Functional assay for assessment of agonistic or antagonistic activity of angiotensin AT2 receptor ligands reveals that EMA401 and PD123319 have agonistic properties.

With the discovery of the protective arm of the renin-angiotensin system (RAS), interest has grown in protective RAS-related receptors such as the angiotensin AT2-receptor [AT2R] as potential new drug targets. While it is known that AT2R couple to Gi, it is also apparent that they do not signal via inhibition of adenylyl cyclase/decrease in cAMP, as do many Gi-coupled receptors. Thus, standard commercially-available assays cannot be applied to test for agonistic or antagonistic properties of AT2R ligands. This lack of standard assays has hampered the development of new drugs targeting the AT2R. Therefore, we aimed at developing a reliable, technically easy assay for the determination of intrinsic activity of AT2R ligands, primarily for distinguishing between AT2R agonists and antagonists. We found that measurement of NO release by DAF-FM fluorescence in primary human aortic endothelial cells (HAEC) or in AT2R-transfected CHO cells is a reliable assay for the characterization of AT2R ligands. While testing the assay, we made several novel findings, including: a) C21 is a full agonist at the AT2R (with the same efficacy as angiotensin II); b) C21 has no intrinsic activity at the receptor Mas; c) AT2R-transfected HEK-293 cells are unresponsive to AT2R stimulation; d) EMA401 and PD123319, which are commonly regarded as AT2R antagonists, are partial agonists at the AT2R. Collectively, we have developed and tested an assay based on the measurement and quantification of NO release in HAEC or in AT2R-CHO cells that is suitable for the characterisation of novel and established AT2R ligands.

Investigating the Bioactive Conformation of Angiotensin II Using Markov State Modeling Revisited with Web-Scale Clustering.

Molecular dynamics simulation is a powerful technique for studying the structure and dynamics of biomolecules in atomic-level detail by sampling their various conformations in real time. Because of the long timescales that need to be sampled to study biomolecular processes and the big and complex nature of the corresponding data, relevant analyses of important biophysical phenomena are challenging. Clustering and Markov state models (MSMs) are efficient computational techniques that can be used to extract dominant conformational states and to connect those with kinetic information. In this work, we perform Molecular Dynamics simulations to investigate the free energy landscape of Angiotensin II (AngII) in order to unravel its bioactive conformations using different clustering techniques and Markov state modeling. AngII is an octapeptide hormone, which binds to the AT1 transmembrane receptor, and plays a vital role in the regulation of blood pressure, conservation of total blood volume, and salt homeostasis. To mimic the water-membrane interface as AngII approaches the AT1 receptor and to compare our findings with available experimental results, the simulations were performed in water as well as in water-ethanol mixtures. Our results show that in the water-ethanol environment, AngII adopts more compact U-shaped (folded) conformations than in water, which resembles its structure when bound to the AT1 receptor. For clustering of the conformations, we validate the efficiency of an inverted-quantized k-means algorithm, as a fast approximate clustering technique for web-scale data (millions of points into thousands or millions of clusters) compared to k-means, on data from trajectories of molecular dynamics simulations with reasonable trade-offs between time and accuracy. Finally, we extract MSMs using various clustering techniques for the generation of microstates and macrostates and for the selection of the macrostate representatives.

Activation pathway of a G protein-coupled receptor uncovers conformational intermediates as targets for allosteric drug design.

G protein-coupled receptors (GPCRs) are the most common proteins targeted by approved drugs. A complete mechanistic elucidation of large-scale conformational transitions underlying the activation mechanisms of GPCRs is of critical importance for therapeutic drug development. Here, we apply a combined computational and experimental framework integrating extensive molecular dynamics simulations, Markov state models, site-directed mutagenesis, and conformational biosensors to investigate the conformational landscape of the angiotensin II (AngII) type 1 receptor (AT1 receptor) - a prototypical class A GPCR-activation. Our findings suggest a synergistic transition mechanism for AT1 receptor activation. A key intermediate state is identified in the activation pathway, which possesses a cryptic binding site within the intracellular region of the receptor. Mutation of this cryptic site prevents activation of the downstream G protein signaling and ß-arrestin-mediated pathways by the endogenous AngII octapeptide agonist, suggesting an allosteric regulatory mechanism. Together, these findings provide a deeper understanding of AT1 receptor activation at an atomic level and suggest avenues for the design of allosteric AT1 receptor modulators with a broad range of applications in GPCR biology, biophysics, and medicinal chemistry.

Assessment of Pregabalin-Induced Cardiotoxicity in Rats: Mechanistic Role of Angiotensin 1-7.

Pregabalin (PRG) possesses great therapeutic benefits in the treatment of epilepsy, neuropathic pain, and fibromyalgia. However, clinical data have reported incidence or exacerbation of heart failure following PRG administration. Experimental data exploring cardiac alterations and its underlying mechanisms are quite scarce. The aim of the present work was to investigate the effect of PRG on morphometric, echocardiographic, neurohumoral, and histopathological parameters in rats. It was hypothesized that alterations in cardiac renin angiotensin system (RAS) might be involved in PRG-induced cardiotoxicity. To further emphasize the role of RAS in the mechanism of PRG-induced cardiotoxicity, the protective potential of diminazene aceturate (DIZE), an ACE2 activator, was investigated. Results showed 44% decrease in ejection fraction and sevenfold increase in plasma N-terminal pro-brain natriuretic peptide. Histopathological examination also showed prominent vacuolar changes and edema in cardiomyocytes. In addition, PRG significantly increased angiotensin II (Ang II), angiotensin converting enzyme (ACE) and angiotensin II type 1 receptor (AT1R) levels, while decreased angiotensin 1-7 (Ang 1-7), angiotensin converting enzyme 2 (ACE2), and Mas receptor (MasR) cardiac levels. DIZE co-administration showed prominent protection against PRG-induced echocardiographic, neurohumoral, and histopathological alterations in rats. In addition, downregulation of ACE/Ang II/AT1R and upregulation of ACE2/Ang 1-7/MasR axes were noted in DIZE co-treated rats. These findings showed, for the first time, the detailed cardiac deleterious effects of PRG in rats. The underlying pathophysiological mechanism is probably mediated via altered balance between the RAS axes in favor to the ACE/Ang II/AT1R pathway. Accordingly, ACE2 activators might represent promising therapeutic agents for PRG-induced cardiotoxicity.

Planejamento e desenvolvimento de ligantes não peptídicos com atividade agonista enviesada no receptor de angiotensina II tipo 1 empregando-se técnicas de modelagem molecular e ensaios in vitro / Drug design of non-peptidic biased agonists for angiotensin II type 1 receptor using molecular modeling techniques and in vitro tests

A insuficiência cardíaca (IC) é uma síndrome de elevada morbimortalidade, correspondendo a um grave problema de saúde pública. Uma das abordagens terapêuticas para IC consiste no uso de antagonistas do receptor de angiotensina II do tipo 1 (AT1R), conhecidos como sartanas. Estudos apontam que uma nova classe de compostos, os agonistas enviesados, é capaz de induzir a sinalização da via da ß-arrestina sem ativação da via da proteína G. Essa seletividade funcional é particularmente interessante, pois a via dependente da proteína G é responsável pelo aumento da pressão arterial, morte celular e fibrose tecidual, levando a hipertrofia cardíaca e progressão da IC. No entanto, a via da ß-arrestina está associada com renovação celular e aumento do inotropismo. Além disso, estudos in vivo sugerem que agonistas enviesados poderiam corresponder a uma terapia superior à dos antagonistas convencionais, que bloqueiam ambas as vias. Apesar do potencial terapêutico, esses compostos possuem estrutura peptídica e, por isso, tem sua administração restrita à via intravenosa. A resolução da estrutura cristalográfica do AT1R permitiu estudos de modelagem molecular mais acurados. Tendo isso em mente, nesse trabalho foram propostos agonistas enviesados de natureza não peptídica para o AT1R por meio de técnicas de modelagem molecular e validação das hipóteses levantadas por ensaios in vitro. Foram realizados estudos de dinâmica molecular com o AT1R (PDB ID: 4YAY) em uma bicamada lipídica e ensaios de ancoramento molecular da angiotensina II (AngII) e do ligante enviesado TRV027. As poses de ancoramento molecular selecionadas foram utilizadas em dinâmicas de complexo, que revelaram diferenças entre os sistemas apo (sem nenhum ligante) e holo (com o ligante no sitio de ligação). Nossos resultados sugerem que o TRV027 induz um padrão exclusivo de ligações de hidrogênio e de estrutura secundária, enquanto que a AngII afeta os resíduos do bolso hidrofóbico do sitio de ligação, principalmente a conformação do Trp2536.48. Com base nas simulações, três farmacóforos foram criados e utilizados de maneira complementar em triagens virtuais na base de dados ZINC15, resultando na seleção de cinco compostos. Um desses compostos apresentou afinidade pelo receptor AT1R e, ainda que estudos complementares de ativação de vias especificas sejam necessários para que o composto possa ser classificado como agonista enviesado, já se constitui em molécula potencialmente promissora. Além disso, esses estudos permitiram a proposição de estruturas inéditas que podem vir a ser hits no processo de desenvolvimento de agonistas enviesados para AT1R. Portanto, como continuidade desse trabalho, essas moléculas serão sintetizadas e investigadas quanto à possível interação com o receptor.

Heart Failure (HF) is a common syndrome with high morbimortality, being considered a serious public health problem. One of the therapeutic approaches for HF consists in the use of the sartan class, which are angiotensin II type 1 receptor (AT1R) antagonists. Recent studies have shown that a new class of compounds, known as biased agonists, is able to induce signaling via ß-arrestin without G-protein activation. This functional selectivity is particularly interesting since G-protein dependent signaling is responsible for cell death and cardiac tissue fibrosis, which leads to cardiac muscle hypertophy and HF progression. On the other hand, ß-arrestin signaling is associated with cellular renewal and increased inotropism. In vivo studies suggests that biased agonists could correspond to a superior therapy over conventional angiotensin II type 1 receptor antagonists, which blocks cell signaling as a whole, however their peptidic structure restricts their use to intravenous administration. Moreover, the AT1R crystal structure determination holds great promise for more accurate molecular modeling studies. With that being said, the aim of this work was to plan and develop new non-peptidic biased agonists for ATR1 employing molecular modeling techniques and in vitro tests for hypothesis validation. Molecular dynamics (MD) simulations of the refined AT1R crystal (PDB ID: 4YAY) embedded in a lipid bilayer and molecular docking studies with angiotensin II (AngII) and TRV027 (biased agonist) were conducted. Selected docking poses from both ligands underwent complex MD simulations revealing differences between apo (ligand free) and holo (ligand in the binding site) systems. Our results suggest that TRV027 induces an exclusive hydrogen bond and secondary structure pattern, while AngII affects the hydrophobic pocket conformation, mainly Trp253. Based on the simulations, three pharmacophore models were created and used in virtual screenings in the ZINC15 database, resulting in the selection of five compounds that were tested in vitro. One of the compounds displayed affinity for AT1R and is a promising molecule. Nonetheless, it needs further pathway activation characterization in order to be a classified as a biased agonist. Furthermore, these results have contributed significantly for the proposition of new structures that could be hits with biased agonist activity for AT1R. Thus, for future works, we point out the necessity for synthesis and characterization of this new compounds

Control of Energy Expenditure by AgRP Neurons of the Arcuate Nucleus: Neurocircuitry, Signaling Pathways, and Angiotensin.

PURPOSE OF REVIEW: Here, we review the current understanding of the functional neuroanatomy of neurons expressing Agouti-related peptide (AgRP) and the angiotensin 1A receptor (AT1A) within the arcuate nucleus (ARC) in the control of energy balance. RECENT FINDINGS: The development and maintenance of obesity involves suppression of resting metabolic rate (RMR). RMR control is integrated via AgRP and proopiomelanocortin neurons within the ARC. Their projections to other hypothalamic and extrahypothalamic nuclei contribute to RMR control, though relatively little is known about the contributions of individual projections and the neurotransmitters involved. Recent studies highlight a role for AT1A, localized to AgRP neurons, but the specific function of AT1A within these cells remains unclear. AT1A functions within AgRP neurons to control RMR, but additional work is required to clarify its role within subpopulations of AgRP neurons projecting to distinct second-order nuclei, and the molecular mediators of its signaling within these cells.

Individualized Angiotensin-Converting Enzyme (ACE)-Inhibitor Therapy in Stable Coronary Artery Disease Based on Clinical and Pharmacogenetic Determinants: The PERindopril GENEtic (PERGENE) Risk Model.

BACKGROUND: Patients with stable coronary artery disease (CAD) constitute a heterogeneous group in which the treatment benefits by angiotensin-converting enzyme (ACE)-inhibitor therapy vary between individuals. Our objective was to integrate clinical and pharmacogenetic determinants in an ultimate combined risk prediction model. METHODS AND RESULTS: Clinical, genetic, and outcomes data were used from 8726 stable CAD patients participating in the EUROPA/PERGENE trial of perindopril versus placebo. Multivariable analysis of phenotype data resulted in a clinical risk score (range, 0-21 points). Three single-nucleotide polymorphisms (rs275651 and rs5182 in the angiotensin-II type I-receptor gene and rs12050217 in the bradykinin type I-receptor gene) were used to construct a pharmacogenetic risk score (PGXscore; range, 0-6 points). Seven hundred eighty-five patients (9.0%) experienced the primary endpoint of cardiovascular mortality, nonfatal myocardial infarction or resuscitated cardiac arrest, during 4.2 years of follow-up. Absolute risk reductions ranged from 1.2% to 7.5% in the 73.5% of patients with PGXscore of 0 to 2. As a consequence, estimated annual numbers needed to treat ranged from as low as 29 (clinical risk score ≥10 and PGXscore of 0) to 521 (clinical risk score ≤6 and PGXscore of 2). Furthermore, our data suggest that long-term perindopril prescription in patients with a PGXscore of 0 to 2 is cost-effective. CONCLUSIONS: Both baseline clinical phenotype, as well as genotype determine the efficacy of widely prescribed ACE inhibition in stable CAD. Integration of clinical and pharmacogenetic determinants in a combined risk prediction model demonstrated a very wide range of gradients of absolute treatment benefit.

Avaliação dos eixos ECA / ANG II / AT1 e ECA-2 / ANG (1-7) / MAS em fibroblastos de gengiva e ligamento periodontal humanos estimulados por IL-1ß e sua contribuição na produção e regulação de citocinas e quimiocinas / Evaluation ACE /ANG II /AT1 and ACE-2 /ANG (1-7) /MAS axis in gingival and periodontal ligament human fibroblasts stimulated with IL-1ß and its contribution in the production of cytokines and chemokines

O Sistema renina-angiotensina (SRA) tem sido relatado como um importante modulador de processos inflamatórios e imunológicos, incluindo a doença periodontal (DP). Estudos sugerem neste sistema um eixo alternativo (ECA-2 /ANG(1-7) /MAS) que atuaria como um contra-regulador de efeitos mediados pelo clássico eixo (ECA /ANGII /AT1). Sabe-se que bactérias periodontopatogênicas, como a Porphyromonas gingivalis (Pg), possuem componentes bioativos de membrana (ex. lipopolissacarídeos-LPS) capazes de induzir uma forte resposta imune no hospedeiro devido à liberação de citocinas nas células, entre elas Interleucina (IL)- 1ß. Neste contexto, fibroblastos são as células mais abundantes nos tecidos periodontais e possuem em sua superfície celular receptores necessários para o reconhecimento da invasão bacteriana, ativando cascatas intracelulares, que levam à produção de citocinas. O objetivo deste estudo foi verificar se os eixos ECA/ ANGII/ AT1 e ECA-2/ ANG(1-7)/ MAS contribuem para a produção e/ ou regulação de citocinas inflamatórias (CI) por fibroblastos de gengiva humana (HGF) e ligamento periodontal humano (HPLF) estimulados por IL-1ß. Após o pré-tratamento com Losartan e Ang (1-7) ou silenciamento mediado por RNA de interferência (RNAi) de AT1, HGF e HPLF foram estimulados por IL-1ß por 3 horas (RNAm) ou 24 horas (proteína). Expressão de RNAm para AT1, MAS, ECA, ECA-2, IL-1ß, TNF-α, IL-6, IL-8, IL-10, TGF-ß, CXCL12, RANK-L e OPG foram avaliados por RT-qPCR e das proteínas IL-6, IL-8, ECA e ECA-2 por ELISA. Foi realizado também Western Blot para detecção de AT1 e ECA nos extratos celulares e dosagem de nitrito no sobrenadante das culturas. Ambos os subtipos de fibroblastos mostraram aumento da expressão de RNAm para AT1, IL-1ß, IL-6, IL-8, TNF-α e OPG, quando estimulados por IL-1ß. No entanto, apenas em HPLF foi observado aumento para MAS, ECA e TGF-ß. Losartan e Ang (1-7) não modularam o transcrito, a secreção de CI e nem a produção de nitrito no sobrenadante das culturas, tanto em HGF como em HPLF. O silenciamento do receptor AT1 reduziu a secreção de IL-6 e IL-8 induzida por IL-1ß em cultura de HGF e HPLF e aumentou a expressão gênica de OPG somente em HGF. Estes resultados sugerem que o silenciamento de AT1, mas não o bloqueio farmacológico deste receptor pelo antagonista Losartan, em HGF e HPLF, pode controlar a produção de IL-6 e IL-8, que por sua vez contribuem para a patogênese periodontal.(AU)

The renin-angiotensin system (RAS) has been reported as an important modulator of inflammatory and immune responses, including periodontal disease (PD). Studies suggest an alternative axis as part of this system (ACE-2 / ANG (1-7) / MAS) that would act as counter-regulatory to the classical axis (ECA / ANGII / AT1). It is known that periodontal bacteria such as Porphyromonas gingivalis (Pg) have bioactive components in their membrane (such as lipopolysaccharide-LPS) capable of inducing a strong immune response in the host due to the release of cytokines in cells, including interleukin (IL) - 1ß. In this regard, fibroblasts are the most abundant cells in periodontal tissues and receptors needed for the recognition of bacterial invasion by activating intracellular cascades that lead to cytokine production. The aim of this study was to determine whether the axes ACE / ANGII / AT1 and ACE-2 / ANG (1-7) / MAS contribute to the production and / or regulation of inflammatory cytokines (IC) by fibroblasts of human gingiva (HGF) and human periodontal ligament (HPLF) stimulated IL-1ß. After pre-treatment with Losartan, Ang (1-7) or silencing mediated by RNA interference (RNAi) of AT1, HGF and HPLF were stimulated by IL-1ß for 3 hours (RNAm) or 24 hours (protein). Expression mRNA for AT1, MAS, ACE, ACE-2, IL-1ß, TNF-α, IL-6, IL-8, IL-10, TGF-ß, CXCL12, RANK-L and OPG was assessed by RT- qPCR and proteins IL-6, IL-8, ACE and ACE-2 by ELISA. Western Blot for the detection of AT1 and ECA and dosage of nitrite was also performed. Experiments stimulated by IL-1ß showed a positive control for gene expression AT1, IL-1ß, IL-6, IL-8, TNF-α and OPG in HGF and HPLF and MAS, ACE and TGF-ß only HPLF. Losartan and Ang (1-7) did not modulate the transcription and secretion of IC and no nitrite production in the culture supernatant of HGF and HPLF. The silencing AT1 reduced IL-6 secretion and IL-8 induced by IL- ß in cultured HGF and HPLF and increased OPG gene expression only HGF. These results suggest that silencing AT1, but not pharmacological blockade of this receptor by Losartan in HPLF and HGF, can control the production of IL-6 and IL-8, which in turn contribute to the pathogenesis of periodontal disease.(AU)

Two distinct calmodulin binding sites in the third intracellular loop and carboxyl tail of angiotensin II (AT(1A)) receptor.

In this study, we present data that support the presence of two distinct calmodulin binding sites within the angiotensin II receptor (AT(1A)), at juxtamembrane regions of the N-terminus of the third intracellular loop (i3, amino acids 214-231) and carboxyl tail of the receptor (ct, 302-317). We used bioluminescence resonance energy transfer assays to document interactions of calmodulin with the AT(1A) holo-receptor and GST-fusion protein pull-downs to demonstrate that i3 and ct interact with calmodulin in a Ca²âº-dependent fashion. The former is a 1-12 motif and the latter belongs to 1-5-10 calmodulin binding motif. The apparent Kd of calmodulin for i3 is 177.0±9.1 nM, and for ct is 79.4±7.9 nM as assessed by dansyl-calmodulin fluorescence. Replacement of the tryptophan (W219) for alanine in i3, and phenylalanine (F309 or F313) for alanine in ct reduced their binding affinities for calmodulin, as predicted by computer docking simulations. Exogenously applied calmodulin attenuated interactions between G protein ßγ subunits and i3 and ct, somewhat more so for ct than i3. Mutations W219A, F309A, and F313A did not alter Gßγ binding, but reduced the ability of calmodulin to compete with Gßγ, suggesting that calmodulin and Gßγ have overlapping, but not identical, binding requirements for i3 and ct. Calmodulin interference with the Gßγ binding to i3 and ct regions of the AT(1A) receptor strongly suggests that calmodulin plays critical roles in regulating Gßγ-dependent signaling of the receptor.

Analysis of [11C]methyl-candesartan kinetics in the rat kidney for the assessment of angiotensin II type 1 receptor density in vivo with PET.

INTRODUCTION: Angiotensin II type 1 (AT(1)) receptors play a key role in the regulation of renal and cardiovascular functions and have been implicated in the pathogenesis of many diseases. The aim of this study was to assess binding of the novel radioligand [(11)C]methyl-candesartan to AT(1) receptors in the rat kidney in vivo with PET. METHODS: Dynamic PET images were acquired for 60 min at baseline, with coinjection of candesartan (5 mg/kg), and after injection of PD123,319 (5 mg/kg). Volumes of distribution (R(C)∙V(T)) were estimated with a two-compartment model, by Logan analysis, and by taking the tissue-to-plasma activity ratio at 50-60 min post-injection. RESULTS: The two-compartment model did not describe the kinetics at baseline adequately and the baseline scans were too short to obtain accurate estimates of R(C)∙V(T) with the Logan approach. Based on the tissue-to-plasma ratios, roughly one-third of V(T) at baseline could be attributed to AT(1) receptor binding. There were no indications of AT(2) receptor binding in the rat kidney. CONCLUSION: It may be possible to detect changes in AT(1) receptor density in the rat kidney in vivo with [(11)C]methyl-candesartan and PET. Imaging AT(1) receptors with PET may provide valuable information on the role of these receptors in the pathogenesis of diseases such as hypertension, diabetic nephropathy, ventricular remodeling, and heart failure.

Postpartum assessment of the renin angiotensin system in women with previous severe, early-onset preeclampsia.

CONTEXT: Women with a history of severe preeclampsia are at an increased risk for the development of vascular disease. OBJECTIVE: We hypothesized that abnormalities in the renin-angiotensin system (RAS) may be a predisposing factor. DESIGN AND SETTING: Physiological assessments were conducted at an academic center. PARTICIPANTS: Sixteen women with previous severe preeclampsia (PPE) were compared with nine previously pregnant controls (PPC) and 11 never-pregnant controls (NPC). INTERVENTIONS: Baseline circulating components of the RAS and expression of angiotensin (ANG) II type I (AT1) and type II (AT2) receptors in the skin were assessed along with the response to simulated orthostatic stress using incremental lower-body negative pressure (LBNP: -15, -25, and -40 mm Hg) and a graded ANG II infusion (1 and 3 ng/kg · min). MAIN OUTCOME MEASURES: Response to LBNP and ANG II was evaluated. RESULTS: RAS components were not different between previously pregnant groups, but were decreased compared with NPC subjects. In response to LBNP, there were significant increases in RAS components in all three groups, but the response to this stimulus was significantly lower and delayed in PPE subjects. Despite the blunted rise in circulating RAS mediators in PPE subjects, their blood pressure was maintained in 88% compared with only 33 and 55% in the PPC and NPC groups, respectively (P = 0.014). All three groups responded to the graded ANG II infusion with an increase in blood pressure that was significantly more pronounced in PPE subjects (P = 0.037) correlating with AT1/AT2 receptor expression. CONCLUSIONS: Alterations in the RAS in formerly preeclamptic patients may contribute to future vascular disease.

Assessment of biological activity of novel peptide analogues of angiotensin IV.

OBJECTIVES: Angiotensin IV (Ang IV) is a metabolite of angiotensin II which acts on specific AT(4) receptors identified as the enzyme insulin regulated aminopeptidase (IRAP). The transduction process of these receptors is unresolved, but Ang IV inhibits the aminopeptidase activity. Ang IV improves cognition in animal models thus there is a desire to develop metabolically stable analogues for further development. METHODS: Peptide analogues of Ang IV were obtained commercially or synthesised. Each peptide was tested in vitro for its ability to inhibit the aminopeptidase activity (IRAP) of mouse brain homogenates and for its effects on isolated rat uterine smooth muscle. KEY FINDINGS: [Des-Val(1) ]-Ang IV, acetylated-Ang IV-amide, Ang IV-amide and [des-His(4) ]-Ang IV all inhibited IRAP. [Sar(1) , Ile(8) ]-Angiotensin II (10 µm) had an effect greater than that of Ang IV or any of the other analogues studied. In isolated uterine smooth muscle, angiotensins II and IV induced contractions, which could be antagonised by an AT(1) -receptor antagonist. None of the novel peptides induced uterine smooth muscle contractions, but [Sar(1) , des Arg(2) -Gly(8) ]-angiotensin II showed significant antagonism of the contractile effects of angiotensin II and carboxyamide-terminated Ang IV-NH(2) showed antagonism of Ang IV-induced contractions. CONCLUSIONS: This study provides five novel inhibitors of IRAP worthy of assessment in behavioural models of learning and memory. The analogues are devoid of AT(1) receptor agonist properties, and the carboxyamide analogue presents an opportunity to elucidate the mechanism of action of Ang IV as, like Ang IV, it inhibits IRAP, but antagonises the effects of Ang IV on isolated smooth muscle.

Assessment of inverse agonism for the angiotensin II type 1 receptor.

The angiotensin II (AngII) type 1 (AT1) receptor is a seven-transmembrane G-protein-coupled receptor that plays a regulatory role in the physiological and pathological processes of the cardiovascular system. AT1 receptor inherently shows constitutive activity even in the absence of AngII, and it is activated not only by AngII but also by AngII-independent mechanisms. Especially, mechanical stress induces cardiac hypertrophy through activation of AT1 receptor without the involvement of AngII. These AngII-independent activities of AT1 receptor can be inhibited by inverse agonists, but not by neutral antagonists. In this chapter, we describe the methods used for biochemical assessment of inverse agonism of a ligand for AT1 receptor. Their applications will improve our understanding of receptor activation and inactivation at a molecular level, and contribute to the development of AT1 receptor blockers possessing superior therapeutic efficacy in cardiovascular diseases.

Assessment of homologous internalization of constitutively active N111G mutant of AT(1) receptor.

Constitutively active mutants (CAMs) of G-protein-coupled receptors mimic the active conformation of the receptor in their ability to activate second messenger systems in the absence of agonist. They have revealed novel properties of drugs that reverse the basal levels of constitutive activity, indicating that the drugs have the inverse agonist activity. Internalization plays an important role in receptor endocytosis and signal transduction. The present chapter provides the investigation of the internalization behavior of CAM N111G of Angiotensin II type 1 (AT(1)) receptor and correlates the result with the mechanism of constitutive activity of the mutant. Both wild-type (WT) and N111G mutant receptors were transiently expressed in COS-7 cells and total inositol phosphate production was measured in presence and absence of the angiotensin II receptor blockers (ARBs). The binding affinities toward agonist and ARBs were also determined. We found that the ARBs have the inverse agonist activity in CAM N111G of AT(1) receptor. The internalization of the mutant, which was much lower than WT receptor, was significantly increased in presence of the ARBs. The results indicate that internalization of CAM N111G of AT(1) receptor is induced by the ARBs, which may be an important characteristic of inverse agonist activities of the ARBs in N111G.

Fixed-dose combination and chronic kidney disease progression: which is the best?

PURPOSE OF REVIEW: Use of combination therapy whether fixed dose or separate pill combinations is becoming more prevalent. Physicians are not routinely trained in using combinations of different antihypertensive medicines. RECENT FINDINGS: Recent outcome trials as well as smaller studies document that meaningful combinations of pharmacologically additive agents help achieve blood pressure (BP) goals faster and reduce outcomes. Clinical trials demonstrate that drugs interfering with the renin-angiotensin system, that is, angiotensin-converting enzyme inhibitors and angiotensin receptor blockers, combined with either calcium antagonists or thiazide-like diuretics and used as first-line antihypertensive therapy in patients whose BP is more than 20/10 mmHg above goal achieve BP goal faster than sequential monotherapy. Additionally, certain combinations may provide better cardiovascular outcomes than other combinations. Lastly, combination therapy has a clear role in helping to reduce cardiovascular and renal risk, when used. SUMMARY: This review provides an update of current evidence regarding the associations of BP control with use of combination antihypertensive therapy to achieve BP goals. A rationale for initial single pill and fixed-dose combination therapy is reviewed. Only one trial with cardiovascular outcome has been performed using a fixed-dose combination and this revealed differences from other trials, including the greatest number of people achieving goal BP and difference in outcomes based on combination used.

Association of A1166C polymorphism in AT(1) receptor gene with baroreflex sensitivity.

The aim of this study was to evaluate the association of A1166C polymorphism in angiotensin II type 1 receptor (AT(1)R) gene with baroreflex sensitivity (BRS in ms/mm Hg; BRSf in mHz/mm Hg) in man. BRS and BRSf were determined by a spectral method in 135 subjects (19-26 years) at a frequency of 0.1 Hz. Genotypes were detected by means of polymerase chain reaction and restriction analysis using enzyme DdeI. We compared BRS and BRSf among genotypes of this polymorphism. The frequency of genotypes of AT(1)R A1166C polymorphism was: 45.9 % (AA, n=62), 45.9 % (AC, n=62), 8.2 % (CC, n=11). Differences in BRS (p<0.05) and BRSf (p<0.01) among genotypes of this single nucleotide polymorphism were found (Kruskal-Wallis: BRS - AA: 7.9+/-3.3, AC: 8.6+/-3.6, CC: 5.9+/-2.3 ms/mm Hg; BRSf - AA: 12.0+/-4.0, AC: 12.0+/-5.0, CC: 8.0+/-3.0 mHz/mm Hg). Compared to carriers of other genotypes (AA+AC) the homozygotes with the less frequent allele (CC) showed significantly lower BRSf (Mann-Whitney: BRSf - AA+AC: 12.0+/-4.0, CC: 8.0+/-3.0 mHz/mm Hg; p<0.01) and borderline lower BRS (BRS - AA+AC: 8.2+/-3.5, CC: 5.9+/-2.5 ms/mm Hg; p=0.07). We found a significant association of A1166C polymorphism in AT(1) receptor gene with baroreflex sensitivity. Homozygosity for the less frequent allele was associated with decreased baroreflex sensitivity.

The influence of six cardiovascular polymorphisms on a first event of ischemic heart disease is modified by sex and age.

OBJECTIVE: To examine the contribution of six cardiovascular polymorphisms to the occurrence of a first event of ischemic heart disease (IHD) in a primary care population with a high prevalence of hypertension. Furthermore, we specified the data for sex and age. METHODS: In this cross sectional case-control study, patients with a first event of IHD (157) and event-free controls (571) were studied. Both the groups were genotyped for the angiotensin II type 1 receptor (A1166C), angiotensinogen (M235 T), angiotensin converting enzyme (4656rpt), endothelial nitric oxide synthase (E298D), G-protein beta3 subunit (C825 T), and alpha-adducin (G460W) polymorphisms. Univariate and multivariate odds ratios (ORs) were calculated to assess the association between a first ischemic event and these polymorphisms. Sliding mean analyses were performed to show age-specific associations. RESULTS: Multivariate ORs indicated a protective association for the carrier status of the T-allele of AGT, overall [OR = 0.69 (0.34-0.90)] and for males [OR = 0.58 (0.27-0.89)]. Sliding mean analyses showed a continuous protective association with IHD of the T-allele of AGT with increasing age in males, whereas in females an increased risk for IHD was observed with a maximum OR of 1.6 at the age of 56 years. CONCLUSION: In this population the T-allele of the AGT polymorphism is protective for a first event of IHD in males.

The contribution of six polymorphisms to cardiovascular risk in a Dutch high-risk primary care population: the HIPPOCRATES project.

This study was designed to examine the contribution of six polymorphisms to the occurrence of cardiovascular disease (CVD) in a Dutch primary care population with a high prevalence of cardiovascular risk factors. In this cross-sectional case-control study, 232 patients with CVD and 571 event-free controls were studied. Patients were genotyped for the AGTR1 (A1166C), AGT (M235T), ACE (4656rpt), NOS3 (E298D), GNB3 (C825T) and ADD1 (G460W) polymorphisms. Univariate and multivariate odds ratios (ORs) were calculated to assess the relationship between genotypes and CVD. Receiver operating characteristic (ROC) analysis was used to quantify the contribution of the polymorphisms to the prediction of CVD. No differences in either genotype or allele frequencies were found between CVD cases and controls. Multivariate analyses, corrected for multiple testing according to Bonferroni, showed significant protective associations for the T-allele of AGT (OR=0.55 (0.34-0.84)) and for the T-allele of ADD1 (OR=0.52 (0.31-0.82)). ROC analysis showed only a very small improvement of CVD risk prediction by adding the six polymorphisms to a model with traditional risk factors. Our data suggest that a major attribution of the six polymorphisms to the cardiovascular risk prediction in a primary care population such as HIPPOCRATES is unlikely.

Assessment of brain AT1-receptor on the nocturnal basal and angiotensin-induced thirst and sodium appetite in ovariectomised rats.

OBJECTIVE: Considering the controversial data regarding the role of the brain renin-angiotensin system (RAS) on the thirst and sodium appetite in ovariectomised rats, we aimed to evaluate the role of the brain angiotensin II (Ang II) AT1-receptor on the nocturnal fluids intake. MATERIALS AND METHODS: Groups of Wistar female rats were ovariectomised and chronically given oestrogen or vehicle to evaluate its influence on effects induced by i.c.v. injection of losartan, Ang I and Ang II. RESULTS: The i.c.v. losartan decreased basal water intake in the ovariectomised group. Ang II but not Ang I-induced nocturnal dipsogenic and natriorexigenic responses in ovariectomised rats. In oestrogen-treated rats, both peptides increased fluids intake. Previously, i.c.v. losartan abolished these effects in all groups. Oestrogen replacement decreased the nocturnal fluids intake, attenuated the losartan and Ang II effects, and highlighted the Ang I response. CONCLUSIONS: The present study has shown for the first time the involvement of AT1-receptor in regulating nocturnal basal water and salt intake in ovariectomised rats. In addition, our data have revealed an unexpected increased brain Ang I-mediated fluid intake in oestrogen-treated ovariectomised compared to ovariectomised rats, which was blocked by previous i.c.v. losartan. Our data have therefore shown that oestrogen influences homeostatic behaviours dependent on brain RAS.