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

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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 IC50of C-ACE with 100 μmol/L Ang I substrate was 1.2 μmol/L and theKiwas 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; IC50values 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 B2receptor. Ang-(1–7) was ineffective directly on the human bradykinin B2receptor 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 B2receptor when the cells were cotransfected with cDNAs of both B2receptor 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 B2receptor 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).

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