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). ...

Angiotensin I-converting enzyme (ACE, kininase II) is a single-chain protein containing two active site domains (named N-and C-domains according to position in the chain). ACE is bound to plasma membranes by its C-terminal hydrophobic transmembrane anchor. Deal fluid, rich in ACE activity, obtained from patients after surgical colectomy was used as the source. Column chromatography, including modified affinity chromatography on lisinopril-Sepharose, yielded homogeneous ACE after only a 45-fold purification. N-terminal sequencing of ileal ACE and partial sequencing of CNBr fragments revealed the presence of an intact N terminus but only a single N-domain active site, ending between residues 443 and 559. Thus, ileal-fluid ACE is a unique enzyme differing from the widely distributed two-domain somatic enzyme or the single C-domain testicular (germinal) ACE. The molecular mass of ileal ACE is 108 kDa and when deglycosylated, the molecular mass is 68 kDa, indicating extensive glycosylation (37% by weight). In agreement with the results reported with recombinant variants of ACE, the ileal enzyme is less C1-dependent than somatic ACE; release of the C-terminal dipeptide from a peptide substrate was optimal in only 10 mM Cl. In addition to hydrolyzing at the C-terminal end of peptides, ileal ACE efficiently cleaved the protected N-terminal tripeptide from the luteinizing hormone-releasing hormone and its congener 6-31 times faster, depending on the Cl concentration, than the C-domain in recombinant testicular ACE. Thus we have isolated an active human ACE consisting of a single N-domain. We suggest that there is a bridge section of about 100 amino acids between the active N-and C-domains of somatic ACE where it may be proteolytically cleaved to liberate the active N-domain. These findings have potential relevance and importance in the therapeutic application of ACE inhibitors.

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Abstract-We measured the cleavage of angiotensin I (Ang I) metabolites by angiotensin I-converting enzyme (ACE) in cultured cells and examined how they augment actions of bradykinin B 2 receptor agonists. Monolayers of Chinese hamster ovary cells transfected to stably express human ACE and bradykinin B 2 receptors coupled to green fluorescent protein (B 2 GFP) or to express only coupled B 2 GFP receptors. We used 2 ACE-resistant bradykinin analogues to activate the B 2 receptors. We used high-performance liquid chromatography to analyze the peptides cleaved by ACE on cell monolayers and found that Ang 1-9 was hydrolyzed 18ϫ slower than Ang I and Ϸ30% slower than Ang 1-7. Ang 1-7 was cleaved to Ang 1-5. Although mol/L concentrations of slowly cleaved substrates Ang 1-7 and Ang 1-9 inhibit ACE, they resensitize the desensitized B 2 GFP receptors in nmol/L concentration, independent of ACE inhibition. This is reflected by release of arachidonic acid through a mechanism involving cross-talk between ACE and B 2 receptors. When ACE was not expressed, the Ang 1-9, Ang 1-7 peptides were inactive. Inhibitors of protein kinase C-␣, phosphatases and Tyr-kinase blocked this resensitization activity, but not basal B 2 activation by bradykinin.

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The presence of high concentrations of membrane-bound carboxypeptidase M in human, baboon, dog, and rat lung was established by employing a variety of techniques. The activity of the enzyme in the membrane-enriched fractions of human, baboon, dog, and rat lung, measured with fluorescent dansyl substrate (DNS-Ala-Arg), was 198, 261, 484, and 153 nmol/h/mg protein, respectively. This activity in the lung was much higher than that found in the heart, liver, or kidney. The enzyme, optimally active around neutral pH, was completely inhibited by 10 microM 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid and was activated by 1 mM CoCl2 to 170%. Antibody to human carboxypeptidase M immunoprecipitated the solubilized carboxypeptidase from human (98%), baboon (81%), and dog (88%) lung membrane fractions. Carboxypeptidase M is attached to lung membranes by a phosphatidylinositol glycan anchor; thus, it is released with bacterial phospholipase C. Membrane fractions from cultured human pulmonary arterial endothelial cells also contained high carboxypeptidase M activity (254 nmol/h/mg protein). A Northern blot of poly(A)+ RNA from various human tissues showed the presence of a high level of carboxypeptidase M mRNA in human lung and placenta. Finally, immunohistochemistry, employing purified antibody to the enzyme, revealed in fluorescent light microscopy that carboxypeptidase M is present in alveolar type I pneumocytes and in macrophages in apparently lower concentration. In contrast, type II alveolar epithelial cells gave negative results. Because carboxypeptidase M cleaves a variety of active peptides (e.g., bradykinin, anaphylatoxins), it may protect the alveolar surface from the effects of these peptides. In addition, carboxypeptidase M could be a marker enzyme for type I cells.