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

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 ¹²⁵I-Ang-(1-12) or ¹²⁵I-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 ¹²⁵I-Ang II formation from ¹²⁵I-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 ¹²⁵I-Ang I substrate (heart: 0.8 ± 0.1 fmol/min/mg; lung: 2.1 ± 0.1 fmol/min/mg). Substrate affinity of ¹²⁵I-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 ¹²⁵I-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 K_m (64 ± 6.3 μM vs 142 ± 17 μM), higher V_max (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 V_max/K_m) for Ang-(1-12) compared to Ang I substrate, respectively. We also investigated ACE mediated hydrolysis of ¹²⁵I-Ang-(1-12) and ¹²⁵I-Ang I in solubilized membrane fractions of the SHR heart and lung. Interestingly, no significant difference in ¹²⁵I-Ang II formation by ACE was detected using either substrate, ¹²⁵I-Ang-(1-12) or ¹²⁵I-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.