Crystal structure of the human carboxypeptidase N (kininase I) catalytic domain.

Human carboxypeptidase N (CPN), a member of the CPN/E subfamily of "regulatory" metallo-carboxypeptidases, is an extracellular glycoprotein synthesized in the liver and secreted into the blood, where it controls the activity of vasoactive peptide hormones, growth factors and cytokines by specifically removing C-terminal basic residues. Normally, CPN circulates in blood plasma as a hetero-tetramer consisting of two 83 kDa (CPN2) domains each flanked by a 48 to 55 kDa catalytic (CPN1) domain. We have prepared and crystallized the recombinant C-terminally truncated catalytic domain of human CPN1, and have determined and refined its 2.1 A crystal structure. The structural analysis reveals that CPN1 has a pear-like shape, consisting of a 319 residue N-terminal catalytic domain and an abutting, cylindrically shaped 79 residue C-terminal beta-sandwich transthyretin (TT) domain, more resembling CPD-2 than CPM. Like these other CPN/E members, two surface loops surrounding the active-site groove restrict access to the catalytic center, offering an explanation for why some larger protein carboxypeptidase inhibitors do not inhibit CPN. Modeling of the Pro-Phe-Arg C-terminal end of the natural substrate bradykinin into the active site shows that the S1' pocket of CPN1 might better accommodate P1'-Lys than Arg residues, in agreement with CPN's preference for cleaving off C-terminal Lys residues. Three Thr residues at the distal TT edge of CPN1 are O-linked to N-acetyl glucosamine sugars; equivalent sites in the membrane-anchored CPM are occupied by basic residues probably involved in membrane interaction. In tetrameric CPN, each CPN1 subunit might interact with the central leucine-rich repeat tandem of the cognate CPN2 subunit via a unique hydrophobic surface patch wrapping around the catalytic domain-TT interface, exposing the two active centers.

Human ACE and bradykinin B2 receptors form a complex at the plasma membrane.

To investigate how angiotensin I-converting enzyme (ACE) inhibitors enhance the actions of bradykinin (BK) on B2 receptors independent of blocking BK inactivation, we expressed human somatic ACE and B2 receptors in CHO cells. Bradykinin and its ACE-resistant analog were the receptor agonists. B2 fused with green fluorescent protein (GFP) and ACE were coprecipitated with antisera to GFP or ACE shown in Western blots. Immunohistochemistry of fixed cells localized ACE by red color and B2-GFP by green. Yellow on plasma membranes of coexpressing cells also indicated enzyme-receptor complex formation. Using ACE-fused cyan fluorescent protein donor and B2-fused yellow fluorescent protein (YFP) acceptor, we registered fluorescence resonance energy transfer (FRET) by the enhanced fluorescence of donor on acceptor photobleaching, establishing close (within 10 nm) positions of B2 receptors and ACE. Bradykinin stimulation cointernalized ACE and B2 receptors. We expressed ACE fused to N terminus of B2 receptors, anchoring only receptors to plasma membranes. Here, in contrast to cells, where both ACE and B2 receptors are separately anchored, ACE inhibitors neither enhance activation of chimeric B2 nor resensitize desensitized B2 receptors. Heterodimer formation between ACE and B2 receptors can be a mechanism for ACE inhibitors to augment kinin activity at cellular level.

Kallikrein activates bradykinin B2 receptors in absence of kininogen.

Kallikreins cleave plasma kininogens to release the bioactive peptides bradykinin (BK) or kallidin (Lys-BK). These peptides then activate widely disseminated B2 receptors with consequences that may be either noxious or beneficial. We used cultured cells to show that kallikrein can bypass kinin release to activate BK B2 receptors directly. To exclude intermediate kinin release or kininogen uptake from the cultured medium, we cultured and maintained cells in medium entirely free of animal proteins. We compared the responses of stably transfected Chinese hamster ovary (CHO) cells that express human B2 receptors (CHO B2) and cells that coexpress angiotensin I-converting enzyme (ACE) as well (CHO AB). We found that BK (1 nM or more) and tissue kallikrein (1-10 nM) both significantly increased release of arachidonic acid beyond unstimulated baseline level. An enzyme-linked immunoassay for kinin established that kallikrein did not release a kinin from CHO cells. We confirmed the absence of kininogen mRNA with RT-PCR to rule out kininogen synthesis by CHO cells. We next tested an ACE inhibitor for enhanced BK receptor activation in the absence of kinin release and synthesized an ACE-resistant BK analog as a control for these experiments. Enalaprilat (1 microM) potentiated kallikrein (100 nM) in CHO AB cells but was ineffective in CHO B2 cells that do not bear ACE. We concluded that kallikrein activated B2 receptors without releasing a kinin. Furthermore, inhibition of ACE enhanced the receptor activation by kallikrein, an action that may contribute to the manifold therapeutic effects of ACE inhibitors.

Angiotensin I-converting enzyme inhibitors block protein kinase C epsilon by activating bradykinin B1 receptors in human endothelial cells.

Angiotensin I-converting enzyme (ACE) inhibitors are widely used to treat patients with cardiovascular and kidney diseases, but inhibition of ACE alone does not fully explain the beneficial effects. We reported that ACE inhibitors directly activate bradykinin B1 receptor at the canonical Zn2+ binding site, leading to prolonged nitric oxide (NO) production in endothelial cells. Protein kinase C (PKC) epsilon, a novel PKC isoform, is up-regulated in myocardium after infarction, suggesting a role in the development of cardiac dysfunction. In cytokine-treated human lung microvascular endothelial cells, B1 receptor activation by ACE inhibitors (enalaprilat, quinaprilat) or peptide ligands (des-Arg10-Lys1-bradykinin, des-Arg9-bradykinin) inhibited PKC epsilon with an IC50 = 7 x 10(-9) M. Despite the reported differences in binding affinity to receptor, the two peptide ligands were equally active, even when inhibitor blocked the cleavage of Lys(1), thus the conversion by aminopeptidase. The synthetic undecapeptide (LLPHEAWHFAR) representing the binding site for ACE inhibitors on human B(1) receptors reduced PKC epsilon inhibition by enalaprilat but not by peptide agonist. A combination of inducible and endothelial NO synthase inhibitors, 1400W [N-(3(aminomethyl) benzyl) acetamidine dihydrochloride] and N omega-nitro-L-arginine (2 microM), significantly reduced inhibition by enalaprilat (100 nM), whereas the NO donor (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl) amino]diazen-1-ium-1,2-diolate (100 microM) inhibited PKC epsilon activity just as the B1 ligands did. In conclusion, NO generated by B1 receptor activation inhibits PKC epsilon.

Hydrolysis of angiotensin peptides by human angiotensin I-converting enzyme and the resensitization of B2 kinin receptors.

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 B2 receptor agonists. Monolayers of Chinese hamster ovary cells transfected to stably express human ACE and bradykinin B2 receptors coupled to green fluorescent protein (B2GFP) or to express only coupled B2GFP receptors. We used 2 ACE-resistant bradykinin analogues to activate the B2 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 18x slower than Ang I and &30% slower than Ang 1-7. Ang 1-7 was cleaved to Ang 1-5. Although micromol/L concentrations of slowly cleaved substrates Ang 1-7 and Ang 1-9 inhibit ACE, they resensitize the desensitized B2GFP 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 B2 receptors. When ACE was not expressed, the Ang 1-9, Ang 1-7 peptides were inactive. Inhibitors of protein kinase C-alpha, phosphatases and Tyr-kinase blocked this resensitization activity, but not basal B2 activation by bradykinin. Ang 1-9 and Ang 1-7 enhance bradykinin activity, probably by acting as endogenous allosteric modifiers of the ACE and B2 receptor complex. Consequently, when ACE inhibitors block conversion of Ang I, other enzymes can still release Ang I metabolites to enhance the efficacy of ACE inhibitors.

Products of angiotensin I hydrolysis by human cardiac enzymes potentiate bradykinin.

Some beneficial effects of ACE inhibitors are attributed to potentiation of bradykinin's actions exerted through its B2 receptor. We investigated them on cultured cells transfected or constitutively expressing both ACE and B2 receptor. The potentiation of bradykinin was indirect and attributed to a crosstalk induced between enzyme and receptor via ACE, a heterodimer formation. While looking for endogenous activators, we investigated the split products of angiotensin I (Ang) Ang 1-9 and 1-7, peptides released by enzymes of human atria and ventricle. Ang 1-9 was liberated by a cathepsin A-type enzyme, Ang 1-7 by a different metallopeptidase-protease. Cathepsin A's presence in heart tissue was shown by deamidating enkephalinamide substrate, by immunoprecipitation and by immunohistochemistry. In immunohistochemistry, cathepsin A was detected in myocytes of atrial tissue. Ang 1-9 and Ang 1-7 potentiated the effect of an ACE-resistant bradykinin analogue on the B2 receptor in transfected cells expressing human ACE and B2, and in human endothelial cells. Ang 1-9 and 1-7 augmented arachidonic acid and NO release by bradykinin. NO liberation by bradykinin from endothelial cells was potentiated at 10nmol/L concentration by Ang 1-9 and Ang 1-7; at higher concentrations, Ang 1-9 was significantly more active. Both peptides had little activity in absence of bradykinin or ACE. Ang 1-9 and 1-7 potentiated bradykinin action on its B2 receptor at much lower concentrations than their IC50 values with ACE. They probably induce conformational changes in the ACE/B2 receptor complex via interaction with ACE.

The kinin system: suggestions to broaden some prevailing concepts.

The existence and importance of the kallikrein-kinin-kininase system, especially in the circulation, has taken over three-quarters of a century to be established. Finding the multiple components derived from renin-angiotensin and their functions stretched over a century [Erdös EG. Perspectives on the early history of angiotensin-converting enzyme-recent follow-ups. In: Giles TD, editor. Angiotensin-converting enzyme (ACE): clinical and experimental insights. Fort Lee: Health Care Communications; 2001, p. 3-16]. Although the discoveries were made independently, it was shown in 1970 that the angiotensin I-converting enzyme (ACE) is identical with kininase II, previously discovered by us, thus, a single protein can regulate either the activation or inactivation of the two peptide products. It followed that inhibitors of ACE can affect both processes [Bhoola KD, Figueroa CD, Worthy K. Bioregulation of kinins: kallikreins, kininogens, and kininases. Pharmacol Rev 1992;44:1-80]. After being engaged for a long time in characterizing the metabolism of various bio-active peptides, we, as well as others, noticed that the effect of ACE inhibitors go beyond simply blocking angiotensin (Ang) II release and bradykinin (BK) inactivation by the enzyme (Kaplan AP, Joseph K, Silverberg M. Pathways for bradykinin formation and inflammatory disease. J Allergy Clin Immunol 2002; 109(2):195-209, Yamada K, Erd6s EG. Kallikrein and prekallikrein of the isolated basolateral membrane of rat kidney. Kidney Int 1982;22:331-7]. It also became apparent to us that in the complex multistep reactions needed to activate the kallikrein-kinin system, there should be some shortcuts-shunts-to accelerate and simplify important processes. Thus, some basic tenets developed after decades of intensive laboratory investigations-and by now generally accepted-can be challenged. For example, it should be considered that the activities of BK and Lys BK (kallidin) can be substantially different, and that sequentially linked reactions, starting with prokallikrein activation and leading to kinin release from kininogen and inhibition of kininases, may be only one way to activate kinin receptors. A summary of some suggested alterations on prevailing concepts is given below.

Neprilysin inhibitors potentiate effects of bradykinin on b2 receptor.

Some beneficial effects of angiotensin-I--converting enzyme (ACE, kininase II) inhibitor therapy are attributed to enhancing the activity of bradykinin on its B(2) receptor. Independent of inhibition of bradykinin hydrolysis, ACE inhibitors enhance the action of bradykinin on its B(2) receptor by inducing crosstalk between ACE and the receptor. We investigated whether inhibitors of another kininase II-type enzyme, neprilysin (neutral endopeptidase 24.11; NEP), could augment bradykinin effects unrelated to blocking its breakdown using a NEP-resistant bradykinin analog as ligand. We used transfected Chinese hamster ovary (CHO) cells stably expressing human B(2) receptor and NEP (CHO/NEP-B(2)) or only B(2) (CHO/B(2)) as control and human pulmonary fibroblasts (IMR90), expressing B(2), but more NEP than ACE. NEP inhibitor phosphoramidon (100 nmol/L), or omapatrilat, which inhibits both NEP and ACE, did not potentiate bradykinin in CHO/B(2) cells. In IMR90 cells, 10 nmol/L bradykinin elevated [Ca(2+)](i) and desensitized the receptor. Adding either 100 nmol/L omapatrilat or phosphoramidon resensitized the receptor to the ligand, which was abolished by receptor blocker HOE 140. Arachidonic acid release by bradykinin from CHO/NEP-B(2) cells was also augmented by 100 nmol/L phosphoramidon or omapatrilat about 3-fold, and again, the inhibitors resensitized the desensitized B(2) receptor. The inhibitors did not potentiate bradykinin when soluble rNEP was added to the medium of CHO/B(2) cells. Similar to ACE, NEP inhibitors potentiated bradykinin independent of inhibiting inactivation. Consequently, omapatrilat could augment bradykinin effects on B(2), when either ACE or NEP is expressed close to receptor on cell membrane.