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 1-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 IC50 of C-ACE with 100 micromol/L Ang I substrate was 1.2 micromol/L and the Ki 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; IC50 values with C-ACE were 0.5 and 0.04 micromol/L. Furthermore, we investigated how Ang-(1-7) acts via bradykinin and the involvement of its B2 receptor. Ang-(1-7) was ineffective directly on the human bradykinin B2 receptor transfected and expressed in Chinese hamster ovary cells. However, Ang-(1-7) potentiated arachidonic acid release by an ACE-resistant bradykinin analogue (1 micromol/L), acting on the B2 receptor when the cells were cotransfected with cDNAs of both B2 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 B2 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).
Deddish
P A
PA
University of Illinois College of Medicine, Chicago 60612-7344, USA.
Marcic
B
B
Jackman
H L
HL
Wang
H Z
HZ
Skidgel
R A
RA
Erdös
E G
EG
D000803:Angiotensin I; D000804:Angiotensin II / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000806:Angiotensin-Converting Enzyme Inhibitors / Q000494:pharmacology; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D001920:Bradykinin / Q000031:analogs & derivatives / Q000378:metabolism; D016466:CHO Cells / Q000187:drug effects / Q000201:enzymology; D002712:Chlorides / Q000494:pharmacology; D006224:Cricetinae; D004794:Enzyme Repression; D006801:Humans; D006868:Hydrolysis; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007703:Peptidyl-Dipeptidase A / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D014162:Transfection
D000806:Angiotensin-Converting Enzyme Inhibitors; D002712:Chlorides; D010446:Peptide Fragments; D000804:Angiotensin II; D016718:Arachidonic Acid; D000803:Angiotensin I; D007703:Peptidyl-Dipeptidase A; C118790:angiotensin I (1-7); D001920:Bradykinin