Primacy of cardiac chymase over angiotensin converting enzyme as an angiotensin-(1-12) metabolizing enzyme

Abstract

We showed previously that rat angiotensin-(1-12) [Ang-(1-12)] is metabolized by chymase and angiotensin converting enzyme (ACE) to generate Angiotensin II (Ang II). Here, we investigated the affinity of cardiac chymase and ACE enzymes for Ang-(1-12) and Angiotensin I (Ang I) substrates. Native plasma membranes (PMs) isolated from heart and lung tissues of adult spontaneously hypertensive rats (SHR) were incubated with radiolabeled (125)I-Ang-(1-12) or (125)I-Ang I, in the absence or presence of a chymase or ACE inhibitor (chymostatin and lisinopril, respectively). Products were quantitated by HPLC connected to an in-line flow-through gamma detector. The rate of (125)I-Ang II formation from (125)I-Ang-(1-12) by chymase was significantly higher (heart: 7.0 ± 0.6 fmol/min/mg; lung: 33 ± 1.2 fmol/min/mg, P < 0.001) when compared to (125)I-Ang I substrate (heart: 0.8 ± 0.1 fmol/min/mg; lung: 2.1 ± 0.1 fmol/min/mg). Substrate affinity of (125)I-Ang-(1-12) for rat cardiac chymase was also confirmed using excess unlabeled Ang-(1-12) or Ang I (0-250 μM). The rate of (125)I-Ang II formation was significantly lower using unlabeled Ang-(1-12) compared to unlabeled Ang I substrate. Kinetic data showed that rat chymase has a lower Km (64 ± 6.3 μM vs 142 ± 17 μM), higher Vmax (13.2 ± 1.3 μM/min/mg vs 1.9 ± 0.2 μM/min/mg) and more than 15-fold higher catalytic efficiency (ratio of Vmax/Km) for Ang-(1-12) compared to Ang I substrate, respectively. We also investigated ACE mediated hydrolysis of (125)I-Ang-(1-12) and (125)I-Ang I in solubilized membrane fractions of the SHR heart and lung. Interestingly, no significant difference in (125)I-Ang II formation by ACE was detected using either substrate, (125)I-Ang-(1-12) or (125)I-Ang I, both in the heart (1.8 ± 0.2 fmol/min/mg and 1.8 ± 0.3 fmol/min/mg, respectively) and in the lungs (239 ± 25 fmol/min/mg and 248 ± 34 fmol/min/mg, respectively). Compared to chymase, ACE-mediated Ang-(1-12) metabolism in the heart was several fold lower. Overall our findings suggest that Ang-(1-12), not Ang I, is the better substrate for Ang II formation by chymase in adult rats. In addition, this confirms our previous observation that chymase (rather than ACE) is the main hydrolyzing enzyme responsible for Ang II generation from Ang-(1-12) in the adult rat heart.

Keywords: Angiotensin I; Angiotensin II; Angiotensin-(1-12); Angiotensin-converting enzyme; Rat cardiac chymase; Renin-angiotensin system.

Fig. 1. Rat ¹²⁵I-Ang-(1-2) and ¹²⁵I-Ang I substrate hydrolysis by PMs isolated from SHR heart

Chromatograms show the ¹²⁵I-Ang II product generation from ¹²⁵I-Ang-(1-12) and ¹²⁵I-Ang I by PMs isolated from the SHR heart. Panel A and B: In the presence of all RAS inhibitors (including chymostatin and lisinopril). Panel C and D: In the absence of chymostatin (chymase inhibitor) only. Panel E and F: In the absence of lisinopril (ACE inhibitor) only. Results are representative of three or more separate metabolism experiments for each group.

Fig. 2. ¹²⁵I-Ang-(1-12) and ¹²⁵I-Ang I substrate hydrolysis by PMs isolated from SHR lung

Chromatograms show the ¹²⁵I-Ang II product generation in the absence of chymostatin (chymase inhibitor) only from (A) ¹²⁵I-Ang-(1-12), and (B) ¹²⁵I-Ang I substrate. Results are representative of three or more separate metabolism experiments for each group.

Fig. 3. Affinity of rat cardiac chymase for Ang-(1-12) and Ang I substrates

The decrease in ¹²⁵I-Ang product formation (%) from rat ¹²⁵I-Ang-(1-12) substrate by PMs incubated in the absence of chymostatin only (chymase inhibitor) with increasing concentrations (0–250 µM) of non-radiolabeled substrates (□) Ang-(1-12) or (■) Ang I. Results are representative of three or more separate experiments for each group.

Fig. 4. Kinetics (K_m and V_max) of rat cardiac chymase for Ang-(1-12) and Ang I substrates

PMs (100 µg) were incubated with increasing concentrations (5–300 µM) of Ang-(1-12) or Ang I substrate in the presence of lisinopril (200 µM, ACE inhibitor) at 37° C for 30 min. The Ang II generated was analyzed by HPLC connected to UV-detector at 215 nm. The K_m and V_max of cardiac chymase for Ang-(1-12) and Ang I substrates were calculated using the Michaelis-Menten equation.