open access publication

Preprint, 2024

Angiotensin-(1-5) is a Potent Endogenous Angiotensin AT2-Receptor Agonist

bioRxiv, Volume 5, 06-28, Page 2024.04.05.588367, 10.1101/2024.04.05.588367

Contributors

Souza-Silva, Igor Maciel 0000-0002-6140-3503 [1] Peluso, A. Augusto [1] Elsaafien, Khalid 0000-0001-9581-918X [2] Nazarova, Antonina L 0000-0002-8480-8861 [3] Assersen, Kasper Bostlund 0000-0002-0889-3689 [1] [4] Rodrigues-Ribeiro, Lucas 0000-0002-4011-637X [5] Mohammed, Mazher 0000-0002-6728-1930 [6] Rodrigues, André Felipe 0000-0002-7219-6896 [7] [8] Nawrocki, Arkadiusz M [1] Jakobsen, Lene Andrup [1] Jensen, Pia Søndergaard [1] De Kloet, Annette Diane 0000-0002-7213-6957 [2] Krause, Eric Gerald 0000-0002-2718-3113 [2] Del Borgo, Mark P [9] Maslov, Ivan O 0000-0002-2322-3819 [9] Widdop, Robert Edward 0000-0002-6515-0435 [9] Santos, Robson Augusto Souza Dos 0000-0001-8738-5852 [5] Bader, Michael 0000-0003-4780-4164 [7] [8] [10] [11] Larsen, Martin Røssel 0000-0001-6203-0123 [1] Verano-Braga, Thiago 0000-0002-7083-2588 [5] Katritch, Vsevolod 0000-0003-3883-4505 [3] Sumners, Collin 0000-0002-9689-2255 [1] [12] Steckelings, Ulrike Muscha M 0000-0002-5430-4275 (Corresponding author) [1]

Affiliations

  1. [1] University of Southern Denmark
    2. [NORA names: SDU University of Southern Denmark; University; Denmark; Europe, EU; Nordic; OECD];
  2. [2] Georgia State University
    3. [NORA names: United States; America, North; OECD];
  3. [3] University of Southern California
    4. [NORA names: United States; America, North; OECD];
  4. [4] Odense University Hospital
    5. [NORA names: Region of Southern Denmark; Hospital; Denmark; Europe, EU; Nordic; OECD];
  5. [5] Universidade Federal de Minas Gerais
    6. [NORA names: Brazil; America, South];

Abstract

Background: The renin-angiotensin system involves many more enzymes, receptors and biologically active peptides than originally thought. With this study, we investigated whether angiotensin-(1-5) [Ang-(1-5)], a 5-amino acid fragment of angiotensin II, has biological activity, and through which receptor it elicits effects. Methods: The effect of Ang-(1-5) (1µM) on nitric oxide release was measured by DAF-FM staining in human aortic endothelial cells (HAEC), or Chinese Hamster Ovary (CHO) cells stably transfected with the angiotensin AT 2 -receptor (AT 2 R) or the receptor Mas. A potential vasodilatory effect of Ang-(1-5) was tested in mouse mesenteric and human renal arteries by wire myography; the effect on blood pressure was evaluated in normotensive C57BL/6 mice by Millar catheter. These experiments were performed in the presence or absence of a range of antagonists or inhibitors or in AT 2 R-knockout mice. Binding of Ang-(1-5) to the AT 2 R was confirmed and the preferred conformations determined by in silico docking simulations. The signaling network of Ang-(1-5) was mapped by quantitative phosphoproteomics. Results: Key findings included: (1) Ang-(1-5) induced activation of eNOS by changes in phosphorylation at Ser1177 eNOS and Tyr657 eNOS and thereby (2) increased NO release from HAEC and AT 2 R-transfected CHO cells, but not from Mas-transfected or non-transfected CHO cells. (3) Ang-(1-5) induced relaxation of preconstricted mouse mesenteric and human renal arteries and (4) lowered blood pressure in normotensive mice - effects which were respectively absent in arteries from AT 2 R-KO or in PD123319-treated mice and which were more potent than effects of the established AT 2 R-agonist C21. (5) According to in silico modelling, Ang-(1-5) binds to the AT 2 R in two preferred conformations, one differing substantially from where the first five amino acids within angiotensin II bind to the AT 2 R. (6) Ang-(1-5) modifies signaling pathways in a protective RAS-typical way and with relevance for endothelial cell physiology and disease. Conclusions: Ang-(1-5) is a potent, endogenous AT 2 R-agonist.

Keywords

AT2 receptor agonist, Ang-(1, Ang-(1-5, C21, C57BL/6 mice, CHO cells, Chinese Hamster Ovary (CHO) cells, DAF-FM, DAF-FM staining, MAS, Millar, Millar catheter, NO release, R-KO, absence, acid, activation of eNOS, active peptides, activity, agonists, amino, amino acids, angiotensin, angiotensin II, angiotensin-(1-5, antagonist, aortic endothelial cells, artery, binding, biological activity, biologically active peptides, biology, blood, blood pressure, catheter, cell physiology, cells, changes, conformation, disease, docking, docking simulations, eNOS, effect, endothelial cell physiology, endothelial cells, enzyme, experiments, findings, fragments of angiotensin II, human aortic endothelial cells, human renal arteries, increased NO release, induced activation, induced relaxation, inhibitors, lowering blood pressure, mice, model, modify signaling pathways, myography, network, nitric oxide release, non-transfected CHO cells, oxide release, pathway, peptide, phosphoproteome, phosphorylation, physiology, preferred conformations, presence, pressure, quantitative phosphoproteomics, receptor Mas, receptors, release, relevance, renal artery, renin-angiotensin system, signal, signaling networks, signaling pathway, simulation, staining, study, system, way, wire, wire myography

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