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A new method for measurement of ACE2 activity in tissues using fluorescent angiotensin peptides (779.9)
Author(s) -
Oliveira Marilene,
Santos Robson
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.779.9
Subject(s) - phosphoramidon , renin–angiotensin system , losartan , chemistry , enzyme , protease , angiotensin ii receptor type 1 , incubation , receptor , angiotensin converting enzyme 2 , biochemistry , angiotensin ii , neprilysin , kidney , captopril , biology , medicine , endocrinology , disease , covid-19 , blood pressure , infectious disease (medical specialty)
The carboxypeptidase ACE2 is a key enzyme of the renin‐angiotensin system, being primarily responsible for the formation of Ang ‐(1‐7 ) by cleavage of the carboxy‐terminal end of Ang II as well as the degradation of Angiotensin A to alamandine. The aim of this study was to develop a new method for measurement of ACE2 activity using the fluorecent Ang A and Ang II and HPLC. To evaluate the activity of ACE2, we used samples of heart, kidney and lung of FVBN wild type mice. Samples were homogenized in buffer for optimal activity of ACE2. Initially, we optimized the conditions of the assay using different concentrations of recombinant human enzyme rhACE2 at different incubation times with the substrate Ang II or Ang A. After the assay optimization, our results showed a linear formation of Ang‐(1‐7) or Alamandine linear with the concentration of RhACE2 and time of incubation. The homogenates of the tissues and the cocktail of inhibitors used for the incubation did not produce fluorescence. With the addition of the protease inhibitors Z ‐prolyl‐ prolinal , Captopril Chymostatin, Phosphoramidon , DL Thiorphan , Amastatin , Bestatin , Benzilsuccinaeo and PCMB, it was possible to inhibit the enzymes related to the SRA except ACE2 ,ensuring the reproducibility of the assay. Losartan also used to prevent the binding of the substrates to AT1 receptors present in homogenates. The ACE2 inhibitor MLN4760,abolished the formation of Ang‐(1‐7) or Alamandine by the homogenates. However, as described previously, DX600 was not able to inhibit ACE2 in mice tissue homogenate. The sensitivity of the assay was higher with the use of Ang A as substrate. This novel non‐isotopic, HPLC‐based method can be used for studies on the functional role of ACE2 in physiological and pathological conditions. Grant Funding Source : INCT‐CNPq, CAPES, FAPEMIG