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). ...
Abstract-Human heart tissue enzymes cleave angiotensin (Ang) I to release Ang 1-9, Ang II, or Ang 1-7. In atrial homogenate preparations, cathepsin A (deamidase) is responsible for 65% of the liberated Ang 1-9. Ang 1-7 was released (88% to 100%) by a metallopeptidase, as established with peptidase inhibitors. Ang II was liberated to about equal degrees by ACE and chymase-type enzymes. Cathepsin A's presence in heart tissue was also proven because it deamidated enkephalinamide substrate by immunoprecipitation of cathepsin A with antiserum to human recombinant enzyme and by immunohistochemistry. In immunohistochemistry, cathepsin A was detected in myocytes of atrial tissue. The products of Ang I cleavage, Ang 1-9 and Ang 1-7, potentiated the effect of an ACE-resistant bradykinin analog and enhanced kinin effect on the B 2 receptor in Chinese hamster ovary cells transfected to express human ACE and B 2 (CHO/AB), and in human pulmonary arterial endothelial cells. Ang 1-9 and 1-7 augmented arachidonic acid and nitric oxide (NO) release by kinin. Direct assay of NO liberation by bradykinin from endothelial cells was potentiated at 10 nmol/L concentration, 2.4-fold (Ang 1-9) and 2.1-fold (Ang 1-7); in higher concentrations, Ang 1-9 was significantly more active than Ang 1-7. Both peptides had traces of activity in the absence of bradykinin. Ang 1-9 and Ang 1-7 potentiated bradykinin action on the B 2 receptor by raising arachidonic acid and NO release at much lower concentrations than their 50% inhibition concentrations (IC 50 s) with ACE.
Part of the beneficial effects of angiotensin I-converting enzyme (ACE) inhibitors are due to augmenting the actions of bradykinin (BK). We studied this effect of enalaprilat on the binding of [3H]BK to Chinese hamster ovary (CHO) cells stably transfected to express the human BK B2 receptor alone (CHO-3B) or in combination with ACE (CHO-15AB). In CHO-15AB cells, enalaprilat (1 mumol/L) increased the total number of low-affinity [3H]BK binding sites on the cells at 37 degrees C, but not at 4 degrees C, from 18.4 +/- 4.3 to 40.3 +/- 11.9 fmol/10(6) cells (P < .05; Kd, 2.3 +/- 0.8 and 5.9 +/- 1.3 nmol/L; n = 4). Enalaprilat preserved a portion of the receptors in high-affinity conformation (Kd, 0.17 +/- 0.08 nmol/L; 8.1 +/- 0.9 fmol/10(6) cells). Enalaprilat decreased the IC50 of [Hyp3-Tyr(Me)8]BK, the BK analogue more resistant to ACE, from 3.2 +/- 0.8 to 0.41 +/- 0.16 nmol/L (P < .05, n = 3). The biphasic displacement curve of the binding of [3H]BK also suggested the presence of high-affinity BK binding sites. Enalaprilat (5 nmol to 1 mumol/L) potentiated the release of [3H]arachidonic acid and the liberation of inositol 1,4,5-trisphosphate (IP3) induced by BK and [Hyp3-Tyr(Me)8]BK. Moreover, enalaprilat (1 mumol/L) completely and immediately restored the response of the B2 receptor, desensitized by the agonist (1 mumol/L [Hyp3-Tyr(Me)8]BK); this effect was blocked by the antagonist, HOE 140. Finally, enalaprilat, but not the prodrug enalapril, decreased internalization of the receptor from 70 +/- 9% to 45 +/- 9% (P < .05, n = 7). In CHO-3B cells, enalaprilat was ineffective. ACE inhibitors in the presence of both the B2 receptor and ACE enhance BK binding, protect high-affinity receptors, block receptor desensitization, and decrease internalization, thereby potentiating BK beyond blocking its hydrolysis.
Bradykinin (BK) and kallidin (Lys-BK), liberated from kininogens by kallikreins, are ligands of the BK B(2) receptor. We investigated whether kallikreins, besides releasing peptide agonist, could also activate the receptor directly. We studied the effect of porcine and human recombinant tissue kallikrein and plasma kallikrein on [Ca(2+)](i) mobilization and [(3)H]arachidonic acid release from cultured cells stably transfected to express human BK B(2) receptor (CHO/B(2), MDCK/B(2), HEK/B(2)), and endothelial cells were used as control cells. As with BK, the actions of kallikrein were blocked by the B(2) antagonist, HOE 140. Kallikrein was inactive on cells lacking B(2) receptor. Kallikrein and BK desensitized the receptor homologously but there was no cross-desensitization. Furthermore, 50 nM human cathepsin G and 50 nM trypsin also activated the receptor; this also was blocked by HOE 140. Experiments excluded a putative kinin release by proteases. [(3)H]AA release by BK was reduced by 40% by added kininase I (carboxypeptidase M); however, receptor activation by tissue kallikrein, trypsin, or cathepsin G was not affected. Prokallikrein and inhibited kallikrein were inactive, suggesting cleavage of a peptide bond in the receptor. Kallikreins were active on mutated B(2) receptor missing the 19 N-terminal amino acids, suggesting a type of activation different from that of thrombin receptor. Paradoxically, tissue kallikreins decreased the [(3)H]BK binding to the receptor with a low K(D) (3 nM) and inhibited it 78%. Thus, kallikreins and some other proteases activate human BK B(2) receptor directly, independent of BK release. The BK B(2) receptor may belong to a new group of serine protease-activated receptors.
Kinin B1 receptor (B1R) expression is induced by injury or inflammatory mediators, and its signaling produces both beneficial and deleterious effects. Kinins cleaved from kininogen are agonists of the B2R and must be processed by a carboxypeptidase to generate B1R agonists des-Arg 9 -bradykinin or desArg 10 Carboxypeptidase M (CPM) 2 was discovered as a glycosylphosphatidylinositol (GPI)-anchored membrane protein with B-type carboxypeptidase activity (1-3) and is a member of the "regulatory" or carboxypeptidase N/E subfamily of metallocarboxypeptidases (4 -8) based on its cDNA sequence (9), genomic structure (10), and x-ray crystal structure (11). The overall structure of CPM is composed of a 295-residue N-terminal catalytic domain, followed by an 86-residue conical -sandwich (transthyretin-like domain) and a unique 25-residue extension to which the GPI anchor is post-translationally attached (11).CPM cleaves only C-terminal Arg or Lys residues, and some of its endogenous substrates include bradykinin, anaphylatoxins C3a, C4a, and C5a, Arg-or Lys-enkephalins, epidermal growth factor, and hemoglobin (2, 7, 12, 13). CPM preferentially cleaves C-terminal Arg as exemplified by the kinetics with Arg 6 -Met 5 -enkephalin (K m ϭ 46 M, k cat ϭ 934 min Ϫ1 ) versus Lys 6 -Met 5 -enkephalin (K m ϭ 375 M, k cat ϭ 663 min Ϫ1 ) (2). Bradykinin exhibits the lowest K m (16 M) of any CPM substrate tested (2), a concentration that is still much higher than the typical physiological concentration of this peptide in the nanomolar range. However, peptidases in vivo typically work at substrate concentrations far below the K m as exemplified by angiotensin I-converting enzyme (ACE), which has the same relatively high K m (16 M) with angiotensin I (14), a major physiological substrate. The development of ACE inhibitors as effective agents for treating hypertension and cardiovascular diseases was based in large part on the critical role of this enzyme in converting angiotensin I to II in vivo (15).The kinin peptides bradykinin and kallidin (Lys-bradykinin) are generated by the proteolytic action of plasma or tissue kallikrein on high or low molecular weight kininogen (16,17). Removal of the C-terminal Arg from bradykinin or kallidin inactivates these peptides as agonists of the constitutively expressed B2 receptor (B2R) (16,17). However, this conversion is a required processing step to generate desArg 9 -bradykinin or des-Arg 10 -kallidin (16, 18), metabolites that have a variety of biological activities mediated by specific activation of a different B1 receptor (B1R) whose expression is induced by inflammatory mediators (19,20). Thus, CPM acts as a cell surface processing enzyme to generate des-Arg-kinin agonists for the B1R, and without this catalytic conversion, B1R signaling could not occur. This is of potential importance in inflammatory or pathological responses. For example, B1R activation stimulates extracellular signal-regulated kinase phosphorylation, prostaglandin production (20), and inducible nitric-oxide synthase-mediate...
Abstract-The beneficial effects of angiotensin I-converting enzyme (ACE) inhibitors go beyond the inhibition of ACE to decrease angiotensin (Ang) II or increase kinin levels. ACE inhibitors also affect kinin B1 and B2 receptor (B1R and B2R) signaling, which may underlie some of their therapeutic usefulness. Key Words: angiotensin I-converting enzyme inhibitors Ⅲ kininase II Ⅲ kinins Ⅲ bradykinin B2 receptor Ⅲ bradykinin B1 receptor Ⅲ allosteric regulation Ⅲ 7-transmembrane G protein-coupled receptor M illions of patients are treated with angiotensin I-converting enzyme (ACE) inhibitors to combat hypertension, congestive heart failure or diabetic renal diseases. 1-4 ACE inhibitors significantly reduce mortality after myocardial infarction 3 and are beneficial in other high risk patients.ACE inhibitors block the metabolism of several peptides by ACE, notably the conversion of angiotensin (Ang) I to II, 5 and the inactivation of bradykinin (BK) 6 -8 or the hemoregulatory tetrapeptide Ac-Ser-Asp-Lys-Pro. 9 The conversion of Ang I to Ang II was first found to occur in horse plasma 5 ; with kidney and human plasma, one of us reported the identity of ACE and kininase II, which we had discovered previously. 6 -8,10,11 Consequently, a single peptidyldipeptidase not only releases the hypertensive Ang II, but also inactivates the hypotensive BK. How much of the therapeutic effectiveness of ACE inhibitors is attributable to blocking Ang II release 12 or to prolonging the short half-life of BK 7 and its congener Lys1-BK (kallidin) has been debated. This is further complicated by the existence of 2 kinin receptors. The first characterized, but incongruously named, B2 receptor (B2R) is activated by native BK or kallidin. 13 The second, so-called B1 receptor (B1R), does not bind native kinins; its ligands are metabolites of BK and kallidin lacking the C-terminal arginine 14 removed by plasma carboxypeptidase (CP)N 15-17 or membrane CPM. 18 -20 Whereas the B2R is widely expressed constitutively, B1R expression is usually induced after noxious stimuli or by inflammatory cytokines, 13,14,[21][22][23] although some cells (bovine lung endothelial or human fibroblasts) express B1Rs constitutively.ACE inhibitors can enhance both B2 and B1R signaling. Blocking kinin inactivation by ACE raises the concentration of intact B2R agonists, which are also the substrates of CPN and -M. This can generate more des-Arg-kinin B1R agonists (Figure). The successful use of antagonists of the Ang II type 1 receptor (AT 1 R) for many of the same indications as ACE inhibitors does not prove ACE inhibitors work only through reducing Ang II as there are complex interrelationships among Ang II, BK and their receptors. Ang II has 2 receptors, AT 1 R and AT 2 R. AT 1 R is blocked by drugs such as losartan, which can shift Ang II actions to the AT 2 R. This switching of receptors further counteracts AT 1 R effects because it leads to the release of mediators such as nitric oxide (NO) and is attributed partially to release of BK to activate B2Rs, a form of "...
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