Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Endothelial nitric oxide synthase interactions with G-protein-coupled receptors.

ABSTRACT: The endothelial nitric oxide synthase (eNOS) is activated in response to stimulation of endothelial cells by a number of vasoactive substances including, bradykinin (BK), angiotensin II (Ang II), endothelin-1 (ET-1) and ATP. In the present study we have used in vitro activity assays of purified eNOS and in vitro binding assays with glutathione S-transferase fusion proteins to show that the capacity to bind and inhibit eNOS is a common feature of membrane-proximal regions of intracellular domain 4 of the BK B2, the Ang II AT1 and the ET-1 ETB receptors, but not of the ATP P2Y2 receptor. Phosphorylation of serine or tyrosine residues in the eNOS-interacting region of the B2 receptor results in a loss of eNOS inhibition due to a decrease in the binding affinity of the receptor domain for the eNOS enzyme. Furthermore, the B2 receptor is transiently phosphorylated on tyrosine residues in cultured endothelial cells in response to BK stimulation. Phosphorylation occurs during the time in which eNOS transiently dissociates from the receptor accompanied by a transient increase in nitric oxide production. Taken together, these data support the hypotheses that eNOS is regulated in endothelial cells by reversible and inhibitory interactions with G-protein-coupled receptors and that these interactions can be modulated by receptor phosphorylation.

SUBMITTER: Marrero MB

PROVIDER: S-EPMC1220558 | biostudies-other | 1999 Oct

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

Gap junction protein Cx37 interacts with endothelial nitric oxide synthase in endothelial cells.

Project description:The gap junction protein connexin37 (Cx37) plays an important role in cell-cell communication in the vasculature. A C1019T Cx37 gene polymorphism, encoding a P319S substitution in the regulatory C terminus of Cx37 (Cx37CT), correlates with arterial stenosis and myocardial infarction in humans. This study was designed to identify potential binding partners for Cx37CT and to determine whether the polymorphism modified this interaction.Using a high-throughput phage display, we retrieved 2 binding motifs for Cx37CT: WHK ... [K,R]XP ... and FHK ... [K,R]XXP ... , the first being more common for Cx37CT-319P and the second more common for Cx37CT-319S. One of the peptides (WHRTPRLPPPVP) showed 77.7% homology with residues 843 to 854 of endothelial nitric oxide synthase (eNOS). In vitro binding of this peptide or of the homologous eNOS sequence to both Cx37CT isoforms was confirmed by cross-linking and surface plasmon resonance. Electrophysiological analysis of Cx37 single channel activity in transfected N2a cells showed that eNOS-like and eNOS(843-854) increased the frequency of events with conductances higher than 300 pS. We demonstrated that eNOS coimmunoprecipitated with Cx37 in a mouse endothelial cell (EC) line (bEnd.3), human primary ECs, and a human EC line transfected with Cx37-319P or Cx37-319S. Cx37 and eNOS colocalized at EC membranes. Moreover, a dose-dependent increase in nitric oxide production was observed in ECs treated with Cx37 antisense.Overall, our data show for the first time a functional and specific interaction between eNOS and Cx37. This interaction may be relevant for the control of vascular physiology both in health and in disease.

| S-EPMC2930827 | biostudies-literature

(-)-epicatechin activation of endothelial cell endothelial nitric oxide synthase, nitric oxide, and related signaling pathways.

Project description:Recent reports indicate that (-)-epicatechin can exert cardioprotective actions, which may involve endothelial nitric oxide synthase (eNOS)-mediated nitric oxide production in endothelial cells. However, the mechanism by which (-)-epicatechin activates eNOS remains unclear. In this study, we proposed to identify the intracellular pathways involved in (-)-epicatechin-induced effects on eNOS, using human coronary artery endothelial cells in culture. Treatment of cells with (-)-epicatechin led to time- and dose-dependent effects that peaked at 10 minutes at 1 mumol/L. (-)-Epicatechin treatment activates eNOS via serine 633 and serine 1177 phosphorylation and threonine 495 dephosphorylation. Using specific inhibitors, we have established the participation of the phosphatidylinositol 3-kinase pathway in eNOS activation. (-)-Epicatechin induces eNOS uncoupling from caveolin-1 and its association with calmodulin-1, suggesting the involvement of intracellular calcium. These results allowed us to propose that (-)-epicatechin effects may be dependent on actions exerted at the cell membrane level. To test this hypothesis, cells were treated with the phospholipase C inhibitor U73122, which blocked (-)-epicatechin-induced eNOS activation. We also demonstrated inositol phosphate accumulation in (-)-epicatechin-treated cells. The inhibitory effects of the preincubation of cells with the calmodulin-dependent kinase II (CaMKII) inhibitor KN-93 indicate that (-)-epicatechin-induced eNOS activation is at least partially mediated via the Ca(2+)/CaMKII pathway. The (-)-epicatechin stereoisomer catechin was only partially able to stimulate nitric oxide production in cells. Together, these results strongly suggest the presence of a cell surface acceptor-effector for the cacao flavanol (-)-epicatechin, which may mediate its cardiovascular effects.

| S-EPMC2874202 | biostudies-literature

Endothelial nitric oxide synthase-derived nitric oxide in the regulation of metabolism.

Project description:Nitric oxide is an endogenously formed gas that acts as a signaling molecule in the human body. The signaling functions of nitric oxide are accomplished through two primer mechanisms: cGMP-mediated phosphorylation and the formation of S-nitrosocysteine on proteins. This review presents and discusses previous and more recent findings documenting that nitric oxide signaling regulates metabolic activity. These discussions primarily focus on endothelial nitric oxide synthase (eNOS) as the source of nitric oxide.

| S-EPMC7531049 | biostudies-literature

Hydrogen sulfide cytoprotective signaling is endothelial nitric oxide synthase-nitric oxide dependent.

Project description:Previous studies have demonstrated that hydrogen sulfide (H2S) protects against multiple cardiovascular disease states in a similar manner as nitric oxide (NO). H2S therapy also has been shown to augment NO bioavailability and signaling. The purpose of this study was to investigate the impact of H2S deficiency on endothelial NO synthase (eNOS) function, NO production, and ischemia/reperfusion (I/R) injury. We found that mice lacking the H2S-producing enzyme cystathionine γ-lyase (CSE) exhibit elevated oxidative stress, dysfunctional eNOS, diminished NO levels, and exacerbated myocardial and hepatic I/R injury. In CSE KO mice, acute H2S therapy restored eNOS function and NO bioavailability and attenuated I/R injury. In addition, we found that H2S therapy fails to protect against I/R in eNOS phosphomutant mice (S1179A). Our results suggest that H2S-mediated cytoprotective signaling in the setting of I/R injury is dependent in large part on eNOS activation and NO generation.

| S-EPMC3939925 | biostudies-literature

AMP-activated protein kinase functionally phosphorylates endothelial nitric oxide synthase Ser633.

Project description:Endothelial nitric oxide synthase (eNOS) plays a central role in maintaining cardiovascular homeostasis by controlling NO bioavailability. The activity of eNOS in vascular endothelial cells (ECs) largely depends on posttranslational modifications, including phosphorylation. Because the activity of AMP-activated protein kinase (AMPK) in ECs can be increased by multiple cardiovascular events, we studied the phosphorylation of eNOS Ser633 by AMPK and examined its functional relevance in the mouse models. Shear stress, atorvastatin, and adiponectin all increased AMPK Thr172 and eNOS Ser633 phosphorylations, which were abolished if AMPK was pharmacologically inhibited or genetically ablated. The constitutively active form of AMPK or an AMPK agonist caused a sustained Ser633 phosphorylation. Expression of gain-/loss-of-function eNOS mutants revealed that Ser633 phosphorylation is important for NO production. The aorta of AMPKalpha2(-/-) mice showed attenuated atorvastatin-induced eNOS phosphorylation. Nano-liquid chromatography/tandem mass spectrometry (LC/MS/MS) confirmed that eNOS Ser633 was able to compete with Ser1177 or acetyl-coenzyme A carboxylase Ser79 for AMPKalpha phosphorylation. Nano-LC/MS/MS confirmed that eNOS purified from AICAR-treated ECs was phosphorylated at both Ser633 and Ser1177. Our results indicate that AMPK phosphorylation of eNOS Ser633 is a functional signaling event for NO bioavailability in ECs.

| S-EPMC2761102 | biostudies-literature

Protein engineering to develop a redox insensitive endothelial nitric oxide synthase.

Project description:The zinc tetrathiolate (ZnS4) cluster is an important structural feature of endothelial nitric oxide synthase (eNOS). The cluster is located on the dimeric interface and four cysteine residues (C94 and C99 from two adjacent subunits) form a cluster with a Zn ion in the center of a tetrahedral configuration. Due to its high sensitivity to oxidants this cluster is responsible for eNOS dimer destabilization during periods of redox stress. In this work we utilized site directed mutagenesis to replace the redox sensitive cysteine residues in the ZnS4 cluster with redox stable tetra-arginines. Our data indicate that this C94R/C99R eNOS mutant is active. In addition, this mutant protein is insensitive to dimer disruption and inhibition when challenged with hydrogen peroxide (H2O2). Further, the overexpression of the C94R/C99R mutant preserved the angiogenic response in endothelial cells challenged with H2O2. The over-expression of the C94R/C99R mutant preserved the ability of endothelial cells to migrate towards vascular endothelial growth factor (VEGF) and preserved the endothelial monolayer in a scratch wound assay. We propose that this dimer stable eNOS mutant could be utilized in the treatment of diseases in which there is eNOS dysfunction due to high levels of oxidative stress.

| S-EPMC4297941 | biostudies-literature

C-reactive protein decreases endothelial nitric oxide synthase activity via uncoupling.

Project description:C-reactive protein (CRP), a cardiovascular risk marker, induces endothelial dysfunction. We have previously shown that CRP decreases endothelial nitric oxide synthase (eNOS) expression and bioactivity in human aortic endothelial cells (HAECs). In this study, we examined the mechanisms by which CRP decreases eNOS activity in HAECs. To this end, we explored different strategies such as availability of tetrahydrobiopterin (BH4)-a critical cofactor for eNOS, superoxide (O(2)(-)) production resulting in uncoupling of eNOS and phosphorylation/dephosphorylation of eNOS. CRP treatment significantly decreased levels of BH4 thereby promoting eNOS uncoupling. Pretreatment with sepiapterin, a BH4 precursor, prevented CRP-mediated effects on BH(4) levels, superoxide production as well as eNOS activity. The gene expression and enzymatic activity of GTPCH1, the first enzyme in the de novo biosynthesis of BH(4), were significantly inhibited by CRP. Importantly, GTPCH1 is known to be regulated by cAMP-mediated pathway. In the present study, CRP-mediated inhibition of GTPCH1 activity was reversed by pretreatment with cAMP analogues. Furthermore, CRP-induced O(2)(-) production was reversed by pharmacologic inhibition and siRNAs to p47 phox and p22 phox. Additionally, CRP treatment significantly decreased the eNOS dimer: monomer ratio confirming CRP-mediated eNOS uncoupling. The pretreatment of cells with NO synthase inhibitor (N-nitro-l-arginine methyl ester [l-NAME]) also prevented CRP-mediated O(2)(-) production further strengthening CRP-mediated eNOS uncoupling. Additionally, CRP decreased eNOS phosphorylation at Ser1177 as well as increased phosphorylation at Thr495. CRP appears to mediate these effects through the Fcgamma receptors, CD32 and CD64. To conclude, CRP uncouples eNOS resulting in increased superoxide production, decreased NO production and altered eNOS phosphorylation.

| S-EPMC2771555 | biostudies-literature

Endothelial nitric oxide synthase in the microcirculation.

Project description:Endothelial nitric oxide synthase (eNOS, NOS3) is responsible for producing nitric oxide (NO)--a key molecule that can directly (or indirectly) act as a vasodilator and anti-inflammatory mediator. In this review, we examine the structural effects of regulation of the eNOS enzyme, including post-translational modifications and subcellular localization. After production, NO diffuses to surrounding cells with a variety of effects. We focus on the physiological role of NO and NO-derived molecules, including microvascular effects on vessel tone and immune response. Regulation of eNOS and NO action is complicated; we address endogenous and exogenous mechanisms of NO regulation with a discussion of pharmacological agents used in clinical and laboratory settings and a proposed role for eNOS in circulating red blood cells.

| S-EPMC4628887 | biostudies-literature

Endothelial nitric-oxide synthase (eNOS) is activated through G-protein-coupled receptor kinase-interacting protein 1 (GIT1) tyrosine phosphorylation and Src protein.

Project description:Nitric oxide (NO) is a critical regulator of vascular tone and plays an especially prominent role in liver by controlling portal blood flow and pressure within liver sinusoids. Synthesis of NO in sinusoidal endothelial cells by endothelial nitric-oxide synthase (eNOS) is regulated in response to activation of endothelial cells by vasoactive signals such as endothelins. The endothelin B (ETB) receptor is a G-protein-coupled receptor, but the mechanisms by which it regulates eNOS activity in sinusoidal endothelial cells are not well understood. In this study, we built on two previous strands of work, the first showing that G-protein βγ subunits mediated activation of phosphatidylinositol 3-kinase and Akt to regulate eNOS and the second showing that eNOS directly bound to the G-protein-coupled receptor kinase-interacting protein 1 (GIT1) scaffold protein, and this association stimulated NO production. Here we investigated the mechanisms by which the GIT1-eNOS complex is formed and regulated. GIT1 was phosphorylated on tyrosine by Src, and Y293F and Y554F mutations reduced GIT1 phosphorylation as well as the ability of GIT1 to bind to and activate eNOS. Akt phosphorylation activated eNOS (at Ser(1177)), and Akt also regulated the ability of Src to phosphorylate GIT1 as well as GIT1-eNOS association. These pathways were activated by endothelin-1 through the ETB receptor; inhibiting receptor-activated G-protein βγ subunits blocked activation of Akt, GIT1 tyrosine phosphorylation, and ET-1-stimulated GIT1-eNOS association but did not affect Src activation. These data suggest a model in which Src and Akt cooperate to regulate association of eNOS with the GIT1 scaffold to facilitate NO production.

| S-EPMC4140289 | biostudies-literature

Vascular and perivascular nitric oxide release and transport: biochemical pathways of neuronal nitric oxide synthase (NOS1) and endothelial nitric oxide synthase (NOS3).

Project description:Nitric oxide (NO) derived from nitric oxide synthase (NOS) is an important paracrine effector that maintains vascular tone. The release of NO mediated by NOS isozymes under various O(2) conditions critically determines the NO bioavailability in tissues. Because of experimental difficulties, there has been no direct information on how enzymatic NO production and distribution change around arterioles under various oxygen conditions. In this study, we used computational models based on the analysis of biochemical pathways of enzymatic NO synthesis and the availability of NOS isozymes to quantify the NO production by neuronal NOS (NOS1) and endothelial NOS (NOS3). We compared the catalytic activities of NOS1 and NOS3 and their sensitivities to the concentration of substrate O(2). Based on the NO release rates predicted from kinetic models, the geometric distribution of NO sources, and mass balance analysis, we predicted the NO concentration profiles around an arteriole under various O(2) conditions. The results indicated that NOS1-catalyzed NO production was significantly more sensitive to ambient O(2) concentration than that catalyzed by NOS3. Also, the high sensitivity of NOS1 catalytic activity to O(2) was associated with significantly reduced NO production and therefore NO concentrations, upon hypoxia. Moreover, the major source determining the distribution of NO was NOS1, which was abundantly expressed in the nerve fibers and mast cells close to arterioles, rather than NOS3, which was expressed in the endothelium. Finally, the perivascular NO concentration predicted by the models under conditions of normoxia was paradoxically at least an order of magnitude lower than a number of experimental measurements, suggesting a higher abundance of NOS1 or NOS3 and/or the existence of other enzymatic or nonenzymatic sources of NO in the microvasculature.

| S-EPMC1987713 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data