Activation of bradykinin B1 receptor by ACE inhibitors

International Immunopharmacology 2 (2002) 1787 – 1793 www.elsevier.com/locate/intimp

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Activation of bradykinin B1 receptor by ACE inhibitors Tatjana Ignjatovic a, Fulong Tan a,b, Viktor Brovkovych a, Randal A. Skidgel a,b, Ervin G. Erdo¨s a,b,* a

Department of Pharmacology (M/C 868), University of Illinois at Chicago College of Medicine, 835 S. Wolcott Avenue, Chicago, IL 60612-7344, USA b Department of Anesthesiology, University of Illinois at Chicago College of Medicine, Chicago, IL 60612-7344, USA

Abstract ACE or kininase II inhibitors are very important, widely used therapeutic agents for the treatment of a variety of diseases. Although they inhibit ACE, thus, angiotensin II release and bradykinin (BK) inactivation, this inhibition alone does not suffice to explain their successful application in medical practice. Enalaprilat and other ACE inhibitors at nanomolar concentrations activate the BK B1 receptor directly in the absence of ACE and the peptide ligands, des-Arg-kinins. The inhibitors activate at the Zn-binding pentameric consensus sequence HEXXH (195 – 199) of B1, a motif also present in the active centers of ACE but absent from the BK B2 receptor. ACE inhibitors, when activating the B1 receptor, elevate intracellular calcium [Ca2 +]i and release NO from cultured cells. Activation by ACE inhibitor was abolished by Ca – EDTA, a B1 receptor antagonist, by a synthetic undecapeptide representing the 192 – 202 sequence in the B1 receptor, and by site-directed mutagenesis of H195 to A. With the exception of the B1 receptor blocker, these agents and the mutation did not affect the actions of the peptide ligand desArg10-Lys1-BK. Ischemia and inflammatory cytokines induce B1 receptors and elevate its expression. Direct activation of the B1 receptor by ACE inhibitors can contribute to their therapeutic efficacy, for example, by releasing NO in vascular beds, or to some of their side effects. D 2002 Published by Elsevier Science B.V. Keywords: Activation; Bradykinin B1 receptor; ACE inhibitor

Angiotensin I converting enzyme inhibitors (ACEIs) are widely used drugs beneficial in the treatment of a variety of cardiovascular and renal diseases [1,2]. They were originally introduced as antihypertensives, but

* Corresponding author. Department of Pharmacology (M/C 868), University of Illinois at Chicago College of Medicine, 835 S. Wolcott Avenue, Chicago, IL 60612-7344, USA. Tel.: +1-312-9969146; fax: +1-312-996-1648. E-mail address: [email protected] (E.G. Erdo¨s).

later, it was realized that they have far reaching effects beyond blood pressure regulation. For example, the Heart Outcomes Prevention Evaluation study (HOPE) conducted in more than 9000 patients showed that ACEI ramipril substantially lowered the rates of death, heart attack, stroke, heart failure and complications related to diabetes mellitus [3]. Ramipril therapy also slowed down the progression of atherosclerosis in humans in the SECURE study [4]. Favorable results of ACEIs also have been reported in other, non-cardiovascular or renal conditions, even in cancer [5].

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A number of reports emphasized that the benefits of ACEIs are mediated through multiple molecular mechanisms [6,7] not just by inhibiting ACE (kininase II), thus suppressing the activation of angiotensin I and blocking the degradation of bradykinin (BK) [8,9]. The actions of ACEIs, when blocked by HOE140, a specific BK B2 receptor blocker, are usually attributed to endogenous BK [9]. In addition, ACEIs also potentiate the effects of exogenous BK and its ACE-resistant analogues on the B2 receptor [10,11]. They probably induce a conformational change on the B2 receptor via ACE and resensitize the receptor desensitized by agonist [12]. In addition, ACEIs may interfere with the targeting of the B2 receptor to caveolin-rich membrane domains [13]. This mode of action of ACEIs is based on interacting with ACE, that leads to an indirect potentiation of BK effect on its receptor and which requires the expression of both ACE and the B2 receptor. In addition, we recently reported a novel mechanism of action of ACEIs, which is independent of ACE. ACEIs in nanomolar concentrations and in the absence of ACE directly activate human and bovine BK B1 receptors on cultured cells [14]. We became interested in the role of the B1 receptor in the cellular, subcellular and molecular modes of actions of these widely employed therapeutic agents. Although ACEIs indirectly enhanced BK effects on its B2 receptor via ACE [10,12], ACEIs had no direct effect on the B2 receptor [10]. The possibility that ACEIs may directly activate the B1 receptor had not been investigated, although conditions of patients treated with ACEIs are frequently associated with an up-regulated expression of B1. The BK B1 receptor belongs to a superfamily of seven-transmembrane domain, G-protein-coupled receptors (GPCR). Its activation stimulates phosphoinositide hydrolysis and increases intracellular calcium [Ca2 +]i through Gaq/11 and phospholipase C [15 – 17]. To follow receptor activation, we first measured the rise in [Ca2 +]i as a marker. To investigate the effects of ACEIs, we used the active form of ACEI enalaprilat and tested it on cultured human fetal lung fibroblasts (IMR-90). These cells are suitable since they express both B1 and B2 receptors. Under basal conditions in most tissues, the B2 receptor is the predominant kinin receptor, whereas B1 is expressed only sparsely [17,18]. Enalaprilat added to the

medium without any exogenous kinin in low concentration (1 nM) markedly increased [Ca2 +]i in IMR-90 cells (Fig. 1). The prolonged elevation of [Ca2 +]i was in contrast to the rather transient and sharp peak that follows B2 receptor activation, but similar to the desArg10-kallidin (DAKD)-mediated response. In general, activation of the B1 receptor produces prolonged, sustained or oscillatory responses. This persistent signaling is due to the lack of desensitization and internalization of the B1 receptor [15,19]. In contrast, the B2 receptor gives transient responses, since this receptor undergoes rapid desensitization and internalization [19,20]. In addition, B1 receptor activation by enalaprilat was inhibited by a B1 receptor blocker (des-Arg10 Leu9-kallidin) but not by a B2 receptor blocker (HOE 140). These initial experiments strongly indicated the existence of a novel mode of action of ACEIs—a direct activation of the B1 receptor in the absence of ACE. Enalaprilat was then tested with other cell types, including those that constitutively express the B1 receptor, bovine pulmonary artery endothelial cells (BPAE) and cells where cytokines induce B1 expression, human lung microvascular endothelial cells. Other cell types were transfected to express the human B1 receptor (Chinese hamster ovary: CHO, human embryonic kidney: HEK 293, African green monkey kidney cells: COS-7 cells). Transfected cells that do not express ACE [10] helped us to further elucidate the mechanism of action of ACEIs. Even in cells lacking ACE, ACEIs still activated the B1 receptor and raised [Ca2 +]i. Thus, we concluded that the activation of the B1 receptor by ACEIs is independent of ACE and clearly differs from the indirect potentiation of B2 receptor responses. This depends on the presence of both ACE and the B2 receptor on the cell surface [10], resulting in induction of an enzyme-receptor cross-talk and heterodimer formation [11,12]. ACEIs have been reported to release NO [21 – 23], and this is a major factor in their therapeutic efficacy, since NO influences a great variety of processes (e.g., vasodilation, platelet aggregation, leukocyte adhesion and vascular smooth muscle mitogenesis) [24,25]. Activation of the B1 receptor by peptide ligand releases NO [26] and we next determined whether ACEIs trigger NO release by activating B1 receptor.

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Fig. 1. Stimulation of [Ca2 +]i transients in IMR-90 fibroblasts. IMR-90 cells were stimulated with des-Arg10-kallidin (DAKD) (A), bradykinin (BK) (B) or enalaprilat (Ept) (C). Notice that enalaprilat effect was blocked by des-Arg10-Leu9-kallidin (DALKD), B1 receptor antagonist (D), but not by HOE 140, B2 receptor antagonist (E). Arrows denote the time of addition of agonists or antagonists. Representative experiments repeated two to four times with 10 – 100 cells in each assay. (Reproduced with permission from Ref. [14]).

To monitor the release of NO from cultured BPAE cells, we used two different methods, electrochemical and fluorescent assays, with similar results. Enalaprilat (10 nM) did indeed release NO and this response was suppressed by des-Arg10-Leu9-kallidin, a selective B1 receptor antagonist (Fig. 2). The prolonged pattern of this response was again similar to that of DAKD, but much different than that of BK acting on the B2 receptor which gave only a transient response. The time course of NO generation triggered by DAKD and enalaprilat resembles the time course of the [Ca2 +]i signals. After establishing that ACEIs directly activate the B1 receptor, we sought to determine the mechanism

and site of action of the inhibitors. Human B1 receptor contains in its second extracellular loop a potential zinc-binding (HEAWH) sequence, which resembles the pentameric consensus sequences in the two active centers of ACE and other members of gluzincin subfamily of zinc-metalloenzymes [27 – 29]. ACEIs bind to the Zn2 + cofactors of ACE either by a SH or COO- group when inhibiting the enzyme [30]. We therefore assumed that this (HEXXH) region in the B1 receptor is the site for activation of the B1 receptor by ACEIs, and addressed this problem using several different approaches. First, point (H195A) mutation of the B1 receptor at the putative zinc-binding site (AEAWH) was tested in

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HEK and COS-7 cells transiently transfected with wild type (WT) or mutated receptor [14]. The H195A mutation abolished only the effect of enalaprilat, while the agonist DAKD remained active. These results were also in good agreement with our observations that enalapril (the esterified pro-drug form of enalaprilat, which cannot bind zinc) is inactive on the B1 receptor. In addition, pretreatment of cells with Ca2EDTA, to bind heavy metals such as zinc on the B1 receptor, also abolished the effect of enalaprilat [14]. We further confirmed these data in

Fig. 3. The potential zinc-binding pentameric consensus sequence in the B1 receptor of various species (observe the difference in dog). This represents a proposed site of attachment of ACEIs to B1 receptor.

another set of experiments, where cells were incubated with synthetic undecapeptide (LLPHEA WHFAR), which incorporates the zinc-binding site of the second extracellular loop of B1, prior to the addition of enalaprilat. The undecapeptide selectively blocked only the effect of enalaprilat but not that of B1 receptor ligand DAKD. This putative zinc-binding motif is present not only in the human B1 receptor we employed but also in other B1 receptors across species (Fig. 3). Whether zinc plays any important role for the activity of the B1 receptor is open to speculation. It appears that zinc is not essential for the activation by peptide ligand; however, the human B1 receptor has high constitutive activity even in the absence of a ligand [31]. The influence of zinc on this spontaneous activity of the receptor remains to be investigated. This zinc-binding motif is absent from the B2 receptor, which is consistent with the inability of ACEIs to directly activate it.

Fig. 2. Stimulation of NO generation. NO production was measured in BPAE cells using a porphyrinic microsensor in real time. (A) Addition of enalaprilat or des-Arg10-kallidin (denoted by the arrows) caused an immediate generation of NO which continued to increase over 20 min. In contrast, BK stimulated a transient increase in NO which returned to baseline by about 5 min. (B) Dose – response curve for enalaprilat. BPAE cells were stimulated with increasing concentrations of enalaprilat and the NO concentration generated at 20 min taken as a measure of the response. Results represent the mean values of five (10 and 100 nM) or two (AM concentration points) independent experiments. (C) B1 receptor blocker inhibits enalaprilat stimulation of NO production. Des-Arg10-kallidin and enalaprilat were added to cells pretreated for 2 min with or without the B1 receptor antagonist, (DALKD) as indicated and the NO concentration generated at 20 min taken as a measure of the response. Shown are the mean values from four or more independent experiments. (Reproduced with permission from Ref. [14]).

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The B1 receptor generally can be rapidly induced under pathological conditions. It was suggested that this highly regulated receptor expression could be an advantage under various stressful conditions, ranging from infection to cardiovascular disorders [17,32 – 34]. Tissue ischemia and myocardial infarction can trigger the induction of the B1 receptor, and activation of B1 receptors could contribute to recovery of ischemic tissues, angiogenesis and wound healing [35,36]. Activation of B1 receptors stimulates angiogenesis through production of NO and up-regulates basic fibroblast growth factor (bFGF, FGF-2) [37]. Therefore, B1 receptors along with vascular endothelial growth factor (VEGF), interleukin 8 (IL-8) and bFGF [38] are induced rapidly in the infarcted parts of myocardium, where they enhance neovascularization and tissue healing. Myocardial infarction is accompanied by an inflammatory response [38], which was originally believed to play only a detrimental, injurious role. However, use of corticosteroids to reduce inflammation actually worsened the condition of patients with acute myocardial infarction, increasing both the size of infarct and incidence of ventricular arrhythmias [39]. In addition, inhibition of a pro-inflammatory transcription factor, NFkB, also inhibits cardioprotection in ischemic preconditioning [40]. Inflammatory mediators may be important in healing and cardiac repair [38]. That would include induction of the B1 receptor. ACEIs administered shortly after myocardial infarction reduce the incidence of death and severe congestive heart failure [41,42]. ACEIs can affect NO synthesis and release in various ways. They potentiate the effects of BK, which is known to release NO through B2 receptor [43]. In addition to increasing production of NO, ACEIs can also decrease its degradation, by lowering angiotensin II and therefore diminishing vascular superoxide production via membrane NADH/NADPH oxidase [44]. In addition to these short-term effects, prolonged treatment with ACEIs also induces expression of endothelial nitric oxide synthase enzyme [45]. Possibly, NO release triggered by direct activation of the B1 receptor also contributes to their advantageous application in the treatment of myocardial infarction and heart failure. The benefits of the administration of ACEIs are numerous and are likely to be mediated by several

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mechanisms. ACE inhibition alone results in many favorable effects mediated via blocking of Ang II generation and potentiation of BK effects. Additional cellular and subcellular actions, such as the direct activation of the B1 receptor, enhance the spectrum of therapeutic possibilities offered by administration of this group of drugs.

Acknowledgements The study was supported in part by NIH-National Heart, Lung and Blood Institute grants #HL36473, #HL58118 to EGE and #HL60678 to RAS. We are grateful to Sara Blaszczak for editorial assistance.

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