Chymase mediates angiotensin-(1-12) metabolism in normal human hearts

Abstract

Identification of angiotensin-(1-12) [Ang-(1-12)] in forming angiotensin II (Ang II) by a non-renin dependent mechanism has increased knowledge on the paracrine/autocrine mechanisms regulating cardiac expression of Ang peptides. This study now describes in humans the identity of the enzyme accounting for Ang-(1-12) metabolism in the left ventricular (LV) tissue of normal subjects. Reverse phase HPLC characterized the products of (125)I-Ang-(1-12) metabolism in plasma membranes (PMs) from human LV in the absence and presence of inhibitors for chymase (chymostatin), angiotensin-converting enzyme (ACE) 1 (lisinopril) and 2 (MLN-4760), and neprilysin (SHC39370). In the presence of the inhibitor cocktail, ≥ 98% ± 2% of cardiac (125)I-Ang-(1-12) remained intact, whereas exclusion of chymostatin from the inhibitor cocktail led to significant conversion of Ang-(1-12) into Ang II. In addition, chymase-mediated hydrolysis of (125)I-Ang I was higher compared with Ang-(1-12). Negligible Ang-(1-12) hydrolysis occurred by ACE, ACE2, and neprilysin. A high chymase activity was detected for both (125)I-Ang-(1-12) and (125)I-Ang I substrates. Chymase accounts for the conversion of Ang-(1-12) and Ang I to Ang II in normal human LV. These novel findings expand knowledge of the alternate mechanism by which Ang-(1-12) contributes to the production of cardiac angiotensin peptides.

Figure 1

¹²⁵I-Ang-(1-12) hydrolysis by Normal Left Ventricular Plasma Membranes. Chromatograms show the hydrolysis of ¹²⁵I-Ang-(1-12) in normal human left ventricular. Panel A: Without chymostatin (chymase-mediated hydrolysis, 30 min), Panel B: Without lisinopril (ACE-mediated hydrolysis, 60 min), Panel C: Without SCH39370 (NEP-mediated hydrolysis, 60 min), and Panel D: Without MLN-4760 (ACE2-mediated hydrolysis, 120 min). Results are representative of three or more separate metabolism experiments for each human sample.

Figure 2

HPLC analysis of ¹²⁵I-Ang II formation (%) from ¹²⁵I-Ang-(1-12) substrate by chymase in the absence or presence of cold Ang-(1-12) or Ang I. As described in Methods, the reaction mixture contained 50 μg of plasma membrane plus ALL RAS inhibitors (except chymostatin) plus substrate incubated in the absence or presence of increasing concentration of cold Ang-(1-12) or Ang I (range: 0–250 μM for 30 min at 37° C.

Figure 3

RAS enzyme activities calculated based on the amount of parent ¹²⁵I-Ang substrate hydrolyzed into products by PMs prepared from human left ventricular tissue with or without the presence of specific RAS inhibitors. Values are Mean ± SEM from parent ¹²⁵I-Ang substrate. Each experiment was done at least three times and the results are the average of six normal human subjects.

Figure 4

Panel A, Chymase protein expression in human left ventricular tissue (n=4).