Endothelial nitric oxide synthase activity is linked to its presence at cell–cell contacts

The enzyme endothelial nitric oxide synthase (eNOS) is essential for vascular integrity. Many studies have demonstrated a link between the localization and activity of eNOS. Here, we studied the influence of cell—cell contact on this link in the microvascular endothelial bEnd.3 cell line. By immunofluorescence microscopy, eNOS localization at the plasma membrane was found to be dependent on cell—cell contact. In particular, eNOS was highly enriched at the intercellular contact sites. Further analysis showed that the pattern of eNOS localization at the plasma membrane resembled that of PECAM-1 (platelet endothelial cell adhesion molecule 1), but not that of the adherens junction proteins VE (vascular endothelial)-cadherin and plakoglobin. eNOS that was localized at the contact sites was, in part, Triton X-100-insoluble, in contrast with eNOS at the Golgi complex, which may indicate an association of eNOS with the actin cytoskeleton. Interestingly, eNOS activity was up-regulated in confluent monolayers compared with subconfluent cells, while there was no difference in eNOS expression. This correlation between cell confluence and eNOS activity was also found when primary bovine aortic endothelial cells were studied. These data imply that cell—cell contact induces the localization of eNOS at intercellular junctions, which is required for agonist-induced eNOS activation.

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Inhibition of MEK/ERK1/2 signalling alters endothelial nitric oxide synthase activity in an agonist-dependent manner

Biochemical Journal ◽

10.1042/bj20060371 ◽

2006 ◽

Vol 398 (2) ◽

pp. 279-288 ◽

Cited By ~ 21

Author(s):

Jacqueline M. Cale ◽

Ian M. Bird

Keyword(s):

Nitric Oxide ◽

Protein Kinase ◽

Nitric Oxide Synthase ◽

Endothelial Nitric Oxide Synthase ◽

Intracellular Trafficking ◽

Enos Activity ◽

Oxide Synthase ◽

Endothelial Nitric Oxide ◽

Nitric Oxide Synthase Activity

eNOS (endothelial nitric oxide synthase) activity is post-translationally regulated in a complex fashion by acylation, protein–protein interactions, intracellular trafficking and phosphorylation, among others. Signalling pathways that regulate eNOS activity include phosphoinositide 3-kinase/Akt, cyclic nucleotide-dependent kinases [PKA (protein kinase A) and PKG], PKC, as well as ERKs (extracellular-signal-regulated kinases). The role of ERKs in eNOS activation remains controversial. In the present study, we have examined the role of ERK1/2 in eNOS activation in HUVEC-CS [transformed HUVEC (human umbilical-vein endothelial cells)] as well as a widely used model for eNOS study, transiently transfected COS-7 cells. U0126 pretreatment of HUVEC-CS potentiated ATP-stimulated eNOS activity, independent of changes in intracellular Ca2+ concentration ([Ca2+]i). In COS-7 cells transiently expressing ovine eNOS, U0126 potentiated A23187-stimulated eNOS activity, but inhibited ATP-stimulated activity. Compensatory changes in phosphorylation of five key eNOS residues did not account for changes in A23187-stimulated activity. However, in the case of ATP, altered phosphorylation and changes in [Ca2+]i may partially contribute to U0126 inhibition of activity. Finally, seven eNOS alanine mutants of putative ERK1/2 targets were generated and the effects of U0126 pretreatment on eNOS activity were gauged with A23187 and ATP treatment. T97A-eNOS was the only construct significantly different from wild-type after U0126 pretreatment and ATP stimulation of eNOS activation. In the present study, eNOS activity was either potentiated or inhibited in COS-7 cells, suggesting agonist dependence for MEK/ERK1/2 signalling [where MEK is MAPK (mitogen-activated protein kinase)/ERK kinase] to eNOS and a complex mechanism including [Ca2+]i, phosphorylation and, possibly, intracellular trafficking.

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Endothelial Nitric Oxide Synthase and Its Negative Regulator Caveolin-1 Localize to Distinct Perinuclear Organelles

Journal of Histochemistry & Cytochemistry ◽

10.1177/002215540205000604 ◽

2002 ◽

Vol 50 (6) ◽

pp. 779-788 ◽

Cited By ~ 36

Author(s):

Roland Govers ◽

Peter van der Sluijs ◽

Elly van Donselaar ◽

Jan-Willem Slot ◽

Ton J. Rabelink

Keyword(s):

Nitric Oxide ◽

Nitric Oxide Synthase ◽

Golgi Complex ◽

Endothelial Nitric Oxide Synthase ◽

Direct Interaction ◽

Negative Regulator ◽

Caveolin 1 ◽

Enos Activity ◽

Oxide Synthase ◽

Endothelial Nitric Oxide

Caveolin-1 is a member of a subset of intracellular proteins that regulate endothelial nitric oxide synthase (eNOS) activity. In caveolae, caveolin-1 inhibits eNOS activity via a direct interaction with the enzyme. Previous work has indicated that both eNOS and caveolin-1 are also localized at the perinuclear Golgi complex. Whether caveolin-1 is involved in eNOS regulation in this cell compartment is unknown. Here we studied the localization of eNOS and caveolin-1 in the perinuclear region of primary bovine aortic endothelial cells. By immunofluorescence microscopy we show that both eNOS and caveolin-1 co-localize with Golgi markers. On treatment of the cells with the microtubule-depolymerizing drug nocodazole, the Golgi complex is scattered and caveolin-1 is found in vesicles at the periphery of the cell, while eNOS is localized at large structures near the nucleus. The nocodazole-induced redistribution of eNOS is similar to that of cis-, medial-, and trans-Golgi markers, while the caveolin-1 redistribution resembles that of sec22, a marker for the intermediate compartment. The localization of eNOS and caveolin-1 at distinct perinuclear compartments that behave differently in the presence of nocodazole indicates that eNOS activity is not regulated by caveolin-1 in the Golgi complex.

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Molecular mechanisms involved in the regulation of the endothelial nitric oxide synthase

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00323.2002 ◽

2003 ◽

Vol 284 (1) ◽

pp. R1-R12 ◽

Cited By ~ 507

Author(s):

Ingrid Fleming ◽

Rudi Busse

Keyword(s):

Nitric Oxide ◽

Nitric Oxide Synthase ◽

Endothelial Nitric Oxide Synthase ◽

Molecular Mechanisms ◽

Fluid Shear Stress ◽

Intracellular Localization ◽

Heat Shock Protein 90 ◽

Enos Activity ◽

Oxide Synthase ◽

Endothelial Nitric Oxide

The endothelial nitric oxide synthase (eNOS), the expression of which is regulated by a range of transcriptional and posttranscriptional mechanisms, generates nitric oxide (NO) in response to a number of stimuli. The physiologically most important determinants for the continuous generation of NO and thus the regulation of local blood flow are fluid shear stress and pulsatile stretch. Although eNOS activity is coupled to changes in endothelial cell Ca2+ levels, an increase in Ca2+ alone is not sufficient to affect enzyme activity because the binding of calmodulin (CaM) and the flow of electrons from the reductase to the oxygenase domain of the enzyme is dependent on protein phosphorylation and dephosphorylation. Two amino acids seem to be particularly important in regulating eNOS activity and these are a serine residue in the reductase domain (Ser1177) and a threonine residue (Thr495) located within the CaM-binding domain. Simultaneous alterations in the phosphorylation of Ser1177 and Thr495 in response to a variety of stimuli are regulated by a number of kinases and phosphatases that continuously associate with and dissociate from the eNOS signaling complex. eNOS associated proteins, such as caveolin, heat shock protein 90, eNOS interacting protein, and possibly also motor proteins provide the scaffold for the formation of the protein complex as well as its intracellular localization.

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Identification of Hypoglycemia-dependent Endothelial Nitric Oxide Synthase O-GlcNAcylation Sites and Regulation of O-GlcNAcylation and Nitric Oxide Production by Hypoglycemia

10.21203/rs.3.rs-22780/v1 ◽

2020 ◽

Author(s):

an he ◽

Shupeng Hu ◽

Qiangzhong Pi ◽

Yongzheng Guo ◽

Yang Long ◽

...

Keyword(s):

Nitric Oxide ◽

Nitric Oxide Synthase ◽

Endothelial Nitric Oxide Synthase ◽

Immunoblot Analysis ◽

Serine Phosphorylation ◽

The Novel ◽

Post Translational Modification ◽

Enos Activity ◽

Oxide Synthase ◽

Endothelial Nitric Oxide

Abstract BackgroundO-GlcNAcylation, an energy-sensitive post-translational modification, plays a major role in endothelial nitric oxide synthase (eNOS) activity regulation. However, the effect of hypoglycemia on eNOS O-GlcNAcylation and whether eNOS exists the novel O-GlcNAcylation sites under hypoglycemia is unknown. Hence, we endeavored to determine the effects of hypoglycemia on eNOS O-GlcNAcylation and the novel O-GlcNAcylation sites of eNOS.MethodBovine aortic endothelial cells (BAECs) and Sprague-Dawley rats were treated by hypoglycemia, and using immunoblotting to measure their eNOS O-GlcNAcylation. eNOS and transfected eNOS were purified by pull-down assay and immunoprecipitation respectively. Novel O-GlcNAcylation sites of eNOS were predicted by HPLC-MS and MS/MS Ion, and determined by immunoblotting. eNOS activity were detected by Elisa and isotope labelling method. ResultsIn BAECs and rats` thoracic aorta, hypoglycemia-associated activation of eNOS was accompanied by an increase in O-GlcNAcylation and had no effect on O-linked serine phosphorylation at residue 1179/1177. Changes in this post-translational modification were associated with increased O-GlcNAc transferase (OGT) activity, and were reversed by AMPK knockdown. Immunoblot analysis of cells expressing His-tagged wild-type human eNOS and human eNOS carrying a mutation at the Ser1177 phosphorylation site confirmed the increase in O-GlcNAcylation in response to hypoglycemia. The observed increase in O-GlcNAcylation indicated that eNOS contains novel O-GlcNAcylation sites that are activated by hypoglycemia. Immunoblot analysis of cells expressing His-tagged human eNOS carrying a mutation at Ser738 and Ser867 confirmed the increase in O-GlcNAcylation in response to hypoglycemia. Contrastingly, in His-tagged human eNOS carrying a mutation at Thr866, O-GlcNAcylation was unaffected by hypoglycemia. Differences among culture conditions were identified using two-way analysis of variance (ANOVA), one-way ANOVA, or unpaired Student’s t-test. ConclusionsHypoglycemia increases eNOS O-GlcNAcylation and activity, potentially via AMPK-OGT pathway, thereby showing the Thr866 as a novel O-GlcNAcylation site involved in hypoglycemia-mediated eNOS activation.

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Electroacupuncture on stomach 36 point (ST‐36) reduces hypertension through endothelial nitric oxide synthase (eNOS) activity in the stomach meridian

The FASEB Journal ◽

10.1096/fasebj.20.5.a1152 ◽

2006 ◽

Vol 20 (5) ◽

Author(s):

Arnaldo Michel Pica ◽

Ricardo G Duran ◽

Walter N Duran ◽

David D. Kim

Keyword(s):

Nitric Oxide ◽

Nitric Oxide Synthase ◽

Endothelial Nitric Oxide Synthase ◽

Enos Activity ◽

Stomach Meridian ◽

Oxide Synthase ◽

Endothelial Nitric Oxide

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New Therapeutic Implications of Endothelial Nitric Oxide Synthase (eNOS) Function/Dysfunction in Cardiovascular Disease

International Journal of Molecular Sciences ◽

10.3390/ijms20010187 ◽

2019 ◽

Vol 20 (1) ◽

pp. 187 ◽

Cited By ~ 41

Author(s):

Andreas Daiber ◽

Ning Xia ◽

Sebastian Steven ◽

Matthias Oelze ◽

Alina Hanf ◽

...

Keyword(s):

Oxidative Stress ◽

Nitric Oxide ◽

Risk Factors ◽

Endothelial Dysfunction ◽

Nitric Oxide Synthase ◽

Endothelial Nitric Oxide Synthase ◽

Enos Activity ◽

Life Style Changes ◽

Oxide Synthase ◽

Endothelial Nitric Oxide

The Global Burden of Disease Study identified cardiovascular risk factors as leading causes of global deaths and life years lost. Endothelial dysfunction represents a pathomechanism that is associated with most of these risk factors and stressors, and represents an early (subclinical) marker/predictor of atherosclerosis. Oxidative stress is a trigger of endothelial dysfunction and it is a hall-mark of cardiovascular diseases and of the risk factors/stressors that are responsible for their initiation. Endothelial function is largely based on endothelial nitric oxide synthase (eNOS) function and activity. Likewise, oxidative stress can lead to the loss of eNOS activity or even “uncoupling” of the enzyme by adverse regulation of well-defined “redox switches” in eNOS itself or up-/down-stream signaling molecules. Of note, not only eNOS function and activity in the endothelium are essential for vascular integrity and homeostasis, but also eNOS in perivascular adipose tissue plays an important role for these processes. Accordingly, eNOS protein represents an attractive therapeutic target that, so far, was not pharmacologically exploited. With our present work, we want to provide an overview on recent advances and future therapeutic strategies that could be used to target eNOS activity and function in cardiovascular (and other) diseases, including life style changes and epigenetic modulations. We highlight the redox-regulatory mechanisms in eNOS function and up- and down-stream signaling pathways (e.g., tetrahydrobiopterin metabolism and soluble guanylyl cyclase/cGMP pathway) and their potential pharmacological exploitation.

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