Mast Cell Peptidases (Carboxypeptidase A and Chymase)‐Mediated Hydrolysis of Human Angiotensin‐(1‐12)

Abstract

IntroductionAngiotensin processing peptidases [carboxypeptidase A (CPA) and chymase] are stored in cardiac mast cell secretory granules and are co‐released into the extracellular environment after activation/degranulation. In the human heart, we showed that chymase is primarily responsible for angiotensin II (Ang II) generation from the alternate substrate angiotensin‐(1‐12) [Ang‐(1‐12)], which is present in human heart. In this study, we investigated whether there are any synergistic effects of CPA and chymase in the processing of Ang‐(1‐12).MethodRadiolabeled human Ang‐(1‐12) [125I‐Ang‐(1‐12)] substrate was incubated with CPA alone (0.325 μg/mL), human recombinant chymase (hrChymase) (0.325 μg/mL) or a combination of CPA + hrChymase (1:1 or 1: ⅓ ratio) in 50 mM Tris‐HCl buffer solution containing 150 mM NaCl (pH 8.0) for 0–15 min at 37 °C. The reaction was stopped by adding an equal volume of 1% phosphoric acid and injected on a C18 column. Eluted 125I‐Ang‐(1‐12) metabolic products were quantified by HPLC connected to in‐line flow‐through gamma detector. To determine the Km and Vmax, CPA and hrChymase were incubated with increasing concentrations (0–300 μM) of human Ang‐(1‐12) substrate for 20 min at 37 °C. The Km and Vmax were calculated using the Michaelis‐Menten equation.ResultsWe found that after 15 min of incubation, CPA sequentially metabolized Ang‐(1‐12) substrate into angiotensin‐(1‐9) [Ang‐(1‐9), 53% (major product)], Ang II (22%) and angiotensin‐(1‐7) [Ang‐(1‐7), 11%]. No further hydrolysis of Ang‐(1‐7) was detected during CPA incubation. In the presence of hrChymase alone, 125I‐Ang‐(1‐12) was directly metabolized into Ang II (89%) and no further hydrolysis of Ang II was detected. In the presence of both CPA + hrChymase (1:1 ratio), the amount of Ang II formation was 68% from 125I‐Ang‐(1‐12) within a 5 min incubation period. The formation of Ang‐(1‐9) and Ang‐(1‐7) were only 11% and 10%, respectively. When CPA + hrChymase were added in 1: ⅓ ratio for 5 min, the Ang II, Ang‐(1‐9) and Ang‐(1‐7) formations were 65%, 27%, 6%, respectively. The Km and Vmax values were 150 ± 5 μM and 384 ± 23 nM/min/mg of CPA and 40 ± 9 μM and 116 ± 20 nM/min/mg of hrChymase.ConclusionsThese findings suggest that compared to CPA, chymase shows a much higher affinity to metabolize Ang‐(1‐12) into Ang II. In addition, Ang II and Ang‐(1‐7) are the end products of chymase and CPA, respectively. Overall, our findings suggest that chymase, rather than CPA, is primarily responsible for Ang II generation from Ang‐(1‐12).Support or Funding InformationThis work was supported by grant from the National Heart, Lung and Blood Institute of the National Institutes of Health (P01 HL‐051952).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.