| 2010 |
S-glutathionylation of eNOS at two conserved cysteine residues in the reductase domain reversibly decreases NO production and increases superoxide generation, uncoupling the enzyme. This modification is increased in hypertensive vessels and is reversed by thiol-specific reducing agents, restoring endothelium-dependent vasodilation. |
In vitro biochemical assay, site-directed mutagenesis of cysteine residues, EPR spectroscopy for superoxide detection, vascular functional studies, and Western blot in hypertensive vessel tissue |
Nature |
High |
21179168
|
| 2007 |
Rho-kinase directly phosphorylates eNOS at Thr495 in vitro, and constitutively active RhoA or Rho-kinase increases this phosphorylation in cells. Thrombin-induced phosphorylation at Thr495 in HUVECs is suppressed by the Rho-kinase inhibitor Y27632, suppressing NO production. |
In vitro kinase assay with constitutively active Rho-kinase, overexpression of constitutively active RhoA/Rho-kinase in COS-7 cells, pharmacological inhibition with Y27632 in HUVECs |
Biochemical and biophysical research communications |
High |
17651694
|
| 2012 |
NO produced by eNOS activates Src kinase, which phosphorylates caveolin-1 at Tyr-14, increasing eNOS/caveolin-1 binding and inhibiting eNOS activity — a negative feedback loop. Phosphomimetic Y14D-Cav-1 increases eNOS/Cav-1 interaction and reduces NO production, whereas phosphodefective Y14F-Cav-1 does not. |
FRET biosensor for eNOS/Cav-1 interaction, coimmunoprecipitation, Src FRET biosensor, eNOS siRNA, NO donor studies, phosphomimetic/phosphodefective Cav-1 mutant overexpression |
Molecular biology of the cell |
High |
22323292
|
| 2018 |
Caveolin-1 stabilizes eNOS expression and regulates its activity; loss of Cav-1 reduces eNOS protein but increases per-molecule eNOS phosphorylation and NO production. eNOS activation leads to Cav-1 S-nitrosylation and destabilization of Cav-1 oligomers, promoting caveola-mediated endocytosis of albumin and insulin. |
Cav-1 siRNA, adenoviral Cav-1-GFP overexpression, Ca2+ ionophore activation, immunofluorescence, S-nitrosylation assay, caveola-mediated endocytosis measurement, eNOS Ser1177 phosphorylation by Western blot |
Molecular biology of the cell |
High |
29563255
|
| 2005 |
PKCalpha isoform specifically stimulates eNOS by increasing phosphorylation at Ser1179 (equivalent to human Ser1177) and NO production in endothelial cells. Inhibition of PKCalpha via siRNA or dominant-negative mutant reduces FGF2-induced Ser1179 phosphorylation and NO production. In vivo PKCalpha transduction increases resting blood flow in an eNOS-dependent manner. |
PKCalpha overexpression and siRNA knockdown in primary endothelial cells, dominant-negative mutant, phosphospecific Western blot, in vivo adenoviral gene transfer to rat femoral arteries with blood flow measurement and L-NAME inhibition |
Circulation research |
High |
16081872
|
| 2006 |
eNOS undergoes differential translocation depending on the agonist: ACh causes eNOS to translocate preferentially to the trans-Golgi network (associated with vasodilation), whereas PAF causes preferential translocation to the cytosol (associated with hyperpermeability), representing a spatial mechanism for discrimination between vascular responses. |
eNOS-GFP stable transfection in ECV-304 cells, lipid raft analysis, immunofluorescence microscopy, endothelial permeability assays |
American journal of physiology. Heart and circulatory physiology |
Medium |
16679407
|
| 2005 |
Hyperhomocysteinemia impairs eNOS activity primarily through PKC activation, which increases phosphorylation of eNOS at Thr495 and decreases eNOS protein expression. A PKC inhibitor (GFX) reverses Hcy-mediated eNOS inactivation and Thr495 phosphorylation in human aortic endothelial cells. |
CBS(-/-) mouse model, aortic ring functional assays, intravital microscopy, eNOS activity assay in MAECs and HAECs, Western blot for phospho-eNOS, PKC inhibitor rescue |
Arteriosclerosis, thrombosis, and vascular biology |
Medium |
16210565
|
| 2006 |
PKCzeta phosphorylates ERK5 at S486, inhibiting ERK5 function and thereby decreasing eNOS protein stability. TNFα stimulates PKCζ to bind ERK5 (shown by Co-IP and mammalian 2-hybrid assay), leading to reduced eNOS protein levels; this is replicated by the ERK5-S486A mutant. |
Coimmunoprecipitation, mammalian 2-hybrid assay, in vitro kinase assay, dominant-negative PKCζ, siRNA, phosphomutant ERK5, Western blot for eNOS protein stability |
Blood |
Medium |
20538799
|
| 2020 |
Endogenous NOS3 resides at two distinct subcellular locations—the plasma membrane and the trans-Golgi network (TGN). Using a targeted DNA-based fluorescent probe (NOckout), NOS3 at the plasma membrane was found to be ~10-fold more active than at the Golgi; however, Golgi NOS3 activity is essential for structural integrity of the Golgi. |
DNA-based fluorescent probe (NOckout) targeted to plasma membrane or TGN in live cells, quantitative NO mapping, loss-of-function experiments |
Nature chemical biology |
High |
32152543
|
| 2008 |
In tumor cells, the PI3K-AKT pathway activates eNOS (NOS3), which S-nitrosylates endogenous wild-type Ras proteins, activating them; this AKT-eNOS-wild-type Ras axis is required for both tumor initiation and maintenance downstream of oncogenic Ras. Blocking AKT-mediated phosphorylation of eNOS inhibits tumor growth. |
Genetic and pharmacological blocking of eNOS phosphorylation in cancer cell lines, mouse tumor models, S-nitrosylation assays for Ras, epistasis analysis |
Nature |
High |
18344980
|
| 2006 |
Fenofibrate activates AMPK in HUVECs and thereby increases eNOS phosphorylation at Ser-1177 and NO production; this effect is not blocked by PKA or PI3K inhibitors, distinguishing AMPK as the relevant kinase in this context. |
Western blot for AMPK and ACC phosphorylation, eNOS Ser1177 phosphorylation, NO measurement, pharmacological inhibitors in HUVECs |
Biochemical and biophysical research communications |
Medium |
16442496
|
| 2010 |
Tbx5 deletion in endocardial cells results in activation of Nos3 (eNOS), which drives increased apoptosis of endocardial and neighboring myocardial cells, causing atrial septal defects. Compound haploinsufficiency of Tbx5 and Nos3 worsens the cardiac phenotype, establishing genetic epistasis where Nos3 acts downstream of Tbx5 in an endocardial cell-survival pathway. |
Conditional Tbx5 knockout in mice, compound Tbx5/Nos3 haploinsufficiency, apoptosis assays, echocardiography, genetic epistasis |
Proceedings of the National Academy of Sciences of the United States of America |
High |
20974940
|
| 2001 |
ecNOS-derived NO mediates the acute (4 h) TNF-alpha-induced increase in endothelial permeability in pulmonary microvessel endothelial monolayers, as shown by antisense knockdown of ecNOS preventing the permeability increase; however, the prolonged (24 h) TNF-induced barrier dysfunction is independent of NO. |
ecNOS antisense oligonucleotide knockdown, NO measurement, albumin permeability assay, NO donor rescue experiment |
American journal of physiology. Lung cellular and molecular physiology |
Medium |
11290515
|
| 2020 |
beta-Arrestin2 forms a complex with GIT1 and eNOS in sinusoidal endothelial cells, directly stimulates eNOS activity and NO production, and this complex is disrupted during liver injury. Overexpression of beta-Arr2 in injured SECs rescues eNOS function; beta-Arr2-mediated GIT1/eNOS complex formation depends on Erk1/2 and Src kinases. |
Coimmunoprecipitation, beta-Arr2 knockout mice with bile duct ligation, adenoviral overexpression rescue, eNOS activity assay, pharmacological kinase inhibition |
Proceedings of the National Academy of Sciences of the United States of America |
High |
32404425
|
| 2019 |
eNOS phosphorylation (as measured by phosphospecific immunoblot) does not necessarily reflect actual NO production. Insulin and VEGF elicit strong eNOS phosphorylation responses but cause no increase in intracellular NO or Ca2+, whereas histamine and ATP promote both phosphorylation and robust NO formation, demonstrating discordance between eNOS phosphorylation and enzyme activation. |
Multispectral biosensor imaging of intracellular NO and Ca2+, chemogenetic eNOS activation, phosphospecific immunoblot in cultured endothelial cells |
Proceedings of the National Academy of Sciences of the United States of America |
High |
31527268
|
| 2017 |
Nox4-driven superoxide generation in aging endothelial cells activates PDI chaperone and induces ER stress, which dissociates HSP90 from eNOS and causes eNOS uncoupling (decreased NO, increased superoxide). Nox4 siRNA or chemical inhibition reverses eNOS uncoupling in aged HUVECs. |
Serial-passage HUVEC aging model, Nox4 siRNA, ER stress inhibitors, HSP90-eNOS coimmunoprecipitation, NO and superoxide measurement |
Free radical biology & medicine |
Medium |
28916474
|
| 2013 |
Phosphorylation of eNOS at S1176 (mouse equivalent of human S1177) controls insulin sensitivity and metabolism: knockin mice with unphosphorylatable S1176A mutation develop insulin resistance, hyperinsulinemia, and adiposity, while phosphomimetic S1176D mice have decreased insulin levels and resistance to high-fat diet weight gain. |
Knockin mouse models (S1176A and S1176D), metabolic phenotyping, glucose/insulin tolerance tests, high-fat diet challenge |
Biochemical and biophysical research communications |
High |
23291238
|
| 2005 |
Laminar shear stress increases eNOS mRNA 3' poly(A) tail length, which prolongs mRNA half-life (from ~6 h to ~18 h) and shifts eNOS mRNA into more translationally active polysome fractions. This nuclear polyadenylation is also induced by hydrogen peroxide and statins. |
Poly(A) tail length analysis, actinomycin D chase for mRNA stability, polysome fractionation, nuclear polyadenylation assay in endothelial cells under shear stress |
Circulation research |
Medium |
15905462
|
| 2005 |
NOSIP (NOS-interacting protein) co-immunoprecipitates with eNOS in lung tissue, indicating a direct protein-protein interaction. NOSIP and eNOS co-localize in ciliated airway epithelial cells and vascular/bronchial smooth muscle, suggesting NOSIP contributes to regulation of eNOS activity and availability in airways. |
Coimmunoprecipitation from lung tissue, RT-PCR, immunohistochemistry |
The journal of histochemistry and cytochemistry |
Medium |
15684328
|
| 1996 |
In cardiac myocytes, NOS3 mRNA and protein are downregulated by cAMP-elevating agents at the transcriptional level (mRNA half-life unchanged). This downregulation is cell-type specific (not seen in cardiac microvascular endothelial cells) and results in accentuated contractile response to beta-adrenergic stimulation and loss of muscarinic cholinergic response. |
In vitro cAMP-elevating drug treatment, in vivo milrinone treatment, Northern/Western blot, mRNA half-life measurement, isolated cardiomyocyte contractility assay |
The Journal of clinical investigation |
Medium |
8621775
|
| 2022 |
Elevated endothelial BACE1 cleaves occludin, causing caveolin-1 membrane accumulation; the increased membrane caveolin-1 binds eNOS and attenuates eNOS activity, resulting in endothelial dysfunction. This pathway is demonstrated in endothelial-specific BACE1 transgenic mice and human hypertensive cerebral microvessels. |
Mass spectrometry substrate identification, immunostaining, BACE1 transgenic mice, BACE1 inhibitor experiments, Western blot for eNOS-caveolin-1 interaction |
Circulation research |
Medium |
35382554
|
| 2023 |
VEGFR2 phosphosite Y1173 activates PLCγ, which triggers Ca2+/PKC-dependent activation of eNOS. eNOS-derived NO promotes tyrosine nitration and activation of Src, which phosphorylates VE-cadherin at Y685, disintegrating endothelial junctions and causing vascular leakage. |
Vegfr2Y1173F/+ knockin mice, Plcg1 endothelial-specific knockout, eNOS activity assay, Src activation measurement, VE-cadherin phosphorylation, vascular permeability assays |
The Journal of clinical investigation |
High |
37651195
|
| 2018 |
OxLDL induces iNOS-dependent S-nitrosylation of eNOS at Cys94 and Cys99 (but not Cys441), which enhances interaction between eNOS and β-catenin, activates β-catenin transcriptional activity, and promotes cell migration and adhesion molecule expression in endothelial cells. Mutation of Cys94/Cys99 partially abolishes these effects. |
eNOS Cys mutagenesis, S-nitrosylation assay, coimmunoprecipitation of eNOS/β-catenin, iNOS-specific inhibitor 1400W, β-catenin reporter assay |
Biochimica et biophysica acta. Molecular basis of disease |
Medium |
29471036
|
| 2022 |
DF-induced endothelial TXNDC5 increases proteasome-mediated degradation of HSF1, reducing HSP90 expression, which accelerates eNOS protein degradation. Endothelium-specific Txndc5 deletion markedly reduces atherosclerosis in ApoE-/- mice. Nanoparticle-delivered CRISPR-Cas9 targeting Txndc5 increases eNOS protein and reduces atherosclerosis in vivo. |
Endothelium-specific Txndc5 knockout in ApoE-/- mice, nanoparticle CRISPR delivery, Western blot for HSF1/HSP90/eNOS stability, proteasome inhibition experiments |
Science advances |
High |
35061532
|
| 2021 |
Hepatocyte-specific eNOS is required for normal mitochondrial fatty acid oxidation and mitophagic responses in liver. Genetic deletion or viral knockdown of hepatocyte eNOS exacerbates hepatic steatosis, inflammation, and mitochondrial dysfunction (reduced respiration, increased H2O2), while hepatocyte eNOS overexpression attenuates Western diet-induced NASH. |
Hepatocyte-specific eNOS genetic deletion and AAV-mediated knockdown/overexpression in mice, mitochondrial respiration, fatty acid oxidation, mitophagy markers (BNIP3, LC3II), Western diet feeding |
Diabetes |
High |
34380696
|
| 2006 |
In the setting of diabetes (db/db mice), eNOS exists in an uncoupled state producing peroxynitrite rather than NO, and genetic overexpression of eNOS in this context exacerbates hepatic ischemia-reperfusion injury. Repletion with BH4 cofactor (or its precursor sepiapterin) re-couples eNOS and is hepatoprotective, establishing BH4 deficiency as the mechanism of diabetic eNOS uncoupling. |
eNOS transgenic and adenoviral overexpression in db/db mice, hepatic I/R injury model, serum ALT measurement, peroxynitrite detection, BH4 and sepiapterin treatment rescue |
Circulation research |
High |
16763164
|
| 2001 |
PI3-kinase mediates isoproterenol (beta2-adrenergic)- and IGF-1-induced ecNOS activation in rat aortic endothelial cells, as shown by increased citrulline production blocked by Wortmannin. Isoproterenol also enhances eNOS association with the Triton X-100-insoluble (membrane raft) fraction, linking PI3K signaling to eNOS membrane localization. |
PI3K activity assay, citrulline production assay, Wortmannin inhibition, Triton X-100 fractionation in rat aortic endothelial cells |
Biochemical and biophysical research communications |
Medium |
11467844
|
| 2006 |
Hydroxyurea induces rapid, transient phosphorylation of eNOS at Ser1177 in a PKA-dependent manner (partially via PKB/Akt), increases cAMP, cGMP, and intracellular Ca2+ in HUVECs, leading to increased NO production. This effect is blocked by competitive NOS inhibitors. |
Western blot for eNOS Ser1177 phosphorylation, PKA and Akt inhibitors, cAMP/cGMP measurement, intracellular Ca2+ imaging, NO measurement, NOS competitive inhibitors in HUVECs and TrHBMEC |
Blood |
Medium |
16527893
|
| 2007 |
FXR (farnesoid X receptor) activation induces eNOS expression in vascular endothelial cells at the transcriptional level. An imperfect inverted repeat DNA motif (IR2, -628 to -641 in rat eNOS promoter) acts as an FXR-responsive element. FXR ligand treatment increases eNOS mRNA, protein, and nitrite/nitrate production; the effect is blocked by actinomycin D. |
Actinomycin D transcription inhibition, eNOS promoter-reporter assay, pharmacological and genetic FXR activation, RT-PCR and Western blot for eNOS, nitrite/nitrate measurement |
Cardiovascular research |
Medium |
18006476
|
| 2016 |
PGC-1alpha promotes eNOS expression and activity through the orphan nuclear receptor ERRalpha. Endothelial-specific PGC-1alpha deletion sensitizes mice to endothelial dysfunction, while overexpression protects against it; protection is abolished by L-NAME or eNOS deletion, placing eNOS downstream of the PGC-1alpha/ERRalpha axis. |
Endothelial-specific PGC-1alpha knockout and transgenic mice, angiotensin-II hypertension model, L-NAME and eNOS KO epistasis, vascular reactivity assays |
Scientific reports |
Medium |
27910955
|
| 2023 |
KLF2 increases eNOS phosphorylation at Ser1177 and eNOS dimerization, and activates the PI3K-Akt pathway and HSP90 to promote eNOS activity. Adenovirus-mediated endothelium-specific KLF2 overexpression enhances endothelium-dependent relaxation and flow-mediated dilation in diabetic mice. |
Adenoviral KLF2 overexpression in mouse aortic endothelium, RNA-sequencing, Western blot for eNOS phosphorylation and dimerization, Akt/HSP90 pathway analysis, vascular function assays in db/db mice |
Diabetes |
Medium |
37347764
|
| 2015 |
Circulating NOS3 from blood cells (distinct from endothelial NOS3) ameliorates maladaptive left ventricular remodeling following reperfused myocardial infarction, reducing scar size and collagen deposition. Bone marrow chimeras devoid of blood-cell NOS3 show worsened LV remodeling compared to chimeras with both endothelial and blood-cell NOS3. |
Bone marrow transplantation chimera approach (NOS3-/- BM into WT mice), 60-min coronary occlusion/reperfusion model, serial echocardiography, pressure catheter hemodynamics, post-mortem histology |
PloS one |
Medium |
25875863
|
| 2017 |
Functionally distinct platelet subpopulations exist: approximately 20% of platelets lack eNOS and consequently fail to produce NO and have downregulated sGC-PKG signaling. eNOS-negative platelets primarily initiate adhesion to collagen and form larger aggregates, while eNOS-positive platelets limit aggregate size via NO generation. |
Flow cytometry to separate eNOS-positive and eNOS-negative platelet subpopulations, flow chamber adhesion assays, aggregation experiments, MMP-2 secretion measurement, integrin αIIbβ3 activation assay |
Cardiovascular research |
Medium |
29016749
|
| 1994 |
Hypoxia reduces eNOS mRNA in human endothelial cells by both decreasing transcription and reducing message stability (40-60% reduction). This leads to decreased NO production, as functional co-culture experiments show hypoxic endothelial cells stimulate significantly less cGMP production by smooth muscle cells. |
Northern blot, nuclear run-on transcription assay, mRNA stability assay, endothelial-smooth muscle co-culture cGMP measurement under normoxia and hypoxia |
The American journal of physiology |
Medium |
7526714
|
| 2018 |
TRPC5 channels activated by ATP-induced IP3 signaling stimulate eNOS/NOS activation and NO-cGMP-PKG signaling in neonatal rat cardiomyocytes. This TRPC5-eNOS axis negatively regulates hypertrophic TRPC3/6-calcineurin/NFAT signaling; pharmacological NOS inhibition potentiates ATP-induced NFAT activity and protein synthesis. |
NOS inhibitors, TRPC5 and TRPC6 selective pharmacological inhibition, NFAT reporter assay, NO measurement, PKG activity assay, protein synthesis measurement in NRCMs |
Frontiers in pharmacology |
Medium |
29872396
|
| 2015 |
eNOS deficiency in mice causes podocyte cellular hypertrophy, mitochondrial abnormalities (increased number/decreased size), mitochondrial DNA mutations (d-17), reduced renal ATP and mitochondrial respiration. Conditioned medium lacking NO induces mitochondrial fragmentation in cultured podocytes, which is rescued by NO donor. |
eNOS knockout mice, ultrastructural analysis, mitochondrial respiration assay in primary podocytes, ATP measurement, NO donor rescue, endothelial conditioned medium experiments |
Free radical biology & medicine |
Medium |
26119782
|
| 2004 |
eNOS (NOS3) protects prostate cancer cells from TRAIL-induced apoptosis. Stable eNOS-expressing PC-3 clones are resistant to TRAIL-induced apoptosis, and NOS inhibition sensitizes LNCaP (high endogenous eNOS/Akt) cells to TRAIL. This protection is mediated downstream of Akt. |
Stable eNOS-overexpressing cell clone generation, NOS inhibitor treatment, apoptosis assays, Akt activity measurement in multiple prostate cancer cell lines |
Cancer letters |
Medium |
15172122
|
| 1998 |
Adventitial expression of recombinant eNOS gene in fibroblasts (via adenoviral vector) restores NO production and bradykinin-induced relaxation in basilar arteries without endothelium, demonstrating that eNOS functional activity in adventitial fibroblasts is sufficient to regulate vascular tone through cGMP production. This effect is blocked by the NOS inhibitor L-NAME. |
Ex vivo adenoviral eNOS gene transfer to canine basilar arteries, endothelium-removal experiments, isometric tension recording, cGMP radioimmunoassay, electron microscopy for protein localization |
Arteriosclerosis, thrombosis, and vascular biology |
Medium |
9714129
|