CHAPTER SIX

Constitutive Activity in the Angiotensin II Type 1 Receptor: Discovery and Applications Hamiyet Unal, Sadashiva S. Karnik1 Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA 1 Corresponding author: e-mail address: [email protected]

Contents 1. 2. 3. 4. 5. 6.

Introduction Discovering Constitutive Activity of AT1 Receptor Mechanism of Constitutive Activation in AT1 Receptor Inverse Agonists and Partial Agonists of AT1 Receptor Constitutive Activity of AT1 Receptor In Vivo Constitutive Activation of AT1 Receptor and Pathophysiology 6.1 Autoantibody activation 6.2 Stretch activation 7. CAM AT1 Receptors as Research Tools 7.1 Angiotensinergic activation of vascular endothelium 7.2 Low-renin hypertension model 7.3 Hypersympathetic vasomotor tone model 7.4 Renal proximal tubular AT1 receptor hyperactivity model 7.5 Adult cardiac myocyte-specific AT1 receptor hyperactivity induction model 8. Conclusion Conflict of Interest Acknowledgments References

156 157 160 162 163 164 164 165 165 165 166 168 169 169 170 171 171 171

Abstract The pathophysiological actions of the renin–angiotensin system hormone, angiotensin II (AngII), are mainly mediated by the AngII type 1 (AT1) receptor, a GPCR. The intrinsic spontaneous activity of the AT1 receptor in native tissues is difficult to detect due to its low expression levels. However, factors such as the membrane environment, interaction with autoantibodies, and mechanical stretch are known to increase G protein signaling in the absence of AngII. Naturally occurring and disease-causing activating mutations have not been identified in AT1 receptor. Constitutively active mutants (CAMs) of AT1 receptor have been engineered using molecular modeling and site-directed mutagenesis approaches among which substitution of Asn111 in the transmembrane helix III

Advances in Pharmacology, Volume 70 ISSN 1054-3589 http://dx.doi.org/10.1016/B978-0-12-417197-8.00006-7

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with glycine or serine results in the highest basal activity of the receptor. Transgenic animal models expressing the CAM AT1 receptors that mimic various in vivo disease conditions have been useful research tools for discovering the pathophysiological role of AT1 receptor and evaluating the therapeutic potential of inverse agonists. This chapter summarizes the studies on the constitutive activity of AT1 receptor in recombinant as well as physiological systems. The impact of the availability of CAM AT1 receptors on our understanding of the molecular mechanisms underlying receptor activation and inverse agonism is described.

ABBREVIATIONS AngII angiotensin II ARB angiotensin receptor blocker AT1 receptor angiotensin II type 1 receptor CAM constitutively active mutant ECL2 extracellular loop 2 GPCR G protein-coupled receptor IP3 inositol trisphosphate RAS renin angiotensin system

1. INTRODUCTION Angiotensin II (AngII) is the octapeptide hormone product of the renin angiotensin system (RAS). AngII action on the cardiovascular, renal, nervous, and endocrine systems is mediated by two high-affinity G proteincoupled receptors (GPCRs), AngII type 1 (AT1) receptor and AngII type 2 receptor, that regulate blood pressure and hydroelectrolytic homeostasis (Oliveira et al., 2007). Most of the physiological actions of AngII (vasoconstriction, aldosterone secretion, and sodium/potassium/water balance) are mediated by the AT1 receptor (De Gasparo, Catt, Inagami, Wright, & Unger, 2000). The AT1 receptor activates a classical Gq/11 protein and phospholipase C pathway to produce second messengers, such as inositol trisphosphate (IP3) and diacylglycerol (DAG), leading to the intracellular calcium flux and activation of protein kinases including the extracellular regulated kinases, ERK1/2. Following initial activation of G protein-mediated signals, the AT1 receptor desensitizes due to GRK-mediated phosphorylation, binds b-arrestin, and enters intracellular trafficking resensitization and recycling pathway. In this phase, AT1 receptor may activate other G proteinindependent signaling pathways that may include activation of tyrosine kinase signaling pathways as well (Hunyady & Catt, 2006). Because the AT1 receptor is a major therapeutic target in treating hypertension and

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cardiovascular and renal diseases, the pharmacology of this receptor has been extensively studied. As a result, many peptide agonists and antagonists and nonpeptide AT1 receptor blockers (ARBs) exist. The molecular basis of AT1 receptor functions is still evolving, and we lack a three-dimensional structural model for the receptor at this time. The AT1 receptor and its ligands function cooperatively to regulate physiological as well as pathological processes. The receptor activity is regulated positively when AngII binds and negatively when ARBs bind as anticipated from classical models of receptor action. Noda et al. (1996) were the first to describe ligand-independent signaling by AT1 receptor due to constitutive activation, an observation that is now reproduced and firmly established (Petrel & Clauser, 2009). Discovery of innate constitutive activity in the delta opioid receptors by Costa and Herz in 1989 (Costa & Herz, 1989) was a transformative event in the pharmacology of GPCRs. The ability of a receptor to spontaneously achieve a signaling conformation resulting in the production of a second messenger without binding natural or pharmacological agonist is defined as constitutive activation. It is also referred to as a “gain-of-function” phenotype. In years following initial description, the importance of constitutive activity concept was firmly established with the characterization of disease-causing mutations in several GPCRs (Costa & Cotecchia, 2005). Site-directed mutagenesis studies discovered mutationinduced constitutive activity in GPCRS leading to a revised classification of pharmacological agents. Terms such as inverse agonists that cause “negative efficacy” were introduced along with classical agonist and antagonist ligands. Mechanistic studies of constitutively active mutant (CAM) receptors led the field to the introduction of a two-state receptor model (R and R*), which has further evolved to accommodate more recent developments in GPCR research (Costa & Cotecchia, 2005; Leff, 1995). Realization of constitutive activation has significantly impacted current understanding of AT1 receptor biology and is changing the view of the robustness of receptor mechanisms that may be ligand-independent. In this chapter, we outline the discovery of intrinsic activity of the AT1 receptor and the application of CAM AT1 receptors in the elucidation of in vivo pathophysiology.

2. DISCOVERING CONSTITUTIVE ACTIVITY OF AT1 RECEPTOR The AT1 receptor displays measurable innate spontaneous activity in the absence of AngII, when 5–10 pmol of receptor/mg total cellular protein

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is expressed in transfected cells. The constitutively active pool of wild-type AT1 receptor is
5% of total in a cell. Hence, it is difficult to detect constitutive activity with the available functional assays in native tissues expressing the AT1 receptor in the femtomole per milligram range. Many factors, such as membrane environment, interacting proteins, receptor autoantibodies, and single-nucleotide polymorphisms, that increase expression can increase constitutive signaling in the absence of AngII. Therefore, the question whether AT1 receptor constitutive activity is of physiological relevance remained. Inspired by early discovery of CAMs linked to endocrine hormone diseases in many GPCR, Davies, Bonnardeaux, Plouin, Corvol, and Clauser (1997) searched for AT1 receptor gene mutations in primary hyperaldosteronism caused by an aldosterone-producing adenoma. AngII is the major activator of aldosterone secretion in the adrenal gland. Hence, constitutively activating somatic mutations in the gene encoding AT1 receptor protein could cause hyperfunctioning adrenal. The 1.1 kilobase coding region (exon 5) of the AT1 receptor gene was analyzed for the presence of mutations using DNA sequencing. Although three silent polymorphisms were detected, no functional mutations were found. It appears unlikely that somatic mutations play a role in primary hyperaldosteronism. In several genome scan studies, SNP variants have been found in the gene for human AT1 receptor but no mutations are reported. This observation might be because phenotype of activating mutations for AT1 receptor might be lethal. Activating mutations have been characterized by site-directed mutagenesis for almost all known GPCRs including the AT1 receptor. The discovery of mutation-induced constitutive activity in the AT1 receptor involved computer modeling and AngII docking analysis, which suggested that Asn111 in TM3 helix (Fig. 6.1) plays a central role in the activation of AT1 receptor by interacting directly with Tyr4 of AngII (Noda et al., 1996). Two residues in AngII, Tyr4 and Phe8, are essential for agonism. Noda et al. demonstrated that Asn111 directly interacts with the Tyr4 side chain of AngII. A decrease in the size of residue side chain at position 111 induces constitutively activated AT1 receptor conformation (R00 ), that is, the Asn111 to Gly substitution in AT1 receptor generates constitutive activation (Fig. 6.2). Expression of this CAM receptor produces increased basal production of IP and AngII addition increases IP production to levels similar to the AngII-activated state [R*] of the wild-type AT1 receptor. Cells stably expressing the CAM AT1 receptor show elevated levels of inositol phosphates and frequent spontaneous intracellular Ca++ oscillations. The Ca(2+) transients triggered with maximal doses of AngII were much weaker in

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RK I GDEAS SN L I M Q I D D S S C 18 P A K F H C S Y E A S RDCR V Q R I G T I I N S R NT RW P G F S N H Y L G T I E N N E Q Y H L Y I L FF 195 I L F CK A M V P VM T Y V D I N A I 101 RH I G L F VA P S A 256 I T L L G 259 T WL S I F I AP 111 L V S Y S PL T Q 199 K I I L S FN H T N P L L AL L I L FV I W Y A F C 112 G V GA S F 200 F G I S LD L V F L F F F 293 F G L A 77 L W I P LA F NS L F L L V I T C 118 L N L I I C I A 74 L I VV L L T L T S I M I I V VK 245 F D S I K VS A I Y T R V Y F F 126 A Y L 125 L V I D L A YM I I DN T K L K VH M W R T P M K R P K S R A R L K L N K K Q K A YE I

274 I

A D I V D TA

Extracellular

MP I T I

C I A Y F N N C L NP L F Y

294 295

G F L G K K F K

Intracellular 305

I P I F Q P Y Y L K K K R A K S H S L N L S T K E V E F C P A P K K T S S S V N D S P R Y S L T SM

CAMs identified by genetic screening CAMs identified by independent reports AngII interacting residues

Figure 6.1 A random mutated library of the AT1 receptor cDNA was screened for constitutive activity by Parnot et al. (2000), who identified several additional mutations. These included not only Asn111 but also mutations of TM2 (Phe77), TM3 (Leu112 and Leu118), TM5 (Leu195), TM6 (Ile245), and TM7 (Leu305). In general, all these CAMs have a modest constitutive activity compared to the N111G mutant. Synergy between different CAMs has been evaluated in order to increase the basal activity. Combining the strongest CAM, that is, N111G, with L305Q and I245T did not further enhance basal activation. However, the combination of N111G and F77A mutations results in an almost fully active CAM receptor (Miura et al., 2008).

Wild type

Response %

100

CAM FA

FA PA

[R*]

NA

[R≤]

PA 50

[R¢]

NA IA 0

Ligand concentration

IA

[R]

Ligand concentration

Figure 6.2 Activation/inhibition of G protein signaling response of the wild-type and CAM mutant AT1 receptor by full agonist (FA), partial agonist (PA), neutral antagonist (NA), and inverse agonist (IA) ligands. Activity-based signaling states indicated are the inactive state of the receptor [R], basal active state of wild-type AT1 receptor [R0 ], basal active state of CAM AT1 receptor [R00 ], and the fully activated state [R*]. The equilibrium between various signaling states is a function of constraint, and the constraint is transiently relaxed by agonists and nontransiently by CAM mutations.

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CAM expressing cells than in wild-type cells because of downregulation of inositol 1,4,5-trisphosphate receptor (IP3R) in cells (Auger-Messier et al., 2004). Constitutive activation is reversed by ARBs, such as candesartan and EXP3174, which can therefore be defined as inverse agonists (Fig. 6.2). Finally, partial agonist peptides become full agonists at this CAM receptor (Fig. 6.2). In further studies, these authors designed several ligands that bound and did not activate wild-type AT1 receptor but fully activated the CAM. Noda et al. concluded that spontaneous occurrence of R00 and R* states is rare in the wild-type AT1 receptor and the Asn111 to Gly mutation increases at least 10-fold the probability of occurrence of R00 and R* states. In concordant studies, constitutive activation of the Asn111 mutants of AT1 receptor was further established (Balmforth, Lee, Warburton, Donnelly, & Ball, 1997; Groblewski et al., 1997; Feng, Miura, Husain, & Karnik, 1998; Miura, Feng, Husain, & Karnik, 1999; Auger-Messier et al., 2003). Furthermore, Auger-Messier had demonstrated that substitution of larger side chains for Asn111 in the AT1 receptor produces a receptor that is difficult to activate (Auger-Messier et al., 2003). Several other mutations of the AT1 receptor are reported to produce weak constitutive activation (see Fig. 6.1). These mutations include Asn295 in TM7 and Asp125 in TM3. A random mutated library of the AT1 receptor cDNA was screened for constitutive activity by Parnot et al. (2000), who identified several additional mutations. These included not only Asn111 but also mutations of TM2 (Phe77), TM3 (Leu112 and Leu118), TM5 (Leu195), TM6 (Ile245), and TM7 (Leu305) (see Fig. 6.1). In general, all these CAMs have a modest constitutive activity compared to the N111G mutant. Synergy between different CAMs has been evaluated in order to increase the basal activity. Combining the strongest CAM, that is, N111G, with L305Q and I245T did not further enhance basal activation. However, the combination of N111G and F77A mutations results in an almost fully active CAM receptor (Miura et al., 2008).

3. MECHANISM OF CONSTITUTIVE ACTIVATION IN AT1 RECEPTOR GPCR activation is a complex mechanism that has two aspects: first, the constraints that govern the inactive state conformation are released, and, second, new interactions that stabilize the active state are formed. Generally, it is believed that most CAMs disrupt the constraints for the inactive state conformation. New interactions forming the activated state conformation

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are not fully mimicked in most CAMs. As a result, signaling, phosphorylation, internalization, and desensitization processes activated by a CAM may partly overlap those by agonist activation of the wild-type receptor. The nature of constraint is best illustrated by the conserved D/ERY motif at the cytoplasmic end of TM3. The constitutive activity that results from mutating the Arg residue of this motif has suggested two potential mechanisms. First, mutation disrupts the constraining interaction with a polar pocket formed by residues in TM1, 2, and 7, and second, mutation disrupts an ionic interaction with a glutamate in TM6. The second model is in better agreement with the structure of the inactive conformation of rhodopsin and was confirmed in b2-adrenergic receptor (Rosenbaum, Rasmussen, & Kobilka, 2009). Thus, constraint represents conserved interhelical interactions that restrict entropy of helices in a GPCR. In the AT1 receptor, this concept is very well elucidated by mutation of Asn111 in TM3 helix. Smaller side chain substitution resulted in constitutive activation, suggesting that the gain-of-function phenotype resulted from the loss of interhelical interactions. Feng et al. (1998) and Nikiforovich, Mihalik, Catt, and Marshall (2005) demonstrated that gain-of-function phenotype also resulted when bulkier residues were introduced in TM3 without mutating Asn111. Increased dynamic conformation of TM helices in the N111G mutant has been systematically mapped (Domazet et al., 2009; Miura & Karnik, 2002; Miura, Zhang, Boros, & Karnik, 2003; Yan et al., 2010). The conformational dynamic change associated with CAMs alters the kinetics of G protein coupling, GRK-mediated receptor phosphorylation, recruitment of b-arrestin, internalization, and recycling of CAM receptors, suggesting a strong G protein-biased signaling induced by CAM receptors (Fig. 6.3). Lee et al. (2007) showed that AngII-bound conformations of the wild-type and the CAM-AT1 receptor are different with regard to coupling preferences to Gq and b-arrestin 1. The wild-type AT1 receptor activates G protein and recruits b-arrestin 1. In contrast, the N111G mutant AT1 receptor preferentially couples to Gq and is inadequate in b-arrestin 1 recruitment. The Ca2+ mobilization by AngII-activated wild-type AT1 receptor was completely blocked by the coexpression of b-arrestin 1, whereas Ca2+ mobilization by AngII-activated N111G-AT1 receptor was not completely abolished by coexpressed b-arrestin 1. This study shows that AngII-activated N111G-AT1 receptor is deficient in recruiting b-arrestin 1 and the CAM preferentially associates with the G protein, Gq. These results are consistent with previous study of Auger-Messier et al. (2003) who showed that CAMs of AT1 receptor exhibit a high-affinity conformation

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Figure 6.3 Schematic representation of signaling and recycling of the wild-type and CAM mutant AT1 receptor. Reversible arrows indicate the relative difference of the recycling state favored by the wild-type and CAM mutant. Thickness of the signaling arrows indicates bias of the CAM mutant AT1 receptor for G protein signaling and rapid intracellular recycling.

that allows more efficient coupling to the G protein. The G protein bias of AT1 receptor CAM is reflected in differences in desensitization and recycling kinetics as well. For instance, agonist-activated wild-type AT1 receptors are rapidly internalized (5–10 min) and slowly recycled (2–3 h) favoring intracellular accumulation. Internalization of the AT1 receptor CAM is partly constitutive and can be robustly promoted by the agonist, leading to preferential intracellular accumulation (Miserey-Lenkei, Parnot, Bardin, Corvol, & Clauser, 2002; Thomas, Qian, Chang, & Karnik, 2000). Treatment with inverse agonists increases cell surface receptors without affecting the total receptor expression, a phenomenon described as “externalization” of CAM receptors. The constitutive internalization process resumes a few minutes after inverse agonist removal. CAMs are thus constitutively recycling from the membrane to the cytosol and vice versa (Bhuiyan & Nagatomo, 2010; Miserey-Lenkei et al., 2002). Altogether, these data show that CAMs of the AT1 receptor do not recapitulate the desensitization process of the ligand-activated wild-type AT1 receptors.

4. INVERSE AGONISTS AND PARTIAL AGONISTS OF AT1 RECEPTOR The N111G mutant AT1 receptor has been a valuable tool in the pharmacological characterization of peptide and the nonpeptide ARBs.

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Le et al. (2003) showed that the ARBs dissociated slowly and behaved as insurmountable antagonist for the wild-type AT1 receptor. In the N111G mutant, a different degree of inverse agonist efficacy was observed for different ARBs, for instance, candesartan > EXP3174 > irbesartan > losartan. However, all ARBs dissociated from the N111G mutant readily, demonstrating that insurmountable property of the ARBs is independent of inverse agonist potential. Most importantly, the availability of CAM mutants of AT1 receptor allowed comparative assessment of efficacy of ARBs in both cell culture and in vivo animal models.

5. CONSTITUTIVE ACTIVITY OF AT1 RECEPTOR IN VIVO Although ex vivo cell culture studies have suggested that the hAT1 receptor inherently possesses spontaneous constitutive activity, whether hAT1 receptor has innate constitutive activity in the complete absence of AngII in vivo has been a question for years since the cloning of the receptor. Activating mutations of the AT1 receptor gene in humans have also not been identified. To elucidate the AngII-independent AT1 receptor activation in vivo, Yasuda et al. (2012) generated hybrid mice overexpressing wildtype hAT1 receptor (200-fold) under the control of a-myosin heavy chain promoter with the angiotensinogen (Agt)-knockout mice. The Agtdeficient parental mice totally lack both circulating and locally produced AngII. The AT1R-TG parental mice overexpressing the hAT1 receptor were shown to induce cardiac remodeling in the presence of endogenous levels of AngII that could be prevented by treatment with the ARB losartan. The wild-type hAT1 receptor overexpressed in the hybrid mice causes systolic dysfunction, progressive chamber dilatation, contractile dysfunction, and interstitial fibrosis compared with normal cardiac structure and function in parental Agt-null mice. The endogenous AT1 receptor did not produce pathology in heart and other tissues in the Agt-null mice. The enhancement of constitutive activity in the hybrid mice hearts is proportional to overexpression of the native hAT1 receptor and treatment with ARBs prevented the pathogenesis. Creation of AT1R-TG/Agt-null hybrids allowed the authors to unequivocally demonstrate the constitutive activity of hAT1 receptor in the hearts of mice in vivo, which till then was shown only in cultured cells. Constitutive activation of hAT1 receptor in the heart tissue was documented by significantly increased distribution of Gq/11 in the cytosol and phosphorylation of extracellular signal-regulated kinases in AT1R-Tg-AgtKO hearts

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compared with each of the parental controls. These molecular changes were associated with pathogenesis. Both activation of G protein signaling and progressive cardiac remodeling was prevented by the AT1 receptor inverse agonist, candesartan. Thus, myocardial overexpression of the wild-type hAT1 receptor increases constitutive activity above the threshold required for initiation of cardiac dysfunction and associated with heart failure pathogenesis. Since treatment with candesartan, the inverse agonist for the AT1 receptor, effectively prevented pathogenesis in this model, the inverse agonist activity of ARBs provides clinical advantage of inhibiting both AngII-dependent and AngII-independent effects. Thus, inverse agonist potential is an important pharmacological parameter defining the beneficial effects on organ protection.

6. CONSTITUTIVE ACTIVATION OF AT1 RECEPTOR AND PATHOPHYSIOLOGY 6.1. Autoantibody activation Allosteric actions of agonistic autoantibodies influence the basal constitutive activity of a GPCR. Autoantibodies for GPCRs alter the conformation of primary ligand binding site indirectly; thus, they mimic some aspects of CAM receptor signaling. In vivo, the combination of an autoantibody and the agonist can display characteristics of a constitutively active receptor (Unal, Jagannathan, & Karnik, 2012). Agonistic autoantibodies for the human AT1 receptor in patients undergoing renal transplantation were shown to lead to allograft rejection. Likewise, diseases such as preeclampsia and vascular allograft rejection are caused by autoantibodies against hAT1 receptor. The AT1 receptor-mediated pathway that contributes to vascular rejection and preeclampsia is activated by IgG1 and IgG3 subclass antibodies (Dragun et al., 2005). Passive antibody transfer induced vasculopathy and hypertension in rodent kidney transplant and pregnancy models (Dechend et al., 2000, 2003; Dragun et al., 2005; Xia, Ramin, & Kellems, 2007; Zhou et al., 2008). Empirical evidence suggests that the autoantibody induces AT1 receptor activation without AngII; however, the mechanism of antibody-mediated receptor activation is not known. Unal, Jagannathan, Bhat, and Karnik (2010) showed that the conformational dynamics of the extracellular loop 2 (ECL2) of the AT1 receptor generates the epitope for AT1 receptor autoantibodies. This allows the antibody to bind and stabilize the activated state of AT1 receptor. Epitope mapping studies in human AT1 receptor have indicated that autoantibodies from preeclampsia patients bind to the epitope “AFHYESQ” (Dragun et al., 2005;

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Zhou et al., 2008), and those from patients with malignant hypertension and refractory, vascular allograft rejection recognize epitopes “ENTNIT” and “AFHYESQ.” Autoantibody binding to ECL2-epitopes may exert strain to activate the AT1 receptor directly, thus providing a molecular basis for the autoantibody action. The affected patients actually benefit from pharmacologic blockade of AT1 receptors. Losartan binding masks both epitopes, providing the basis for protection of patients harboring AT1 receptordirected autoantibodies. Autoantibodies have been identified for the many GPCRs (Unal et al., 2012). Most of the autoantibodies recognize epitopes on the ECL2 of the receptor structure. They appear to influence receptor activity often and can cause pathologies such as neuroendocrine disorders, cardiomyopathy, and Chagas’ disease. These autoimmune disorders can be counteracted with inverse agonist treatments as has been evaluated in AT1 receptor pathologies.

6.2. Stretch activation Mechanical stress has been shown to be a novel mechanism that activates AT1 receptor independent of AngII (Yasuda, Akazawa, Qin, Zou, & Komuro, 2008; Zou et al., 2004). Mechanical stress is the primary stimulus for cardiac hypertrophy due to increased afterload in vivo, in isolated heart Langendorff preparations, and also when cardiomyocytes in culture are subjected to passive stretch. Pretreatment of cardiomyocytes with ARBs significantly attenuated hypertrophic, responses induced by stretching (Sadoshima, Xu, Slayter, & Izumo, 1993; Yamazaki et al., 1995). Mechanical stress can allosterically increase constitutive activation of the AT1 receptor. Stretch-insensitive cells, such as HEK293, gain stretch-sensitive ERK1/2 activation when transfected with AT1 receptor, which is selectively blocked by ARBs such as candesartan. Besides AT1 receptor, the bradykinin B2 receptor in endothelial cells has been shown to be stretchsensitive (Chachisvilis, Zhang, & Frangos, 2006). Constitutive activation of GPCRs by mechanical stretch is not a general phenomenon but specific to some GPCRs including the AT1 receptor.

7. CAM AT1 RECEPTORS AS RESEARCH TOOLS 7.1. Angiotensinergic activation of vascular endothelium Ramchandran et al. (2006) generated transgenic mice targeting expression of the constitutively active N111G mutant AT1 receptor only in vascular

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endothelium by using the endothelial-specific Tie-1 promoter. Functional AT1 receptors are present on endothelial cells, but their overall importance in physiology is unclear. Demonstrating whether endothelial cell activation by AngII modulates contraction of vascular smooth muscle cells in the vasculature is a technically challenging problem because of the low density of AT1 receptor in endothelial cells and the relative dominance of AT1 receptor in smooth muscle cells in the vessels. Furthermore, the currently available AT1 receptor antagonists do not discriminate AT1 receptor in endothelial and smooth muscle cells. Classical AngII-infusion studies would affect both smooth muscle cells and endothelial cells without selectivity. By directing transgenic overexpression of the constitutively active N111G mutant AT1 receptor, they were able to selectively mimic angiotensinergic activation of only the endothelial cells. Their results demonstrated that actions of AT1 receptor on endothelium in vivo contribute to steady-state blood pressure regulation and its impairment may cause hypertension (Table 6.1). The enhanced angiotensinergic signal in the endothelium in these transgenic mice resulted in hypotension and bradycardia. The pressor response of carotid artery to acute infusion of AngII was significantly reduced. Expression of endothelial nitric oxide synthase was increased in the endothelial cells. As a result, the production of hypotensive mediators, nitric oxide and cyclic guanosine monophosphate, was increased in the blood of the mice causing the phenotypes. Hypotension and bradycardia observed in the TG mice could be rescued by treatment with an AT1 receptor-selective inverse agonist. These results imply that the AngII action through the AT1 receptor on endothelial cells is antagonistic to vasoconstriction in general and this action moderates the magnitude of functional response to AngII in the smooth muscle cells. This control mechanism in vivo most likely is a determinant of altered hemodynamic regulation involved in endothelial dysfunction in hypertensive cardiovascular disease.

7.2. Low-renin hypertension model Billet et al. (2007) created the mouse AT1a receptor gene knockin model of the constitutively active N111S mutant with a C-terminal deletion. The C-terminal deletion reduces receptor internalization and desensitization. Thus, the effective signaling by the CAM receptor on the plasma membrane increased; at the same time, the cellular sequestration or toxicity of the CAM receptor in vivo is minimal. This is a unique model system for evaluating the role of the renin–angiotensin system. Until the publication of this work,

Table 6.1 Regulation of function and pathology by localized AT1 receptor activity Tissue function Overexpression pathology

Vascular endothelium (Ramchandran Moderate blood vessel contraction, et al., 2006) contribute to steady-state blood pressure regulation

– Hypotension and bradycardia – Reduced pressor response to AngII infusion – Increased expression of endothelial nitric oxide synthase – Increased production of hypotensive mediators, nitric oxide and cyclic guanosine monophosphate

Moderate and protracted increase of System-wide: liver, heart, kidney, adrenal gland, and aorta (Billet et al., AT1 function in the whole body, change in blood pressure and 2007) aldosterone/renin ratio

– – – – – – –

Rostral ventrolateral medulla (Allen et al., 2006)

Modulates the activity of adjacent neurons to change blood pressure

– Increase in blood pressure – Increase in sympathetic vasomotor tone

Renal proximal tubules (Li et al., 2011)

Regulates systemic blood pressure, without activating the circulating renin angiotensin system

– Increase in baseline blood pressure – No difference in the blood pressure in response to AngII infusion

Cardiac myocytes (Ainscough et al., 2009)

In adult myocardium, AT1 receptor – Adverse ventricular remodeling – Increased interstitial fibrosis, dilatation of the left expression levels promote ventricle, and impaired cardiac function compensatory hypertrophy with normal function, whereas increased – Dilatation, reduced ejection fraction, and increased fibrosis in the absence of change in systemic blood ligand stimulation induces cardiac pressure dysfunction

Increased pressor response to AngII infusion Increase in blood pressure Renal fibrosis and cardiac fibrosis Diastolic dysfunction No cardiac hypertrophy Low renin and aldosterone levels Decreased baroreflex

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physiological contributions of renin–angiotensin system have been investigated by overexpression and gene knockout of its components in animals. Because most disease states arise due to moderate but protracted change in gene function, data elucidating the effect of the constitutive activation of a component of the RAS would imitate progression of disease in vivo. The homozygous mice expressing this CAM receptor recapitulate characteristics anticipated from in vitro studies. The pressor response to infused AngII is more sensitive and longer-lasting. A moderate and stable increase in BP, 20 mmHg, was observed for these mice. Most importantly, these mice develop early and progressive renal fibrosis and cardiac fibrosis and diastolic dysfunction, but there is no significant cardiac hypertrophy in these mice. The renin production is unusually low and aldosterone levels are normal in the homozygous mice. This phenotype, together with the hypersensitivity to AngII infusion, resembles what is classically described in primary hyperaldosteronism, in which there is an abnormal aldosterone/renin ratio. Thus, the new mouse model demonstrated that cardiac and renal fibrosis may occur, perhaps by local action of the AT1a receptor in the tissue, without a severe increase of BP. This mouse model will be useful for investigating the role of AngII in target organs similar to some forms of human hypertension (Table 6.1). In a follow-up study, this hypertensive model was further characterized for neuromodulation of the increased blood pressure and heart rate (PalmaRigo et al., 2010). The authors reported a decreased baroreflex in the CAM AT1 receptor knockin mice compared with normal mice. It is likely that the blood pressure elevation of the mutant mice results from the amplification of the effects of AngII at different central and peripheral sites.

7.3. Hypersympathetic vasomotor tone model Allen et al. (2006) used adenoviruses encoding either the wild-type AT1a receptor or the CAM mutant N111G-AT1a receptor to determine the effect of sustained increases in AT1a receptor density and activity in rostral ventrolateral medulla of Wistar-Kyoto (WKY) rats. The AT1a receptors are expressed within the rostral ventrolateral medulla, and microinjections of AngII into this region increase sympathetic vasomotor tone, which is inhibited by inverse agonists of the receptor. Adenoviral infection was limited to the rostral ventrolateral medulla and receptor expression was sustained for
10 days. The transgene was also expressed in glia but not in neurons of the rostral ventrolateral medulla. Rats receiving the wild-type

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AT1a receptor showed no change in blood pressure. In contrast, rats injected with the CAM receptor displayed an increase in blood pressure for 3–4 days and returned to basal levels. Thus, an increased AT1a receptor activity is a primary determinant of efferent drive from rostral ventrolateral medulla. The authors concluded, most importantly, that constitutive receptor signaling in glia of the rostral ventrolateral medulla not the neurons modulates the activity of adjacent neurons to change blood pressure (Table 6.1). The capacity to “switch-on” the renin–angiotensin system by delivering a constitutive receptor demonstrated in this study is important. This experimental paradigm is useful to examine whether the blood pressure responses to dehydration or hypertonic saline are enhanced. Together, these results demonstrate that adenoviral-induced expression of CAM transgenes is a powerful means of assessing our understanding of local renin–angiotensin system activation in vivo as well as homeostatic mechanisms that spring to reset regulation of blood pressure.

7.4. Renal proximal tubular AT1 receptor hyperactivity model Li et al. (2011) hypothesized that modulating the action of locally in the renal proximal tubules would alter systemic blood pressure. They used a proximal tubule-specific, androgen-dependent, promoter construct (KAP2) to drive overexpression of the CAM mutant N111G-AT1 receptor transgene. Androgen administration of female transgenic mice caused a robust induction of the transgene in the kidney and increased baseline blood pressure. The circulating renin–angiotensin system activity was unaltered in the transgenic mice. In addition, there was no difference in the blood pressure response to infused dose of AngII (Table 6.1). These data provide evidence that local activation of AT1 receptor in the renal proximal tubule is a regulator of systemic blood pressure, without activating the circulating renin– angiotensin system.

7.5. Adult cardiac myocyte-specific AT1 receptor hyperactivity induction model Although cardiac hypertrophy is an established independent risk factor in humans, cardiac hypertrophy and pathological remodeling in response to local RAS activity within adult myocardium are not distinguished from those caused by systemic pressure overload. Ainscough et al. (2009) generated a doxycycline-inducible N111G transgenic mouse model, which

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expresses the CAM receptor in cardiac myocytes when doxycycline is removed from drinking water in the adult mice. Transgenic mice expressing wild-type or the CAM AT1 receptor were compared after 4 weeks of doxycycline induction. In this study, the authors exploited AngIV, a partial agonist of wild type but a full agonist of the CAM AT1 receptor to investigate the myocardial growth, remodeling, and functional responses to AT1 receptor stimulation, specifically in adult cardiomyocytes, under normal conditions following AngIV infusion. The expression of wild-type or N111G-AT1 receptor induced from the onset of adolescence produced enhanced myocyte growth and associated with cardiac hypertrophy in the adult. This was not associated with change in blood pressure or heart rate and did not progress to pathological remodeling or heart failure. However, selective activation of CAM receptors by AngIV peptide infusion induced adverse ventricular remodeling within 4 weeks. This was characterized by increased interstitial fibrosis, dilatation of the left ventricle, and impaired cardiac function (Table 6.1). This study demonstrated that local AT1 receptor activity in adult myocardium promotes compensated cardiac hypertrophy with normal function, whereas short-term increased ligand stimulation induces cardiac dysfunction with dilatation, reduced ejection fraction, and increased fibrosis in the absence of change in systemic blood pressure.

8. CONCLUSION Thus, the CAM AT1 receptors have been extremely useful and powerful in establishing local RAS activity in different tissue pathogenesis. These studies established that controlled upregulation of local AT1 receptor activity mimics various in vivo disease conditions. Tissue- or cell-specific activation of the renin–angiotensin system can have effects on systemic arterial pressure without altering the levels of circulating angiotensin peptides. In classical models of endocrine regulation, abnormal change in the efficacy or level of the hormone is thought to cause pathology. With regard to pathologies of the RAS, the focus of therapeutic strategies has been on controlling circulating and local AngII levels. Upregulation of AT1 receptor in stressed hearts and vessels in response to various hormones, cytokines, inflammation, or metabolic stress could proportionally enhance constitutive activity of the AT1 receptor and accelerate the progression of disease in these tissues. Clinically, the angiotensin-converting enzyme (ACE) inhibitors and ARBs share many of the same clinical benefits on the treatment of

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hypertension and heart failure. Benefits beyond lowering blood pressure are anticipated by blockade of constitutive activity of the receptor directly through inverse agonists of AT1 receptor. ARBs do exert additional cardiovascular benefits resulting in vasodilation, growth inhibition, and nitric oxide production through their effects on endothelial function, oxidative stress and antioxidant properties, platelet function, and ventricular remodeling. Benefits from ACE inhibitors may occur by different mechanism, due to their effects through tissue remodeling. With regard to side effect profiles, ARBs are better tolerated than ACE inhibitors (Chrysant, Chrysant, Chrysant, & Shiraz, 2010; Probstfield & O’Brien, 2010). In general, the inverse agonists are even better therapeutics than neutral antagonists in treating diseases caused by genetic variations and constitutively activating mutations of GPCRs. Although a hormone-negative condition in vivo may never be the cause of many cardiovascular diseases, the importance of constitutive activity of a native GPCR in disease pathogenesis is a real possibility to consider in selecting treatment strategy. Transgenic mice expressing CAM GPCRs have been developed as animal models of human diseases. These models will be useful research tools for discovering and evaluating comparative potencies of inverse agonists.

CONFLICT OF INTEREST The authors have no conflicts of interest to declare.

ACKNOWLEDGMENTS This work was supported in part by National Institutes of Health RO1 Grant HL57470 (to S. K.) and National Research Service Award HL007914 (to H. U.).

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