title: 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.
authors.affiliation: General Surgery, Wake Forest University School of Medicine, Winston-Salem, NC, USA. Electronic address: sahmad@wakehealth.edu.
authors.forename: Sarfaraz
authors.initials: S
authors.lastname: Ahmad
authors.affiliation: General Surgery, Wake Forest University School of Medicine, Winston-Salem, NC, USA; Hypertension and Vascular Research Center, Wake Forest University School of Medicine, Winston-Salem, NC, USA.
authors.forename: Jasmina
authors.initials: J
authors.lastname: Varagic
authors.affiliation: Hypertension and Vascular Research Center, Wake Forest University School of Medicine, Winston-Salem, NC, USA.
authors.forename: Jessica L
authors.initials: JL
authors.lastname: VonCannon
authors.affiliation: Hypertension and Vascular Research Center, Wake Forest University School of Medicine, Winston-Salem, NC, USA; Anesthesiology, Wake Forest University School of Medicine, Winston-Salem, NC, USA; Internal Medicine/Molecular Medicine, Wake Forest University School of Medicine, Winston-Salem, NC, USA.
authors.forename: Leanne
authors.initials: L
authors.lastname: Groban
authors.affiliation: Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL, USA.
authors.forename: James F
authors.initials: JF
authors.lastname: Collawn
authors.affiliation: Division of Cardiovascular Disease, Department of Medicine, University of Alabama at Birmingham, Birmingham VA Medical Center, Birmingham, AL, USA.
authors.forename: Louis J
authors.initials: LJ
authors.lastname: Dell'Italia
authors.affiliation: General Surgery, Wake Forest University School of Medicine, Winston-Salem, NC, USA; Internal Medicine/Nephrology, Wake Forest University School of Medicine, Winston-Salem, NC, USA; Internal Medicine/Cardiovascular Medicine, Wake Forest University School of Medicine, Winston-Salem, NC, USA.
authors.forename: Carlos M
authors.initials: CM
authors.lastname: Ferrario
mesh_terms: D000803:Angiotensin I / Q000378:metabolism*; D000804:Angiotensin II / Q000378:metabolism*; D000808:Angiotensinogen / Q000378:metabolism*; D000818:Animals; D053818:Chymases / Q000378:metabolism*; D008297:Male; D009206:Myocardium / Q000201:enzymology* / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism*; D007703:Peptidyl-Dipeptidase A / Q000378:metabolism*; D051381:Rats; D011918:Rats, Inbred SHR
chemical_list: D010446:Peptide Fragments; C515557:proangiotensin-12, rat; D000808:Angiotensinogen; D000804:Angiotensin II; D000803:Angiotensin I; D007703:Peptidyl-Dipeptidase A; D053818:Chymases
keywords: Angiotensin I; Angiotensin II; Angiotensin-(1-12); Angiotensin-converting enzyme; Rat cardiac chymase; Renin-angiotensin system