Angiotensin-converting enzyme (ACE) inhibitors have different selectivity for bradykinin binding sites of human somatic ACE

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Abstract

The angiotensin-converting enzyme (ACE) has two natural substrates and two catalytic domains: one cleaving angiotensin I and one inactivating bradykinin. The aim of this study was to investigate the comparative binding affinity of ACE inhibitors for the two binding sites of human endothelial ACE. In vitro binding assays were performed to test the ability of bradykinin, angiotensin I, or various ACE inhibitors (enalaprilat, perindoprilat, quinaprilat, ramiprilat, and trandolaprilat) to displace a saturating concentration of [125I]351A, a radiolabeled lisinopril analogue, from ACE binding sites. The calculated IC50 values for the ACE inhibitors were in the nanomolar range, while those for the natural substrates were in the micromolar range. The bradykinin/angiotensin I selectivity ratios calculated from double displacement experiments were: perindoprilat, 1.44; ramiprilat, 1.16; quinaprilat, 1.09; trandolaprilat, 1.08; enalaprilat, 1.00. The ACE inhibitors generally had higher affinity for the bradykinin than the angiotensin I binding sites, supporting the idea that these agents are primarily inhibitors of bradykinin degradation, and secondarily inhibitors of angiotensin II production. Perindoprilat had the highest selectivity for bradykinin versus angiotensin I binding sites, and enalaprilat has the lowest. These results indicate that there are differences in the affinity of ACE inhibitors toward sites for bradykinin degradation, which could lead to differences in efficacy in cardiovascular disease.

Introduction

Human somatic angiotensin-converting enzyme (ACE) is a type I membrane protein that is most likely derived from the duplication of an ancestral gene, and comprises two homologous extracellular catalytic domains (the N- and C-terminal domains), each of which contains a putative active site (Soubrier et al., 1988, Dive et al., 1999). Somatic ACE is ubiquitous in human tissue and plasma, in contrast to the shorter germinal isoform of ACE, which is found only in the male testes and has only one active site.

ACE cleaves the C-terminal dipeptide of various oligopeptides, including bradykinin and angiotensin I. It can be regarded as either the enzyme kininase II, due to its action on bradykinin degradation, thereby increasing vasodilation, or as angiotensin I-converting enzyme, due to its inhibition of the conversion of angiotensin I into the vasoconstricting octapeptide angiotensin II (Erdos, 2006).

ACE inhibitors are a class of drugs that modulate the activity of ACE. As a consequence, they alter the balance between the vasoconstrictive, salt-retentive, and hypertrophic properties of angiotensin II, and the vasodilatory and natriuretic properties of bradykinin (Brown and Vaughan, 1998, Zisman, 1998, Dzau et al., 2002). Bradykinin promotes vasodilation by stimulating the production of nitric oxide (NO) and other relaxing factors in the endothelium (Moncada et al., 1991, Moncada and Higgs, 1993) (Table 1). ACE inhibitors are widely used in the treatment of cardiovascular and renal disease, and for the secondary prevention of coronary artery disease (Fox, 2003, Bertrand, 2004, Ferrari, 2005, Pfeffer and Frohlich, 2006, ESC, 2006).

These agents were developed well before the discovery that ACE has two catalytic domains, and there is little available information on the relative actions and affinities of bradykinin and angiotensin I on the two domains. The aim of this study was to compare the affinity of five different ACE inhibitors (namely, enalapril, perindopril, quinapril, ramipril, and trandolapril) for the two binding sites of human endothelial ACE by means of in vitro binding studies. We chose these particular ACE inhibitors because they are among the most commonly used in clinical practice, and also because they have been investigated in large-scale international clinical trials in the setting of coronary artery disease with differing outcomes (Yusuf et al., 2000, Pitt et al., 2001, Fox, 2003, Braunwald et al., 2004).

Section snippets

Reagents

Chemicals, cell culture reagents, angiotensin I, and bradykinin were from Sigma Aldrich (Milan, Italy). Collagenase type I was from Worthington (DBA Italia, Segrate, Italy), Plasmocin™ from InvivoGen (Labogen, Rho, Italy), and gelatin from Difco (Becton Dickinson Italia, Buccinasco, Italy). Pooled human serum was kindly provided by the Transfusion Center of the Brescia Hospital. All the ACE inhibitors investigated here are administered as prodrugs, and so the in vitro studies were performed

Single displacement experiments

Single displacement experiments were performed with two ligands in order to test the specific affinity of various ACE inhibitors for ACE binding sites. In these experiments, [125I]351A binding was assessed in the presence of various single ACE inhibitors, and the appropriate IC50 value was interpolated from competition curves.

All the ACE inhibitors examined had IC50 values in the nanomolar range, with the following order of affinity quinaprilat > trandolaprilat > ramiprilat > perindoprilat > 

Discussion

The main finding of this study is that ACE inhibitors possess different selectivity for the bradykinin versus the angiotensin I binding sites of human ACE. To our knowledge, this is the first time that ACE inhibitors have been compared for their selectivity for ACE binding sites in vitro. Among the ACE inhibitors investigated, perindoprilat has the highest affinity for the bradykinin binding site versus the angiotensin I binding site.

ACE has two functionally active domains, known as the N- and

Conclusion

The ACE inhibitor class is not homogeneous in terms of affinity for ACE and selectivity for bradykinin and angiotensin I binding sites. At equivalent doses and for the same angiotensin I affinity, perindoprilat has a much greater affinity for the bradykinin binding sites than the other ACE inhibitors studied here. Because the bradykinin/angiotensin I selectivity ratio is higher, then it is reasonable to conclude that the capacity for enzymatic activity at the bradykinin binding sites is also

Acknowledgements

The authors wish to thank the staff of the delivery room and the Transfusion Centre of the Brescia University Hospital for kindly providing human umbilical cords and serum, and Servier for supplying drugs and radiolabeled ligands. The study was supported by an unrestricted grant from Servier, France.

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