The Angiotensin II type 2 receptor (AT2R) promotes vasodilation by nitric oxide (NO) release from endothelial cells. However, the mechanisms underlying the AT2R-induced stimulation of endothelial NO synthase (eNOS) is still not completely understood. Therefore, we investigated whether in addition to the known AT2R-mediated phosphorylation of eNOS at Ser1177, activation of phosphatases and dephosphorylation of eNOS at Tyr657 and Thr495 are also involved. Human aortic endothelial cells (HAEC) were stimulated with the AT2R-agonist Compound 21 (C21) (1 µM) in the presence or absence of either PD123319 (10 µM; AT2R antagonist), l-NG-Nitroarginine methyl ester (l-NAME) (10 µM; eNOS inhibitor), MK-2206 (100 nM; protein kinase B (Akt) inhibitor) sodium fluoride (NaF) (1 nM; serine/threonine phosphatase inhibitor) or sodium orthovanadate (Na3VO4) (10 nM; tyrosine phosphatase inhibitor). NO release was estimated by quantifying 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM) fluorescence. The phosphorylation status of activating (eNOS-Ser1177) or inhibitory eNOS residues (eNOS-Tyr657, eNOS-Thr495) was determined by Western blotting. Phosphorylation of Akt at Ser473 was measured to estimate Akt activity. AT2R stimulation significantly increased NO release from HAEC, which was blocked by PD123319, l-NAME and both phosphatase inhibitors. Intracellular calcium transients were not changed by C21. AT2R stimulation resulted in phosphorylation of eNOS-Ser1177 and dephosphorylation of eNOS-Tyr657 and eNOS-Thr495. Phosphorylation at eNOS-Ser1177 was prevented by inhibition of Akt with MK-2206. From these data, we conclude that AT2R stimulation in human endothelial cells increases eNOS activity through phosphorylation of activating eNOS residues (eNOS-Ser1177) by Akt, and through dephosphorylation of inactivating eNOS residues (eNOS-Tyr657, eNOS-Thr495) by serine/threonine and tyrosine phosphatases, thus increasing NO release.
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A representation of the intestinal microflora. In Clinical Science volume 132 (issue 7), Rajani and Jia review recent research on the effect of bacterial metabolites on host metabolism (microbiota-host co-metabolism) associated with conditions such as obesity, cardiovascular disease, diabetes and non-alcoholic fatty liver disease (pages 791-811). Then, in issue 8, Lezutekong et al. (pages 901-904) provide a commentary on a recent research by Kim et al. in Clinical Science that demonstrates a crucial link between gut microbiota and bacterial metabolites such as butyrate, gut leakiness, and hypertension. These and other articles from the journal are featured in a themed collection on the topic of the microbiome and chronic disease.
Research Article|
April 06 2018
Identification of protein phosphatase involvement in the AT2 receptor-induced activation of endothelial nitric oxide synthase
A. Augusto Peluso;
A. Augusto Peluso
1IMM - Department of Cardiovascular and Renal Research, University of Southern Denmark, Odense, Denmark
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Jesper Bork Bertelsen;
Jesper Bork Bertelsen
1IMM - Department of Cardiovascular and Renal Research, University of Southern Denmark, Odense, Denmark
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Kenneth Andersen;
Kenneth Andersen
1IMM - Department of Cardiovascular and Renal Research, University of Southern Denmark, Odense, Denmark
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Tenna Pavia Mortsensen;
Tenna Pavia Mortsensen
1IMM - Department of Cardiovascular and Renal Research, University of Southern Denmark, Odense, Denmark
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Pernille B. Hansen;
Pernille B. Hansen
1IMM - Department of Cardiovascular and Renal Research, University of Southern Denmark, Odense, Denmark
2Cardiovascular and Metabolic Disease, IMED Biotech Unit, AstraZeneca, Gothenburg, Sweden
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Colin Sumners;
Colin Sumners
3Department of Physiology and Functional Genomics, University of Florida, Gainesville, FL, U.S.A.
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Michael Bader;
Michael Bader
4Max Delbrück Center for Molecular Medicine, Berlin, Germany
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Robson A. Santos;
Robson A. Santos
5National Institute of Science and Technology in Nanobiopharmaceutics, Department of Physiology and Biophysics, Federal University of Minas Gerais (UFMG), Belo Horizonte, Brazil
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Ulrike Muscha Steckelings
1IMM - Department of Cardiovascular and Renal Research, University of Southern Denmark, Odense, Denmark
Correspondence: Ulrike Muscha Steckelings ([email protected])
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Publisher: Portland Press Ltd
Received:
December 27 2017
Revision Received:
March 03 2018
Accepted:
March 14 2018
Accepted Manuscript online:
March 14 2018
Online ISSN: 1470-8736
Print ISSN: 0143-5221
© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society
2018
Clin Sci (Lond) (2018) 132 (7): 777–790.
Article history
Received:
December 27 2017
Revision Received:
March 03 2018
Accepted:
March 14 2018
Accepted Manuscript online:
March 14 2018
Citation
A. Augusto Peluso, Jesper Bork Bertelsen, Kenneth Andersen, Tenna Pavia Mortsensen, Pernille B. Hansen, Colin Sumners, Michael Bader, Robson A. Santos, Ulrike Muscha Steckelings; Identification of protein phosphatase involvement in the AT2 receptor-induced activation of endothelial nitric oxide synthase. Clin Sci (Lond) 16 April 2018; 132 (7): 777–790. doi: https://doi.org/10.1042/CS20171598
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