Altered endothelial nitric oxide synthase (eNOS) activity in senescent rat heart is due to translocation of caveolin 1

2002 ◽  
Vol 34 (6) ◽  
pp. A18
Author(s):  
T. Damy ◽  
P. Ratajczak ◽  
P. Oliviéro ◽  
J. Boczkowski ◽  
C. Heymes ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Endothelial Nitric Oxide Synthase ◽  
Rat Heart ◽  
Caveolin 1 ◽  
Enos Activity ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

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 Caveolin-1 mediates endotoxin inhibition of endothelin-1-induced endothelial nitric oxide synthase activity in liver sinusoidal endothelial cells

2009 ◽  
Vol 297 (5) ◽  
pp. G930-G939 ◽  
Author(s):  
Willson Kwok ◽  
Sang Ho Lee ◽  
Cathy Culberson ◽  
Katarzyna Korneszczuk ◽  
Mark G. Clemens
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Nitric Oxide Synthase ◽  
Molecular Mechanism ◽  
Endothelial Nitric Oxide Synthase ◽  
Liver Sinusoidal Endothelial Cells ◽  
Caveolin 1 ◽  
Enos Activity ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Endothelin-1 (ET-1) plays a key role in the regulation of endothelial nitric oxide synthase (eNOS) activation in liver sinusoidal endothelial cells (LSECs). In the presence of endotoxin, an increase in caveolin-1 (Cav-1) expression impairs ET-1/eNOS signaling; however, the molecular mechanism is unknown. The objective of this study was to investigate the molecular mechanism of Cav-1 in the regulation of LPS suppression of ET-1-mediated eNOS activation in LSECs by examining the effect of caveolae disruption using methyl-β-cyclodextrin (CD) and filipin. Treatment with 5 mM CD for 30 min increased eNOS activity (+255%, P < 0.05). A dose (0.25 μg/ml) of filipin for 30 min produced a similar effect (+111%, P < 0.05). CD induced the perinuclear localization of Cav-1 and eNOS and stimulated NO production in the same region. Readdition of 0.5 mM cholesterol to saturate CD reversed these effects. Both the combined treatment with CD and ET-1 (CD + ET-1) and with filipin and ET-1 stimulated eNOS activity; however, pretreatment with endotoxin (LPS) abrogated these effects. Following LPS pretreatment, CD + ET-1 failed to stimulate eNOS activity (+51%, P > 0.05), which contributed to the reduced levels of eNOS-Ser1177 phosphorylation and eNOS-Thr495 dephosphorylation, the LPS/CD-induced overexpression and translocation of Cav-1 in the perinuclear region, and the increased perinuclear colocalization of eNOS with Cav-1. These results supported the hypothesis that Cav-1 mediates the action of endotoxin in suppressing ET-1-mediated eNOS activation and demonstrated that the manipulation of caveolae produces significant effects on ET-1-mediated eNOS activity in LSECs.

scholarly journals The Key Role of Caveolin-1 in Estrogen-Mediated Regulation of Endothelial Nitric Oxide Synthase Function in Cerebral Arterioles In vivo

2001 ◽  
Vol 21 (8) ◽  
pp. 907-913 ◽  
Author(s):  
Hao-Liang Xu ◽  
Elena Galea ◽  
Roberto A. Santizo ◽  
Verna L. Baughman ◽  
Dale A. Pelligrino
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Functional Activity ◽  
Endothelial Nitric Oxide Synthase ◽  
Down Regulation ◽  
Caveolin 1 ◽  
Cerebral Arterioles ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

The marked impairment in cerebrovascular endothelial nitric oxide synthase (eNOS) function that develops after ovariectomy may relate to the observation that the abundance of cerebral vascular eNOS and its endogenous inhibitor, caveolin-1, vary in opposite directions with chronic changes in estrogen status. The authors endeavored, therefore, to establish a link between these correlative findings by independently manipulating, in ovariectomized female rats, eNOS and caveolin-1 expression, while monitoring agonist (acetylcholine)-stimulated eNOS functional activity. In the current study, the authors showed that individually neither the up-regulation of eNOS (through simvastatin treatment), nor the down-regulation of caveolin-1 (through antisense oligonucleotide administration) is capable of restoring eNOS function in pial arterioles in vivo in these estrogen-depleted rats. Only when eNOS up-regulation and caveolin-1 down-regulation are combined is activity normalized. These results establish a mechanistic link between the estrogen-associated divergent changes in the abundance of caveolin-1 and eNOS protein and eNOS functional activity in cerebral arterioles.

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 Interaction between Caveolin-1 and the Reductase Domain of Endothelial Nitric-oxide Synthase

1998 ◽  
Vol 273 (35) ◽  
pp. 22267-22271 ◽  
Author(s):  
Sanjay Ghosh ◽  
Ratan Gachhui ◽  
Carol Crooks ◽  
Chaoqun Wu ◽  
Michael P. Lisanti ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Endothelial Nitric Oxide Synthase ◽  
Caveolin 1 ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide ◽  
Reductase Domain

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.

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