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 mol/L Ang I substrate was 1.2 mol/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 mol/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 mol/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).(Hypertension. 1998;31:912-917.)

Abstract-We studied the enhancement of the effects of bradykinin B 2 receptor agonists by agents that react with active centers of angiotensin-converting enzyme (ACE) independent of enzymatic inactivation. The potentiation and the desensitization and resensitization of B 2 receptor were assessed by measuring [ 3 H]arachidonic acid release and [Ca 2ϩ ] i mobilization in Chinese hamster ovary cells transfected to express human ACE and B 2 receptor, or in endothelial cells with constitutively expressed ACE and receptor. Administration of bradykinin or its ACE-resistant analogue desensitized the receptor, but it was resensitized (arachidonic acid release or [Ca 2ϩ T herapy with angiotensin I-converting enzyme (ACE) inhibitors initially was aimed at lowering elevated blood pressure. 1 By now, however, it has gained much wider applications in combating heart and kidney diseases, such as congestive heart failure and diabetic nephropathy, involving millions of patients. 2-7 Inhibitors of ACE affect both angiotensin II (Ang II) and bradykinin metabolism by blocking the production of the vasoconstrictor peptide and inactivating the vasodilator peptide, 8 but these actions alone do not completely explain, for example, the beneficial effects of ACE inhibitors on the heart. These effects are not only due to lowering systemic blood pressure and peripheral vascular resistance. In laboratory experiments, many of the improvements in cardiac function brought about by ACE inhibitors are blocked by the bradykinin B 2 receptor antagonist Hoe 140. 9 -14 We have observed, on the isolated atria 15 and ileum 16 of guinea pig, that ACE inhibitors potentiate the actions of bradykinin indirectly at the receptor level. Using cultured Chinese hamster ovary (CHO) cells cotransfected with the cDNA of human ACE and B 2 receptor, we showed that ACE inhibitors augment the release of signal transduction products by bradykinin independent of inhibiting the degradation of bradykinin but have no direct effect on the B 2 receptor. 17 On the basis of accumulated evidence, it was suggested that the above effects and the resensitization of the receptor, desensitized by an agonist, are due to a crosstalk between ACE and the B 2 receptor on the plasma membrane of the cells. We have also reported that angiotensin-(1-7), a substrate cleaved by the N-domain active site of ACE and an inhibitor of the C-domain active site in vitro, potentiates bradykinin at the receptor level in a manner similar to that of ACE inhibitors. 18 The present report extends and reconfirms the previous observations, mainly in different cells, by using ACE inhibitors, inhibitory and noninhibitory monoclonal and polyclonal antibodies, a mutated ACE molecule, and endogenous peptide and snake venom peptide substrates of ACE,8,19 to show

To investigate further the relationship of angiotensin I-converting enzyme (ACE) inhibitors to activation of the B 2 bradykinin (BK) receptor, we transfected Chinese hamster ovary cells to stably express the human receptor and either wild-type ACE (WT-ACE), an ACE construct with most of the cytosolic portion deleted (Cytdel-ACE), or ACE with a glycosylphosphatidylinositol (GPI) anchor replacing the transmembrane and cytosolic domains (GPI-ACE). BK or its ACE-resistant analogue were the agonists. All activities (arachidonic acid release and calcium mobilization) were blocked by the B 2 antagonist HOE 140. B 2 was desensitized by repeated administration of BK but resensitized to agonist by ACE inhibitors in the cells expressing both B 2 and either WT-ACE or Cyt-del-ACE. In GPI-ACE expressing cells, the B 2 receptor was still activated by the agonists, but ACE inhibitors did not resensitize. Pretreatment with filipin returned the sensitivity to inhibitors. In immunocytochemistry, GPI-ACE showed patchy, uneven distribution on the plasma membrane that was restored by filipin. Thus, ACE inhibitors were inactive as long as GPI-ACE was sequestered in cholesterol-rich membrane domains. WT-ACE and B 2 receptor in Chinese hamster ovary cells co-immunoprecipitated with antibody to receptor, suggesting an interaction on the cell membrane. ACE inhibitors augment BK effects on receptors indirectly only when enzyme and receptor molecules are sterically close, possibly forming a heterodimer.Renin was discovered over a century ago (1), and kallikrein was discovered about 25 years later (2). Many of the cascading events initiated by these proteases are integrated by angiotensin I-converting enzyme (ACE) 1 or kininase II (3) as it activates angiotensin I to angiotensin II (4) and inactivates kinins (5).Subsequently, it became obvious that inhibitors of ACE affect the metabolism of both peptides (6). The successful clinical applications of ACE inhibitors have gone far beyond controlling elevated blood pressure (7, 8), but questions remain regarding which of the beneficial effects are due to inhibiting angiotensin II activation and which are caused by blocking the enzymatic breakdown of bradykinin (BK) or kallidin. The very extensive clinical applications of ACE inhibitors, not only in treating hypertension but also in treating cardiac conditions, (e.g. congestive heart failure or after myocardial infarction), and in diabetic nephropathies (9 -11), have kept attention focused on this issue. In laboratory experiments and in some clinical studies (12, 13), many effects of ACE inhibitors were abolished by the BK B 2 receptor blocker HOE 140. Although it was assumed that these effects were due to inhibiting the inactivation of BK, early bioassays already indicated that substances that did not prolong the half-life of BK still potentiated its actions on the isolated guinea pig ileum (14). Experiments on isolated guinea pig atria demonstrated that ACE inhibitors can resensitize the heart tissue desensitized by a B 2 receptor agonist (15). ...