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Discovered more than 30 years ago, the angiotensin AT2 receptor (AT2R) has evolved from a binding site with unknown function to a firmly established major effector within the protective arm of the renin-angiotensin system (RAS) and a target for new drugs in development. The AT2R represents an endogenous protective mechanism that can be manipulated in the majority of preclinical models to alleviate lung, renal, cardiovascular, metabolic, cutaneous, and neural diseases as well as cancer. This article is a comprehensive review summarizing our current knowledge of the AT2R, from its discovery to its position within the RAS and its overall functions. This is followed by an in-depth look at the characteristics of the AT2R, including its structure, intracellular signaling, homo- and heterodimerization, and expression. AT2R-selective ligands, from endogenous peptides to synthetic peptides and nonpeptide molecules that are used as research tools, are discussed. Finally, we summarize the known physiological roles of the AT2R and its abundant protective effects in multiple experimental disease models and expound on AT2R ligands that are undergoing development for clinical use. The present review highlights the controversial aspects and gaps in our knowledge of this receptor and illuminates future perspectives for AT2R research. SIGNIFICANCE STATEMENT: The angiotensin AT2 receptor (AT2R) is now regarded as a fully functional and important component of the renin-angiotensin system, with the potential of exerting protective actions in a variety of diseases. This review provides an in-depth view of the AT2R, which has progressed from being an enigma to becoming a therapeutic target.

Aims These studies evaluate whether angiotensin type-2 receptors (AT2Rs) that are expressed on γ-aminobutyric acid (GABA) neurons in the nucleus of the solitary tract (NTS) represent a novel endogenous blood pressure lowering mechanism. Methods and results Experiments combined advanced genetic and neuroanatomical techniques, pharmacology, electrophysiology and optogenetics in mice to define the structure and cardiovascular-related function of NTS neurons that contain AT2R. Using mice with Cre-recombinase directed to the AT2R gene, we discovered that optogenetic stimulation of AT2R-expressing neurons in the NTS increases GABA release and blood pressure. To evaluate the role of the receptor, per se, in cardiovascular regulation, we chronically delivered C21, a selective AT2R agonist, into the brains of normotensive mice and found that central AT2R activation reduces GABA-related gene expression and blunts the pressor responses induced by optogenetic excitation of NTS AT2R neurons. Next, using in situ hybridization, we found that the levels of Agtr2 mRNAs in GABAergic NTS neurons rise during experimentally-induced hypertension, and we hypothesized that this increased expression may be exploited to ameliorate the disease. Consistent with this, final experiments revealed that central administration of C21 attenuates hypertension, an effect that is abolished in mice lacking AT2R in GABAergic NTS neurons. Conclusions These studies unveil novel hindbrain circuits that maintain arterial blood pressure, and reveal a specific population of AT2R that can be engaged to alleviate hypertension. The implication is that these discrete receptors may serve as an access point for activating an endogenous depressor circuit. Translational perspective Hypertension is a widespread health problem and risk factor for cardiovascular disease and stroke. Although treatment options exist, many patients suffer from resistant hypertension, which is associated with enhanced sympathetic drive. Thus, many available therapeutics focus on dampening pressor mechanisms. The present studies take the alternative approach of treating hypertension by exploiting an endogenous depressor mechanism.

It is common knowledge that the renin-angiotensin system (RAS), in particular angiotensin II acting through the angiotensin AT1-receptor (AT1R), is pivotal for the regulation of blood pressure (BP) and extracellular volume. More recent findings have revealed that the RAS is far more complex than initially thought and that it harbours additional mediators and receptors, which are able to counteract and thereby fine-tune AT1R-mediated actions. This review will focus on the angiotensin AT2-receptor (AT2R), which is one of the "counter-regulatory" receptors within the RAS. It will review and discuss data related to the role of the AT2R in regulation of BP and focus on the following 3 questions: Do peripheral AT2R have an impact on BP regulation, and, if so, does this effect become apparent only under certain conditions? Are central nervous system AT2R involved in regulation of BP, and, if so, which brain areas are involved and what are the mechanisms? Does dysfunction of AT2R contribute to the pathogenesis of hypertension in preeclampsia?

A high-fat diet (HFD) induces an increase in arterial pressure and a decrease in baroreflex function, which may be associated with increased expression of angiotensin type 1 receptor (AT1R) and pro-inflammatory cytokine genes and reduced expression of the angiotensin type 2 receptor (AT2R) gene within the nucleus of the solitary tract (NTS), a key area of the brainstem involved in cardiovascular control. Thus, in the present study, we evaluated the changes in arterial pressure and gene expression of components of the renin-angiotensin system (RAS) and neuroinflammatory markers in the NTS of rats fed a HFD and treated with either an AT1R blocker or with virus-mediated AT2R overexpression in the NTS. Male Holtzman rats (300-320 g) were fed either a standard rat chow diet (SD) or HFD for 6 weeks before commencing the tests. AT1R blockade in the NTS of HFD-fed rats attenuated the increase in arterial pressure and the impairment of reflex bradycardia, whereas AT2R overexpression in the NTS only improved the baroreflex function. The HFD also increased the hypertensive and decreased the protective axis of the RAS and was associated with neuroinflammation within the NTS. The expression of angiotensin-converting enzyme and neuroinflammatory components, but not AT1R, in the NTS was reduced by AT2R overexpression in this site. Based on these data, AT1R and AT2R in the NTS are differentially involved in the cardiovascular changes induced by a HFD. Chronic inflammation and changes in the RAS in the NTS may also account for the cardiovascular responses observed in HFD-fed rats.

PURPOSE OF REVIEW. To review recent data that suggest opposing effects of brain angiotensin type-1 (AT(1)R) and type-2 (AT(2)R) receptors on blood pressure (BP). Here, we discuss recent studies that suggest pro-hypertensive and pro-inflammatory actions of AT(1)R, and anti-hypertensive and anti-inflammatory actions of AT(2)R. Further, we propose mechanisms for the interplay between brain angiotensin receptors and neuroinflammation in hypertension. RECENT FINDINGS. The renin-angiotensin system (RAS) plays an important role in regulating cardiovascular physiology. This includes brain AT(1)R and AT(2)R, both of which are expressed in or adjacent to brain regions that control BP. Activation of AT(1)R within those brain regions mediate increases in BP and cause neuroinflammation, which augments the BP increase in hypertension. The fact that AT(1)R and AT(2)R have opposing actions on BP, suggests that AT(1)R and AT(2)R may have similar opposing actions on neuroinflammation. However, the mechanisms by which brain AT(1)R and AT(2)R mediate neuroinflammatory responses remain unclear. SUMMARY. The interplay between brain angiotensin receptor subtypes and neuroinflammation exacerbates or protects against hypertension.

Significant neuroprotective effects of angiotensin II type 2 (AT 2 ) receptor (AT 2 receptor) agonists in ischemic stroke have been previously demonstrated in multiple studies. However, the routes of agonist application used in these pre-clinical studies, direct intracerebroventricular (ICV) and systemic administration, are unsuitable for translation into humans; in the latter case because AT 2 receptor agonists are blood-brain barrier (BBB) impermeable. To circumvent this problem, in the current study we utilized the nose-to-brain (N2B) route of administration to bypass the BBB and deliver the selective AT 2 receptor agonist Compound 21 (C21) to näive rats or rats that had undergone endothelin 1 (ET-1)-induced ischemic stroke. The results obtained from the present study indicated that C21 applied N2B entered the cerebral cortex and striatum within 30 min in amounts that are therapeutically relevant (8.4-9 nM), regardless of whether BBB was intact or disintegrated. C21 was first applied N2B at 1.5 h after stroke indeed provided neuroprotection, as evidenced by a highly significant, 57% reduction in cerebral infarct size and significant improvements in Bederson and Garcia neurological scores. N2B-administered C21 did not affect blood pressure or heart rate. Thus, these data provide proof-of-principle for the idea that N2B application of an AT 2 receptor agonist can exert neuroprotective actions when administered following ischemic stroke. Since N2B delivery of other agents has been shown to be effective in certain human central nervous system diseases, the N2B application of AT 2 receptor agonists may become a viable mode of delivering these neuroprotective agents for human ischemic stroke patients.

The discovery of the first selective, small-molecule ATR receptor (AT2R) agonist compound 21 (C21) (8) that is now extensively studied in a large variety of in vitro and in vivo models is described. The sulfonylcarbamate derivative 8, encompassing a phenylthiofen scaffold is the drug-like agonist with the highest affinity for the AT2R reported to date (Ki = 0.4 nM). Structure-activity relationships (SAR), regarding different biaryl scaffolds and functional groups attached to these scaffolds and with a particular focus on the impact of various para substituents displacing the methylene imidazole group of 8, are discussed. Furthermore, the consequences of migration of the methylene imidazole group and presumed structural requirements for ligands that are aimed as AT2R agonists (e.g. 8) or AT2R antagonists (e.g. 9), respectively, are briefly addressed. A summary of the pharmacological actions of C21 (8) is also presented.

The angiotensin AT2 -receptor is a main receptor of the protective arm of the renin-angiotensin-system. Understanding of this unconventional G-protein coupled receptor has significantly advanced during the past decade, largely because of the availability of a selective non-peptide AT2 -receptor agonist, which allowed the conduct of a multitude of studies in animal disease models. This article reviews such preclinical studies that in their entirety provide strong evidence for an anti-fibrotic effect mediated by activation of the AT2 -receptor. Prevention of the development of fibrosis by AT2 -receptor stimulation has been demonstrated in lung, heart, blood vessels, kidney, pancreas and skin. In lung, AT2 -receptor stimulation was even able to reverse existing fibrosis. The article further discusses intracellular signalling mechanisms mediating the AT2 -receptor coupled anti-fibrotic effect, including activation of phosphatases and subsequent interference with pro-fibrotic signalling pathways, induction of matrix-metalloproteinases, and hetero-dimerisation with the AT1 -receptor, the TGF-βRII-receptor or the RXFP1-receptor for relaxin. Knowledge of the anti-fibrotic effects of the AT2 -receptor is of particular relevance, because drugs targeting this receptor have entered clinical development for indications involving fibrotic diseases. This article is protected by copyright. All rights reserved.

Brain angiotensin-II (Ang-II) type-1 receptors (AT1Rs), which exert profound effects on normal cardiovascular, fluid, and metabolic homeostasis, are overactivated in and contribute to chronic sympathoexcitation and hypertension. Accumulating evidence indicates that the activation of Ang-II type-2 receptors (AT2Rs) in the brain exerts effects that are opposite to those of AT1Rs, lowering blood pressure, and reducing hypertension. Thus, it would be interesting to understand the relative cellular localization of AT1R and AT2R in the brain under normal conditions and whether this localization changes during hypertension. Here, we developed a novel AT1aR-tdTomato reporter mouse strain in which the location of brain AT1aR was largely consistent with that determined in the previous studies. This AT1aR-tdTomato reporter mouse strain was crossed with our previously described AT2R-eGFP reporter mouse strain to yield a novel dual AT1aR/AT2R reporter mouse strain, which allowed us to determine that AT1aR and AT2R are primarily localized to different populations of neurons in brain regions controlling cardiovascular, fluid, and metabolic homeostasis. Using the individual AT1aR-tdTomato reporter mice, we also demonstrated that during hypertension induced by the administration of deoxycorticosterone acetate-salt, there was no shift in the expression of AT1aR from neurons to microglia or astrocytes in the paraventricular nucleus, a brain area important for sympathetic regulation. Using AT2R-eGFP reporter mice under similar hypertensive conditions, we demonstrated that the same was true of AT2R expression in the nucleus of the solitary tract (NTS), an area critical for baroreflex control. Collectively, these findings provided a novel means to assess the colocalization of AT1R and AT2R in the brain and a novel view of their cellular localization in hypertension.

Angiotensin-(1-7) [Ang-(1-7)] is an endogenous, heptapeptide hormone acting through the Mas receptor (MasR), with antiproliferative and antiangiogenic properties. Recent studies have shown that Ang-(1-7) has an antiproliferative action on lung adenocarcinoma cells and prostate cancer cells. In this study, we report that MasR levels were significantly upregulated in nasopharyngeal carcinoma (NPC) specimens and NPC cell lines. Viral vector–mediated expression of Ang-(1-7) dramatically suppressed NPC cell proliferation and migration in vitro. These effects were completely blocked by the specific Ang-(1-7) receptor antagonist A-779, suggesting that they are mediated by the Ang-(1-7) receptor Mas. In this study, Ang-(1-7) not only caused a significant reduction in the growth of human nasopharyngeal xenografts, but also markedly decreased vessel density, suggesting that the heptapeptide inhibits angiogenesis to reduce tumor size. Mechanistic investigations revealed that Ang-(1-7) inhibited the expression of the proangiogenic factors VEGF and PlGF. Taken together, the data suggest that upregulation of MasR could be used as a diagnostic marker of NPC and Ang-(1-7) may be a novel therapeutic agent for nasopharyngeal cancer therapy because it exerts significant antiangiogenic activity. Mol Cancer Ther; 15(1); 37–47. ©2015 AACR.

Summary The aim of this study was to examine whether macrophage migration inhibitory factor (MIF) could exaggerate inflammatory response in a mouse model of experimental autoimmune uveitis (EAU) and to explore the underlying mechanism. Mutant serotype 8 adeno-associated virus (AAV8) (Y733F)-chicken β-actin (CBA)-MIF or AAV8 (Y733F)-CBA-enhanced green fluorescent protein (eGFP) vector was delivered subretinally into B10.RIII mice, respectively. Three weeks after vector delivery, EAU was induced with a subcutaneous injection of a mixture of interphotoreceptor retinoid binding protein (IRBP) peptide with CFA. The levels of proinflammatory cytokines were detected by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). Retinal function was evaluated with electroretinography (ERG). We found that the expression of MIF and its two receptors CD74 and CD44 was increased in the EAU mouse retina. Compared to AAV8.CBA.eGFP-injected and untreated EAU mice, the level of proinflammatory cytokines, the expression of Notch1, Notch4, delta-like ligand 4 (Dll4), Notch receptor intracellular domain (NICD) and hairy enhancer of split-1 (Hes-1) increased, but the ERG a- and b-wave amplitudes decreased in AAV8.CBA.MIF-injected EAU mice. The Notch inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT) reduced the expression of NICD, Hes-1 and proinflammatory cytokines. Further, a MIF antagonist ISO-1 attenuated intraocular inflammation, and inhibited the differentiation of T helper type 1 (Th1) and Th17 in EAU mice. We demonstrated that over-expression of MIF exaggerated ocular inflammation, which was associated with the activation of the Notch signalling. The expression of both MIF and its receptors are elevated in EAU mice. Over-expression of MIF exaggerates ocular inflammation, and this exaggerated inflammation is associated with the activation of the Notch signalling and Notch pathway. Our data suggest that the MIF–Notch axis may play an important role in the pathogenesis of EAU. Both the MIF signalling pathways may be promising targets for developing novel therapeutic interventions for uveitis.

In most situations, the angiotensin AT2-receptor (AT2R) mediates physiological actions opposing those mediated by the AT1-receptor (AT1R), including a vasorelaxant effect. Nevertheless, experimental evidence vastly supports that systemic application of AT2R-agonists is blood pressure neutral. However, stimulation of AT2R locally within the brain or the kidney apparently elicits a systemic blood pressure lowering effect. A systemic effect of AT2R stimulation on blood pressure can also be achieved, when the prevailing effect of continuous background AT1R-stimulation is attenuated by low-dose AT1R blockade. Despite a lack of effect on blood pressure, AT2R stimulation still protects from hypertensive end-organ damage. Current data and evidence therefore suggest that AT2R agonists will not be suitable as future anti-hypertensive drugs, but that they may well be useful for end-organ protection in combination with established anti-hypertensives.

Angiotensin-II acts at its type-1 receptor (AT1R) in the brain to regulate body fluid homeostasis, sympathetic outflow and blood pressure. However, the role of the angiotensin type-2 receptor (AT2R) in the neural control of these processes has received far less attention, largely because of limited ability to effectively localize these receptors at a cellular level in the brain. The present studies combine the use of a bacterial artificial chromosome transgenic AT2R-enhanced green fluorescent protein (eGFP) reporter mouse with recent advances in in situ hybridization (ISH) to circumvent this obstacle. Dual immunohistochemistry (IHC)/ISH studies conducted in AT2R-eGFP reporter mice found that eGFP and AT2R mRNA were highly co-localized within the brain. Qualitative analysis of eGFP immunoreactivity in the brain then revealed localization to neurons within nuclei that regulate blood pressure, metabolism, and fluid balance (e.g., NTS and median preoptic nucleus [MnPO]), as well as limbic and cortical areas known to impact stress responding and mood. Subsequently, dual IHC/ISH studies uncovered the phenotype of specific populations of AT2R-eGFP cells. For example, within the NTS, AT2R-eGFP neurons primarily express glutamic acid decarboxylase-1 (80.3 ± 2.8 %), while a smaller subset express vesicular glutamate transporter-2 (18.2 ± 2.9 %) or AT1R (8.7 ± 1.0 %). No co-localization was observed with tyrosine hydroxylase in the NTS. Although AT2R-eGFP neurons were not observed within the paraventricular nucleus (PVN) of the hypothalamus, eGFP immunoreactivity is localized to efferents terminating in the PVN and within GABAergic neurons surrounding this nucleus. These studies demonstrate that central AT2R are positioned to regulate blood pressure, metabolism, and stress responses.

Activation of the angiotensin II type 2 receptor (AT2R) by administration of Compound 21 (C21), a selective AT2R agonist, induces neuroprotection in models of ischemic stroke in young adult animals. The mechanisms of this neuroprotective action are varied, and may include direct and indirect effects of AT2R activation. Our objectives were to assess the long-term protective effects of post-stroke C21 treatments in a clinically-relevant model of stroke in aged rats and to characterize the cellular localization of AT2Rs in the mouse brain of transgenic reporter mice following stroke. Intraperitoneal injections of C21 (0.03mg/kg) after ischemic stroke induced by transient monofilament middle cerebral artery occlusion resulted in protective effects that were sustained for up to at least 3-weeks post-stroke. These included improved neurological function across multiple assessments and a significant reduction in infarct volume as assessed by magnetic resonance imaging. We also found AT2R expression to be on neurons, not astrocytes or microglia, in normal female and male mouse brains. Stroke did not induce altered cellular localization of AT2R when assessed at 7 and 14 days post-stroke. These findings demonstrate that the neuroprotection previously characterized only during earlier time points using stroke models in young animals is sustained long-term in aged rats, implying even greater clinical relevance for the study of AT2R agonists for the acute treatment of ischemic stroke in human disease. Further, it appears that this sustained neuroprotection is likely due to a mix of both direct and indirect effects stemming from selective activation of AT2Rs on neurons or other cells besides astrocytes and microglia.

The goal of the present review is to examine the evidence for beneficial actions of manipulation of the RAS (renin–angiotensin system) in stroke, with particular focus on Ang-(1–7) [angiotensin-(1–7)] and its receptor Mas. The RAS appears to be highly involved in the multifactorial pathophysiology of stroke. Blocking the effects of AngII (angiotensin II) at AT1R (AngII type 1 receptor), through the use of commonly prescribed ACE (angiotensin-converting enzyme) inhibitors or AT1R blockers, has been shown to have therapeutic effects in both ischaemic and haemorrhagic stroke. In contrast with the deleterious actions of over activation of AT1R by AngII, stimulation of AT2Rs (AngII type 2 receptors) in the brain has been demonstrated to elicit beneficial effects in stroke. Likewise, the ACE2/Ang-(1–7)/Mas axis of the RAS has been shown to have therapeutic effects in stroke when activated, countering the effects of the ACE/AngII/AT1R axis. Studies have demonstrated that activating this axis in the brain elicits beneficial cerebral effects in rat models of ischaemic stroke, and we have also demonstrated the cerebroprotective potential of this axis in haemorrhagic stroke using stroke-prone spontaneously hypertensive rats and collagenase-induced striatal haemorrhage. The mechanism of cerebroprotection elicited by ACE2/Ang-(1–7)/Mas activation includes anti-inflammatory effects within the brain parenchyma. The major hurdle to overcome in translating these results to humans is devising strategies to activate the ACE2/Ang-(1–7)/Mas cerebroprotective axis using post-stroke treatments that can be administered non-invasively. Abbreviations: ACE, angiotensin-converting enzyme; ACEi, ACE inhibitor(s); aCSF, artificial cerebrospinal fluid; Ang-(1–7), angiotensin-(1–7); AngII, angiotensin II; ARB, AT1R blocker; AT1R, AngII type 1 receptor; AT2R, AngII type 2 receptor; BBB, blood–brain barrier; BP, blood pressure; COX-2, cyclo-oxygenase 2; DIZE, diminazene aceturate; eNOS, endothelial nitric oxide synthase; EPC, endothelial progenitor cell; ERK1/2, extracellular-signal-regulated kinase 1/2; ET-1, endothelin-1; HPβCD, hydroxypropyl-β-cyclodextran; HSC, haematopoetic stem cell; ICV, intracerebroventricular(ly); IL, interleukin; iNOS, inducible nitric oxide synthase; MCAO, middle cerebral artery occlusion; NF-κB, nuclear factor κB; RAS, renin–angiotensin system; SHR, spontaneously hypertensive rat; spSHR, stroke-prone SHR; TNF-α, tumour necrosis factor-α

1. In recent years it has been firmly established that apart from the classic renin-angiotensin system (RAS) comprising angiotensin (Ang) II, angiotensin converting enzyme (ACE; responsible for AngII generation) and the angiotensin AT(1) receptor (AT(1)R), there also exist protective arms of the RAS that comprise the angiotensin AT(2) receptor (AT(2)R), Ang-(1-7), ACE2 (mainly responsible for Ang-(1-7) synthesis) and Mas, the receptor for Ang-(1-7). 2. Stimulation of AT(2)R promotes neuronal differentiation, neurite outgrowth and axonal regeneration, which results in an acceleration of repair and improved functional outcome after injury of peripheral nerves or the spinal cord. 3. Stimulation of AT(2)R and the receptor Mas has been shown to reduce infarct size and ameliorate neurological deficits in various animal models of stroke. The underlying mechanisms of action are comprised of activation of direct neurotrophic, anti-inflammatory and anti-oxidant pathways, as well as the augmentation of cerebral blood flow. 4. Cognitive function is improved by AT(2)R stimulation due, at least in part, to increased cerebral blood flow. There is indirect evidence that Ang-(1-7) could also play a role in protection against cognitive decline, but studies confirming this have not yet been published. 5. In view of the data reviewed in this article, it can be assumed that the protective arms of the RAS are putative targets in the treatment of neurological diseases, which involve tissue damage or cognitive impairment.

Previously we demonstrated that viral-mediated increased expression of the anti-inflammatory cytokine interleukin-10 within the paraventricular nucleus of the hypothalamus significantly reduces blood pressure in normal rats made hypertensive by infusion of angiotensin II. However, the exact cellular locus of this interleukin-10 action within the paraventricular nucleus is unknown. In the present study we tested whether interleukin-10 exerts direct effects at its receptors located on hypothalamic neurons to offset the neuronal excitatory actions of angiotensin II via its type 1 receptors. The results indicated the presence of immunoreactive interleukin-10 receptors on neurons in normal rat paraventricular nucleus and that receptors for this cytokine were also expressed in neurons cultured from the hypothalamus. Patch-clamp electrophysiological recordings from these cultures revealed that extracellular application of interleukin-10 alone did not exert any alterations in neuronal membrane delayed rectifier or transient potassium currents. However, angiotensin II elicited a significant decrease in delayed rectifier potassium current, an effect that was abolished by interleukin-10 application. Since decreases in delayed rectifier potassium current contribute to increased neuronal excitability, this result is consistent with a direct inhibitory action of interleukin-10 on angiotensin-induced excitation of hypothalamic neurons. As such, these data are the first indication of a neuronal locus of action of interleukin-10 to temper the actions of angiotensin II in the hypothalamus.

Obesity is associated with increased levels of angiotensin-II (Ang-II), which activates angiotensin type 1a receptors (AT1a) to influence cardiovascular function and energy homeostasis. To test the hypothesis that specific AT1awithin the brain control these processes, we used the Cre/lox system to delete AT1afrom the paraventricular nucleus of the hypothalamus (PVN) of mice. PVN AT1adeletion did not affect body mass or adiposity when mice were maintained on standard chow. However, maintenance on a high-fat diet revealed a gene by environment interaction whereby mice lacking AT1ain the PVN had increased food intake and decreased energy expenditure that augmented body mass and adiposity relative to controls. Despite this increased adiposity, PVN AT1adeletion reduced systolic blood pressure, suggesting that this receptor population mediates the positive correlation between adiposity and blood pressure. Gene expression studies revealed that PVN AT1adeletion decreased hypothalamic expression of corticotrophin-releasing hormone and oxytocin, neuropeptides known to control food intake and sympathetic nervous system activity. Whole-cell patch-clamp recordings confirmed that PVN AT1adeletion eliminates responsiveness of PVN parvocellular neurons to Ang-II, and suggest that Ang-II responsiveness is increased in obese wild-type mice. Central inflammation is associated with metabolic and cardiovascular disorders and PVN AT1adeletion reduced indices of hypothalamic inflammation. Collectively, these studies demonstrate that PVN AT1aregulate energy balance during environmental challenges that promote metabolic and cardiovascular pathologies. The implication is that the elevated Ang-II that accompanies obesity serves as a negative feedback signal that activates PVN neurons to alleviate weight gain.

The role of the angiotensin type-2 receptor (AT2R) in the neural control of cardiovascular homeostasis and hypertension is not well-characterized. Using a BAC transgenic AT2R-enhanced green fluorescent protein (eGFP) reporter mouse, dense localization of AT2R to GABA neurons was found within the nucleus of the solitary tract (NTS) of the hindbrain, an area important for regulating baroreflex function and blood pressure. This localization was confirmed by RNAscope fluorescence in situ hybridization. Considering that GABA is pressor in the intermediate NTS (intNTS), and that its effects are enhanced in models of neurogenic hypertension such as SHR and deoxycorticosterone acetate (DOCA)-salt rats, we tested the hypothesis that AT2R on GABA neurons in the NTS constitute a counter-regulatory, blood pressure lowering mechanism. In support of this idea, we have demonstrated that: (i) Optogenetic stimulation of GABA neurons in the intNTS of normal mice elicited a significant increase in blood pressure; (ii) mRNA levels for both the GABA synthetic enzyme Gad1 and for the AT2R are significantly increased in the intNTS of DOCA-salt hypertensive mice; (iii) Intracerebroventricular (ICV) infusion of the AT2R agonist Compound 21 (C21; 7.5 ng/h) into normotensive or DOCA-salt hypertensive mice elicited a significant reduction of systolic blood pressure, an effect that was much larger in hypertensive (126.2 ± 5.0 v. 139.8 ± 3.2 mmHg; n = 14; p = 0.02) than in normotensive (119.8 ± 2.6 v. 126.5 ± 2.5 mmHg; n = 16; p = 0.04) mice and was accompanied by decreased levels of the GABA synthetic enzymes Gad1 and Gad2 in the intNTS; (iv) Finally, the crucial involvement of GABA neurons in the blood pressure lowering effect of AT2R was proven by the lack of any effect of ICV C21 in DOCA-salt hypertensive mice containing a selective knockout of AT2R from GABA neurons. These novel data provide strong evidence for an anti-hypertensive action of AT2R within the intNTS, an effect that is exerted through decreases in GABA transmission.

We hypothesized that moderate cardiac-selective overexpression of the angiotensin II type 2 receptor (AT2R) would protect the myocardium from ischaemic injury after a myocardial infarction (MI) induced by coronary artery ligation. For in vitro studies, adenoviral vector expressing genomic DNA of AT2R and enhanced green fluorescence protein (EGFP) was used to overexpress AT2R in rat neonatal cardiac myocytes. Expression of AT2R, measured by real-time PCR and immunostaining, demonstrated efficient transduction of AT2R in a dose-dependent pattern. The AT2R constitutively induced apoptosis in rat neonatal cardiac myocytes in dose-dependent patterns. For in vivo studies, 4 × 1010 vector genomes (vg) of recombinant adeno-associated virus serotype 9 (rAAV9)-chicken β actin promoter-AT2R was injected into the left ventricle of 5-day-old Sprague–Dawley rats. At 6 weeks of age, hearts were harvested and expression of AT2R determined by real-time PCR and Western blotting. Expression was increased onefold over control hearts, and no apoptosis was detected. Two subsequent in vivo studies were performed. In a prevention study, 4 × 1010 vg of rAAV9-CBA-AT2R was injected into the left ventricle of 5-day-old Sprague–Dawley rats and MI was induced at 6 weeks of age. For a post-treatment study, 4 × 1010 vg of rAAV9-CBA-AT2R was administrated to the peri-infarcted myocardium area immediately after MI in 6-week-old animals. For both in vivo studies, cardiac functions were assessed using echocardiography and haemodynamic measurements 4 weeks after coronary artery ligation. In the in vivo studies, the rats subjected to MI showed significant decreases in fractional shortening and rate of change of left ventricular pressure, with increased left ventricular end-diastolic pressure and ventricular hypertrophy. For the prevention study, the moderate cardiac-selective overexpression of AT2R attenuated these MI-induced impairments and also caused a decrease in ventricular wall thinning. In the post-treatment study, the overexpression of AT2R partly reversed the MI-induced cardiac dysfunction. Myocardial infarction also induced the upregulation of angiotensin II type 1 receptor, angiotensin-converting enzyme and collagen I mRNA expression, all of which were attenuated by the overexpression of AT2R. It is concluded that moderate cardiac-selective overexpression of AT2R protects heart function from ischaemic injury, which may be mediated, at least in part, through modulation of components of the cardiac renin–angiotensin system and collagen levels in the myocardium.

It is a well-established fact that components of the renin–angiotensin system (RAS) play a critical role in the progression of heart failure. Pharmacotherapeutic interventions with either angiotensin-converting enzyme (ACE) inhibitors (Haywood et al. 1997) or angiotensin type 1 receptor blockers (Steckelings et al. 2005; Ferreira et al. 2009) have demonstrated significant protection against myocardial infarction and heart failure in experimental animal models as well as in patients. Several studies have suggested that the beneficial effects observed with angiotensin-converting enzyme inhibitors are not only due to the reduction in the formation of the detrimental angiotensin II (Ang II), but are also due to significant elevation in the levels of angiotensin-(1–7) (Ang-(1–7); Keidar et al. 2007). Correlative studies have also shown that ACE2 and Ang-(1–7) levels are increased in the heart following myocardial infarction in both humans and rats (Averill et al. 2003; Burrell et al. 2005). A growing body of evidence indicates that Ang-(1–7) plays an active role in maintaining cardiovascular homeostasis, thus providing protection against heart diseases. This conclusion is supported by the following findings. Angiotensin-(1–7) has been shown to attenuate isoprenaline-induced cardiac hypertrophy (Santos et al. 2004). Chronic administration of Ang-(1–7) has been shown to improve coronary artery perfusion and endothelial function in a rat model for heart failure (Loot et al. 2002). Angiotensin-(1–7) was shown to reduce the incidence and the duration of postischaemic reperfusion arrhythmias in the isolated rat heart (Ferreira et al. 2001; De Mello, 2004). Intravenous infusion of Ang-(1–7) resulted in improved contractile function in rat hearts (Sampaio et al. 2003). Angiotensin-(1–7) has been demonstrated to attenuate cardiac remodelling significantly in terms of reducing myocyte hypertrophy and interstitial fibrosis (Santos et al. 2004; Tallant et al. 2005; Grobe et al. 2006, 2007b; Mercure et al. 2008; Ferreira et al. 2010; Giani et al. 2010). Furthermore, AVE0991, a synthetic mimetic of Ang-(1–7), has been shown to exert cardioprotective effects (Ferreira et al. 2007). Although Ang-(1–7) exerts beneficial effects against a range of cardiovascular diseases, the in vivo efficacy of this peptide is severely hampered owing to rapid degradation by peptidases. The half-life for Ang-(1–7) is very short and dependent on species. For example, following intravenous administration the half-life of Ang-(1–7) in humans is approximately 30 min (Kono et al. 1986; Rodgers et al. 2006), whereas in rodents it is approximately 20 s (Iusuf et al. 2008). To overcome this limitation, we have overexpressed Ang-(1–7) in the rodent heart shortly after birth, as we previously demonstrated (Methot et al. 2001; Diez-Freire et al. 2006), to evaluate its cardioprotective effects against myocardial infarction. Also, we hypothesize that Ang-(1–7) may exert its beneficial effects against myocardial infarction by restoring the balance between the deleterious ACE–Ang II–angiotensin II type 1 receptor (AT1R) axis and the beneficial ACE2–Ang-(1–7)–Mas axis of the RAS.

Activation of angiotensin-converting enzyme 2 (ACE2), production of angiotensin-(1–7) [Ang-(1–7)] and stimulation of the Ang-(1–7) receptor Mas exert beneficial actions in various peripheral cardiovascular diseases, largely through opposition of the deleterious effects of angiotensin II via its type 1 receptor. Here we considered the possibility that Ang-(1–7) may exert beneficial effects against CNS damage and neurological deficits produced by cerebral ischaemic stroke. We determined the effects of central administration of Ang-(1–7) or pharmacological activation of ACE2 on the cerebral damage and behavioural deficits elicited by endothelin-1 (ET-1)-induced middle cerebral artery occlusion (MCAO), a model of cerebral ischaemia. The results of the present study demonstrated that intracerebroventricular infusion of either Ang-(1–7) or an ACE2 activator, diminazine aceturate (DIZE), prior to and following ET-1-induced MCAO significantly attenuated the cerebral infarct size and neurological deficits measured 72 h after the insult. These beneficial actions of Ang-(1–7) and DIZE were reversed by co-intracerebroventricular administration of the Mas receptor inhibitor, A-779. Neither the Ang-(1–7) nor the DIZE treatments altered the reduction in cerebral blood flow elicited by ET-1. Lastly, intracerebroventricular administration of Ang-(1–7) significantly reduced the increase in inducible nitric oxide synthase mRNA expression within the cerebral infarct that occurs following ET-1-induced MCAO. This is the first demonstration of cerebroprotective properties of the ACE2–Ang-(1–7)–Mas axis during ischaemic stroke, and suggests that the mechanism of the Ang-(1–7) protective action includes blunting of inducible nitric oxide synthase expression.

Recent discovery of (pro)renin receptor (PRR) has revolutionized the field of renin–angiotensin system (RAS) physiology and has underscored the critical role of the local and tissue RAS in the development and establishment of cardiac, vascular and renal pathophysiology linked to hypertension and diabetes (Ichihara et al. 2007; Nguyen, 2007). (Pro)renin receptor is a 350 amino acid protein with a single transmembrane domain that binds and actives prorenin. Prorenin contains a ‘handle’ region that conceals the active site of renin (Suzuki et al. 2003). Prorenin binding to PRR results in non-proteolytic activation of the enzyme by inducing a conformational change at the ‘handle’ region, exposing the active site (Nguyen et al. 2002; Danser et al. 2007). In addition, renin binding to the PRR increases its intrinsic activity. Thus, prorenin bound to PRR is many-fold more efficient in the conversion of angiotensinogen to angiotensin I, subsequently leading to increased angiotensin II (Ang II) at the tissue level (Nguyen et al. 2002; Nguyen, 2007). Studies have established that PRR plays a dual role in the regulation of RAS activity: it binds to prorenin/renin to facilitate the formation of Ang I and II, and the binding initiates an intracellular signal transduction pathway involving mitogen-activated protein kinases (MAPK). The latter action is presumably associated with increased synthesis of profibrotic molecules such as plasminogen activator inhibitor-1 (PAI-1), fibronectin, collagen and transforming growth factor-β (TGF-β; Huang et al. 2006; Danser et al. 2007; Nguyen, 2007). The importance of PRR in the cardiovascular system is further evident from pathophysiological and transgenic studies. For example: (i) inhibition of PRR by a ‘handle’ region peptide has been shown to protect the heart and kidney against tissue damage induced by hypertension (Ichihara et al. 2004, 2006); (ii) activation of PRR selectively promotes pathological retinal neovascularization (Satofuka et al. 2007); and (iii) overexpression of PRR in rats results in elevated blood pressure and increased plasma aldosterone (Burckle et al. 2006). Collectively, these observations indicate that PRR may be a critical member of the RAS in the maintenance of normal cardiovascular physiology. In spite of its emerging involvement and importance in cardiac, renal and vascular pathophysiology, nothing is known about the role of PRR in the brain. This is particularly relevant in view of the fact that the brain RAS is integral in neural control of cardiovascular functions and its hyperactivity is linked to hypertension (Veerasingham & Raizada, 2003; Peterson et al. 2006; Osborn et al. 2007). Although the presence of all the components of the RAS is unquestionable in the brain, their cellular distribution and the interplay of different cell types (neurons, glia, etc.) in the generation of Ang II and the access of this hormone to the Ang II type 1 (AT1) receptor in cardiovascular-relevant neurons remains elusive. In addition, the presence of low levels of renin in the brain has been a consistent enigma in establishing an independent role of the brain RAS (Dzau et al. 1986; Baltatu et al. 1998). Discovery of the PRR is of great significance in this regard and may be critical in solving the puzzle of an intrinsic RAS in the brain and delineating the mechanism of neuronal Ang II actions. Thus, our objective in the present study was to determine whether neurons within the brain express a functional PRR, in at attempt to elucidate the role of the brain RAS in cardiovascular functions.

Our previous studies demonstrated that peripheral overexpression of angiotensin II (ANG II) type 2 receptors (AT2R) prevents hypertension-induced cardiac hypertrophy and remodeling without altering high blood pressure. This, coupled with the observations that AT2R play a role in the antihypertensive actions of ANG II type 1 receptor (AT1R) blockers (ARBs), led us to propose that peripheral overexpression of AT2R would improve the antihypertensive action of losartan (Los) in Sprague-Dawley (SD) rats made hypertensive via chronic infusion of ANG II. Here we utilized adenoviral vector-mediated AT2R gene transfer to test this hypothesis. A single intracardiac injection of adenoviral vector containing genomic AT2R (G-AT2R) DNA and enhanced green fluorescent protein (EGFP) gene controlled by cytomegalovirus (CMV) promoters (Ad-G-AT2R-EGFP; 5 × 109 infectious units) into adult SD rats produced robust AT2R overexpression in cardiovascular tissues (kidney, lung, heart, aorta, mesenteric artery, and renal artery) that persisted for 3–5 days postinjection. By 7 days post viral injection, the overexpressed AT2R are reduced toward basal values in certain tissues (lung, kidney, and heart) and are undetectable in others (kidney and blood vessels). In two separate protocols, we demonstrated that the hypotensive effect of Los (0.125, 0.5, and 1.0 mg/kg iv) was significantly greater in the AT2R-overexpressing animals (−40.7 ± 4.3, −41.8 ± 4.8, and −48.1 ± 2.6 mmHg, respectively) compared with control vector (Ad-CMV-EGFP)-treated rats (−12.4 ± 2.2, −20.2 ± 3.4, and −27.3 ± 3.4 mmHg, respectively). These results provide support for a depressor role of AT2R and the proposal that combined AT2R agonist and ARB treatment may be an improved therapeutic strategy for controlling hypertension.

Pulmonary fibrosis (PF) is a fatal disease characterized by scarring of the lung tissue. A dysregulated renin angiotensin system (RAS) has been implicated in the pathology of PF. Recent reports suggest a beneficial role for the angiotensin type II receptor (AT2R) in cardiopulmonary disorders; however, the effects of AT2R in PF have not been studied. We have taken advantage of a recently identified AT2R agonist, Compound 21 (C21), to evaluate its actions in the bleomycin (BLM) model of PF. PF was induced by a single intra-tracheal instillation of 2.5mg/kg of BLM. Following BLM administration, a subset of animals was treated with C21 (0.03mg/kg, ip, daily) for 2 weeks (prevention protocol). In a subsequent study, C21 was administered after three days of BLM injection (reversal protocol). For both prevention and reversal protocols, hemodynamic parameters were measured, and tissues collected after 15 days of BLM injection. BLM caused significant increase in right ventricular systolic pressure (RVSP) and induced maladaptive cardiac remodeling. However, C21 treatment considerably reduced BLM-induced elevation in RVSP (Con: 29+1; BLM: 47+2; BLM+C21(P): 37+2; BLM+C21(R): 41+3 mmHg), attenuated ventricular hypertrophy (Con: 0.28+0.01; BLM: 0.41+0.01; BLM+C21(P): 0.35+0.01; BLM+C21(R): 0.37+0.02) and improved right heart function in both prevention (P) and reversal (R) protocols. Histopathological assessment of hematoxylin and eosin stained lung sections also showed decreased collagen accumulation in the C21 treated group. Collectively, our results suggest that the AT2R agonist, C21 exerts potent antifibrotic effects and may hold promise for patients with PF.

Much evidence indicates that pro-inflammatory effects of the renin-angiotensin system within the hypothalamus, including microglial activation and production of pro-inflammatory cytokines, play a role in chronic neurogenic hypertension. Our objective here was to examine whether angiotensin-(1-7) [Ang-(1-7)], a protective component of the renin-angiotensin system, exerts direct actions at microglia to counteract these pro-inflammatory effects. Mas, the Ang-(1-7) receptor, was shown to be present on cultured hypothalamic microglia. Treatment of these cells with Ang-(1-7) (100-1000 nM, 3-12 h) elicited significant decreases in basal levels of mRNAs for the pro-inflammatory cytokines interleukin-1β (IL-1β) and tumor-necrosis factor α (TNFα) and of the microglia-macrophage marker CD11b, and increases in basal levels of the anti-inflammatory cytokine interleukin-10. Incubation of microglial cultures with (pro)renin (PRO) (10-50 nM; 6 h) elicited significant increases in mRNAs for IL-1β, TNFα and CD11b. The effects of PRO (10 nM) on IL-1β and TNFα mRNAs, and TNFα protein, were significantly attenuated by co-treatment with Ang-(1-7) (100 nM). Lastly, these actions of Ang-(1-7) were abolished by the Mas antagonist A-779, and were associated with reductions in NF-κB subunit expression. Collectively, these data provide the first evidence that Ang-(1-7) can exert direct effects at microglia to lower baseline and counteract PRO-induced increases in pro-inflammatory cytokines. Renin-Angiotensin system mediated microglial activation and pro-inflammatory cytokine production within the hypothalamus are components of the chronic neuroinflammation associated with 'neurogenic' hypertension. We demonstrated that angiotension-(1-7) acting via its receptor Mas on hypothalamic microglia lessens baseline and (pro)renin-induced increases in pro-inflammatory cytokine production by these cells. This is the first evidence that angiotensin-(1-7) has direct anti-inflammatory effects via microglia.

The discovery of beneficial neuroprotective effects of the angiotensin converting enzyme 2-angiotensin-(1-7)-Mas axis [ACE2-Ang-(1-7)-Mas] in ischemic and hemorrhagic stroke has spurred interest in a more complete characterization of its mechanisms of action. Here, we summarize findings that describe the protective role of the ACE2-Ang-(1-7)-Mas axis in stroke, along with a focused discussion on the potential mechanisms of neuroprotective effects of Ang-(1-7) in stroke. The latter incorporates evidence describing the actions of Ang-(1-7) to counter the deleterious effects of angiotensin II (AngII) via its type 1 receptor, including anti-inflammatory, anti-oxidant, vasodilatory, and angiogenic effects, and the role of altered kinase-phosphatase signaling. Interactions of Mas with other receptors, including bradykinin receptors and AngII type 2 receptors are also considered. A more complete understanding of the mechanisms of action of Ang-(1-7) to elicit neuroprotection will serve as an essential step toward research into potential targeted therapeutics in the clinical setting.

In this chapter, we explore the beneficial actions in stroke elicited by the activation of the angiotensin-converting enzyme 2/angiotensin-(1-7)/receptor Mas axis of the renin–angiotensin system. The neuroprotective efficacy in stroke of targeted manipulation of this axis is becoming well-established, thanks to findings from an increasing number of studies, which use a variety of stroke models and activating compounds. As part of a discussion of the effects of angiotensin-(1-7) in the brain and other tissues, we also describe several possible mechanisms by which the receptor Mas exerts its neuroprotective effects. Finally, we identify important steps to the clinical translation of these promising findings from the bench to the bedside.

Glutamate, the major excitatory neurotransmitter, is critical for normal brain development and function. Both extremes of glutamate receptor activity are detrimental for the brain. Glutamate's role in excitotoxicity has driven the search for receptor antagonists as neuroprotective agents, most of which have failed to achieve clinical, i.e. efficacious and safe, neuroprotection. High selectivity and potency provide potential explanations for this failure. For example, targeting individual glutamate receptor subtypes leaves other pathways of glutamatergic excitotoxicity intact. Furthermore, potent depression of glutamate receptor activity causes clinical side effects, such as the symptoms of schizophrenia produced by NMDA receptor antagonists. To produce efficacious neuroprotection devoid of significant side effects, it may be necessary to normalize the function of all components of the glutamatergic system, instead of blocking a single type of glutamate receptors. Halogenated derivatives of aromatic amino acids modulate glutamatergic activity via multiple pre- and postsynaptic actions with moderate efficacy. In addition, these compounds may trap hydroxyl radicals and facilitate hydroxyl radical-impaired glutamate uptake. Their balanced polyvalent action may overcome the limitations of previously tested glutamatergic agents and provide a basis for their use in the treatment of neurological and neuropsychiatric disorders. The properties of this class of compounds and relevant patents are reviewed in this article.

Previous evidence has suggested that brain catecholamine levels are important in the regulation of central angiotensin II receptors. In the present study, the effects of norepinephrine and 3,4-dihydroxyphenylethylamine (dopamine) on angiotensin II receptor regulation in neuronal cultures from rat hypothalamus and brainstem have been examined. Both catecholamines elicit significant decreases in [125I]angiotensin II-specific binding to neuronal cultures prepared from normotensive rats, effects that are dose dependent and that are maximal within 4-8 h of preincubation. Saturation and Scatchard analyses revealed that the norepinephrine-induced decrease in the binding is due to a decrease in the number of angiotensin II receptors in neuronal cultures, with little effect on the receptor affinity. Norepinephrine has no significant actions on [125I]angiotensin II binding in cultures prepared from spontaneously hypertensive rats. The downregulation of angiotensin II receptors by norepinephrine or dopamine is blocked by alpha 1-adrenergic and not by other adrenergic antagonists, a result suggesting that this effect is initiated at the cell surface involving alpha 1-adrenergic receptors. This is further supported by our data indicating a parallel downregulation of specific alpha 1-adrenergic receptors elicited by norepinephrine. In summary, these results show that norepinephrine and dopamine are able to alter the regulation of neuronal angiotensin II receptors by acting at alpha 1-adrenergic receptors, which is a novel finding.

The cellular characteristics of the beta-adrenoreceptor in glial and neuronal cells from the newborn rat brain were determined by (-)-[125I]iodocyanopindolol binding. In membranes from both cell types, the binding was saturable and from competition assays the potency series of (-)-isoproterenol greater than (-)-epinephrine = (-)-norepinephrine greater than (+)-isoproterenol was observed. 5'-Guanylyl-imidodiphosphate reduced the affinity of (-)-isoproterenol for the beta-adrenoreceptor from glial cells but had no effect on agonist affinity in neuronal cells. Chronic treatment of both cell types with (-)-isoproterenol reduced the receptor content and the capacity of the agonist to increase the cellular cyclic AMP content. However, the receptor recovery after chronic agonist treatment was faster in glial cells (72 h) than neuronal cells (120 h) and was blocked by cycloheximide. Treatment of both types with the irreversible beta-blocker bromoacetylalprenololmentane (2 microM) reduced the receptor content by 78% but no receptor recovery was observed for 120 h after the initial receptor loss. The data indicated that the majority of beta-adrenoreceptors in both cell types are the beta-1 subtype, but show some differences in receptor-agonist interactions. Furthermore, these CNS cells may be useful models for regulatory studies on the beta-adrenoreceptor.

Angiotensin II exerts its physiological effects by activating multiple subtypes of its receptor such as AT 1a -, AT 1b -, and AT 2 -receptors. Because of a high degree of similarity among these G-protein–coupled receptors, it has been difficult to assign diverse physiological actions of angiotensin II through these receptor subtypes. We have developed small interfering RNAs to selectively inhibit the expression of the AT 1a receptor (AT 1a R) subtype. A dsRNA, AT 1 47, was found to be highly selective and efficient in reducing the levels of AT 1a R subtype. Transfection of AT 1a R-expressing CHO cells with dsRNA AT 1 47 resulted in an 80% decrease in the AT 1a R expression. In contrast, dsRNA AT 1 47 showed no significant effects on both AT 1b R and AT 2 R subtypes. Thus, AT 1 47 provides us with a powerful tool to selectively silence this subtype of receptor to investigate its role in cardiovascular physiology.

Angiotensin II (Ang II) elicits Ang II type 1 receptor (AT1-R)-mediated increases in neuronal firing within the hypothalamus and brainstem that are ultimately responsible for physiological actions such as increased blood pressure and fluid intake. Although there is a growing literature on the intracellular mechanisms that mediate the actions of Ang II via AT1-R in neurons, little is known about the mechanisms that diminish or “switch-off” the neuronal chronotropic action of Ang II. In the present study, we identified macrophage migration inhibitory factor (MIF) as an intracellular inhibitor of the actions of Ang II in neurons. The evidence is as follows. First, Ang II, acting via AT1-R, increases the intracellular levels of MIF in neurons cultured from rat hypothalamus and brainstem. Second, elevation of intracellular MIF by Ang II prevents further chronotropic actions of this peptide. Third, intracellular application of exogenous recombinant MIF abolishes the Ang II-induced chronotropic action in neurons. Finally, intracellular application of the MIF peptide fragment MIF-(50-65), which harbors the thiol oxidoreductase property of the MIF molecule, mimics the inhibitory actions of MIF on Ang II-stimulated neuronal firing. Thus, this study is the first to demonstrate the existence of an intracellular negative regulator of Ang II-induced actions in neurons and indicates that MIF may act as a physiological brake for the chronotropic effects of Ang II in rat neurons.

Most of our knowledge of the function of the angiotensin type 2 receptor (AT(2)R) has been obtained from transgenic mouse models. The aim of the present study was to investigate the role of the AT(2)R in normotensive Sprague-Dawley (SD) rats by using antisense gene transfer technology to 'knockdown' this specific receptor subtype. A retroviral vector containing full-length AT(2)R antisense cDNA (AT(2)R-AS) was constructed and the effectiveness of the transduction of AT(2)R-AS was studied in vitro. In subsequent in vivo studies, 5-day-old normotensive SD rats received a single intracardiac bolus (25 microl) of AT(2)R-AS viral particles. When animals reached adulthood, direct blood pressure (BP), and both pressor and dipsogenic responses to angiotensin II were investigated. Long-lasting expression of the AT(2)R-AS transcript and a reduction in mRNA and binding of the AT(2)R was observed in vitro. Expression of AT(2)R-AS transcript was maintained for 90 days in heart, kidney, lung and brain, indicating a high degree of transgene transduction in vivo. As adults, systolic BP and the pressor responses to angiotensin were significantly elevated in AT(2)R-AS-treated rats. However, AT(2)R-AS-treated rats displayed significantly reduced dipsogenic responses to both angiotensin and water deprivation. Collectively, these data demonstrate that a single neonatal injection of the retroviral vector containing antisense to the AT(2) receptors in rats results in similar cardiovascular and dipsogenic responses as reported in AT(2)R knockout mice. The actions of the AT(2) receptors appear to be antagonistic to the cardiovascular actions of the AT(1) receptors, whereas AT(1) and AT(2) receptors appear to act synergistically in the regulation of water intake.

Angiotensin II (Ang II), acting at Ang II type 1 receptors (AT1Rs), increases the firing rate of neurons from Wistar-Kyoto (WKY) rat brain via protein kinase C (PKC)- and calcium-calmodulin kinase II (CaMKII)-dependent mechanisms. The objectives of this study were twofold; first, to compare the Ang-II-stimulated increase in firing of neurons from WKY and spontaneous hypertensive rats (SHR) and second, to elucidate the signaling mechanisms involved. Action potentials were measured in neurons cultured from SHR and WKY rat brains using the whole cell configuration of the patch-clamp technique in the current-clamp mode. Ang II (100 nM) caused three- and sixfold increases in neuronal firing rate in WKY rat and SHR neurons, respectively; effects that were abolished by the AT1R antagonist Losartan (1 μM). Co-administration of calphostin C (10 μM, a PKC inhibitor) and KN-93 (10 μM, a CaMKII inhibitor) completely blocked this Ang II action in WKY rat neurons, while they caused only a ∼50% attenuation in SHR neurons. The residual increase in firing rate produced by Ang II in SHR neurons was blocked by inhibitors of phosphatidylinositol 3 kinase (PI3-kinase), either LY 294002 (10 μM) or wortmannin (100 nM). These observations suggest that a PI3-kinase signaling pathway may be responsible for the enhanced chronotropic effect produced by Ang II in SHR neurons.

Angiotensin II (Ang II) modulates, via Ang II type 1 (AT(1)) receptors, the activity of brain catecholaminergic neurons. Here we utilized catecholaminergic CATH.a cells to define the effects of Ang II on delayed rectifier K(+) current (I(Kv)), one of the factors that determines changes in neuronal activation. Receptor binding analyses demonstrated the presence of AT(1) receptors in CATH.a cells. Whole cell voltage clamp experiments in these cells revealed that Ang II (100nM) produced a significant inhibition of I(Kv), that was abolished by the AT(1) receptor blocker, losartan (1 microM), or by inhibition of phospholipase C (PLC) with U73122 (10 microM). Furthermore, this action of Ang II was completely abolished by co-inhibition of protein kinase C (PKC) and calcium/calmodulin protein kinase II (CaMKII). These results demonstrate that Ang II produces an inhibition of I(Kv) in CATH.a cells, via an intracellular pathway that includes PLC, PKC, and CaMKII.

Epinephrine (Epi) mediates various physiological effects via alpha2A-adrenergic receptors (alpha2A-ARs). Studies in mice with a point mutation in the gene for alpha2A-AR have shown that these receptors are responsible for the centrally mediated depressor effects of alpha2-AR agonists. These studies underscore the importance of understanding the basic cellular mechanisms involved in the expression of alpha2A-ARs, of which little is known. We use astroglia cultured from the hypothalamus and brainstem of adult Sprague-Dawley rats as a model system in which to study factors that regulate alpha2A-AR expression. These cells contain alpha2-ARs, which are predominately of the alpha2A-AR subtype. Our studies have shown that Epi causes a dose- and time-dependent decrease in steady-state levels of alpha2A-AR mRNA and number of alpha2A-ARs, effects that are mediated via alpha1- and beta-adrenergic receptors (alpha1-ARs and beta-ARs). These effects of Epi on alpha2A-AR mRNA and alpha2A-AR number are mimicked by activation of protein kinase C or increases in cellular cyclic AMP, which are intracellular messengers activated by alpha1-ARs and beta-ARs, respectively. Taken together, these results indicate that expression of alpha2A-ARs is regulated in a heterologous manner by Epi, via alpha1-AR- and beta-AR-mediated intracellular pathways.

Evidence indicates that angiotensin II type 2 receptors (AT2R) exert cerebroprotective actions during stroke. A selective non-peptide AT2R agonist, Compound 21 (C21), has been shown to exert beneficial effects in models of cardiac and renal disease, as well as hemorrhagic stroke. Here, we hypothesize that C21 may exert beneficial effects against cerebral damage and neurological deficits produced by ischemic stroke. We determined the effects of central and peripheral administration of C21 on the cerebral damage and neurological deficits in rats elicited by endothelin-1 induced middle cerebral artery occlusion (MCAO), a model of cerebral ischemia. Rats infused centrally (intracerebroventricular) with C21 before endothelin-1 induced MCAO exhibited significant reductions in cerebral infarct size and the neurological deficits produced by cerebral ischemia. Similar cerebroprotection was obtained in rats injected systemically (intraperitoneal) with C21 either before or after endothelin-1 induced MCAO. The protective effects of C21 were reversed by central administration of an AT2R inhibitor, PD123319. While C21 did not alter cerebral blood flow at the doses used here, peripheral post-stroke administration of this agent significantly attenuated the MCAO-induced increases in inducible nitric oxide synthase, chemokine (C-C) motif ligand 2 and C-C chemokine receptor type 2 mRNAs in the cerebral cortex, indicating that the cerebroprotective action is associated with an anti-inflammatory effect. These results strengthen the view that AT2R agonists may have potential therapeutic value in ischemic stroke, and provide the first evidence of cerebroprotection induced by systemic post stroke administration of a selective AT2R agonist.

Previously, we determined that angiotensin II (Ang II) elicits an Ang II type 2 (AT2) receptor–mediated increase of neuronal delayed rectifier K+( I KV) current in neuronal cultures from newborn rat hypothalamus and brain stem. This requires generation of lipoxygenase (LO) metabolites of arachidonic acid (AA) and activation of serine/threonine phosphatase type 2A (PP-2A). Enhancement of I KV results in a decrease in net inward current during the action potential (AP) upstroke as well as shortening of the refractory period, which may lead to alterations in neuronal firing rate. Thus, in the present study, we used whole-cell current clamp recording methods to investigate the AT2 receptor–mediated effects of Ang II on the firing rate of cultured neurons from the hypothalamus and brain stem. At room temperature, these neurons exhibited spontaneous APs with an amplitude of 77.72 ± 2.7 mV ( n = 20) and they fired at a frequency of 0.8 ± 0.1 Hz ( n = 11). Most cells had a prolonged early after-depolarization that followed an initial fully developed AP. Superfusion of Ang II (100 nM) plus losartan (LOS, 1 μM) to block Ang II type 1 receptors elicited a significant chronotropic effect that was reversed by the AT2 receptor inhibitor PD 123,319 (1 μM). LOS alone had no effect on any of the parameters measured. The chronotropic effect of Ang II was reversed by the general LO inhibitor 5,8,11,14-eicosatetraynoic acid (10 μM) or by the selective PP-2A inhibitor okadaic acid (1 nM) and was mimicked by the 12-LO metabolite of AA 12-(S)-hydroxy-(5Z, 8Z, 10E, 14Z)-eicosatetraynoic acid. These data indicate that Ang II elicits an AT2 receptor–mediated increase in neuronal firing rate, an effect that involves generation of LO metabolites of AA and activation of PP-2A.

Angiotensin II (Ang II) elicits an Ang II type 2 (AT2) receptor–mediated increase in voltage-dependent delayed rectifier K+current ( IKV) in neurons cultured from newborn rat hypothalamus and brain stem. In previous studies, we have determined that this effect of Ang II is mediated via a Gi protein, activation of phospholipase A2(PLA2), and generation of arachidonic acid (AA). AA is rapidly metabolized within cells via lipoxygenases (LO), cyclooxygenase (COX) or p450 monooxygenase enzymes, and the metabolic products are known regulators of K+currents and channels. Thus in the present study, we have investigated whether the AT2receptor–mediated effects of Ang II on neuronal IKVrequire AA metabolism and if so, which metabolic pathways are involved. The data presented here indicate that the stimulatory actions of Ang II and AA on neuronal IKVare attenuated by selective blockade of 12-LO enzymes. However, the effects of Ang II are not altered by blockade of 5-LO or p450 monooxygenase enzymes. Furthermore, the actions of Ang II are mimicked by a 12-LO metabolite of AA, but 5-LO metabolites such as leukotriene B4and C4do not alter neuronal IKV. These data indicate that the AT2receptor–mediated stimulation of neuronal IKVis partially mediated through 12-LO metabolites of AA.

The effector hormone of the renin-angiotensin system, angiotensin II, plays a major role in cardiovascular regulation. In rats, both angiotensin receptor subtypes, AT 1 and AT 2 , are up-regulated after myocardial infarction but previous studies failed to identify the cell types which express the AT 2 receptor in the heart. To address this question we established a single-cell reverse transcriptase-polymerase chain reaction for AT 1 and AT 2 receptors to determine whether these receptor subtypes are expressed in adult rat cardiomyocytes before and 1 day after myocardial infarction. By laser-assisted cell picking, section profiles of single cells without genomic DNA contamination were isolated. After dividing samples into two identical aliquots, polymerase chain reaction amplification for AT 1 and AT 2 receptors was carried out and polymerase chain reaction products were subjected to gel electrophoresis. Compared to control ( n = 4) and sham-operated animals ( n = 4), the number of cardiomyocytes expressing the AT 1 receptor mRNA 1 day after myocardial infarction ( n = 4) was not changed (42% and 33% versus 45%, respectively). On the other hand, AT 2 receptor mRNA was expressed in 8% and 13%, respectively, of cardiomyocytes gained from control ( n = 4) and sham-operated animals ( n = 4) and in 14% isolated after myocardial infarction ( n = 4). These results demonstrate for the first time that the AT 2 receptor is expressed in adult cardiomyocytes in vivo . They further suggest that the previously observed up-regulation of cardiac AT 1 and AT 2 receptors after myocardial infarction involves cell types other than cardiomyocytes.

Angiotensin II (Ang II) acts at specific receptors located on neurons in the hypothalamus and brain stem to elicit alterations in blood pressure, fluid intake, and hormone secretion. These actions of Ang II are mediated via Ang II type 1 (AT1) receptors and involve modulation of membrane ionic currents and neuronal activity. In previous studies we utilized neurons cultured from the hypothalamus and brain stem of newborn rats to investigate the AT1receptor–mediated effects of Ang II on neuronal K+currents. Our data indicate that Ang II decreases neuronal delayed rectifier (Kv) current, and that this effect is partially due to activation of protein kinase C (PKC), specifically PKCα. However, the data also indicated that another Ca2+-dependent mechanism was also involved in addition to PKC. Because Ca2+/calmodulin-dependent protein kinase II (CaM KII) is a known modulator of K+currents in neurons, we investigated the role of this enzyme in the AT1receptor–mediated reduction of neuronal Kv current by Ang II. The reduction of neuronal Kv current by Ang II was attenuated by selective inhibition of either calmodulin or CaM KII and was mimicked by intracellular application of activated (autothiophosphorylated) CaM KIIα. Concurrent inhibition of CaM KII and PKC completely abolished the reduction of neuronal Kv by Ang II. Consistent with these findings is the demonstration that Ang II increases CaM KII activity in neuronal cultures, as evidenced by increased levels of autophosphorylated CaM KIIα subunit. Last, single-cell reverse transcriptase (RT)-PCR analysis revealed the presence of AT1receptor-, CaM KIIα-, and PKCα subunit mRNAs in neurons that responded to Ang II with a decrease in Kv current. The present data indicate that the AT1receptor–mediated reduction of neuronal Kv current by Ang II involves a Ca2+/calmodulin/CaM KII pathway, in addition to the previously documented involvement of PKC.

Abstract —Angiotensin II (Ang II) has powerful modulatory actions on cardiovascular function that are mediated by specific receptors located on neurons within the hypothalamus and brain stem. Incubation of neuronal cocultures of rat hypothalamus and brain stem with Ang II elicits an Ang II type 1 (AT 1 ) receptor–mediated inhibition of total outward K + current that contributes to an increase in neuronal firing rate. However, the exact K + conductance(s) that is inhibited by Ang II are not established. Pharmacological manipulation of total neuronal outward K + current revealed a component of K + current sensitive to quinine, tetraethylammonium, and 4-aminopyridine, with IC 50 values of 21.7 μmol/L, 1.49 mmol/L, and 890 μmol/L, respectively, and insensitive to α-dendrotoxin (100 to 500 nmol/L), charybdotoxin (100 to 500 nmol/L), and mast cell degranulating peptide (1 μmol/L). Collectively, these data suggest the presence of Kv2.2 and Kv3.1b. Biophysical examination of the quinine-sensitive neuronal K + current demonstrated a macroscopic conductance with similar biophysical properties to those of Kv2.2 and Kv3.1b. Ang II (100 nmol/L), in the presence of the AT 2 receptor blocker PD123,319, elicited an inhibition of neuronal K + current that was abolished by quinine (50 μmol/L). Reverse transcriptase–polymerase chain reaction analysis confirmed the presence of Kv2.2 and Kv3.1b mRNA in these neurons. However, Western blot analyses demonstrated that only Kv2.2 protein was present. Coexpression of Kv2.2 and the AT 1 receptor in Xenopus oocytes demonstrated an Ang II–induced inhibition of Kv2.2 current. Therefore, these data suggest that inhibition of Kv2.2 contributes to the AT 1 receptor–mediated reduction of neuronal K + current and subsequently to the modulation of cardiovascular function.

Mammalian brain contains high densities of angiotensin II (Ang II) type 1 (AT1) receptors, localized mainly to specific nuclei within the hypothalamus and brainstem regions. Neuronal AT1 receptors within these areas mediate the stimulatory actions of central Ang II on blood pressure, water and sodium intake, and vasopressin secretion, effects that involve the modulation of brain noradrenergic pathways. This review focuses on the intracellular events that mediate the functional effects of Ang II in neurons, via AT1 receptors. The signaling pathways involved in short-term changes in neuronal activity, membrane ionic currents, norepinephrine (NE) release, and longer-term neuromodulatory actions of Ang II are discussed. It will be apparent from this discussion that the signaling pathways involved in these events are often distinct.

Angiotensin II (Ang II) type 2 (AT2) receptors are highly expressed in neonate brain and may have a role in developmental processes such as apoptosis. Concurrent activation of c-Jun N-terminal kinase (JNK) and inhibition of Erk mitogen-activated protein kinase activities is important for apoptosis in many cells, and we previously demonstrated that stimulation of AT2 receptors causes decreased mitogen-activated protein kinase activity in neurons cultured from newborn rat hypothalamus and brain stem. Using such cultures we have employed terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling and internucleosomal DNA fragmentation to assess the role of AT2 receptors in neuronal apoptosis. Ang II (100 nM; 4-72 h) alone produced no significant neuronal apoptosis, and AT2 receptor activation did not stimulate JNK activity. However, exposure of cultures to UV radiation (6 J/m2/sec for 4 sec) to stimulate JNK elicited neuronal apoptosis that was significantly enhanced by Ang II, an effect that was abolished by the AT2 receptor antagonist PD 123,319 (1 microM) or the serine/threonine phosphatase inhibitor okadaic acid (3 nM). Additionally, Ang II enhanced the UV radiation-induced decrease in the levels of the DNA repair enzyme poly-(ADP-ribose) polymerase. These data indicate that Ang II, via AT2 receptors and activation of a serine/threonine phosphatase, contributes to neuronal apoptosis.

The brain contains both angiotensin II (Ang II) type 1 (AT1) and Ang II type 2 (AT2) receptors. Neuronal AT1 receptors mediate the stimulatory actions of Ang II on blood pressure, water and salt intake, and secretion of vasopressin. In contrast, neuronal AT2 receptors have been implicated in the stimulation of apoptosis and as being antagonistic to AT1 receptors. The physiological actions of Ang II in the brain, whether mediated by AT1 or AT2 receptors, involve changes in neuronal activity that are initiated by changes in the activity of membrane ionic currents and channels. This review focusses on the intracellular signalling pathways that couple neuronal AT1 and AT2 receptors to changes in the activity of membrane K+ and Ca2+ currents and channels. As will become clear from our discussion, the signalling pathways that are modulated by neuronal AT1 and AT2 receptors are quite distinct.

The objective of this review is to examine the role of neuronal angiotensin II (Ang II) receptors in vitro. Two types of G protein-coupled Ang II receptors have been identified in cardiovascularly relevant areas of the brain: the AT 1 and the AT 2 . We have utilized neurons in culture to study the signaling mechanisms of AT 1 and AT 2 receptors. Neuronal AT 1 receptors are involved in norepinephrine (NE) neuromodulation. NE neuromodulation can be either evoked or enhanced . Evoked NE neuromodulation involves AT 1 receptor-mediated, losartan-dependent, rapid NE release, inhibition of K + channels and stimulation of Ca 2+ channels. AT 1 receptor-mediated enhanced NE neuromodulation involves the Ras-Raf-MAP kinase cascade and ultimately leads to an increase in NE transporter, tyrosine hydroxylase and dopamine β-hydroxylase mRNA transcription. Neuronal AT 2 receptors signal via a G i protein and are coupled to activation of PP2A and PLA 2 and stimulation of K + channels. Finally, putative cross-talk pathways between AT 1 and AT 2 receptors will be discussed.