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Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) is a critical mediator of vascular function. eNOS is tightly regulated at various levels, including transcription, co- and posttranslational modifications, and by various protein–protein interactions. Using stable isotope labeling with amino acids in cell culture (SILAC) and mass spectrometry (MS), we identified several eNOS interactors, including the protein plasminogen activator inhibitor-1 (PAI-1). In cultured human umbilical vein endothelial cells (HUVECs), PAI-1 and eNOS colocalize and proximity ligation assays demonstrate a protein–protein interaction between PAI-1 and eNOS. Knockdown of PAI-1 or eNOS eliminates the proximity ligation assay (PLA) signal in endothelial cells. Overexpression of eNOS and HA-tagged PAI-1 in COS7 cells confirmed the colocalization observations in HUVECs. Furthermore, the source of intracellular PAI-1 interacting with eNOS was shown to be endocytosis derived. The interaction between PAI-1 and eNOS is a direct interaction as supported in experiments with purified proteins. Moreover, PAI-1 directly inhibits eNOS activity, reducing NO synthesis, and the knockdown or antagonism of PAI-1 increases NO bioavailability. Taken together, these findings place PAI-1 as a negative regulator of eNOS and disruptions in eNOS–PAI-1 binding promote increases in NO production and enhance vasodilation in vivo. [ABSTRACT FROM AUTHOR]

Background: Arteries chronically constricted in culture remodel to smaller diameters. Conversely, elevated luminal shear stress (SS) promotes outward remodeling of arteries in vivo and prevents inward remodeling in culture in a nitric oxide synthase (NOS)-dependent manner. Objectives: To determine whether SS-induced prevention of inward remodeling in cultured arteries is specifically eNOS-dependent and requires dilation, and whether SS alters the expression of eNOS and other genes potentially involved in remodeling. Methods: Female mouse thoracodorsal arteries were cannulated, pressurized to 80 mm Hg, and cultured for 2 days with low SS (<7 dyn/cm2), high SS (≥15 dyn/cm2), high SS + L-NAME (NOS inhibitor, 10–4 M), or high SS in arteries from eNOS–/– mice. In separate arteries cultured 1 day with low or high SS, eNOS and connexin (Cx) 37, Cx40, and Cx43 mRNA were assessed with real-time PCR. Results: High SS caused little change in passive diameters after culture (–4.7 ± 2.0%), which was less than low SS (–18.9 ± 1.4%; p < 0.0001), high SS eNOS–/– (–18.0 ± 1.5; p < 0.001), or high SS + L-NAME (–12.0 ± 0.6%; nonsignificant) despite similar constriction during culture. Cx37 mRNA expression was increased (p < 0.05) with high SS, but other gene levels were not different. Conclusions: eNOS is involved in SS-induced prevention of inward remodeling in cultured small arteries. This effect does not require NO-mediated dilation. SS increased Cx37. [ABSTRACT FROM AUTHOR]

Endothelial NOS (eNOS), and its product NO, are vital components of the control of vasomotor function and cardiovascular homeostasis. In the present review, we will take a deep dive into eNOS enzymology, function and mechanisms regulating endothelial NO. The mechanisms regulating eNOS and NO synthesis discussed here include alterations to transcriptional, post-translational modifications and protein-protein regulations. Also, we will discuss the phenotypes associated with various eNOS mutants and the consequences of a disrupted eNOS/NO cascade, highlighting the importance of eNOS function and vascular homeostasis. LINKED ARTICLES: This article is part of a themed section on Nitric Oxide 20 Years from the 1998 Nobel Prize. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.2/issuetoc. [ABSTRACT FROM AUTHOR]

Ocular neovascularization is a devastating pathology of numerous ocular diseases and is a major cause of blindness. Caveolin-1 (Cav-1) plays important roles in the vascular system. However, little is known regarding its function and mechanisms in ocular neovascularization. Here, using comprehensive model systems and a cell permeable peptide of Cav-1, cavtratin, we show that Cav-1 is a critical player in ocular neovascularization. The genetic deletion of Cav-1 exacerbated and cavtratin administration inhibited choroidal and retinal neovascularization. Importantly, combined administration of cavtratin and anti-VEGF-A inhibited neovascularization more effectively than monotherapy, suggesting the existence of other pathways inhibited by cavtratin in addition to VEGF-A. Indeed, we found that cavtratin suppressed multiple critical components of pathological angiogenesis, including inflammation, permeability, PDGF-B and endothelial nitric oxide synthase expression (eNOS). Mechanistically, we show that cavtratin inhibits CNV and the survival and migration of microglia and macrophages via JNK. Together, our data demonstrate the unique advantages of cavtratin in antiangiogenic therapy to treat neovascular diseases. [ABSTRACT FROM AUTHOR]

The PI3K/Akt pathway is necessary for several key endothelial cell (EC) functions, including cell growth, migration, survival, and vascular tone. However, existing literature supports the idea that Akt can be either pro- or antiangiogenic, possibly due to compensation by multiple isoforms in the EC when a single isoform is deleted. Thus, biochemical, genetic, and proteomic studies were conducted to examine isoform-substrate specificity for Akt1 vs. Akt2. In vitro, Akt1 preferentially phosphorylates endothelial nitric oxide synthase (eNOS) and promotes NO release, whereas non-physiological overexpression of Akt2 can bypass the loss of Akt1. Conditional deletion of Akt1 in the EC, in the absence or presence of Akt2, retards retinal angiogenesis, implying that Akt1 exerts a nonredundant function during physiological angiogenesis. Finally, proteomic analysis of Akt substrates isolated from Akt1- or Akt2-deficient ECs documents that phosphorylation of multiple Akt substrates regulating angiogenic signaling is reduced in Akfl-deficient, but not Akt2-deficient, ECs, including eNOS and Fork-head box proteins. Therefore, Akt1 promotes angiogenesis largely due to phosphorylation and regulation of important downstream effectors that promote aspects of angiogenic signaling. [ABSTRACT FROM AUTHOR]

The role of endothelium-derived nitric oxide (NO) in acute inflammation is not known. Here, we examine acute inflammation in congenic endothelial NO synthase-deficient (eNOS-/- mice. Intraplantar injection of carrageenan induces a biphasic inflammatory response. The early phase (0-6 h) is largely eliminated, and the secondary phase (24-96 h) is markedly reduced in eNOS-/- but not WT mice. Inhibition of phosphatidylinositol 3-kinase or hsp90, pathways upstream of eNOS activation, also reduces carrageenan-stimulated edema formation. To separate the ability of eNOS to regulate leukocyte trafficking vs. vascular permeability, zymosanstimulated leukocyte infiltration and protein extravasation were assessed in WT and eNOS-/- mice. Zymosan increases inflammatory cell extravasation to the same extent in WT and eNOS' mice, whereas the extravasation of plasma protein is lower in eNOS' mice. Inhibition of phosphatidylinositol 3-kinase and hsp90 also blocks protein leakage, but not leukocyte influx. These data collectively support the critical role for eNOS in regulating the magnitude of the acute inflammatory response and show that eNOS is critical for regulating microcirculatory endothelial barrier function in vivo. [ABSTRACT FROM AUTHOR]

In endothelia, NO is synthesized by endothelial NO synthase (eNOS), which is negatively regulated by caveolin-1 (Cay-1), the primary coat protein of caveolae. We show that delivery of Cay-I amino acids 82-101 (Cay) fused to an internalization sequence from Antennapedia (AP) blocks NO release in vitro and inflammation and tumor angiogenesis in vivo. To characterize the molecular mechanism by which the AP-Cav peptide and Cay-I mediate eNOS inhibition, we subdivided the Cay portion of AP-Cav into three domains (Cay-A, -B, and -C), synthesized five overlapping peptides (AP-Cav-A, -AB, -B, -BC, and -C), and tested their effects on eNOS- dependent activities. Peptides containing the Cay-B domain (amino acids 89-95) induced time- and dose-dependent inhibition of eNOS-dependent NO release in cultured endothelial cells, NO- dependent inflammation in the ear, and hydraulic conductivity in isolated venules. Alanine scanning of AP-Cav-B revealed that Thr-90 and -91 (T90,91) and Phe-92 (F92) are crucial for AP-Cav-B- and AP-Cav-mediated inhibition of eNOS. Mutation of F92 to A92 in the Cay-1 cDNA caused the loss of eNOS inhibitory activity compared with wild-type Cay-1. These data highlight the impor- tance of amino acids 89-95 and particularly F92 in mediating eNOS inhibition by AP-Cav and Cay-1. [ABSTRACT FROM AUTHOR]

Caveolae are the sites in the cell membrane responsible for concentrating an array of signaling molecules critical for cell function. Recent studies have begun to identify the functions of caveolin-1, the 22-kDa caveolar protein that oligomerizes and inserts into the cytoplasmic face of the plasma membrane. Caveolin-1 appears to regulate caveolar internalization by stabilizing caveolae at the plasma membrane rather than controlling the shape of the membrane invagination. Because caveolin-1 is a scaffolding protein, it has also been hypothesized to function as a "master regulator" of signaling molecules in caveolae. Deletion of the caveolin-1 gene in mice resulted in cardiac hypertrophy and lung fibrosis, indicating its importance in cardiac and lung development. In the endothelium, caveolin-1 regulates nitric oxide signaling by binding to and inhibiting endothelial nitric oxide synthase (eNOS). Increased cytosolic Ca[sup 2+] or activation of the kinase Akt leads to eNOS activation and its dissociation from caveolin-1. Caveolae have also been proposed as the vesicle carriers responsible for transcellular transport (transcytosis) in endothelial cells. Transcytosis, the primary means of albumin transport across continuous endothelia, occurs by fission of caveolae from the membrane. This event is regulated by tyrosine phosphorylation of caveolin-1 and dynamin. As Ca[sup 2+] influx channels and pumps are localized in caveolae, caveolin-1 is also an important determinant of Ca[sup 2+] signaling in endothelial cells. Many of these findings were presented in San Diego, CA, at the 2003 Experimental Biology symposium "Caveolin Regulation of Endothelial Function" and are reviewed in this summary. [ABSTRACT FROM AUTHOR]

Histamine increases the permeability of capillaries and venules but little is known of its precapillary actions on the control of tissue perfusion. Using gene ablation and pharmacological interventions, we tested whether histamine could increase muscle blood flow through stimulating nitric oxide (NO) release from microvascular endothelium. Vasomotor responses to topical histamine were investigated in second-order arterioles in the superfused cremaster muscle of anesthetized C57BL6 mice and null platelet endothelial cell adhesion molecule-1 (PECAM-1[sup -/-]) and null endothelial NO synthase (eNOS[sup -/-]) mice aged 8-12 wk. Neither resting (17 ± 1 µm) nor maximum diameters (36 ± 2 µm) were different between groups, nor was the constrictor response (∼5 ± 1 µm) to elevating superfusate oxygen from 0 to 21%. For arterioles of C57BL6 and PECAM1[sup -/-] mice, cumulative addition of histamine to the superfusate produced vasodilation (1 nM-1 µM; peak response, 9 ± 1 µm) and then vasoconstriction (10-100 µM; peak response, 12 ± 2 µm). In eNOS[sup -/-] mice, histamine produced only vasoconstriction. In C57BL6 and PECAM-1[sup -/-] mice, vasodilation was abolished with N[sup ω]-nitro-L-arginine (30 µM); in all mice, vasoconstriction was abolished with nifedipine (1 µM). Vasomotor responses were eliminated with pyrilamine (1 µM; H1 receptor antagonist) yet remained intact with cimetidine (1 µM; H[sub 2] receptor antagonist). These findings illustrate that the biphasic vasomotor response of mouse cremaster arterioles to histamine is mediated through H[sub 1] receptors on endothelium (NO-dependent vasodilation) as well as smooth muscle (Ca[sup 2+] entry and constriction). Thus histamine can increase as well as decrease muscle blood flow, according to local concentration. However, when NO production is compromised, only vasoconstriction and flow reduction occur. [ABSTRACT FROM AUTHOR]

Studies the potential contribution and mechanisms of endogenous nitric oxide produced by the low-output endothelial nitric oxide synthase in protecting cells against hydrogen peroxide stress. Effect of nitric oxide on cell viability after hydrogen peroxide exposure; Effect of nitric oxide on energy metabolism; Effect of nitric oxide on mitochondrial respiration and cell viability.

Normalization of tumor vasculature is an emerging strategy to improve cytotoxic therapies. Here we show that eliminating nitric oxide (NO) production from tumor cells via neuronal NO synthase silencing or inhibition establishes perivascular gradients of NO in human glioma xenografts in mice and normalizes the tumor vasculature, resulting in improved tumor oxygenation and response to radiation treatment. Creation of perivascular NO gradients may be an effective strategy for normalizing abnormal vasculature. [ABSTRACT FROM AUTHOR]

Discusses the role of protein-protein interaction in the regulation of nitric oxide synthases (NOS). Protein binding; Nitric oxide (NO) signalling pathway; Activity and spatial organization of NO synthesis; Trafficking of NOS.

A hallmark of inflammation is increased vascular permeability. Increases in vascular permeability and the migration of inflammatory cells are linked to complex interactions of inflammatory mediators with the vascular endothelium. Normally, endothelial nitric oxide synthase (eNOS) produces a tonic amount of nitric oxide (NO), which is responsible for the homeostasis between the endothelium and surrounding tissues. However, most agonists that act on endothelial cells cause a series of post-translational modifications that influence eNOS activity. Furthermore, stimulation by shear stress, autacoids or growth factors either induces eNOS or shifts it to a more active state, which produces a burst of NO. Here, we highlight recent findings about eNOS and propose how new pharmacological tools can be used to dissect the involvement and contribution of eNOS to inflammatory responses. [Copyright &y& Elsevier]

Liver sinusoidal endothelial cell (LSEC) dysfunction has been reported in alcohol-related liver disease, yet it is not known whether LSECs metabolize alcohol. Thus, we investigated this, as well as the mechanisms of alcohol-induced LSEC dysfunction and a potential therapeutic approach for alcohol-induced liver injury. Primary human, rat and mouse LSECs were used. Histone deacetylase 6 (HDAC6) was overexpressed specifically in liver ECs via adeno-associated virus (AAV)-mediated gene delivery to decrease heat shock protein 90 (Hsp90) acetylation in ethanol-fed mice. LSECs expressed CYP2E1 and alcohol dehydrogenase 1 (ADH1) and metabolized alcohol. Ethanol induced CYP2E1 in LSECs, but not ADH1. Alcohol metabolism by CYP2E1 increased Hsp90 acetylation and decreased its interaction with endothelial nitric oxide synthase (eNOS) leading to a decrease in nitric oxide (NO) production. A non-acetylation mutant of Hsp90 increased its interaction with eNOS and NO production, whereas a hyperacetylation mutant decreased NO production. These results indicate that Hsp90 acetylation is responsible for decreases in its interaction with eNOS and eNOS-derived NO production. AAV8-driven HDAC6 overexpression specifically in liver ECs deacetylated Hsp90, restored Hsp90's interaction with eNOS and ameliorated alcohol-induced liver injury in mice. Restoring LSEC function is important for ameliorating alcohol-induced liver injury. To this end, blocking acetylation of Hsp90 specifically in LSECs via AAV-mediated gene delivery has the potential to be a new therapeutic strategy. Alcohol metabolism in liver sinusoidal endothelial cells (LSECs) and the mechanism of alcohol-induced LSEC dysfunction are largely unknown. Herein, we demonstrate that LSECs can metabolize alcohol. We also uncover a mechanism by which alcohol induces LSEC dysfunction and liver injury, and we identify a potential therapeutic strategy to prevent this. [Display omitted] • LSECs express CYP2E1 and ADH1 and metabolize alcohol. • Ethanol causes Hsp90 acetylation, which decreases NO production in LSECs. • HDAC6 overexpression in LSECs restores Hsp90 function and NO production. • HDAC6 overexpression in LSECs ameliorates alcohol-induced liver injury in mice. [ABSTRACT FROM AUTHOR]

Background:Hypoxia and consequent production of vascular endothelial growth factor A (VEGFA)promote blood vessel leakiness and edema in ocular diseases. Anti-VEGFA therapeutics mayaggravate hypoxia; therefore, therapy development is needed.Methods:Oxygen-induced retinopathy was used as a model to test the role of nitric oxide (NO) inpathological neovascularization and vessel permeability. Suppression of NO formation wasachieved chemically using L-NMMA, or genetically, in endothelial NO synthase serine to alanine(S1176A) mutant mice.Results:Suppression of NO formation resulted in reduced retinal neoangiogenesis. Remainingvascular tufts exhibited reduced vascular leakage through stabilized endothelial adherens junctions,manifested as reduced phosphorylation of vascular endothelial (VE)-cadherin Y685 in a c-Src-dependent manner. Treatment with a single dose of L-NMMA in established retinopathy restoredthe vascular barrier and prevented leakage.Conclusions:We conclude that NO destabilizes adheren junctions, resulting in vascularhyperpermeability, by converging with the VEGFA/VEGFR2/c-Src/VE-cadherin pathway.Funding:This study was supported by the Swedish Cancer foundation (19 0119 Pj ), the SwedishResearch Council (2020-01349), the Knut and Alice Wallenberg foundation (KAW 2020.0057) and aFondation Leducq Transatlantic Network of Excellence Grant in Neurovascular Disease (17 CVD03). KAW also supported LCW with a Wallenberg Scholar grant (2015.0275). WCS was supportedby Grants R35 HL139945, P01 HL1070205, AHA MERIT Award. DV was supported by grants fromthe Deutsche Forschungsgemeinschaft, SFB1450, B03, and CRU342, P2. [ABSTRACT FROM AUTHOR]

Background: Atherosclerosis is driven by synergistic interactions between pathological, biomechanical, inflammatory, and lipid metabolic factors. Our previous studies demonstrated that absence of caveolin-1 (Cav1)/caveolae in hyperlipidemic mice strongly inhibits atherosclerosis, which was attributed to activation of endothelial nitric oxide (NO) synthase (eNOS) and increased production of NO and reduced inflammation and low-density lipoprotein trafficking. However, the contribution of eNOS activation and NO production in the athero-protection of Cav1 and the exact mechanisms by which Cav1/caveolae control the pathogenesis of diet-induced atherosclerosis are still not clear.Methods: Triple-knockout mouse lacking expression of eNOS, Cav1, and Ldlr were generated to explore the role of NO production in Cav1-dependent athero-protective function. The effects of Cav1 on lipid trafficking, extracellular matrix remodeling, and vascular inflammation were studied both in vitro and in vivo with a mouse model of diet-induced atherosclerosis. The expression of Cav1 and distribution of caveolae regulated by flow were analyzed by immunofluorescence staining and transmission electron microscopy.Results: We found that absence of Cav1 significantly suppressed atherogenesis in Ldlr-/-eNOS-/- mice, demonstrating that athero-suppression is independent of increased NO production. Instead, we find that the absence of Cav1/caveolae inhibited low-density lipoprotein transport across the endothelium and proatherogenic fibronectin deposition and disturbed flow-mediated endothelial cell inflammation. Consistent with the idea that Cav1/caveolae may play a role in early flow-dependent inflammatory priming, distinct patterns of Cav1 expression and caveolae distribution were observed in athero-prone and athero-resistant areas of the aortic arch even in wild-type mice.Conclusions: These findings support a role for Cav1/caveolae as a central regulator of atherosclerosis that links biomechanical, metabolic, and inflammatory pathways independently of endothelial eNOS activation and NO production. [ABSTRACT FROM AUTHOR]

Abstract: Phosphorylation of endothelial nitric oxide synthase (eNOS) is an important regulator of its enzymatic activity. We generated knockin mice expressing phosphomimetic (SD) and unphosphorylatable (SA) eNOS mutations at S1176 to study the role of eNOS phosphorylation. The single amino acid SA mutation is associated with hypertension and decreased vascular reactivity, while the SD mutation results in increased basal and stimulated endothelial NO production. In addition to these vascular effects, modulation of the S1176 phosphorylation site resulted in unanticipated effects on metabolism. The eNOS SA mutation results in insulin resistance, hyperinsulinemia, adiposity, and increased weight gain on high fat. In contrast, the eNOS SD mutation is associated with decreased insulin levels and resistance to high fat-induced weight gain. These results demonstrate the importance of eNOS in regulation of insulin sensitivity, energy metabolism, and bodyweight regulation, and suggest eNOS phosphorylation as a novel target for the treatment of obesity and insulin resistance. [Copyright &y& Elsevier]

The heterogeneous localization of endothelial nitricoxide synthase (eNOS) on the Golgi complex versus the plasma membrane has made it difficult to dissect the regulation of each pool of enzyme. Here, we generated fusion proteins that specifically target the plasma membrane or cytoplasmic aspects of the Golgi complex and have assessed eNOS activation. Plasma membrane-targeted eNOS constructs were constitutively active, phosphorylated, and responsive to transmembrane calcium fluxes, yet were insensitive to further activation by Aktmediated phosphorylation. In contrast, cis-Golgi complex-targeted eNOS behaved similarly to wild-type eNOS and was less sensitive to calcium-dependent activation and highly responsive to Akt-dependent phosphorylation compared with plasma membrane versions. In plasma membrane- and Golgi complex-targeted constructs, Ser1179 is critical for NO production. This study provides clear evidence for functional roles of plasma membrane- and Golgi complex-localized eNOS and supports the concept that proteins thought to be regulated and to function exclusively in the plasma membrane of cells can indeed signal and be regulated in internal Golgi membranes. [ABSTRACT FROM AUTHOR]

Endothelial nitric.oxide synthase (eNOS), the enzyme responsible for production of endothelial NO, is under tight and complex regulation. Proper cellular localization of eNOS is critical for optimal coupling of extracellular stimulation with NO production. In addition, the molecular chaperone Hsp90 interacts with eNOS and positively regulates eNOS activity. Hsp90 is modulated by physical interaction with its co-chaperones. CHIP (carboxyl terminus of Hsp70-interacting protein) is such a co-chaperone that remodels the Hsp90 heterocomplex and causes protein degradation of some Hsp90 substrates through the ubiquitin-protein isopeptide ligase activity of CHIP. Here we show that CHIP incorporated into the eNOS·Hsp90 complex and specifically decreased soluble eNOS levels in transiently transfected COS cells. Surprisingly, in contrast to the effects of the Hsp90 inhlbitor geldanamycin, which induces eNOS ubiquitylation and its subsequent protein degradation, CHIP did not target eNOS for ubiquitylation and proteasome-dependent degradation. Instead, CHIP partitioned soluble eNOS into an insoluble and inactive cellular compartment, presumably through its co-chaperone activity. This effect seems to be due to displacement of eNOS from the Golgi apparatus, which is otherwise required for trafficking of eNOS to the plasmalemma and subsequent activation. Consistent with observations from overexpression studies, eNOS localization to the membrane and activity were increased in mouse lung endothelial cells lacking CHIP. Taken together, these results demonstrate a novel co-chaperone-dependent mechanism through which eNOS trafficking is regulated and suggest a potentially generalized role for CHIP in protein trafficking through the Golgi compartment. [ABSTRACT FROM AUTHOR]

There is evidence that endothelial nitric-oxide synthase (eNOS) is regulated by reciprocal dephosphorylation of Thr[sup 497] and phosphorylation of Ser[sup 1179]. To examine the interrelationship between these sites, cells were transfected with wild-type (WT), T497A, T497D, S1179D, and T497A/S1179D eNOS and activity, NO release and eNOS localization were assessed. Although eNOS T497A, Sl179D and T497A/S1179D eNOS had greater enzymatic activity than did WT eNOS in lysates, basal production of NO from cells was markedly reduced in cells transfected with T497A and T497A/S1179D eNOS but augmented in cells transfected with S1179D eNOS. Stimulating cells with ATP or ionophore normalized the loss of function seen with T497A and T497A/S1179D eNOS to levels observed with WT and SI 179D eNOS, respectively. Despite these functional differences, the localization of eNOS mutants were similar to WT. Because both T497A and T497A/S1179D eNOS exhibited higher enzyme activity but reduced production of NO, we examined whether these mutations were "uncoupling" NO synthesis. T497A and T497A/S1179D eNOS generated 2-3 times more superoxide anion than WT eNOS, and both basal and stimulated interactions of T497A/S1179D eNOS with hsp90 were reduced in co-immunoprecipitation experiments. Thus, the phosphorylation/dephosphorylation of Thr[sup 497] may be an intrinsic switch mechanism that determines whether eNOS generates NO versus superoxide in cells. [ABSTRACT FROM AUTHOR]

Shear stress stimulates NO production involving the Ca2+-independent mechanisms in endothelial cells. We have shown that exposure of bovine aortic endothelial cells (BAEC) to shear stress stimulates phosphorylation of eNOS at S635 and S1179 by the protein kinase A- (PKA-) dependent mechanisms. We examined whether phosphorylation of S635 of eNOS induced by PKA stimulates NO production in a calcium-independent manner. Expression of a constitutively active catalytic subunit of PKA (Cqr) in BAEC induced phosphorylation of S635 and S1179 residues and dephosphorylation of T497. Additionally, Cqr expression stimulated NO production, which could not be prevented by treating cells with the intracellular calcium chelator BAPTA-AM. To determine the role of each eNOS phosphorylation site in NO production, HEK-293 cells transfected with eNOS point mutants whereby S116, T497, S635, and S1179 were mutated to either A or D. Maximum NO production from S635D-expressing cells was significantly higher than that of either wild type or S635A in both basal and elevated [Ca2+]i conditions. More interestingly, S635D cells produced NO even when [Ca2+]i was nearly depleted by BAPTA-AM. We confirmed these results obtained in HEK-293 cells in BAEC transfected with S635D, S635A, or wild-type eNOS vector. These findings suggest that, once phosphorylated at S635 residue, eNOS produces NO without requiring any changes in [Ca2+]i. PKA-dependent phosphorylation of eNOS S635 and subsequent basal NO production in a Ca2+-independent manner may play an important role in regulating vascular biology and pathophysiology. [Copyright &y& Elsevier]

Examines the influence of individual serine phosphorylation sites in endothelial nitric-oxide synthase (eNOS). Focus on basal and stimulated NO release, cooperative phosphorylation and co-association with hsp90 and Akt; Effect of serine phosphorylation site mutation to alanine on the phospho-state of other sites; Promotion of a protein-protein interaction from the mutation of serine to alanine.

Demonstrates that 17β-estradiol rapidly activates endothelial nitric oxide synthase through Src kinase in human endothelial cells. Indication that the PP2-inhibitable kinase is upstream of PI3-kinase and Akt; Blockade of 17β-estradiol-induced Akt phosphorylation by Src family kinase specific inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine.

Although native LDL (n-LDL) is well recognized for inducing endothelial cell (EC) dysfunction, the mechanisms remain unclear. One hypothesis is n-LDL increases caveolin-1 (Cav-1), which decreases nitric oxide (⋅NO) production by binding endothelial nitric oxide synthase (eNOS) in an inactive state. Another is n-LDL increases superoxide anion (O2•−), which inactivates ⋅NO. To test these hypotheses, EC were incubated with n-LDL and then analyzed for ⋅NO, O2•−, phospho-eNOS (S1179), eNOS, Cav-1, calmodulin (CaM), and heat shock protein 90 (hsp90). n-LDL increased NOx by more than 4-fold while having little effect on A23187-stimulated nitrite production. In contrast, n-LDL decreased cGMP under basal and A23187-stimulated conditions and increased O2•− by a mechanism that could be inhibited by L-nitroargininemethylester (L-NAME) and BAPTA/AM. n-LDL increased phospho-eNOS by 149%, eNOS by ∼34%, and Cav-1 by 28%, and decreased the association of hsp90 with eNOS by 49%. n-LDL did not appear to alter eNOS distribution between membrane fractions (∼85%) and cytosol (∼15%). Only 3–6% of eNOS in membrane fractions was associated with Cav-1. These data support the hypothesis that n-LDL increases O2•−, which scavenges ⋅NO, and suggest that n-LDL uncouples eNOS activity by decreasing the association of hsp90 as an initial step in signaling eNOS to generate O2•−. [Copyright &y& Elsevier]