N-DOMAIN-SPECIFIC SUBSTRATE AND C-DOMAIN INHIBITORS OF ANGIOTENSIN-CONVERTING ENZYME ANGIOTENSIN-(1-7) AND KETO-ACE

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 hydrolys is 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 a pproximately one half the rate of bradykinin but negligibly by C-ACE. It inhibits C-ACE, however, at an order of magnitude lower concentrati on than N-ACE; the IC50 of C-ACE with 100 mu mol/L Ang I substrate was 1.2 mu mol/L and the K-i was 0.13. While searching for a specific inh ibitor of a single active site of ACE, we found that keto-ACE inhibite d bradykinin and Ang I hydrolysis by C-ACE in approximately a 38- to 4 7-times lower concentration than by N-ACE; IC50 values with C-ACE were 0.5 and 0.04 mu mol/L. Furthermore, we investigated how Ang-(1-7) act s via bradykinin and the involvement of its B-2 receptor. Ang-(1-7) wa s ineffective directly on the human bradykinin B-2 receptor transfecte d and expressed in Chinese hamster ovary cells. However, Ang-(1-7) pot entiated arachidonic acid release by an ACE-resistant bradykinin analo gue (1 mu mol/L), acting on the B-2 receptor when the cells were cotra nsfected 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-documente d kininlike actions of Ang-(1-7).