takahisa Murata
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Bile Acids ◽  
Inflammatory Responses ◽  
Vascular Endothelial Cells ◽  
Lithocholic Acid ◽  
Signal Pathways ◽  
No Production ◽  
Vascular Endothelial ◽  
Anti Inflammatory

Objective TGR5, a membrane-bound, G-protein-coupled receptor for bile acids, is known to be involved in regulation of energy homeostasis and inflammation. However, little is known about the function of TGR5 in vascular endothelial cells. In the present study, we examined whether TGR5 agonism represents anti-inflammatory effects in vascular endothelial cells focusing on nitric oxide (NO) production. Methods and Results In human umbilical vein endothelial cells (HUVECs), treatment with taurolithocholic acid (TLCA), which has the highest affinity to TGR5 among various bile acids, significantly reduced tumor necrosis factor (TNF)-α-induced vascular cell adhesion molecule (VCAM)-1 protein expression and adhesion of human monocytes, U937. These effects were abrogated by a NO synthase (NOS) inhibitor, N G -Monomethyl-L-arginine (L-NMMA). In bovine aortic endothelial cells (BAECs), treatment with TLCA as well as lithocholic acid, which also has high affinity to TGR5, significantly increased the NO production. In contrast, deoxycholic acid and chenodeoxycholic acid, which possess low affinity to TGR5, did not affect the NO production. Gene depletion of TGR5 by siRNA transfection abolished TLCA-induced NO production in BAECs. TLCA-induced NO production was also observed in HUVECs measured as intracellular cGMP accumulation. We next investigated the signal pathways responsible for the TLCA-induced NO production in endothelial cells. Treatment with TLCA increased endothelial NOS (eNOS) ser1177 phosphorylation in HUVECs. This response was accompanied by increased Akt ser473 phosphorylation and intracellular Ca 2+ ([Ca 2+ ] i ). Treatment with phosphoinositide 3-kinase (PI3K) inhibitor, LY294002, or blockade of calcium channel with La 3+ , significantly decreased TLCA-induced eNOS ser1177 phosphorylation and subsequent NO production. Conclusion These results indicate that TGR5 agonism can mediate anti-inflammatory responses by suppressing VCAM-1 expression and monocytes adhesion to endothelial cells. This function is dependent on NO production via Akt activation and [Ca 2+ ] i increase.

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 Androgen Receptor-Dependent Activation of Endothelial Nitric Oxide Synthase in Vascular Endothelial Cells: Role of Phosphatidylinositol 3-Kinase/Akt Pathway

Endocrinology ◽  
2010 ◽  
Vol 151 (4) ◽  
pp. 1822-1828 ◽  
Author(s):  
Jing Yu ◽  
Masahiro Akishita ◽  
Masato Eto ◽  
Sumito Ogawa ◽  
Bo-Kyung Son ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Androgen Receptor ◽  
Pi3 Kinase ◽  
Maximal Response ◽  
No Production ◽  
Dependent Manner ◽  
Small Interference Rna ◽  
Enos Phosphorylation ◽  
Interference Rna

The mechanisms of testosterone-induced vasodilatation are not fully understood. This study investigated the effect of testosterone on nitric oxide (NO) synthesis and its molecular mechanism using human aortic endothelial cells (HAEC). Testosterone at physiological concentrations (1–100 nm) induced a rapid (15–30 min) increase in NO production, which was associated with phosphorylation and activation of endothelial NO synthase (eNOS). Then, the involvement of the androgen receptor (AR), which is abundantly expressed in HAEC, was examined. The effect of testosterone on eNOS activation and NO production were abolished by pretreatment with an AR antagonist nilutamide and by transfection with AR small interference RNA. In contrast, testosterone-induced eNOS phosphorylation was unchanged by pretreatment with an aromatase inhibitor or by transfection with ERα small interference RNA. 5α-Dihydrotestosterone, a nonaromatizable androgen, also stimulated eNOS phosphorylation. Next, the signaling cascade that leads to eNOS phosphorylation was explored. Testosterone stimulated rapid phosphorylation of Akt in a time- and dose-dependent manner, with maximal response at 15–60 min. The rapid phosphorylation of eNOS or NO production induced by testosterone was inhibited by Akt inhibitor SH-5 or by phosphatidylinositol (PI) 3-kinase inhibitor wortmannin. Co-immunoprecipitation assays revealed a testosterone-dependent interaction between AR and the p85α subunit of PI3-kinase. In conclusion, testosterone rapidly induces NO production via AR-dependent activation of eNOS in HAEC. Activation of PI3-kinase/Akt signaling and the direct interaction of AR with p85α are involved, at least in part, in eNOS phosphorylation.

Modulation of the l-arginine/nitric oxide signalling pathway in vascular endothelial cells

2004 ◽  
Vol 71 ◽  
pp. 143-156 ◽  
Author(s):  
Amanda W. Wyatt ◽  
Joern R. Steinert ◽  
Giovanni E. Mann
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Amino Acid ◽  
Protein Kinase ◽  
Signalling Pathway ◽  
No Production ◽  
Dependent Manner ◽  
Amino Acid Transport System ◽  
Membrane Hyperpolarization ◽  
Cationic Amino Acid

Nitric oxide (NO) is synthesized from l-arginine, and in endothelial cells influx of l-arginine is mediated predominantly via Na+-independent cationic amino acid transporters. Constitutive, Ca2+-calmodulin-sensitive eNOS (endothelial nitric oxide synthase) metabolizes l-arginine to NO and l-citrulline. eNOS is present in membrane caveolae and the cytosol and requires tetrahydrobiopterin, NADPH, FAD and FMN as additional cofactors for its activity. Supply of l-arginine for NO synthesis appears to be derived from a membrane-associated compartment distinct from the bulk intracellular amino acid pool, e.g. near invaginations of the plasma membrane referred to as 'lipid rafts' or caveolae. Co-localization of eNOS and the cationic amino acid transport system y+ in caveolae in part explains the 'arginine paradox', related to the phenomenon that in certain disease states eNOS requires an extracellular supply of l-arginine despite having sufficient intracellular l-arginine concentrations. Vasoactive agonists normally elevate [Ca2+]i (intracellular calcium concentration) in endothelial cells, thus stimulating NO production, whereas fluid shear stress, 17ϐ-oestradiol and insulin cause phosphorylation of the serine/threonine protein kinase Akt/protein kinase B in a phosphoinositide 3-kinase-dependent manner and activation of eNOS at basal [Ca2+]i levels. Adenosine causes an acute activation of p42/p44 mitogen-activated protein kinase and NO release, with membrane hyperpolarization leading to increased system y+ activity in fetal endothelial cells. In addition to acute stimulatory actions of D-glucose and insulin on l-arginine transport and NO synthesis, gestational diabetes, intrauterine growth retardation and pre-eclampsia induce phenotypic changes in the fetal vasculature, resulting in alterations in the l-arginine/NO signalling pathway and regulation of [Ca2+]i. These alterations may have significant implications for long-term programming of the fetal cardiovascular system.

Shear Stress Induces ATP-Independent Transient Nitric Oxide Release From Vascular Endothelial Cells, Measured Directly With a Porphyrinic Microsensor

1995 ◽  
Vol 77 (2) ◽  
pp. 284-293 ◽  
Author(s):  
Anthony J. Kanai ◽  
Harold C. Strauss ◽  
George A. Truskey ◽  
Anne L. Crews ◽  
Saul Grunfeld ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Shear Stress ◽  
Vascular Endothelial Cells ◽  
Vascular Endothelial ◽  
Nitric Oxide Release

scholarly journals High glucose-induced IKK-Hsp-90 interaction contributes to endothelial dysfunction

2009 ◽  
Vol 296 (1) ◽  
pp. C182-C192 ◽  
Author(s):  
Sumathy Mohan ◽  
Ryszard Konopinski ◽  
Bo Yan ◽  
Victoria E. Centonze ◽  
Mohan Natarajan
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Endothelial Dysfunction ◽  
High Glucose ◽  
Vascular Endothelial Cells ◽  
Heat Shock Protein 90 ◽  
No Production ◽  
Enos Activity ◽  
Hsp 90 ◽  
Endothelial Nitric Oxide

A decline in the bioavailability of nitric oxide (NO) that causes endothelial dysfunction is a hallmark 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 κB kinases (IKK)α, β, and γ] in nonvascular 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 (3.5 ± 0.65-fold) and translational (1.97 ± 0.17-fold) expression as well as the catalytic activity of IKKβ (2.45 ± 0.4-fold). Both IKKβ and eNOS could be coimmunoprecipitated with Hsp-90. Inhibition of Hsp-90 with geldanamycin (2 μM) or Radicicol (20 μM) mitigated (0.45 ± 0.04-fold and 0.93 ± 0.16-fold, respectively) HG induced-IKKβ activity (2.5 ± 0.42-fold). Blocking of IKKβ expression by IKK inhibitor II (15 μM wedelolactone) or small interferring RNA (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.

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