Regulation of endothelial nitric oxide synthase by the actin cytoskeleton

2003 ◽  
Vol 284 (6) ◽  
pp. C1542-C1549 ◽  
Author(s):  
Yunchao Su ◽  
Sophia Edwards-Bennett ◽  
Michael R. Bubb ◽  
Edward R. Block
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Endothelial Nitric Oxide Synthase ◽  
Plasma Membranes ◽  
Enos Activity ◽  
Swinholide A ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide ◽  
Texas Red ◽  
Enos Protein

In the present study, the association of endothelial nitric oxide synthase (eNOS) with the actin cytoskeleton in pulmonary artery endothelial cells (PAEC) was examined. We found that the protein contents of eNOS, actin, and caveolin-1 were significantly higher in the caveolar fraction of plasma membranes than in the noncaveolar fraction of plasma membranes in PAEC. Immunoprecipitation of eNOS from lysates of caveolar fractions of plasma membranes in PAEC resulted in the coprecipitation of actin, and immunoprecipitation of actin from lysates of caveolar fractions resulted in the coprecipitation of eNOS. Confocal microscopy of PAEC, in which eNOS was labeled with fluorescein, F-actin was labeled with Texas red-phalloidin, and G-actin was labeled with deoxyribonuclease I conjugated with Texas red, also demonstrated an association between eNOS and F-actin or G-actin. Incubation of purified eNOS with purified F-actin and G-actin resulted in an increase in eNOS activity. The increase in eNOS activity caused by G-actin was much higher than that caused by F-actin. Incubation of PAEC with swinholide A, an actin filament disruptor, resulted in an increase in eNOS activity, eNOS protein content, and association of eNOS with G-actin and in a decrease in the association of eNOS with F-actin. The increase in eNOS activity was higher than that in eNOS protein content in swinholide A-treated cells. In contrast, exposure of PAEC to phalloidin, an actin filament stabilizer, caused decreases in eNOS activity and association of eNOS with G-actin and increases in association of eNOS with F-actin. These results suggest that eNOS is associated with actin in PAEC and that actin and its polymerization state play an important role in the regulation of eNOS activity.

Expression of endothelial nitric oxide synthase in the postnatal developing porcine kidney

2001 ◽  
Vol 280 (5) ◽  
pp. R1269-R1275 ◽  
Author(s):  
Michael J. Solhaug ◽  
Usa Kullaprawithaya ◽  
Xui Q. Dong ◽  
Ke-Wen Dong
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Protein Content ◽  
Endothelial Nitric Oxide Synthase ◽  
Enos Expression ◽  
Developmentally Regulated ◽  
Enos Mrna ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide ◽  
Enos Protein

The postnatal pattern of renal endothelial nitric oxide synthase (eNOS) is unknown. The purpose of this study was to characterize eNOS expression during maturation and compare this to neuronal NOS (nNOS). The experiments measured whole kidney eNOS mRNA expression by RT-PCR and protein content by Western blot, as well as cortical and medullary protein content in piglets at selected postnatal ages and in adult pigs. Whole kidney eNOS mRNA was compared with nNOS. Whole kidney eNOS expression decreased from the newborn to its lowest at 7 days, returning by 14 days to adult levels. This eNOS mRNA pattern contrasted with nNOS, which was highest at birth, and progressively decreased to its lowest level in the adult. At birth, cortical eNOS protein was greater than medullary, contrasting with the adult pattern of equivalent levels. In conclusion eNOS is developmentally regulated during early renal maturation and may critically participate in renal function during this period. The eNOS developmental pattern differs from nNOS, suggesting that these isoforms may have different regulatory factors and functional contributions in the postnatal kidney.

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

2006 ◽  
Vol 398 (2) ◽  
pp. 279-288 ◽  
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.

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

2002 ◽  
Vol 50 (6) ◽  
pp. 779-788 ◽  
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.

scholarly journals Locally Different Endothelial Nitric Oxide Synthase Protein Levels in Ascending Aortic Aneurysms of Bicuspid and Tricuspid Aortic Valve

2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
Salah A. Mohamed ◽  
Arlo Radtke ◽  
Roza Saraei ◽  
Joern Bullerdiek ◽  
Hajar Sorani ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Aortic Valve ◽  
Endothelial Nitric Oxide Synthase ◽  
Aortic Aneurysms ◽  
Tricuspid Aortic Valve ◽  
Protein Levels ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide ◽  
Enos Protein

Aims. Dysregulated expression of the endothelial nitric oxide synthase (eNOS) is observed in aortic aneurysms associated with bicuspid aortic valve (BAV). We determined eNOS protein levels in various areas in ascending aortic aneurysms.Methods and Results. Aneurysmal specimens were collected from 19 patients, 14 with BAV and 5 with tricuspid aortic valve (TAV). ENOS protein levels were measured in the outer curve (convexity), the opposite side (concavity), the distal and above the sinotubular junction (proximal) aneurysm. Cultured aortic cells were treated with NO synthesis inhibitor L-NAME and the amounts of 35 apoptosis-related proteins were determined. In patients with BAV, eNOS levels were significantly lower in the proximal aorta than in the concavity and distal aorta. ENOS protein levels were also lower in the convexity than in the concavity. While the convexity and distal aorta showed similar eNOS protein levels in BAV and TAV patients, levels were higher in TAV proximal aorta. Inhibition of NO synthesis in aneurysmal aortic cells by L-NAME led to a cytosolic increase in the levels of mitochondrial serine protease HTRA2/Omi.Conclusion. ENOS protein levels were varied at different areas of the aneurysmal aorta. The dysregulation of nitric oxide can lead to an increase in proapoptotic HTRA2/Omi.

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

2003 ◽  
Vol 284 (1) ◽  
pp. R1-R12 ◽  
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.

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

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.

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

2019 ◽  
Vol 20 (1) ◽  
pp. 187 ◽  
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.

scholarly journals Endothelial Nitric Oxide Synthase Expression Is Progressively Increased in Primary Cerebral Microvascular Endothelial Cells During Hyperbaric Oxygen Exposure

2009 ◽  
Vol 2 (1) ◽  
pp. 7-13 ◽  
Author(s):  
Xiongfei Xu ◽  
Zhongzhuang Wang ◽  
Quan Li ◽  
Xiang Xiao ◽  
Qinglin Lian ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Nitric Oxide Synthase ◽  
Hyperbaric Oxygen ◽  
Endothelial Nitric Oxide Synthase ◽  
Microvascular Endothelial Cells ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide ◽  
Enos Protein

Exposure to hyperbaric oxygen (HBO) can lead to seizures. Many studies have demonstrated that there exist a very close relationship between the alteration of cerebral blood flow (CBF) and the onset of seizures. Nitric oxide (NO) may play a key role in the change of CBF during exposure, and modulation of endothelial nitric oxide synthase (eNOS)-derived NO by HBO is responsible for early vasoconstriction, whereas late HBO-induced vasodilation depends upon a large amount of NO from both eNOS and neuronal nitric oxide synthase (nNOS). To investigate the effect of HBO on the activity and expression of eNOS in cerebral microvascular endothelial cells (CMEC) in vitro, primarily cultured CMEC from neonatal rats were exposed to oxygen at 500 kPa [5 atmosphere absolute (ATA)] for 10, 20, 30, 60 and 120 minutes (min), then eNOS activity, protein and mRNA contents in cells were detected. Our results showed that immediately after exposure, 30, 60 and 120 min HBO exposures did not alter NOS activity. When detected no matter immediately or six hours (h) after exposure, these exposures also did not alter eNOS protein and mRNA levels. However, when detected 24 h after exposure, 30, 60 and 120 min exposures upregulated eNOS protein content by 39%, 60% and 40% respectively. 10 and 20 min exposures upregulated eNOS mRNA content by about 15%, while 30, 60 and 120 min exposures upregulated it by about 20–30%. The increased eNOS protein and mRNA contents at 24 h after exposure may reflect new protein synthesis for eNOS. Our studies showed that with the exposing protocols we used, HBO did induce eNOS expression increase in CMEC. However, compared with the decrease of CBF in vivo, which occurred in a relative short time after rat was exposed to HBO above 4 ATA, the responses of eNOS in CMEC in vitro were a little slow. Thus we considered that for the vasodilation in the late period of HBO exposure before seizure, the effect of NO produced by eNOS was limited.

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