The Golgi Association of Endothelial Nitric Oxide Synthase Is Necessary for the Efficient Synthesis of Nitric Oxide

Sessa, William C.; Garca-Cardea, Guillermo; Liu, Jianwei; Keh, Agnes; Pollock, Jennifer S.; Bradley, John R.; Thiru, S; Braverman, Irwin M.; Desai, Kaushik

doi:10.1074/jbc.270.30.17641

Cited by 236 publications

(223 citation statements)

References 21 publications

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“…Mechanisms underlying eNOS dysfunction in diabetes include: (i) decreased expression of eNOS; (ii) decreased phosphorylation of eNOS; (iii) increased degradation of nitric oxide, secondary to enhanced superoxide production in diabetes; and (iv) changes in expression of cofactors in the eNOS complex that are important for regulating eNOS activity [22,23,24]. The present study is the first to link chronic elevated glucose with modulation of EC eNOS mRNA levels through mitochondrial production of ROS with subsequent activation of AP-1.…”

Section: Discussionmentioning

confidence: 99%

Hyperglycaemia-induced superoxide production decreases eNOS expression via AP-1 activation in aortic endothelial cells

et al. 2004

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Aims/hypothesis. Hyperglycaemia is a primary cause of vascular complications in diabetes. A hallmark of these vascular complications is endothelial cell dysfunction, which is partly due to the reduced production of nitric oxide. The aim of this study was to investigate the regulation of endothelial nitric oxide synthase (eNOS) activity by acute and chronic elevated glucose. Methods. Human aortic endothelial cells were cultured in 5.5 mmol/l (NG) or 25 mmol/l glucose (HG) for 4 h, 1 day, 3 days or 7 days. Mouse aortic endothelial cells were freshly isolated from C57BL/6J control and diabetic db/db mice. The expression and activity of eNOS were measured using quantitative PCR and nitrite measurements respectively. The binding of activator protein-1 (AP-1) to DNA in nuclear extracts was determined using electrophoretic mobility-shift assays. Results. Acute exposure (4 h) of human aortic endothelial cells to 25 mmol/l glucose moderately increased eNOS activity and eNOS mRNA and protein expression. In contrast, chronic exposure to elevated glucose (25 mmol/l for 7 days) reduced total nitrite levels (46% reduction), levels of eNOS mRNA (46% reduction) and eNOS protein (65% reduction). In addition, AP-1 DNA binding activity was increased in chronic HGcultured human aortic endothelial cells, and this effect was reduced by the specific inhibition of reactive oxygen species production through the mitochondrial electron transport chain. Mutation of AP-1 sites in the human eNOS promoter reversed the effects of HG. Compared with C57BL/6J control mice, eNOS mRNA levels in diabetic db/db mouse aortic endothelial cells were reduced by 60%. This decrease was reversed by the overexpression of manganese superoxide dismutase using an adenoviral construct. Conclusions/interpretation. In diabetes, the expression and activity of eNOS is regulated through glucosemediated mitochondrial production of reactive oxygen species and activation of the oxidative stress transcription factor AP-1. Abbreviations: AP-1, activator protein-1 · EC, endothelial cell · eNOS, endothelial nitric oxide synthase · FBS, fetal bovine serum · HG, high glucose · MnSOD, manganese superoxide dismutase · MOI, multiplicity of infection · NF-κB, nuclear factor-κB · NG, normal glucose · ROS, reactive oxygen species · UCP-1, uncoupling protein-1

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Section: Discussionmentioning

confidence: 99%

Hyperglycaemia-induced superoxide production decreases eNOS expression via AP-1 activation in aortic endothelial cells

et al. 2004

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“…21 For fluorescence images, confluent cells between the second and fourth passages were plated in 6-well plates. CHO-transfected and CHO-nontransfected cells were preincubated in Hanks' balanced salt solution containing 4,5-diaminofluorescein-diacetate (Molecular Probes; 10 Ϫ6 mol/L; 20 minutes).…”

Section: No Measurementmentioning

confidence: 99%

Angiotensin-(1-7) Through Receptor Mas Mediates Endothelial Nitric Oxide Synthase Activation via Akt-Dependent Pathways

et al. 2007

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Abstract-Angiotensin-(1-7) [Ang-(1-7)] causes endothelial-dependent vasodilation mediated, in part, by NO release.However, the molecular mechanisms involved in endothelial NO synthase (eNOS) activation by Ang-(1-7) remain unknown. Using Chinese hamster ovary cells stably transfected with Mas cDNA (Chinese hamster ovary-Mas), we evaluated the underlying mechanisms related to receptor Mas-mediated posttranslational eNOS activation and NO release. We further examined the Ang-(1-7) profile of eNOS activation in human aortic endothelial cells, which constitutively express the Mas receptor. Chinese hamster ovary-Mas cells and human aortic endothelial cell were stimulated with Ang-(1-7; 10 Ϫ7 mol/L; 1 to 30 minutes) in the absence or presence of A-779 (10 Ϫ6 mol/L). Additional experiments were performed in the presence of the phosphatidylinositol 3-kinase inhibitor wortmannin (10 Ϫ6 mol/L). Changes in eNOS (at Ser1177/Thr495 residues) and Akt phosphorylation were evaluated by Western blotting. NO release was measured using both the fluorochrome 2,3-diaminonaphthalene and an NO analyzer. Ang-(1-7) significantly stimulated eNOS activation (reciprocal phosphorylation/dephosphorylation at Ser1177/Thr495) and induced a sustained Akt phosphorylation (PϽ0.05). Concomitantly, a significant increase in NO release was observed (2-fold increase in relation to control). These effects were blocked by A-779. Wortmannin suppressed eNOS activation in both Chinese hamster ovary-Mas and human aortic endothelial cells. Our findings demonstrate that Ang-(1-7), through Mas, stimulates eNOS activation and NO production via Akt-dependent pathways. These novel data highlight the importance of the Ang-(1-7)/Mas axis as a putative regulator of endothelial function. T he renin-angiotensin system is a crucial regulator of cardiovascular homeostasis. Most physiological effects of angiotensin (Ang) II are mediated via Ang II type 1 (AT 1 ) receptors (AT 1 R), with Ang II type 2 (AT 2 ) receptors (AT 2 R) counteracting AT 1 R actions. 1 Growing evidence indicates that the Ang peptide Ang-(1-7) plays an important role in the renin-angiotensin system. 2 This heptapeptide is formed by Ang-converting enzyme-dependent and Ang-converting enzyme-independent pathways. Much attention has been given recently to its formation through hydrolysis of Ang II by the ectoenzyme Ang-converting enzyme 2, which is present in many organs. 3 Ang-(1-7) opposes many Ang II-stimulated actions. Ang-(1-7), acting through the G protein-coupled receptor (GPCR) Mas, releases NO and prostaglandins causing vasodilation, inhibition of cell growth, and opposition of AT 1 R-mediated Ang II vasoconstrictor and proliferative effects. 2 Overactivity of the renin-angiotensin system, as observed in cardiovascular diseases, and the lack of balance among its peptides may reduce NO bioavailability leading to endothelial dysfunction. 4,5 NO plays a critical role in endothelial function, maintaining vasodilator tone, inhibiting platelet aggregation and adhesion, and modulating vascular smooth ...

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“…Cell medium was processed for the measurement of nitrite (NO 2 Ϫ ), the stable breakdown product of NO in aqueous solution, by NO-specific chemiluminescence using a NO analyzer (Ionics Instruments, Boulder, CO; Sessa et al, 1995). Transfected COS-7 cells were processed for the measurement of nitrite, 48 h after transfection.…”

Section: No Releasementioning

confidence: 99%

Src-mediated Phosphorylation of Hsp90 in Response to Vascular Endothelial Growth Factor (VEGF) Is Required for VEGF Receptor-2 Signaling to Endothelial NO Synthase

Duval

Bœuf

Huot

et al. 2007

MBoC

130

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Nitric oxide (NO) release from endothelial cells, via endothelial NO synthase (eNOS) activation, is central to the proangiogenic actions of vascular endothelial growth factor (VEGF). VEGF signaling to eNOS is principally mediated byan Akt-dependent phosphorylation of eNOS and by increased association of eNOS to the molecular chaperone, heatshock protein 90 kDa (Hsp90). Herein, we report that VEGFR-2 activation induces tyrosine phosphorylation of VEGF receptor 2 (VEGFR-2)-associated Hsp90␤. Tyrosine phosphorylation of Hsp90␤ in response to VEGF is dependent on internalization of the VEGFR-2 and on Src kinase activation. Furthermore, we demonstrate that c-Src directly phosphorylates Hsp90 on tyrosine 300 residue and that this event is essential for VEGF-stimulated eNOS association to Hsp90 and thus NO release from endothelial cells. Our work identifies Y300 phosphorylation of Hsp90 as a novel regulated posttranslational modification of the chaperone and demonstrates its importance in the proangiogenic actions of VEGF, namely by regulating NO release from endothelial cells. INTRODUCTIONVascular endothelial growth factor (VEGF) is a potent proangiogenic cytokine that activates three specific receptor tyrosine kinases (RTK): VEGF receptor (VEGFR)-1, -2, and -3. Gene knockout studies revealed that VEGF and VEGFRs are essential for the development of the vasculature in mouse embryos (Fong et al., 1995;Shalaby et al., 1995;Carmeliet et al., 1996;Dumont et al., 1998). In addition to its functional roles in vasculogenesis, VEGF plays key roles in adult physiological and pathological angiogenesis such as wound healing and tumor vascularization. VEGFR-2 mediates most of the functional and biochemical effects of VEGF in endothelial cells including proliferation, migration, survival, and nitric oxide (NO) release. These effects are produced through the activation of multiple signaling pathways, such as the phosphoinositide-3 kinase (PI-3 kinase)/ Akt, p38 mitogen-activated protein kinase (MAPK), phospholipase C (PLC)-␥, and Erk/MAPK, after the activation of VEGFR-2 (Rousseau et al., 1997;Takahashi and Shibuya, 1997;Gerber et al., 1998;Takahashi et al., 2001). VEGF-mediated activation of the Ser/Thr kinase Akt leads to phosphorylation of serine 1179 on bovine endothelial NO synthase (eNOS; Ser 1177 in the human form), which increases eNOS catalytic activity and results in release of NO from endothelial cells (Fulton et al., 1999;Dimmeler et al., 1999). In turn, NO released from endothelium contributes to the proangiogenic effects of VEGF. Indeed, VEGF-mediated vascular permeability, angiogenesis, and endothelial cell precursor mobilization are markedly impaired in eNOS-deficient mice (Fukumura et al., 2001;Aicher et al., 2003).Heat-shock protein 90 kDa (Hsp90) is a multifunctional molecular chaperone, and its role in the prevention of protein aggregation and in the refolding of denatured proteins has been studied in much detail (Whitesell and Lindquist, 2005). Hsp90 is also a regulator of the activity of signaling molecules su...

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The Golgi Association of Endothelial Nitric Oxide Synthase Is Necessary for the Efficient Synthesis of Nitric Oxide

Cited by 236 publications

References 21 publications

Hyperglycaemia-induced superoxide production decreases eNOS expression via AP-1 activation in aortic endothelial cells

Hyperglycaemia-induced superoxide production decreases eNOS expression via AP-1 activation in aortic endothelial cells

Angiotensin-(1-7) Through Receptor Mas Mediates Endothelial Nitric Oxide Synthase Activation via Akt-Dependent Pathways

Src-mediated Phosphorylation of Hsp90 in Response to Vascular Endothelial Growth Factor (VEGF) Is Required for VEGF Receptor-2 Signaling to Endothelial NO Synthase

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