N-Domain–Specific Substrate and C-Domain Inhibitors of Angiotensin-Converting Enzyme

Abstract

Abstract —We used the isolated N- and C-domains of the angiotensin I–converting enzyme (N-ACE and C-ACE; ACE; kininase II) to investigate the hydrolysis of the active 1–7 derivative of angiotensin (Ang) II and inhibition by 5- S -5-benzamido-4-oxo-6-phenylhexanoyl- l -proline (keto-ACE). Ang-(1–7) is both a substrate and an inhibitor; it is cleaved by N-ACE at approximately one half the rate of bradykinin but negligibly by C-ACE. It inhibits C-ACE, however, at an order of magnitude lower concentration than N-ACE; the IC ₅₀ of C-ACE with 100 μmol/L Ang I substrate was 1.2 μmol/L and the K _i was 0.13. While searching for a specific inhibitor of a single active site of ACE, we found that keto-ACE inhibited bradykinin and Ang I hydrolysis by C-ACE in approximately a 38- to 47-times lower concentration than by N-ACE; IC ₅₀ values with C-ACE were 0.5 and 0.04 μmol/L. Furthermore, we investigated how Ang-(1–7) acts via bradykinin and the involvement of its B ₂ receptor. Ang-(1–7) was ineffective directly on the human bradykinin B ₂ receptor transfected and expressed in Chinese hamster ovary cells. However, Ang-(1–7) potentiated arachidonic acid release by an ACE-resistant bradykinin analogue (1 μmol/L), acting on the B ₂ receptor when the cells were cotransfected with cDNAs of both B ₂ receptor and ACE and the proteins were expressed on the plasma membrane of Chinese hamster ovary cells. Thus like other ACE inhibitors, Ang-(1–7) can potentiate the actions of a ligand of the B ₂ receptor indirectly by binding to the active site of ACE and independent of blocking ligand hydrolysis. This potentiation of kinins at the receptor level can explain some of the well-documented kininlike actions of Ang-(1–7).