angiotensin i–converting enzyme inhibitors are allosteric

Angiotensin I–Converting Enzyme Inhibitors Are AllostericEnhancers of Kinin B1 and B2 Receptor Function

Ervin G. Erdos, Fulong Tan, Randal A. Skidgel

Abstract—The beneficial effects of angiotensin I-converting enzyme (ACE) inhibitors go beyond the inhibition of ACE todecrease angiotensin (Ang) II or increase kinin levels. ACE inhibitors also affect kinin B1 and B2 receptor (B1R andB2R) signaling, which may underlie some of their therapeutic usefulness. They can indirectly potentiate the actions ofbradykinin (BK) and ACE-resistant BK analogs on B2Rs to elevate arachidonic acid and NO release in laboratoryexperiments. Studies indicate that ACE inhibitors and some Ang metabolites increase B2R functions as allostericenhancers by inducing a conformational change in ACE. This is transmitted to B2Rs via heterodimerization with ACEon the plasma membrane of cells. ACE inhibitors are also agonists of the B1R, at a Zn-binding sequence on the secondextracellular loop that differs from the orthosteric binding site of the des-Arg-kinin peptide ligands. Thus, ACEinhibitors act as direct allosteric B1R agonists. When ACE inhibitors enhance B2R and B1R signaling, they augmentNO production. Enhancement of B2R signaling activates endothelial NO synthase, yielding a short burst of NO;activation of B1Rs results in a prolonged high output of NO by inducible NO synthase. These actions, outside inhibitingpeptide hydrolysis, may contribute to the pleiotropic therapeutic effects of ACE inhibitors in various cardiovasculardisorders. (Hypertension. 2010;55:214-220.)

Key Words: angiotensin I–converting enzyme inhibitors � kininase II � kinins � bradykinin B2 receptor� bradykinin B1 receptor � allosteric regulation � 7-transmembrane G protein–coupled receptor

Millions of patients are treated with angiotensin I–con-verting enzyme (ACE) inhibitors to combat hyperten-

sion, congestive heart failure or diabetic renal diseases.1–4

ACE inhibitors significantly reduce mortality after myocar-dial infarction3 and are beneficial in other high risk patients.

ACE inhibitors block the metabolism of several peptidesby ACE, notably the conversion of angiotensin (Ang) I to II,5

and the inactivation of bradykinin (BK)6–8 or the hemoregu-latory tetrapeptide Ac-Ser-Asp-Lys-Pro.9 The conversion ofAng I to Ang II was first found to occur in horse plasma5;with kidney and human plasma, one of us reported theidentity of ACE and kininase II, which we had discoveredpreviously.6 – 8,10,11 Consequently, a single peptidyl-dipeptidase not only releases the hypertensive Ang II, but alsoinactivates the hypotensive BK. How much of the therapeuticeffectiveness of ACE inhibitors is attributable to blockingAng II release12 or to prolonging the short half-life of BK7

and its congener Lys1-BK (kallidin) has been debated. This isfurther complicated by the existence of 2 kinin receptors. Thefirst 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; itsligands are metabolites of BK and kallidin lacking theC-terminal arginine14 removed by plasma carboxypeptidase(CP)N15–17 or membrane CPM.18–20 Whereas the B2R is

widely expressed constitutively, B1R expression is usuallyinduced after noxious stimuli or by inflammatory cyto-kines,13,14,21–23 although some cells (bovine lung endothelialor human fibroblasts) express B1Rs constitutively.

ACE inhibitors can enhance both B2 and B1R signaling.Blocking kinin inactivation by ACE raises the concentrationof intact B2R agonists, which are also the substrates of CPNand -M. This can generate more des-Arg-kinin B1R agonists(Figure). The successful use of antagonists of the Ang II type1 receptor (AT1R) for many of the same indications as ACEinhibitors does not prove ACE inhibitors work only throughreducing Ang II as there are complex interrelationshipsamong Ang II, BK and their receptors. Ang II has 2 receptors,AT1R and AT2R. AT1R is blocked by drugs such as losartan,which can shift Ang II actions to the AT2R. This switching ofreceptors further counteracts AT1R effects because it leads tothe release of mediators such as nitric oxide (NO) and isattributed partially to release of BK to activate B2Rs, a formof “crosstalk.”12,24,25 Intricate Ang II and kinin receptorinterrelationships were also indicated in animal experimentswhere both kinin B1 and B2Rs share in the favorablecardiovascular effects of AT1R blockade.26,27

Despite the established role of Ang and kinin receptors inACE inhibitor effects, much remains puzzling. For example,BK levels in plasma, even after ACE inhibitors, are lower

Received October 5, 2009; first decision October 22, 2009; revision accepted December 7, 2009.From the Department of Pharmacology, University of Illinois College of Medicine, Chicago, Ill.Correspondence to Ervin G. Erdos, Department of Pharmacology (M/C 868), University of Illinois College of Medicine, 835 S Wolcott, Chicago, IL

60612. E-mail [email protected]© 2010 American Heart Association, Inc.

Hypertension is available at http://hyper.ahajournals.org DOI: 10.1161/HYPERTENSIONAHA.109.144600

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than the effective doses of exogenous peptide. Crosstalkbetween AT2Rs and B2Rs does not necessarily require therelease of BK by kallikrein, because kallikrein and otherproteases can directly activate the B2R.28–31 Suggestions thatACE inhibitors lead to an “accumulation” of BK are notplausible.

We suggest that ACE inhibitors have actions that gobeyond blocking covalent peptide bond hydrolysis and mayexplain some of their therapeutic effectiveness. As summa-rized below, we propose that these drugs are also allostericeffectors of B1Rs and B2Rs, functioning as direct allostericagonists of B1Rs and as indirect allosteric enhancers of kininactivity on B2Rs via interactions with ACE.

ACE Inhibitors Are Allosteric Enhancers ofB2R Signaling Via ACE-B2R Interactions on

the Cell SurfaceRegarding allosterism, Monod et al32 concluded that inregulatory proteins, indirect interactions between distinctspecific binding sites explain their regulatory function. Thepresentation of Monod and Jacob at a meeting in 1961 wasconsidered revolutionary.33 Proteins could recognize morethan one molecular partner at an unrelated second site,

forming the basis for allosteric effects.32 Put another way, the2 sites can “talk” to each other, and, as we understand it, this“talking” is mediated by conformational change. Accordingto the International Union of Pharmacology Committee onReceptor Nomenclature,34 allosteric enhancers are modula-tors of receptor function that enhance orthosteric ligandaffinity and/or agonist efficacy, while having no effect ontheir own. They can indirectly bias receptor signaling toendogenous agonists by allosteric modification of the recep-tor.35,36 G protein–coupled receptors, such as the B2R, areprototypes of allosteric proteins that can exist in manydifferent conformational states. Allosteric transition involvesisomerization and stabilization of one of the many conforma-tional states of the receptor, each of which may have adifferent affinity for a ligand and/or preferentially activatedifferent signal transduction pathways. Thus, allosteric en-hancers may affect only a subset of the full signaling pathwayof a receptor; this “collateral efficacy” can lead to new drugdiscovery.35

Investigations into allosteric modulators of G protein–coupled receptors have focused on small molecule ligands,37

but we propose that ACE serves this function with the B2R.Thus, ACE inhibitors enhance mediator release (eg, NO,

ACEN-domain

ACE

BK B2Ragonist

CPM or CPNdes-Arg9-BK

B1Ragonist ACE

C-domain

Inhibitor

BK1-7(Inactive)

Active site “lid”

N-domain

ConformationalChange

HEAWH

ACE Inhibitor

1ACE

ACEInhibitor

Resensitization/Potentiation

C-domain

HEAWH

ACEInhibitor 23

B2 Receptor

B1 Receptor

N-domain

C-domain

Figure. How ACE inhibitors can enhance kinin signaling. (1) ACE inhibitors block the degradation of B2R agonist kinins, thus enhancingB2R signaling. Prolonging kinin half-life increases substrate concentration for carboxypeptidase (CP) M or N to generate des-Arg-kinins,agonists of the B1R. (2) Human ACE associates with B2Rs in a heterodimer on cell membranes. In the crystal structure, the N and Cdomains of ACE have a deep active site cleft and a “lid” region that restricts access of substrates or inhibitors before a conformationalchange. The 2 domains exhibit negative cooperativity; binding ACE inhibitor to one domain can alter the conformation of the otherdomain. Transmission of the conformational change of ACE to the associated B2R is likely mediated by the C domain. Enhanced medi-ator release or resensitization of the B2R is the consequence. (Modified from Figure 11.3 in Erdos EG, Tan F, Skidgel RA. Kinin recep-tors and ACE inhibitors: an interrelationship. In: DeMello WC, Frohlich ED, eds. Renin Angiotensin System and Cardiovascular Disease.New York: Humana Press; 2009:135–150 by permission of Springer Science � Business Media, LLC 2009.) (3) In the third mode ofaction, ACE inhibitors are direct allosteric agonists of B1Rs. The canonical zinc binding motif in the second extracellular loop (HEAWH)is important for ACE inhibitors to activate B1Rs but not for des-Arg-kinin ligands. In human endothelial cells under inflammatory condi-tions, ACE inhibitors activate B1Rs that in turn posttranslationally activate iNOS producing a prolonged high output of NO.

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prostaglandins or endothelium-derived hyperpolarizing fac-tor)13,38 not only by protecting BK from degradation but alsoindirectly by binding to ACE, which allosterically enhancesB2R activation by orthosteric ligand BK (Figure).

The first clinically tested ACE inhibitor, teprotide, camefrom snake venom peptides that potentiated BK action onisolated organs.39 Although the Bothrops jararaca venompeptides BPP5a and BPP9a inhibited ACE,39 with otherBK-potentiating peptides or analogs, ACE inhibition, kinininactivation, and BK potentiation did not correlate.40 Thisindicated an additional mechanism(s). For example, addingan ACE inhibitor to the isolated guinea pig ileum whenisotonic contraction to BK was, at its maximum, rapidlydoubled its magnitude.41 This was not attributable to blockingBK inactivation because its t1/2 was 12 to 15 minutes in theileal preparation. On guinea pig atria, enalaprilat enhancedthe positive inotropic effect of BK, but not by blockinginactivation.42 Using different techniques, investigators foundthat ACE inhibitors potentiated BK actions in tissue prepa-rations and not by inhibition of BK degradation. This led tothe supposition that ACE inhibitors acted on B2Rs or stim-ulated a crosstalk between ACE and the B2R.42–47

Experiments with cultured cells led us to conclude thatACE inhibitors induce a conformational change in ACE thatis transmitted to the B2R owing to their close contact,resulting in potentiation or reactivation of receptor signaling.For example, enalaprilat preserved the B2R in high affinityform that increased arachidonic acid release.46 The inhibitoralso resensitized B2Rs, which had been desensitized to BK,and reduced its internalization. B2Rs are desensitized afterprolonged exposure to kinins and become unresponsive to asecond dose of ligand. Thus, resensitization by ACE inhibi-tors causes the B2R to react again to agonist already presentin the medium. The resensitization of B2R by ACE inhibitorwas confirmed with porcine endothelial cells and attributed toblocking sequestration of receptor into caveolin-rich mem-branes.45 These and numerous additional experiments indi-cate that ACE inhibitors can act through ACE as indirectallosteric enhancers of BK effects on B2R, resulting inincreased mediator release.46–53

More proof of these indirect effects of ACE inhibitors onB2R signaling was obtained with BK analogs such as HT-BK(�50% resistant to cleavage by ACE)41,42,46,48 or other almostcompletely resistant BK analogs.49–53 Generally, ACE onlycleaves oligopeptides of less than 13 residues efficiently,46,50,52

but B2R agonists can be larger molecules. For example,BK coupled at the N terminus to soluble dextran54 orLys1-BK dansylated at the � and � amino groups of Lys1remained B2R agonists not appreciably cleaved by ACE.52

Another ACE- resistant B2R agonist has a nonpeptidebond [Phe8�(CH2-NH)Arg9] at the C terminus.55 Even withthese BK analogs, ACE inhibitors still augmented B2R signalingobviously without preventing enzymatic degradation.

ACE inhibitor enhancement of B2R action involves sig-naling pathways different from those stimulated by B2Ragonists alone. For example, B2R activation by BK is notaffected by protein kinase C and phosphatase inhibitors, butthey blocked the resensitization of the B2R to BK by an ACEinhibitor.49,52 ACE inhibitors also decreased B2R phosphor-

ylation,47,49 suggesting the involvement of phosphorylationand dephosphorylation of the B2R in this response. Thetyrosine kinase inhibitor genistein also blocked B2R resensi-tization caused by ACE inhibitors or Ang1–7, and Ang1–9

peptides,52 indicating that these endogenous peptides are alsoallosteric enhancers of B2R function (see below).

For ACE inhibitors to augment BK responses, ACE and theB2R must be coexpressed on the plasma membrane, closeenough to transmit allosteric effects. For example, ACEinhibitors did not potentiate BK effects in cells lacking ACEor when purified soluble ACE was added to the medium.56

Furthermore, B2R expression can modulate ACE activity.57

We used several techniques to show that ACE and B2Rsinteract.56 ACE and the B2R coimmunoprecipitated andconfocal microscopy revealed that they were colocalized onthe membrane. Using B2Rs tagged at C terminus with yellowfluorescent protein (acceptor) and ACE labeled at the Cterminus with donor cyan fluorescent protein, fluorescenceresonance energy transfer (FRET) indicated that the fluoro-phores attached to ACE and B2R were within 10 nm on themembrane.56

Most of the ACE molecule exists free in the extracellularspace (ratio of extracellular to transmembrane and cytosolicresidues �25:1), quite different from the human B2R, whichhas only a short free N-terminal domain (ratio of N-domain totransmembrane and cytosolic residues is �1:12). To establishwhether the extracellular domains are also close enough forFRET, we labeled B2R with an N-terminal yellow fluorescentprotein and ACE with an N-terminal cyan fluorescent proteinand detected significant FRET, indicating the extracellularportions are also within 10 nm (Z. Chen, R.D. Minshall, F.T.,E.G.E., R.A.S., 2009, data to be published).

The formation of ACE/B2R heterodimers on the mem-brane should be a bimolecular reaction dependent on reactantconcentrations. If ACE is in excess, ACE inhibitors couldmore effectively enhance the activation of B2R by kinins,whereas if cells express many more B2Rs than ACE, ACEinhibitors would not be effective.56

The precise interaction sites and orientation of B2Rs andACE on the membrane are still unknown, but biochemicaland structural features provide clues. Somatic ACE has 2active sites contained in N and C domains, connected by abridge section.58 The short transmembrane anchor is followedby a cytosolic tail (Figure).59 The cytosolic and transmem-brane domains of ACE are not required for interaction andpotentiation of B2R responses.53 However, for ACE tointeract with the B2Rs, ACE has to be membrane anchoredfor proper orientation. A chimeric B2R molecule with ACEfused to its N terminus had ACE activity and was a functionalB2R, but ACE inhibitors did not potentiate B2R responses.56

Finally, with an ACE construct containing the C domainactive site but lacking most of the N domain, ACE inhibitorstill potentiated B2R responses.60 This suggests that theextracellular C domain interacts with B2Rs (Figure). This isconsistent with the ACE crystal structure and modelingindicating 2 possible orientations for N and C domains,61

placing the N domain either �36 Å or 72 Å from themembrane. Based on the crystal structure of the �-adrenergic

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receptor,62 extracellular domains of the B2R could extendmaximally �20 Å above the membrane.

Supportive evidence indicates that binding of ACE inhib-itors alters the conformation of ACE, a necessary movementfor allosteric modification of the B2R. For example, ACEinhibitors induce phosphorylation of the C-terminal tail ofACE, thereby activating signal transduction pathways toincrease expression of proteins such as COX-2.63 The 2 activedomains of ACE have different specificities64 and exhibitnegative cooperativity65; binding of ACE inhibitor to onedomain alters the conformation of the second domain, ren-dering it inaccessible to a second inhibitor. The crystalstructures of the C- or N- domain revealed a deep active sitecleft closed to exterior by a “lid”; consequently, a conforma-tional change would be required for the access of substrate orinhibitor to the enzyme.61,66,67 Modeling of the ACE structureindicates intrinsic flexibility around the active site, suggestinga hinge mechanism to open it for substrate/inhibitor bind-ing.67 Thus, inhibitor binding to ACE does change its con-formation, which can be transmitted to the B2R owing to theirclose association on the membrane,56 resulting in allostericenhancement of B2R signaling (Figure).

ACE Inhibitors and Kinin B1RsACE inhibitors affect the 2 kinin receptors differently.21–23,46,47,50

The human B1R is directly activated by ACE inhibitors, even inabsence of ACE expression (Figure).21–23,68 ACE inhibitorsactivate B1Rs to release NO, most consistently in culturedhuman endothelial cells via inducible NO synthase (iNOS), inthe same range as the B1R ligands des-Arg10-kallidin ordes-Arg9-BK (10�9 to 10�6 mol/L).21–23 Although des-Arg10-kallidin has higher affinity for the B1R than des-Arg9-BK, the2 are about equally active on human B1Rs23 owing to theirefficacies. Enalaprilat, quinalaprilat, ramiprilat, and captoprilare active B1R agonists; lisinopril is not at the same concen-trations, probably because of its positively charged �-NH2.22

In ACE, the canonical pentamer sequence (HEXXH)containing zinc-binding residues in the N- and C-domainactive sites are important for inhibitor binding.59 In metal-lopeptidases, all 3 zinc-binding residues (2 His and 1 Glu) donot have to be sequential provided they are sterically close(within �3 Å) in the 3D structure. The second extracellularloop of the human B1R has the same consensus sequence(HEAWH; residues 195 to 199) required for ACE inhibitor(but not peptide ligands) to activate B1Rs (Figure).22 Thus,allosteric ACE inhibitor activity is blocked by agents ormutations not affecting orthosteric des-Arg-kinin activity. Forexample, a synthetic undecapeptide containing the pentamer(LLPHEAWHFAR; residues 192 to 202), blocked B1Ractivation by enalaprilat but not by des-Arg-kinin.22,23 Muta-tion of H195 to Ala in human B1R did not affect peptideagonist action, but the effect of enalaprilat was much reduced(Ignjatovic et al22 and F.T., E.G.E., R.A.S., 2009, data to bepublished).

B1R activation can increase inflammation, pain, and fibro-sis in diabetic cardiomyopathy,13,14,69 but it is also beneficialafter myocardial infarction in rats or mice.27,70,71 IncreasedNO synthesis, owing to B1R activation,21,72 may also con-tribute to the therapeutic effects of ACE inhibitors after MI

and protect cardiomyocytes.73 NO release, after ACE inhib-itor activation of B1R, inhibited protein kinase C�23 that canbenefit the failing heart.74 B1R signaling was recently re-ported to prevent homing of encephalitogenic T-lymphocytesinto the CNS, which was enhanced in B1R�/� mice.75 CPM,closely associated with myelin centrally and peripherally,76

should contribute by generating B1R ligands. The reportmentioned that ACE inhibitor also suppresses inflammationin the CNS.75

More Considerations About B2 and B1RsWithout carboxypeptidases, endogenous orthosteric B1R li-gands could not be generated and B1R signaling would notoccur. CPM and B1Rs interact on the cell membrane77 andbased on the crystal structure and modeling of CPM,20 itsactive site would be properly oriented along the membraneto deliver agonist effectively to B1R. In bovine or humanendothelial cells, B2R agonists cause B1R-dependent re-lease of calcium or generation of NO,77,78 which alsodepended on CPM.

Activation of B1 and B2Rs can promote inflammation orintensify pain13,14 but can also improve the functions of thefailing heart or kidney.4,12,13,26,27,70,79 B1 and B2Rs bothactivate NO synthesis, but B2R agonists stimulate transientendothelial NOS–derived NO, whereas B1R activation leadsto prolonged high-output NO via iNOS.21,22,72 ACE inhibitorsdo not activate B1Rs in blood vessels lacking endothelium,where peptide ligands are vasoconstrictor.14

ACE inhibitors can potentiate kallikrein-mediated stimula-tion of B2Rs, independent of kinin release,29,30 but afterprekallikrein activation.80 Plasma prekallikrein may also beallosterically activated by prolylcarboxypeptidase81 or heatshock protein 90.82 This could result from induction of aconformational change in prekallikrein, exposing it to anotherprotease or to trace autocatalytic activity, yielding activatedkallikrein.83,84

Endogenous B2R EnhancersEndogenous peptides, such as Ang derivatives Ang1–7 andAng1–9, can also augment orthosteric BK effects on B2Rs.52,85

Ang1–9 is released from Ang I by a carboxypeptidase86 or bycathepsin A (deamidase).85,87,88 Ang1–9, a relatively stableintermediate, is also liberated by human heart tissue.85,88

Ang1–7 is cleaved from Ang I by human neprilysin89 and fromAng II by ACE290,91 and prolylcarboxypeptidase.92 Ang1–7

counteracts Ang II actions for example by improving barore-ceptor reflex and decreasing vascular and smooth musclegrowth. Ang1–7 activates the Mas receptor and also potenti-ates BK effects in vivo.91 Both Ang1–9 and Ang1–7 can inhibitACE, but they augment BK effects on B2Rs at orders ofmagnitude lower concentrations in cultured cells than theirIC50 values.52,85 Thus, Ang1–7 and Ang1–9 could antagonizeAng II effects in vivo, also as allosteric enhancers of the B2R.

PerspectivesWe did not, and could not, aim to complete the history ofACE inhibitors and leave no major questions unanswered, buthave sought to summarize some modes of actions that maycontribute to the efficacy of these drugs. The complexities




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make it difficult to interpret their effects as attributable onlyto a single mediator. ACE cleaves other active peptidesbesides Ang I and BK and ACE inhibitors enhance responsesof kinin receptors beyond blocking kinin catabolism.29,46,93,94

Exogenous ACE inhibitors and endogenous Ang1–7 andAng1–9 peptides are indirect allosteric enhancers of B2Ractivation by the orthosteric peptide ligands. They augmentcollateral efficacy by inducing conformation changes viaACE and B2R complexes on cell plasma membranes. Thisleads to enhanced release of mediators such as NO, endothe-lium-derived hyperpolarizing factor,38 or prostaglandins.13

ACE inhibitors are also direct activators of B1Rs at anallosteric site that differs from the orthosteric site of peptideligands. The consequence is a prolonged high-output NOproduction by iNOS in human endothelial cells.22,23,72 Fi-nally, ACE inhibitors can potentiate direct actions of kal-likrein on the B2R in the absence of kinin release.29,30,95

Sources of FundingThis work was supported by National Institutes of Health grantsHL60678, DK 41431, and HL36473.

DisclosuresNone.

References1. Gavras HP, Faxon DP, Berkoben J, Brunner HR, Ryan TJ. Angiotensin

converting enzyme inhibition in patients with congestive heart failure.Circulation. 1978;58:770–776.

2. Solomon SD, Rice MM, K AJ, Jose P, Domanski M, Sabatine M, GershBJ, Rouleau J, Pfeffer MA, Braunwald E. Renal function and effec-tiveness of angiotensin-converting enzyme inhibitor therapy in patientswith chronic stable coronary disease in the Prevention of Events withACE inhibition (PEACE) trial. Circulation. 2006;114:26–31.

3. Yusuf S, Sleight P, Pogue J, Bosch J, Davies R, Dagenais G. Effects ofan angiotensin-converting-enzyme inhibitor, ramipril, on cardiovascularevents in high-risk patients. The Heart Outcomes Prevention EvaluationStudy Investigators. N Engl J Med. 2000;342:145–153.

4. Pfeffer MA, Frohlich ED. Improvements in clinical outcomes with theuse of angiotensin-converting enzyme inhibitors: cross-fertilizationbetween clinical and basic investigation. Am J Physiol Heart CircPhysiol. 2006;291:H2021–H2025.

5. Skeggs LT Jr, Kahn JR, Shumway NP. The preparation and function ofthe hypertensin-converting enzyme. J Exper Med. 1956;103:295–299.

6. Erdos EG, Yang HYT. An enzyme in microsomal fraction of kidney thatinactivates bradykinin. Life Sci. 1967;6:569–574.

7. Yang HY, Erdos EG, Levin Y. Characterization of a dipeptide hydrolase(kininase II: angiotensin I converting enzyme). J Pharmacol Exp Ther.1971;177:291–300.

8. Yang HYT, Erdos EG. Second kininase in human blood plasma. Nature.1967;215:1402–1403.

9. Peng H, Carretero OA, Vuljaj N, Liao TD, Motivala A, Peterson EL,Rhaleb NE. Angiotensin-converting enzyme inhibitors: a new mechanismof action. Circulation. 2005;112:2436–2445.

10. Yang HYT, Erdos EG, Levin Y. A dipeptidyl carboxypeptidase thatconverts angiotensin I and inactivates bradykinin. Biochim Biophys Acta.1970;214:374–376.

11. Igic R, Erdos EG, Yeh HSJ, Sorrels K, Nakajima T. The angiotensin Iconverting enzyme of the lung. Circ Res. 1972;31:II-51–II-61.

12. Liu Y-H, Yang X-P, Sharov VG, Nass O, Sabbah HN, Peterson E,Carretero OA. Effects of angiotensin-converting enzyme inhibitors andangiotensin II type 1 receptor antagonists in rats with heart failure. Roleof kinins and angiotensin II type 2 receptors. J Clin Invest. 1997;99:1926–1935.

13. Bhoola KD, Figueroa CD, Worthy K. Bioregulation of kinins: kallikreins,kininogens, and kininases. Pharmacol Rev. 1992;44:1–80.

14. Regoli D, Barabe J. Pharmacology of bradykinin and related kinins.Pharmacol Rev. 1980;32:1–46.

15. Erdos EG, Sloane EM. An enzyme in human blood plasma that inacti-vates bradykinin and kallidins. Biochem Pharmacol. 1962;11:585–592.

16. Keil C, Maskos K, Than M, Hoopes JT, Huber R, Tan F, Deddish PA,Erdos EG, Skidgel RA, Bode W. Crystal structure of the human car-boxypeptidase N (kininase I) catalytic domain. J Mol Biol. 2007;366:504–516.

17. Skidgel RA, Erdos EG. Structure and function of human plasma car-boxypeptidase N, the anaphylatoxin inactivator. Int Immunopharmacol.2007;7:1888–1899.

18. Skidgel RA, Johnson AR, Erdos EG. Hydrolysis of opioid hexapeptidesby carboxypeptidase N. Presence of carboxypeptidase in cell membranes.Biochem Pharmacol. 1984;33:3471–3478.

19. Skidgel RA. Carboxypeptidase M. In: Barrett AJ, Rawlings ND,Woessner JF, eds. Handbook of Proteolytic Enzymes. 2nd ed. London:Academic Press; 2004:851–854.

20. Reverter D, Maskos K, Tan F, Skidgel RA, Bode W. Crystal structure ofhuman carboxypeptidase M, a membrane-bound enzyme that regulatespeptide hormone activity. J Mol Biol. 2004;338:257–269.

21. Ignjatovic T, Stanisavljevic S, Brovkovych V, Skidgel RA, Erdos EG.Kinin B1 receptors stimulate nitric oxide production in endothelial cells:signaling pathways activated by angiotensin I-converting enzyme inhib-itors and peptide ligands. Mol Pharmacol. 2004;66:1310–1316.

22. Ignjatovic T, Tan F, Brovkovych V, Skidgel RA, Erdos EG. Novel modeof action of angiotensin I converting enzyme inhibitors. Direct activationof bradykinin B1 receptor. J Biol Chem. 2002;277:16847–16852.

23. Stanisavljevic S, Ignjatovic T, Deddish PA, Brovkovych V, Zhang K,Erdos EG, Skidgel RA. Angiotensin I-converting enzyme inhibitors blockprotein kinase C epsilon by activating bradykinin B1 receptors in humanendothelial cells. J Pharmacol Exp Ther. 2006;316:1153–1158.

24. Carey RM. Cardiovascular and renal regulation by the angiotensin type 2receptor: the AT2 receptor comes of age. Hypertension. 2005;45:840–844.

25. Tschope C, Schultheiss HP, Walther T. Multiple interactions between therenin-angiotensin and the kallikrein-kinin systems: role of ACE inhibitionand AT1 receptor blockade. J Cardiovasc Pharmacol. 2002;39:478–487.

26. Duka A, Kintsurashvili E, Duka I, Ona D, Hopkins TA, Bader M,Gavras I, Gavras H. Angiotensin-converting enzyme inhibition afterexperimental myocardial infarct: role of the kinin B1 and B2receptors. Hypertension. 2008;51:1352–1357.

27. Xu J, Carretero OA, Shesely EG, Rhaleb NE, Yang JJ, Bader M, YangXP. The kinin B1 receptor contributes to the cardioprotective effect ofangiotensin-converting enzyme inhibitors and angiotensin receptorblockers in mice. Exp Physiol. 2009;94:322–329.

28. Hecquet C, Tan F, Marcic BM, Erdos EG. Human bradykinin B2 receptoris activated by kallikrein and other serine proteases. Mol Pharmacol.2000;58:828–836.

29. Biyashev D, Tan F, Chen Z, Zhang K, Deddish PA, Erdos EG, Hecquet C.Kallikrein activates bradykinin B2 receptors in absence of kininogen.Am J Physiol Heart Circ Physiol. 2006;290:H1244–H1250.

30. Chao J, Yin H, Gao L, Hagiwara M, Shen B, Yang ZR, Chao L. Tissuekallikrein elicits cardioprotection by direct kinin B2 receptor activationindependent of kinin formation. Hypertension. 2008;52:715–720.

31. Vandell AG, Larson N, Laxmikanthan G, Panos M, Blaber SI, Blaber M,Scarisbrick IA. Protease-activated receptor dependent and independentsignaling by kallikreins 1 and 6 in CNS neuron and astroglial cell lines.J Neurochem. 2008;107:855–870.

32. Monod J, Wyman J, Changeux JP. On the nature of allosteric transitions:a plausible model. J Mol Biol. 1965;12:88–118.

33. Bray D. Wetware. A Computer in Every Living Cell. New Haven, Conn:Yale University Press; 2009:61.

34. Neubig RR, Spedding M, Kenakin T, Christopoulos A. InternationalUnion of Pharmacology Committee on Receptor Nomenclature and DrugClassification. XXXVIII. Update on terms and symbols in quantitativepharmacology. Pharmacol Rev. 2003;55:597–606.

35. Kenakin T. Collateral efficacy in drug discovery: taking advantage of thegood (allosteric) nature of 7TM receptors. Trends Pharmacol Sci. 2007;28:407–415.

36. Kenakin TP. Seven transmembrane receptors as nature’s prototype allo-steric protein: de-emphasizing the geography of binding. Mol Pharmacol.2008;74:541–543.

37. Valant C, Sexton PM, Christopoulos A. Orthosteric/allosteric bitopicligands: going hybrid at GPCRs. Mol Interv. 2009;9:125–135.

38. Campbell WB, Harder DR. Endothelium-derived hyperpolarizing factorsand vascular cytochrome P450 metabolites of arachidonic acid in theregulation of tone. Circ Res. 1999;84:484–488.




ownloaded from


39. Ferreira SH, Bartelt DC, Greene LJ. Isolation of bradykinin-potentiatingpeptides from Bothrops jararaca venom. Biochemistry. 1970;9:2583–2593.

40. Mueller S, Gothe R, Siems WD, Vietinghoff G, Paegelow I, Reissmann S.Potentiation of bradykinin actions by analogues of the bradykinin poten-tiating nonapeptide BPP9alpha. Peptides. 2005;26:1235–1247.

41. Minshall RD, Nedumgottil SJ, Igic R, Erdos EG, Rabito SF. Potentiationof the effects of bradykinin on its receptor in the isolated guinea pigileum. Peptides. 2000;21:1257–1264.

42. Minshall RD, Erdos EG, Vogel SM. Angiotensin I-converting enzymeinhibitors potentiate bradykinin’s inotropic effects independently ofblocking its inactivation. Am J Cardiol. 1997;80:132A–136A.

43. Auch-Schwelk W, Bossaller C, Claus M, Graf K, Grafe M, Fleck E. ACEinhibitors are endothelium dependent vasodilators of coronary arteriesduring submaximal stimulation with bradykinin. Cardiovasc Res. 1993;27:312–317.

44. Hecker M, Porsti I, Bara AT, Busse R. Potentiation by ACE inhibitors ofthe dilator response to bradykinin in the coronary microcirculation: inter-action at the receptor level. Br J Pharmacol. 1994;111:238–244.

45. Benzing T, Fleming I, Blaukat A, Muller-Esterl W, Busse R. Angioten-sin-converting enzyme inhibitor ramiprilat interferes with the seques-tration of the B2 kinin receptor within the plasma membrane of nativeendothelial cells. Circulation. 1999;99:2034–2040.

46. Minshall RD, Tan F, Nakamura F, Rabito SF, Becker RP, Marcic B,Erdos EG. Potentiation of the actions of bradykinin by angiotensin Iconverting enzyme (ACE) inhibitors. The role of expressed human bra-dykinin B2 receptors and ACE in CHO cells. Circ Res. 1997;81:848–856.

47. Marcic B, Deddish PA, Jackman HL, Erdos EG. Enhancement of bra-dykinin and resensitization of its B2 receptor. Hypertension. 1999;33:835–843.

48. Erdos EG, Deddish PA. The kinin system: suggestions to broaden someprevailing concepts. Int Immunopharmacol. 2002;2:1741–1746.

49. Marcic BM, Erdos EG. Protein kinase C and phosphatase inhibitors blockthe ability of angiotensin I-converting enzyme inhibitors to resensitize thereceptor to bradykinin without altering the primary effects of bradykinin.J Pharmacol Exp Ther. 2000;294:605–612.

50. Erdos EG, Marcic BM. Kinins, receptors, kininases and inhibitors–wheredid they lead us? Biol Chem. 2001;382:43–47.

51. Marcic BM, Deddish PA, Jackman HL, Erdos EG, Tan F. Effects of theN-terminal sequence in the N-domain of ACE on the properties of itsC-domain. Hypertension. 2000;36:116–121.

52. Chen Z, Tan F, Erdos EG, Deddish PA. Hydrolysis of angiotensinpeptides by human angiotensin I-converting enzyme and the resensiti-zation of B2 kinin receptors. Hypertension. 2005;46:1368–1373.

53. Marcic B, Deddish PA, Skidgel RA, Erdos EG, Minshall RD, Tan F.Replacement of the transmembrane anchor in angiotensin I-convertingenzyme (ACE) with a glycosylphosphatidylinositol tail affects activationof the B2 bradykinin receptor by ACE inhibitors. J Biol Chem. 2000;275:16110–16118.

54. Odya CE, Levin Y, Erdos EG, Robinson CJG. Soluble dextran complexesof kallikrein, bradykinin, and enzyme inhibitors. Biochem Pharmacol.1978;27:173–179.

55. Drapeau G, Rhaleb N-E, Dion S, Jukic D, Regoli D. [Phe8 �(CH2-NH)Arg9]bradykinin, a B2 receptor selective agonist which is not brokendown by either kininase I or kininase II. Eur J Pharmacol. 1988;155:193–195.

56. Chen Z, Deddish PA, Minshall RD, Becker RP, Erdos EG, Tan F. HumanACE and bradykinin B2 receptors form a complex at the plasmamembrane. FASEB J. 2006;20:2261–2270.

57. Sabatini RA, Guimaraes PB, Fernandes L, Reis FC, Bersanetti PA, MoriMA, Navarro A, Hilzendeger AM, Santos EL, Andrade MC, Chagas JR,Pesquero JL, Casarini DE, Bader M, Carmona AK, Pesquero JB. ACEactivity is modulated by kinin B2 receptor. Hypertension. 2008;51:689–695.

58. Deddish PA, Wang J, Michel B, Morris PW, Davidson NO, Skidgel RA,Erdos EG. Naturally occurring active N-domain of human angiotensin Iconverting enzyme. Proc Natl Acad Sci U S A. 1994;91:7807–7811.

59. Corvol P, Eyries M, Soubrier F. Peptidyl-dipeptidase A/angiotensinI-converting enzyme. In: Barrett AJ, Rawlings ND, Woessner JF, eds.Handbook of Proteolytic Enzymes. Vol 1. 2nd ed. San Diego, Calif:Academic Press; 2004:332–346.

60. Deddish PA, Wang L-X, Jackman HL, Michel B, Wang J, Skidgel RA,Erdos EG. Single-domain angiotensin converting enzyme (kininase II):

characterization and properties. J Pharmacol Exp Ther. 1996;279:1582–1589.

61. Corradi HR, Schwager SL, Nchinda AT, Sturrock ED, Acharya KR.Crystal structure of the N domain of human somatic angiotensinI-converting enzyme provides a structural basis for domain-specific in-hibitor design. J Mol Biol. 2006;357:964–974.

62. Cherezov V, Rosenbaum DM, Hanson MA, Rasmussen SG, Thian FS,Kobilka TS, Choi HJ, Kuhn P, Weis WI, Kobilka BK, Stevens RC.High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor. Science. 2007;318:1258–1265.

63. Fleming I, Kohlstedt K, Busse R. New fACEs to the renin-angiotensinsystem. Physiology (Bethesda). 2005;20:91–95.

64. Jaspard E, Wei L, Alhenc-Gelas F. Differences in the properties andenzymatic specificities of the two active sites of angiotensin I-convertingenzyme (kininase II). Studies with bradykinin and other natural peptides.J Biol Chem. 1993;268:9496–9503.

65. Binevski PV, Sizova EA, Pozdnev VF, Kost OA. Evidence for thenegative cooperativity of the two active sites within bovine somaticangiotensin-converting enzyme. FEBS Lett. 2003;550:84–88.

66. Natesh R, Schwager SL, Sturrock ED, Acharya KR. Crystal structure ofthe human angiotensin-converting enzyme-lisinopril complex. Nature.2003;421:551–554.

67. Watermeyer JM, Sewell BT, Schwager SL, Natesh R, Corradi HR,Acharya KR, Sturrock ED. Structure of testis ACE glycosylation mutantsand evidence for conserved domain movement. Biochemistry. 2006;45:12654–12663.

68. Donnini S, Solito R, Giachetti A, Granger HJ, Ziche M, Morbidelli L.Fibroblast growth factor-2 mediates angiotensin-converting enzyme in-hibitor-induced angiogenesis in coronary endothelium. J Pharmacol ExpTher. 2006;319:515–522.

69. Westermann D, Walther T, Savvatis K, Escher F, Sobirey M, Riad A,Bader M, Schultheiss HP, Tschope C. Gene deletion of the kinin receptorB1 attenuates cardiac inflammation and fibrosis during the developmentof experimental diabetic cardiomyopathy. Diabetes. 2009;58:1373–1381.

70. Tschope C, Spillmann F, Altmann C, Koch M, Westermann D, Dhayat N,Dhayat S, Bascands JL, Gera L, Hoffmann S, Schultheiss HP, Walther T.The bradykinin B1 receptor contributes to the cardioprotective effects ofAT1 blockade after experimental myocardial infarction. Cardiovasc Res.2004;61:559–569.

71. Xu J, Carretero OA, Sun Y, Shesely EG, Rhaleb N-E, Liu Y-H, Liao T-D,Yang JJ, Bader M, Yang X-P. Role of the B1 kinin receptor in theregulation of cardiac function and remodeling after myocardial infarction.Hypertension. 2005;45:747–753.

72. Zhang Y, Brovkovych V, Brovkovych S, Tan F, Lee B-S, Sharma T,Skidgel RA. Dynamic receptor-dependent activation of induciblenitric-oxide synthase by ERK-mediated phosphorylation of Ser745. J BiolChem. 2007;282:32453–32461.

73. McMurray JJV, Pfeffer MA, Swedberg K, Dzau VJ. Which inhibitor ofthe renin-angiotensin system should be used in chronic heart failure andacute myocardial infarction? Circulation. 2004;110:3281–3288.

74. Inagaki K, Koyanagi T, Berry NC, Sun L, Mochly-Rosen D. Pharmaco-logical inhibition of epsilon-protein kinase C attenuates cardiac fibrosisand dysfunction in hypertension-induced heart failure. Hypertension.2008;51:1565–1569.

75. Schulze-Topphoff U, Prat A, Prozorovski T, Siffrin V, Paterka M, Herz J,Bendix I, Ifergan I, Schadock I, Mori MA, Van Horssen J, Schroter F,Smorodchenko A, Han MH, Bader M, Steinman L, Aktas O, Zipp F.Activation of kinin receptor B1 limits encephalitogenic T lymphocyterecruitment to the central nervous system. Nat Med. 2009;15:788–793.

76. Nagae A, Deddish PA, Becker RP, Anderson CH, Abe M, Skidgel RA,Erdos EG. Carboxypeptidase M in brain and peripheral nerves. J Neu-rochem. 1992;59:2201–2212.

77. Zhang X, Tan F, Zhang Y, Skidgel RA. Carboxypeptidase M and kininB1 receptors interact to facilitate efficient B1 signaling from B2 agonists.J Biol Chem. 2008;283:7994–8004.

78. Sangsree S, Brovkovych V, Minshall RD, Skidgel RA. Kininase I-typecarboxypeptidases enhance nitric oxide production in endothelial cells bygenerating bradykinin B1 receptor agonists. Am J Physiol Heart CircPhysiol. 2003;284:H1959–H1968.

79. Carretero OA. Novel mechanism of action of ACE and its inhibitors.Am J Physiol Heart Circ Physiol. 2005;289:H1796–H1797.

80. Takada Y, Skidgel RA, Erdos EG. Purification of human prokallikrein.Identification of the site of activation by the metalloproteinase ther-molysin. Biochem J. 1985;232:851–858.




ownloaded from


81. Schmaier AH, McCrae KR. The plasma kallikrein-kinin system: its evo-lution from contact activation. J Thromb Haemost. 2007;5:2323–2329.

82. Joseph K, Tholanikunnel BG, Kaplan AP. Heat shock protein 90 cat-alyzes activation of the prekallikrein-kininogen complex in the absence offactor XII. Proc Natl Acad Sci U S A. 2002;99:896–900.

83. Pungercar JR, Caglic D, Sajid M, Dolinar M, Vasiljeva O, Pozgan U,Turk D, Bogyo M, Turk V, Turk B. Autocatalytic processing of pro-cathepsin B is triggered by proenzyme activity. FEBS J. 2009;276:660–668.

84. Joseph K, Tholanikunnel BG, Kaplan AP. Factor XII-independentcleavage of high-molecular-weight kininogen by prekallikrein and inhi-bition by C1 inhibitor. J Allergy Clin Immunol. 2009;124:143–149.

85. Jackman HL, Massad MG, Sekosan M, Tan F, Brovkovych V, MarcicBM, Erdos EG. Angiotensin 1-9 and 1-7 release in human heart: role ofcathepsin A. Hypertension. 2002;39:976–981.

86. Goldstein SM, Kaempfer CE, Kealey JT, Wintroub BU. Human mast cellcarboxypeptidase. Purification and characterization. J Clin Invest. 1989;83:1630–1636.

87. Jackman HL, Tan F, Tamei H, Beurling-Harbury C, Li X-Y, Skidgel RA,Erdos EG. A peptidase in human platelets that deamidates tachykinins:probable identity with the lysosomal “protective protein.” J Biol Chem.1990;265:11265–11272.

88. Erdos EG, Jackman HL, Brovkovych V, Tan F, Deddish PA. Products ofangiotensin I hydrolysis by human cardiac enzymes potentiate bra-dykinin. J Mol Cell Cardiol. 2002;34:1569–1576.

89. Gafford JT, Skidgel RA, Erdos EG, Hersh LB. Human kidney “enkeph-alinase,” a neutral metalloendopeptidase that cleaves active peptides.Biochemistry. 1983;22:3265–3271.

90. Garabelli PJ, Modrall JG, Penninger JM, Ferrario CM, Chappell MC.Distinct roles for angiotensin-converting enzyme 2 and carboxypeptidaseA in the processing of angiotensins within the murine heart. Exp Physiol.2008;93:613–621.

91. Maia LG, Ramos MC, Fernandes L, de Carvalho MH, Campagnole-Santos MJ, Souza dos Santos RA. Angiotensin-(1-7) antagonist A-779attenuates the potentiation of bradykinin by captopril in rats. J CardiovascPharmacol. 2004;43:685–691.

92. Odya CE, Marinkovic D, Hammon KJ, Stewart TA, Erdos EG. Purifi-cation and properties of prolylcarboxypeptidase (angiotensinase C) fromhuman kidney. J Biol Chem. 1978;253:5927–5931.

93. Danser AH, Tom B, de Vries R, Saxena PR. L-NAME-resistantbradykinin-induced relaxation in porcine coronary arteries is NO-de-pendent: effect of ACE inhibition. Br J Pharmacol. 2000;131:195–202.

94. Sivieri DO Jr, Bispo-da-Silva LB, Oliveira EB, Resende AC, SalgadoMC. Potentiation of bradykinin effect by angiotensin-converting enzymeinhibition does not correlate with angiotensin-converting enzyme activityin the rat mesenteric arteries. Hypertension. 2007;50:110–115.

95. Koch M, Bonaventura K, Spillmann F, Dendorfer A, Schultheiss HP,Tschope C. Attenuation of left ventricular dysfunction by an ACE inhib-itor after myocardial infarction in a kininogen-deficient rat model. BiolChem. 2008;389:719–723.




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Ervin G. Erdös, Fulong Tan and Randal A. SkidgelB2 Receptor Function

Converting Enzyme Inhibitors Are Allosteric Enhancers of Kinin B1 and−Angiotensin I

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