Abstract
Since angiotensin-(1-12) [Ang-(1-12)] is a non-renin dependent alternate precursor for the generation of cardiac Ang peptides in rat tissue, we investigated the metabolism of Ang-(1-12) by plasma membranes (PM) isolated from human atrial appendage tissue from nine patients undergoing cardiac surgery for primary control of atrial fibrillation (MAZE surgical procedure). PM was incubated with highly purified 125I-Ang-(1-12) at 37°C for 1 h with or without renin-angiotensin system (RAS) inhibitors [lisinopril for angiotensin converting enzyme (ACE), SCH39370 for neprilysin (NEP), MLN-4760 for ACE2 and chymostatin for chymase; 50 µM each]. 125I-Ang peptide fractions were identified by HPLC coupled to an inline γ-detector. In the absence of all RAS inhibitor, 125I-Ang-(1-12) was converted into Ang I (2±2%), Ang II (69±21%), Ang-(1-7) (5±2%), and Ang-(1-4) (2±1%). In the absence of all RAS inhibitor, only 22±10% of 125I-Ang-(1-12) was unmetabolized, whereas, in the presence of the all RAS inhibitors, 98±7% of 125I-Ang-(1-12) remained intact. The relative contribution of selective inhibition of ACE and chymase enzyme showed that 125I-Ang-(1-12) was primarily converted into Ang II (65±18%) by chymase while its hydrolysis into Ang II by ACE was significantly lower or undetectable. The activity of individual enzyme was calculated based on the amount of Ang II formation. These results showed very high chymase-mediated Ang II formation (28±3.1 fmol×min−1×mg−1, n = 9) from 125I-Ang-(1-12) and very low or undetectable Ang II formation by ACE (1.1±0.2 fmol×min−1×mg−1). Paralleling these findings, these tissues showed significant content of chymase protein that by immunocytochemistry were primarily localized in atrial cardiac myocytes. In conclusion, we demonstrate for the first time in human cardiac tissue a dominant role of cardiac chymase in the formation of Ang II from Ang-(1-12).
Highlights
It is well established that the renin-angiotensin system (RAS) is a major regulatory hormonal network influencing cardiovascular homeostasis, blood pressure, and fluid and electrolyte balance
We show that human atrial tissue samples obtained from patients undergoing cardiac interventions for correction of atrial fibrillation expresses ir-Ang-(1-12) and converts 125I-Ang-(1-12) into angiotensin peptides
The study demonstrates that chymase is the main metabolic pathway by which this extended form of Ang I serves as a substrate for the generation of Ang II in human atrial samples
Summary
It is well established that the renin-angiotensin system (RAS) is a major regulatory hormonal network influencing cardiovascular homeostasis, blood pressure, and fluid and electrolyte balance. HPLC of human 125I-Ang-(1-12) metabolic products generated by plasma membrane (50 mg) isolated from human atrial appendage incubated with or without the presence of RAS inhibitors at 37uC for 60 min. To reassure ourselves that the results obtained with the chymostatin inhibitor reflected a selective inhibition of chymase, we further evaluated the hydrolysis of the colorimetric substrate (N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide; N-suc-AAPF-pNA) by human plasma membrane in the absence and in the presence of chymostatin. The ir-chymase bands were normalized with bactin and relative chymase protein expression in the human atrial tissue was consistence with the HPLC results of chymase activity to generate Ang II products from 125I-Ang-(1-12) (Figure 3). Negative controls without primary antibody show no staining for chymase (Figure 5)
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