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 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 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 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.

Role of Tissue Renin-angiotensin System and the Chymase/angiotensin-( 1-12) Axis in the Pathogenesis of Diabetic Retinopathy

“…Our studies show that human chymase converts Ang-(1-12) and Ang I directly into Ang II with greater efficiency and selectivity than the ACE [85, 92]. More recently Ahmad et al, (2016) demonstrated that chymase has a higher affinity for the Ang-(1-12) compared to Ang I to generate Ang II product in both human and rodents [81]. …”

“…Thus, the therapeutic approaches to prevent the progression of DR in diabetic people using the ACE inhibitor require further exploration. Our recent studies show that the chymase/Ang-(1-12) axis is primarily responsible for the generation of cellular Ang II rather than the ACE [81, 82]. These findings further suggest that the combination therapy (chymase and ACE inhibitions), compared to ACE inhibition alone, might be more beneficial to prevent the DR progression in diabetic people.…”

“…Growing number of studies support a role of a local RAS in retinal damage and progression of retinopathy. Recent studies by our group suggest that intracellular Ang II generation may occur primarily by chymase rather than ACE [81, 92]. Moreover, the affinity of chymase to generate intracellular Ang II is several folds higher than that of ACE in circulation.…”

Renin angiotensin aldosterone inhibition in the treatment of cardiovascular disease

“…A preliminary report from our laboratory implicates kallikrein or an aprotinin-sensitive enzyme for the cleavage of AGT into Ang-(1-12) [39]. While the potential contribution of Ang-(1-25) as an Ang II forming substrate is confined to one published paper where the substrate was identified from the urine of normal subjects [28], more extensive research on Ang-(1-12) has led to the conclusion that this extended form of angiotensin I (Ang I) is truly a functionally endogenous Ang II generating substrate that is predominantly expressed in tissues serving as an intracrine source for direct Ang II production [40]. Ang-(1-12) expression and content is augmented in the heart of spontaneous hypertensive rats (SHR) [41], the left ventricle of rats expressing the human AGT gene [42], and the enlarged left atrial appendage of subjects with left heart disease and left atrial enlargement [43].…”

“…As illustrated in Figure 1, Ang II formation from Ang-(1-12) represents a non-renin dependent pathway [34, 49]. Ang-(1-12) affinity for chymase is several orders of magnitude higher than for ACE [40]; studies of Ang-(1-12) metabolism in plasma membranes isolated from human left atrial [50] or left ventricular tissues [51] showed chymase as the primary Ang II forming enzyme. A robust but often neglected literature implicates chymase as the primary Ang II forming enzyme from Ang I in humans [35–37, 52].…”