AT Receptor Stimulation Research Articles

Abstract P450: AT2 Receptor Stimulation Reduces Ischemic Brain Damage Through AT2 Receptor-interacting Protein Signal

Objectives: We reported the preventive effects of angiotensin II type 2 (AT2) receptor stimulation on ischemic brain damage. Moreover, we have cloned ATIP (AT2 receptor interacting protein) as a protein interacting specifically with the C-terminal tail of the AT2 receptor, and suggest that ATIP should play key roles in diverse mechanisms of AT2 receptor actions. However, the effect of ATIP on ischemic brain damage is unclear. Therefore, we investigated the effects of the ATIP and compound 21 (C21), a selective non-peptide AT2 receptor agonist, on focal cerebral ischemia. Method: Ten-week-old male ATIP-transgenic (ATIP-Tg) and littermate (WT) mice were subjected to middle cerebral artery occlusion (MCAO) with silicon-coated micro-filament. C21 (10 μg/kg/day) was administered 2 weeks before MCAO. Twenty-four hours after MCAO, ischemic volume was determined and depicted as mm 3 . Expression of methyl methanesulfonate sensitive 2 (MMS2) mRNA as a neuroprotective factor was determined by real-time RT-PCR. Cerebral blood flow (CBF) before and after MCA occlusion were measured by laser speckle flowmetry. Collateral circulation was evaluated by the perfusion of India ink. Results: Systolic blood pressure did not differ among all groups. There was no significant difference in ischemic size without C21 treatment between two strains (WT, 80 mm 3 ; ATIP-Tg, 75 mm 3 ). Treatment with C21 significantly decreased ischemic size in both strains (WT, 66 mm 3 ; ATIP-Tg, 50 mm 3 ). Interestingly, this protective effect of C21 was more marked in ATIP-Tg compared with WT mice (WT, 16% (66 of 80); ATIP-Tg, 27% (50 of 75) reduction, respectively). MMS2 expression increased in ipsilateral hemisphere of ATIP-Tg mice compared with contralateral hemisphere. There were no significant differences in CBF of core region of ischemic area, among all groups. However, the reduction of CBF in penumbra region after MCA occlusion was attenuated in ATIP-Tg mice with C21 administration. Treatment with C21 tended to increase the cerebral collateral number before MCA occlusion in ATIP-Tg mice not in WT. Conclusions: These results suggested that ATIP could enhance the cerebral protective effects of AT 2 receptor stimulation at least in part due to the increase of CBF and MMS2 expression after ischemia.

Crosstalk between angiotensin and the nonamyloidogenic pathway of Alzheimer's amyloid precursor protein

The association between hypertension and an increased risk for Alzheimer's disease (AD) and dementia is well established. Many data suggest that modulation of the renin-angiotensin system may be meaningful for the prevention and therapy of neurodegenerative disorders, in particular AD. Proteolytic cleavage of the amyloid precursor protein (APP) by α-secretase precludes formation of neurotoxic Aβ peptides and is expected to counteract the development of AD. An established approach for the up-regulation of α-secretase cleavage is the activation of G protein-coupled receptors (GPCRs). Therefore, our study aimed to analyze whether stimulation of angiotensin AT1 or AT2 receptors stably expressed in HEK cells influence the nonamyloidogenic pathway of APP processing. Treatment of both receptors with angiotensin II clearly showed that only activation of the AT1 receptor increased several fold the α-secretase-mediated shedding of APP. This effect was completely abolished by treatment with the AT1 receptor-specific antagonist telmisartan. Using the BIM-46187 inhibitor, we demonstrate that the Gαq protein-mediated pathway is involved in this stimulation process. Stimulation of AT1 receptors with the β-arrestin-biased agonist SII was ineffective regarding α-secretase-mediated APP shedding. This result discloses that only the G protein-dependent pathway is involved in the Ang II-induced APP shedding. Blocking of Gβγ subunits by the inhibitor gallein completely prevented constitutive and Ang II-induced APP shedding. Our findings provide evidence that induction of APP shedding via Ang II/AT1 receptor stimulation is effected by G protein activation with Gβγ subunits playing important roles.

Abstract WP106: Attenuation of Stroke Damage by Angiotensin II Type 2 Receptor Stimulation via Peroxisome Proliferator-Activated Receptor-gamma Activation

Introduction and hypothesis: Brain renin–angiotensin system plays a role in ischemic brain damage after stroke. It is known that stimulation of angiotensin II type 2 (AT 2 ) receptor has a protective role in ischemic brain injury. We demonstrated that AT 2 receptor stimulation by compound 21 (C21), direct AT 2 receptor agonist, inhibited vascular intimal proliferation with activation of peroxisome proliferator-activated receptor-gamma (PPAR-γ). Agents with PPAR-γ agonistic effect have been demonstrated to be neuroprotective in brain ischemia. However, whether direct AT 2 receptor stimulation has a protective effect on ischemic brain injury via PPAR-γ activation is still unknown. Thus, we investigated the beneficial effect of C21 on ischemic brain damage by AT 2 receptor stimulation via PPAR-γ activation. Methods: Eight-week-old male C57BL/6J and human renin and human angiotensinogen double-transgenic (hRN/hANG-Tg) mice were subjected to middle cerebral artery (MCA) occlusion. Before MCA occlusion, they were administered C21 with or without GW9662, a PPAR-γ antagonist, for 2 weeks. Ischemic size, inflammation and oxidative stress were assessed 24 hours after MCA occlusion. Cerebral blood flow was measured in the core and periphery of the MCA territory before, immediately after, 1 hour and 24 hours after MCA occlusion. Results: Administration of C21 with or without GW9662 had no significant effect on blood pressure. Ischemic brain area at 24 hours after MCA occlusion was significantly enlarged in hRN/hANG-Tg mice, compared with C57BL/6J mice. Treatment with C21 decreased the ischemic area compared with the no-treatment group, with an increase in cerebral blood flow, both in C57BL/6J and hRN/hANG-Tg mice. Co-administration of GW9662 partially attenuated the protective effect of C21 on ischemic size, via an increment in the expression of inflammatory cytokines and superoxide production, although GW9662 treatment alone had no significant effect on ischemic size. Conclusions: These results suggest that direct AT 2 receptor stimulation has a beneficial effect on stroke partly due to activation of PPAR-γ.

Stimulation of AT1 Angiotensin Receptors Activates CaV1.2 L-Type Ca2+ Channels through Beta-Arrestin2 and Casein Kinase 2 in Immature Cardiomyocytes

Angiotensin II (AngII) plays important roles in cardiovascular regulation in the perinatal period. Here, we examined the effect of AngII on cardiac CaV1.2 L-type Ca2+ channels. As assessed in the whole-cell configuration of the patch-clamp method, 2-hour treatment of AngII (3 μM) doubled the activity of CaV1.2 channels in mouse neonatal ventricular cardiomyocytes (NVCM) and an atrial myocyte line HL-1, but not in adult ventricular cardiomyocytes (AVCM). An AT1 receptor antagonist, candesartan (10 μM) and knockdown of β-arrestin2, but not an AT2 receptor antagonist, PD123319 (3 μM) or knockdown of β-arrestin1 or Gq/11 significantly suppressed the AngII-mediated activation of CaV1.2 channels in HL-1 cardiomyocytes. To elucidate possible involvement of protein kinases in this system, we examined the effects of array of kinase inhibitors in NVCM. Then, we found that a casein kinase 2 (CK2) inhibitor, quinalizarin (3 μM) almost completely inhibited the AngII-mediated activation of CaV1.2 channels. Knockdown of CK2α, α’ or β subunit in HL-1 cardiomyocytes indicated requirement of CK2 α’ and β but not α in this response. Immunoblotting revealed that the expression levels of CK2α’ was 7 and 4.5 times higher in NVCM and HL-1 cardiomyocytes than in AVCM, respectively. In tsA201 cells, overexpression of CK2α’β significantly increased the activity of recombinant CaV1.2 channels composed of C-terminally truncated α1C, the distal C-terminus of α1C, β2C and α2δ1 subunits; however, it failed to activate CaV1.2 channels containing α1C whose threonine 1704 was substituted with alanine. These results indicate that stimulation of AT1 receptors activates CaV1.2 L-type Ca2+ channels through β-arrestin2 and casein kinase 2 in immature but not adult cardiomyocytes. This response may play an important role in cardiovascular regulation in the perinatal period.

Immunologic Effects of the Renin-Angiotensin System.

Inappropriate activation of the renin-angiotensin system (RAS) exacerbates renal and vascular injury. Accordingly, treatment with global RAS antagonists attenuates cardiovascular risk and slows the progression of proteinuric kidney disease. By reducing BP, RAS inhibitors limit secondary immune activation responding to hemodynamic injury in the target organ. However, RAS activation in hematopoietic cells has immunologic effects that diverge from those of RAS stimulation in the kidney and vasculature. In preclinical studies, activating type 1 angiotensin (AT1) receptors in T lymphocytes and myeloid cells blunts the polarization of these cells toward proinflammatory phenotypes, protecting the kidney from hypertensive injury and fibrosis. These endogenous functions of immune AT1 receptors temper the pathogenic actions of renal and vascular AT1 receptors during hypertension. By counteracting the effects of AT1 receptor stimulation in the target organ, exogenous administration of AT2 receptor agonists or angiotensin 1-7 analogs may similarly limit inflammatory injury to the heart and kidney. Moreover, although angiotensin II is the classic effector molecule of the RAS, several RAS enzymes affect immune homeostasis independently of canonic angiotensin II generation. Thus, as reviewed here, multiple components of the RAS signaling cascade influence inflammatory cell phenotype and function with unpredictable and context-specific effects on innate and adaptive immunity.

Abstract WP290: AT2 Receptor Stimulation Reduces Stroke Size in Concert With AT2 Receptor-Interacting Protein

Objective: We previously reported that the ischemic brain area was significantly larger in angiotensin II type 2 (AT 2 ) receptor-deficient mice after middle cerebral artery (MCA) occlusion compared to wild-type mice. We have cloned ATIP (AT 2 receptor interacting protein) as a protein interacting specifically with the C-terminal tail of the AT 2 receptor, and suggest that ATIP might play key roles in diverse mechanisms of AT 2 receptor signaling. However, the effect of ATIP on brain damage has not been clarified. We investigated the possibility that ATIP could enhance the protective effects of compound 21 (C21), a selective direct non-peptidic AT 2 receptor agonist, on focal cerebral ischemia. Method: Ten week-old male ATIP-transgenic (ATIP-Tg) and littermate (WT) mice were subjected to permanent MCA occlusion with silicon-coated micro-filament. C21 (10 μg/kg/day) was administered 2 weeks before MCA occlusion. Twenty-four hours after MCA occlusion, ischemic area and neurological deficit was assessed. Cerebral blood flow (CBF) was measured by laser speckle flowmetry. Expression of methyl methanesulfonate sensitive 2 (MMS2) as a neuroprotective factor were measured by real-time RT-PCR. Collateral circulation was evaluated by the perfusion of India ink. Results: Systolic blood pressure did not differ between ATIP-Tg and WT mice with or without C21. There was no significant difference in ischemic size without C21 treatment between two strains. Treatment with C21 decreased ischemic size and improved neurological deficit in both strains. These protective effects by C21 were more marked in ATIP-Tg mice compared with WT mice. Treatment of C21 did not affect CBF in the core region of ischemic area after MCA occlusion in both strains; however, the reduction of CBF in penumbra region was markedly attenuated in ATIP-Tg mice treated with C21. MMS2 expression increased in ipsilateral hemisphere of ATIP-Tg mice compared with contralateral hemisphere. C21 treatment tended to increase collateral number in ATIP-Tg mice. Conclusions: These results suggested that ATIP could enhance the cerebral protective effects of AT 2 receptor stimulation at least in part due to the improvement of CBF and increase of MMS2 expression.

Activation of the cell membrane angiotensin AT2 receptors in human leiomyosarcoma cells induces differentiation and apoptosis by a PPARγ - dependent mechanism.

Angiotensin II (Ang II), the main effector peptide of the renin-angiotensin system (RAS), acting on AT1 and AT2 receptors participates in the regulation of proliferation, differentiation and apoptosis in tumour cells. The peroxisome-proliferator activated receptor γ (PPARγ) and its ligands exert anti-tumour effects in various human cancer cell lines. The present study investigates the effects initiated by AT1- and AT2 receptor stimulation in SK-UT-1 cells, a human leiomyosarcoma cell line, and clarifies the role of the PPARγ in the AT2 receptor-induced differentiation and apoptosis.Selective stimulation of AT1- and AT2 receptors was achieved by incubation of the cells with Ang II (10-6 M) in the presence of the selective AT2 receptor antagonist, PD 123177 (10-6 M) and the AT1 receptor antagonist, losartan (10-5 M), respectively, the selective PPARγ antagonist, GW 9662, was used at concentration 10-6 M. The expression of smooth muscle cell differentiation markers, SM22α and calponin, was analysed at RNA- and protein levels using RT PCR and Western blot, which was also used to quantify Bcl-2-, Bax- and cleaved caspase-3 proteins. The translocation of the AT2-receptor interacting protein 1 (ATIP1) to the nuclei was studied by Western blot and immunofluorescence staining. The mitochondrial status and the metabolic activity in response to AT1- and AT2 receptor activation were assessed by the quantification of 99mTc - sestamibi and 2´-deoxy-2´-[18F]fluoro-D-glucose uptake.AT1 receptor stimulation did not exert any profound effects in quiescent SK-UT-1 cells. The effects induced by Ang II acting on AT2 receptors were time-dependent. A short, 3 - 6 h lasting stimulation promotes differentiation, i.e increases in the mRNA- and protein levels of SM22α and calponin, whereas a sustained stimulation for 48 h activates the intrinsic apoptotic pathway, as evidenced by reduced cell numbers, down-regulation of the anti-apoptotic Bcl-2 protein and increased levels of the Bax protein and cleaved caspase-3. The effects were reversed by the PPARγ antagonist, GW 9662, clearly implying a PPARγ-dependent mechanism. Our results also demonstrate a co-localisation of the AT2-receptor interacting protein, ATIP1, and the PPARγ in nuclei of SK-UT-1 cells and an accumulation of ATIP1 in the nuclear fraction in response to AT2 receptor stimulation. The regulation of the differentiation and apoptosis via the AT2 receptor favours an important functional role of this receptor in quiescent, slow-cycling SK-UT-1 cells and provides the rationale for the use of AT1 receptor antagonists for the treatment of human leiomyosarcomas.

SY 12-2 ACE INHIBITORS AND ARBS

Blockade of the renin-angiotensin-aldosterone system (RAAS) can be accomplished at the level of the angiotensin-generating enzymes renin and angiotensin-converting enzyme (ACE; using renin inhibitors or ACE inhibitors), the type 1 angiotensin II (AT1) receptor or mineralocorticoid receptor (MR; using angiotensin receptor blockers [ARBs] or MR blockers) and/or renin release (using beta-blockers). Several of these drugs are often combined-for example in heart failure-but such approaches may ultimately lead to RAAS annihilation with adverse consequences such as hypotension, renal dysfunction and hyperkalaemia. The biochemical consequences of each type of blockade are different. For instance, ACE inhibitors will lower angiotensin II, thus no longer allowing stimulation of both AT1 and type 2 angiotensin II (AT2) receptors, while ARBs raise angiotensin II, allowing selective stimulation of the unoccupied AT2 receptor. This might be of particular importance in women, in whom the protective AT2 receptor pathway is believed to be upregulated. Multiple clinical trials have compared the various types of RAAS blockers and/or their combination. This talk will summarize the current evidence with regard to similarities and differences between ACE inhibitors and ARBs, also considering their side-effect profile, dose and combination with other RAAS blockers.

Angiotensin AT2-receptor stimulation improves survival and neurological outcome after experimental stroke in mice.

This study investigated the effect of post-stroke, direct AT2-receptor (AT2R) stimulation with the non-peptide AT2R-agonist compound 21 (C21) on infarct size, survival and neurological outcome after middle cerebral artery occlusion (MCAO) in mice and looked for potential underlying mechanisms. C57/BL6J or AT2R-knockout mice (AT2-KO) underwent MCAO for 30min followed by reperfusion. Starting 45min after MCAO, mice were treated once daily for 4days with either vehicle or C21 (0.03mg/kg ip). Neurological deficits were scored daily. Infarct volumes were measured 96h post-stroke by MRI. C21 significantly improved survival after MCAO when compared to vehicle-treated mice. C21 treatment had no impact on infarct size, but significantly attenuated neurological deficits. Expression of brain-derived neurotrophic factor (BDNF), tyrosine kinase receptor B (TrkB) (receptor for BDNF) and growth-associated protein 43 (GAP-43) were significantly increased in the peri-infarct cortex of C21-treated mice when compared to vehicle-treated mice. Furthermore, the number of apoptotic neurons was significantly decreased in the peri-infarct cortex in mice treated with C21 compared to controls. There were no effects of C21 on neurological outcome, infarct size and expression of BDNF or GAP-43 in AT2-KO mice. From these data, it can be concluded that AT2R stimulation attenuates early mortality and neurological deficits after experimental stroke through neuroprotective mechanisms in an AT2R-specific way. Key message • AT2R stimulation after MCAO in mice reduces mortality and neurological deficits.• AT2R stimulation increases BDNF synthesis and protects neurons from apoptosis.• The AT2R-agonist C21 acts protectively when applied post-stroke and peripherally.

Combined Angiotensin Receptor Modulation in the Management of Cardio-Metabolic Disorders.

Cardiovascular and metabolic disorders, such as hypertension, insulin resistance, dyslipidemia or obesity are linked with chronic low-grade inflammation and dysregulation of the renin–angiotensin system (RAS). Consequently, RAS inhibition by ACE inhibitors or angiotensin AT1 receptor (AT1R) blockers is the evidence-based standard for cardiovascular risk reduction in high-risk patients, including diabetics with albuminuria. In addition, RAS inhibition reduces the new onset of diabetes mellitus. Yet, the high and increasing prevalence of metabolic disorders, and the high residual risk even in properly treated patients, calls for additional means of pharmacological intervention. In the past decade, the stimulation of the angiotensin AT2 receptor (AT2R) has been shown to reduce inflammation, improve cardiac and vascular remodeling, enhance insulin sensitivity and increase adiponectin production. Therefore, a concept of dual AT1R/AT2R modulation emerges as a putative means for risk reduction in cardio-metabolic diseases. The approach employing simultaneous RAS blockade (AT1R) and RAS stimulation (AT2R) is distinct from previous attempts of double intervention in the RAS by dual blockade. Dual blockade abolishes the AT1R-linked RAS almost completely with subsequent risk of hypotension and hypotension-related events, i.e. syncope or renal dysfunction. Such complications might be especially prominent in patients with renal impairment or patients with isolated systolic hypertension and normal-to-low diastolic blood pressure values. In contrast to dual RAS blockade, the add-on of AT2R stimulation does not exert significant blood pressure effects, but it may complement and enhance the anti-inflammatory and antifibrotic/de-stiffening effects of the AT1R blockade and improve the metabolic profile. Further studies will have to investigate these putative effects in particular for settings in which blood pressure reduction is not primarily desired.

A novel role of endothelium in activation of latent pro-membrane type 1 MMP and pro-MMP-2 in rat aorta.

Aortic stiffness is an independent risk factor for progression of cardiovascular diseases. Degradation of elastic fibres in aorta due to angiotensin II (ANGII)-stimulated overactivation of latent membrane type 1 matrix metalloproteinase (MT1MMP) and matrix metalloproteinase-2 (MMP2) is regarded to represent an important cause of aortic stiffness. Therefore, clarification of the causal mechanisms triggering the overactivation of these MMPs is of utmost importance. This study addresses the endothelium as a novel key activator of latent pro-MT1MMP and pro-MMP2 in rat aorta. Using a co-culture model of rat aortic endothelial cells (ECs) and smooth muscle cells (SMCs), we found that ANGII stimulation resulted in activation of latent pro-MT1MMP and pro-MMP2 in SMCs exclusively when co-cultured with ECs (assessed with western blot and gelatin zymography, respectively). EC-specific AT1 receptor stimulation triggered endothelin-1 release and paracrine action on SMCs. Endothelin-1 increased expression and activity of pro-protein convertase furin in SMCs via endothelin receptor type A (assessed with qPCR and furin activity assay, respectively). Consequently, furin acted in two ways. First, it increased the activation of latent pro-MT1MMP and, second, it activated pro-αvβ3 integrin. Both pathways led to overactivation of latent pro-MMP2. In vitro findings in the co-culture model were fully consistent with the ex vivo findings obtained in isolated rat aorta. We propose that the endothelium under ANGII stimulation acts as a novel and key activator of latent pro-MT1MMP and pro-MMP2 in SMCs of rat aorta. Therefore, endothelium may critically contribute to pathophysiology of aortic stiffness.

The Role of Endothelial Dysfunction in Aortic Aneurysms.

Abdominal aortic aneurysm is a vascular disease which, despite the fact that it shares common risk factors with atherosclerosis, develops in parallel but as a partly independent process, through different pathogenic mechanisms. The pathogenic mechanisms involve metalloproteinase and collagenase activation, median and adventitial degradation, elastin lysis, vascular smooth cells transformation and apoptosis, collagen production and lysis imbalance combined with excessive inflammatory infiltration. Endothelial cells respond to a number of stimulating factors, including smoking, hypertension and AT1 receptor stimulation and non-uniform distribution of wall stress. Their ability to produce NO is crucial in order to adapt. Endothelial cells contribute to AAA development due to increased oxidative stress which is partly mediated by impaired NO bioavailability due to endothelial dysfunction and NADPH oxidase overexpression. In addition, they express several molecules among which adherence molecules, selectins, endothelin-1, regulating inflammatory infiltration and oxidative stress. Inflammatory cells consist of monocytes, polymorphonuclear neutrophils and lymphocytes and they are involved in the degrading process in the aortic wall by secreting proteolytic enzymes or by releasing interleukins which mediate the inflammation response. Endothelial dysfunction and arterial stiffness reflect on indices like FMD, carotid-femoral PWV and augmentation index, sometimes with controversial results. At present, surgical treatment is the only option provided in patients with large AAA, in particular. Focusing on the emerging role of endothelial cells in AAA pathology may contribute in creating new therapeutic options in a disease which has not yet a well-accepted, implemented pharmaceutical treatment.

Abstract P143: The Angiotensin AT2 Receptor Agonist Compound 21 Is a Low Affinity Thromboxane A2 Receptor Antagonist

Objective: The purpose of this study was to investigate potential vasorelaxant effects of angiotensin AT2-receptor stimulation by the specific AT2-receptor agonist C21 in pericardial resistance arteries from cardiovascular disease patients. Methods: Parietal pericardium was obtained during coronary artery bypass-grafting and/or valve replacement in patients. Pericardial arteries from pigs and mesenteric arteries from C57BL-6J and AT2R deficient (AT2R-/y) mice were used for comparison. Isolated arteries were mounted onto wire myographs, pre-contracted with either K+, phenylephrine, endothelin-1 (ET-1) or the thromboxane agonist U46619, and the relaxing effect of C21 (0.1nM - 10μM) was investigated in the absence or presence of 3nM valsartan (AT1R antagonist), 10μM PD123319 or 10μM M132 (AT2R antagonists). Results: C21 significantly relaxed ET-1 contracted porcine arteries (Emax -37 ± 6 %; -0.87 vs -0.45 N/m relaxation; N=6; P<0.05, two-way ANOVA) and phenylephrine contracted mouse mesenteric arteries (Emax -52 ± 10 %; -0.26 vs -0.09 N/m relaxation; N=6; P<0.01, two-way ANOVA), and this was inhibited by PD123319 or M132 and absent in arteries from AT2R-/y. C21 significantly induced vasorelaxation in U46619 contracted vessels from all species and vascular beds (e.g. in human pericardial arteries: Emax -78 ± 10 %; -0.67 vs -0.07 N/m relaxation; N=17; P<0.01, two-way ANOVA), and this effect was not blocked by AT2R antagonists and still present in AT2R-/y. C21 further inhibited U46619 induced platelet aggregation. An Arrestin Biosensor Assay revealed that C21 binds to the TXA2 receptor with a Ki of 3.74 μM. Conclusion: Depended on species, vascular bed and contractile stimulus, C21 relaxes resistance-sized arteries by AT2R stimulation or by TXA2 antagonism. These data together with the low affinity binding of C21 to the TXA2 receptor and its effect on platelet aggregation strongly suggest that C21 is not only a high affinity, selective AT2R agonist, but also a low affinity TXA2 receptor antagonist.

Comparative analyses of downstream signal transduction targets modulated after activation of the AT1 receptor by two β-arrestin-biased agonists.

G protein-coupled receptors (GPCRs) are involved in essentially all physiological processes in mammals. The classical GPCR signal transduction mechanism occurs by coupling to G protein, but it has recently been demonstrated that interaction with β-arrestins leads to activation of pathways that are independent of the G protein pathway. Also, it has been reported that some ligands can preferentially activate one of these signaling pathways; being therefore called biased agonists for G protein or β-arrestin pathways. The angiotensin II (AngII) AT1 receptor is a prototype GPCR in the study of biased agonism due to the existence of well-known β-arrestin-biased agonists, such as [Sar1, Ile4, Ile8]-AngII (SII), and [Sar1, D-Ala8]-AngII (TRV027). The aim of this study was to comparatively analyze the two above mentioned β-arrestin-biased agonists on downstream phosphorylation events and gene expression profiles. Our data reveal that activation of AT1 receptor by each ligand led to a diversity of activation profiles that is far broader than that expected from a simple dichotomy between “G protein-dependent” and “β-arrestin-dependent” signaling. We observed clusters of activation profiles common to AngII, SII, and TRV027, as well as downstream effector activation that are unique to AngII, SII, or TRV027. Analyses of β-arrestin conformational changes after AT1 receptor stimulation with SII or TRV027 suggests that the observed differences could account, at least partially, for the diversity of modulated targets observed. Our data reveal that, although the categorization “G protein-dependent” vs. “β-arrestin-dependent” signaling can be of pharmacological relevance, broader analyses of signaling pathways and downstream targets are necessary to generate an accurate activation profile for a given ligand. This may bring relevant information for drug development, as it may allow more refined comparison of drugs with similar mechanism of action and effects, but with distinct side effects.

Compound 21, a selective agonist of angiotensin AT2 receptors, prevents endothelial inflammation and leukocyte adhesion in vitro and in vivo.

Angiotensin AT2 receptors are upregulated in disease states such as atherosclerosis and blockade of the AT2 receptors exacerbates plaque formation. Direct stimulation of these receptors is anti-atherogenic but the mechanisms and pathways involved remain unknown. We examined the effect of direct AT2 receptor stimulation with Compound 21 (C21) on the leukocyte adhesion cascade in vitro, right through to plaque formation in vivo. Effects of C21 on TNFα-induced inflammation were assessed in human umbilical vein endothelial cells (HUVECs), activation of monocytes, polarisation of monocyte-derived macrophages and in intact mouse aortae. C21 attenuated TNFα-induced: monocyte adhesion to cultured HUVECs, adhesion molecule expression and abolished TNFα-induced ROS production. TNFα-induced NFκB translocation from the cytoplasm to the nucleus, essential for cytokine production, was prevented by C21. C21 did not influence monocyte activation or macrophage polarisation but did reduce TNFα and IL-6 mRNA expression in M1 macrophages. The anti-inflammatory effects of C21 were abolished by an AT2 receptor antagonist confirming that the effects of C21 were AT2 receptor-mediated. Also, leukocyte adhesion and cytokine gene expression, induced by high-fat diet (HFD), was attenuated in ApoE(-/-) mice treated with C21. Plaque size and stability were improved with C21 treatment with increased smooth muscle cell composition and decreased lipid size, compared with HFD-saline treated mice. C21 prevented TNFα-induced and HFD-induced vascular inflammation in vitro and in vivo. Our data provide strong evidence that the anti-atherosclerotic actions of C21 were due to vascular anti-inflammatory effects, mediated by AT2 receptors.

Hypotensive and sympathoinhibitory responses to selective central AT2 receptor stimulation in spontaneously hypertensive rats.

The type2 angiotensin receptor (AT2R) has been suggested to counterbalance the type1 angiotensin receptor (AT1R) in the central regulation of blood pressure and sympathetic tone. In the present study we investigated the blood pressure responses to stimulation of central AT2Rs by the selective agonist Compound 21in conscious spontaneously hypertensive rats (SHRs) and normotensive Wistar Kyoto rats (WKY rats). We also assessed the impact on noradrenaline [norepinephrine (NE)] plasma levels, autonomic function, spontaneous baroreflex sensitivity, and the possible involvement of the nitric oxide (NO) pathway and the AT1Rs. Chronic intracerebroventricular Compound 21 infusion lowered blood pressure and NE plasma levels in both rat strains. The night-time hypotensive effect was greater in SHRs compared with WKY rats. Compound 21 improved spontaneous baroreflex sensitivity more in SHRs than in WKY rats. These effects were abolished by co-administration of the AT2R antagonist PD123319 or the NO synthase inhibitor Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME). Central AT1R blockade did not enhance the hypotensive response to Compound 21. Chronic selective stimulation of central AT2Rs lowers blood pressure through sympathoinhibition, and improves spontaneous baroreflex sensitivity more in SHRs than in WKY rats. These responses appear to require a functioning central NO pathway, but are not modified by central AT1R blockade. Collectively, the data demonstrate specific beneficial effects of stimulation of central AT2Rs in hypertension associated with increased sympathetic tone, and suggest that central AT2Rs may represent a potential new therapeutic target for the treatment of neurogenic hypertension.

Stimulation of the Angiotensin II AT2 Receptor is Anti-inflammatory in Human Lipopolysaccharide-Activated Monocytic Cells.

Recently, AT2 receptors have been discovered on the surface of human immunocompetent cells such as monocytes. Data on regulative properties of this receptor on the cellular immune response are poor. We hypothesized that direct stimulation of the AT2 receptor mediates anti-inflammatory responses in these cells. Human monocytic THP-1 and U937 cells were stimulated with lipopolysaccharide (LPS) and the selective AT2 receptor agonist Compound 21 (C21). Expression of pro- and anti-inflammatory cytokines IL-6, IL-10, tumor necrosis factor-α (TNFα), and IL-1β were analyzed on both the transcriptional and the translational level over course of time. Treatment with C21 attenuated the expression of TNFα, IL-6, and IL-10 after LPS challenge in both cell lines in a time- and dose-dependent manner. We conclude that selective AT2 receptor stimulation acts anti-inflammatory in human monocytes. Modulation of cytokine response by AT2 receptor activation might be a beneficial and novel treatment concept in inflammatory conditions.

Angiotensin AT2 receptor stimulation inhibits activation of NADPH oxidase and ameliorates oxidative stress in rotenone model of Parkinson's disease in CATH.a cells

Oxidative stress has long been considered as a major contributing factor in the pathogenesis of Parkinson's disease (PD). The brain has an independent local renin–angiotensin system (RAS). Angiotensin II (Ang II) activates NADPH-dependent oxidases, which are a major source of superoxide and are upregulated in major aging-related diseases such as hypertension and neurodegenerative disease. In this study, we firstly examined whether CGP42112, an AT2 receptor (AT2R) agonist, may exert direct protective effects on the rotenone-induced CATH.a cell injury in vitro. We used CATH.a cell line to evaluate changes in cultured dopaminergic neuron levels of superoxide dismutase (SOD), glutathione (GSH) and reactive oxygen species (ROS). We also evaluated expression of NADPH oxidase, AT1 and AT2 receptors in treated with phosphate buffer saline (PBS), rotenone, Ang II, AT2R agonist CGP42112, or AT2R antagonist PD123319, alone and combined (n=6, each group). Quantitative reverse transcriptase PCR (qRT-PCR) and western blot were used to determine messenger RNA (mRNA) and protein levels of the AT1, AT2 receptors and NADPH oxidase. ROS generation was determined by the dichlorodihydrofluorescein diacetate fluorescent probe assay. The levels of SOD and GSH were measured by using available kits. In our study, CGP42112 (100nM) significantly reduced rotenone-induced oxidative stress and elevated the total SOD activity and GSH level. In addition, CGP42112 significantly increased AT2R expression and attenuated Ang II-induced NADPH oxidase activation, and these effects were completely abolished by the AT2R antagonist, PD123319 (1μM). Our results suggest that CGP42112 attenuates rotenone-induced oxidative stress in CATH.a neuron via activating AT2R and suppressing NADPH oxidase expression.

Angiotensin II mobilizes intracellular calcium and activates pannexin-1 channels in rat carotid body type II cells via AT1 receptors.

Key points A locally generating, angiotensin II (ANG II) system is present in the rat carotid body (CB) and up‐regulation of this system occurs in certain pathophysiological situations, enhancing sympathetic activity. Here, we show that, similar to chemoreceptor type I cells, glial‐like type II cells also express functional AT1Rs, stimulation of which causes release of Ca2+ from intracellular stores. ANG II–AT1R signalling in type II cells activates an inward current carried by pannexin‐1 (Panx‐1) channels which are known to act as conduits for release of ATP, a key CB excitatory neurotransmitter. Combined effects of ANG II and ATP, which also activates Panx‐1 currents via P2Y2 receptors, were synergistic; chelating intracellular Ca2+ with BAPTA prevented Panx‐1 current activation. We propose that the excitatory function of ANG II in the CB involves dual actions at both type I and type II cells. AbstractA local angiotensin‐generating system is present in the carotid body (CB) and increased angiotensin II (ANG II) signalling contributes to enhanced CB excitation in chronic heart failure (CHF) and after chronic or intermittent hypoxia. ANG II actions have thus far been attributed solely to stimulation of AT1 receptors (AT1Rs) on chemoreceptor type I cells. Here, we show that in dissociated rat CB cultures, ANG II also stimulates glial‐like type II cells, identified by P2Y2‐receptor‐induced intracellular Ca2+ elevation (Δ[Ca2+]i). ANG II induced a dose‐dependent (EC50 ∼8 nm), robust Δ[Ca2+]i in type II cells that was reversibly abolished by the AT1R blocker losartan (1 μm). The ANG II‐induced Δ[Ca2+]i persisted in Ca2+‐free medium but was sensitive to store depletion with cyclopiazonic acid (1 μm). Similar to P2Y2 receptor agonists, ANG II (20–1000 nm) activated pannexin‐1 (Panx‐1) current that was reversibly abolished by carbenoxolone (5 μm). This current arose with a variable delay and was reversibly inhibited by losartan. Repeated application of ANG II often led to current run‐down, attributable to AT1R desensitization. When applied to the same cell the combined actions of ANG II and ATP on Panx‐1 current were synergistic. Current induced by either ligand was inhibited by BAPTA‐AM (1 μm), suggesting that intracellular Ca2+ signalling contributed to Panx‐1 channel activation. Because open Panx‐1 channels release ATP, a key CB excitatory neurotransmitter, it is plausible that paracrine stimulation of type II cells by ANG II contributes to enhanced CB excitability, especially in pathophysiological conditions such as CHF and sleep apnoea.

Angiotensin AT2 receptor stimulation is anti-inflammatory in lipopolysaccharide-activated THP-1 macrophages via increased interleukin-10 production

Macrophages have an important role in the pathogenesis of hypertension and associated end-organ damage via the activation of the Toll-like receptors, such as Toll-like receptor-4 (TLR4). Accumulating evidence suggests that the angiotensin AT2 receptor (AT2R) has a protective role in pathological conditions involving inflammation and tissue injury. We have recently shown that AT(2)R stimulation is renoprotective, which occurs in part via increased levels of anti-inflammatory interleukin-10 (IL-10) production in renal epithelial cells; however, the role of AT(2)R in the inflammatory activity of macrophages is not known. The present study was designed to investigate whether AT(2)R activation stimulates an anti-inflammatory response in TLR4-induced inflammation. The effects of the anti-inflammatory mechanisms that occurred following pre-treatment with the AT(2)R agonist Compound 21 (C21) (1 μmol ml(-1)) on the cytokine profiles of THP-1 macrophages after activation by lipopolysaccharide (LPS) (1 μg ml(-1)) were studied. The AT(2)R agonist dose-dependently attenuated LPS-induced tumor necrosis factor-α (TNF-α) and IL-6 production but increased IL-10 production. IL-10 was critical for the anti-inflammatory effects of AT(2)R stimulation because the IL-10-neutralizing antibody dose-dependently abolished the AT(2)R-mediated decrease in TNF-α levels. Further, enhanced IL-10 levels were associated with a sustained, selective increase in the phosphorylation of extracellular signal-regulated kinase (ERK1/2) but not p38 mitogen-activated protein kinase (MAPK). Blocking the activation of ERK1/2 before C21 pre-treatment completely abrogated this increased IL-10 production in response to the AT(2)R agonist C21, while there was a partial reduction in IL-10 levels following the inhibition of p38. We conclude that AT(2)R stimulation exerts a novel anti-inflammatory response in THP-1 macrophages via enhanced IL-10 production as a result of sustained, selective ERK1/2 phosphorylation, which may have protective roles in hypertension and associated tissue injury.