Robson A.S. Santos, Melissa Tainan Silva Dias, Amanda de Sá Martins de Bessa, Carolina Fonseca de Barros, Matheus F. Itaborahy, Filipe Alex da Silva, Sthefanie Chaves de Almeida Gonçalves, Lucas Rodrigues‐Ribeiro, Kamylle Silva Ferraz, Ana Paula Davel, Natália Ribeiro Cabacinha Nóbrega, Bruno Durante da Silva, Sérgio Scalzo, Pedro Alves Soares Vaz de Castro, João Batista Rodrigues Dutra, Ivana S. Lula, Isadora Zhong Liang Ferreira Feng, Uri Flegler Vieira-Machado, Ana Caroline Ventris de Godoy, A. Monteiro, Marcos Eliezeck, Bruno Sanches, André Monteiro, Gabriela da Silva Magalhães, Nícia Pedreira Soares, Danilo Augusto Alves Pereira, Júlia Rezende Ribeiro, Maria Luiza Dias-Pinto, Leandro Eziquiel de Souza, Amanda Silva, Daisy Motta‐Santos, Michael Bäder, Natália Alenina, Luciano dos Santos Aggum Capettini, Marco Antônio Peliky Fontes, Andréa Siqueira Haibara, Daniel Campos Villela, Thiago Verano‐Braga, Maria Cláudia Irigoyen, Fernanda Ribeiro Marins, Carlos H. Castro, Ana Cristina Simões e Silva, Sílvia Guatimosim, M. Fátima Leite, Maria José Campagnole‐Santos
BACKGROUND: The renin-angiotensin system comprises a biochemical cascade that hydrolyzes angiotensinogen into several different bioactive peptides, which can activate different receptors, promoting plenty of specific effects. This study aimed to evaluate the presence of the putative product of alamandine, the pentapeptide Ala-(1-5) (alamandine-[1-5]), in the circulation and its biological activity. METHODS: To accomplish this, we have used mass spectrometry (Matrix-assisted Laser Desorption/Ionization Time-of-Flight/Time-of-Flight mass spectrometry [MALDI/TOF/TOF], Liquid Chromatography-Tandem Mass Spectrometry [LC-MS/MS]) and several methodologies, including isolated blood vessels, isolated perfused hearts, isolated cardiomyocytes, blood pressure recording in freely moving normotensive and spontaneously hypertensive rats, high-resolution echocardiography, central administration (intracerebroventricular infusion and microinjection in the insular cortex), cell culture (endothelial cells and G-protein–coupled receptors-transfected Chinese Hamster Ovary cells), and wild-type and Mas (Mas receptor proto-oncogene), MrgD (Mas-related G-protein–coupled receptor subtype D), or AT2 (angiotensin II type 2) receptor-deficient mice. RESULTS: We show that Ala-(1-5) is present in the circulation of healthy humans and rodents and promotes many biological central and peripheral actions. A major role for ACE (angiotensin-converting enzyme) activity in the formation of Ala-(1-5) from alamandine in the circulation was observed using plasma samples from angiotensinogen-knockout mice. Ala-(1-5) increases baroreflex sensitivity and produces a long-lasting (≈6 hours) antihypertensive effect in spontaneously hypertensive rats, associated with a significant reduction in cardiac output. Additionally, Ala-(1-5) decreases inotropism in isolated perfused hearts and reduces contractility in cardiomyocytes. In Chinese Hamster Ovary cells-transfected cells, Ala-(1-5) can bind and stimulate NO production through all receptors from the renin-angiotensin system protective arm (Mas, MrgD, and AT 2 receptors). On the other hand, the Ala-(1-5) effects on cardiomyocytes and mouse aortic rings were abolished only by MrgD genetic deletion, but not by Mas or AT2 receptor knockout. CONCLUSIONS: Our data demonstrate that Ala-(1-5) is a newly identified peptide within the renin-angiotensin system, with strong blood pressure–lowering effects that vary in mechanisms of action among different tissues. Ala-(1-5) has distinct characteristics that differentiate it from the conventional renin-angiotensin system pathways responsible for reducing blood pressure.