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Background The renin‐angiotensin system plays a crucial role in human physiology, and its main hormone, angiotensin, activates 2 G‐protein–coupled receptors, the angiotensin type‐1 and type‐2 receptors, in almost every organ. However, controversy exists about the location, distribution, and expression levels of these receptors. Concerns have been raised over the low sensitivity, low specificity, and large variability between lots of commercially available antibodies for angiotensin type‐1 and type‐2 receptors, which makes it difficult to reconciliate results of different studies. Here, we describe the first non–antibody‐based sensitive and specific targeted quantitative mass spectrometry assay for angiotensin receptors. Methods and Results Using a technique that allows targeted analysis of multiple peptides across multiple samples in a single mass spectrometry analysis, known as TOMAHAQ (triggered by offset, multiplexed, accurate mass, high resolution, and absolute quantification), we have identified and validated specific human tryptic peptides that permit identification and quantification of angiotensin type‐1 and type‐2 receptors in biological samples. Several peptide sequences are conserved in rodents, making these mass spectrometry assays amenable to both preclinical and clinical studies. We have used this method to quantify angiotensin type‐1 and type‐2 receptors in postmortem frontal cortex samples of older adults (n=28) with Alzheimer dementia. We correlated levels of angiotensin receptors to biomarkers classically linked to renin‐angiotensin system activation, including oxidative stress, inflammation, amyloid‐β load, and paired helical filament‐tau tangle burden. Conclusions These robust high‐throughput assays will not only catalyze novel mechanistic studies in the angiotensin research field but may also help to identify patients with an unbalanced angiotensin receptor distribution who would benefit from angiotensin receptor blocker treatment.

Kruppel-like factor 2 (KLF2) regulates endothelial cell metabolism; endothelial dysfunction is associated with hypertension and is a predictor of atherosclerosis development and cardiovascular events. Here, we investigated the role of KLF2 in hypertensive nephropathy by regulating KLF2 expression in human primary glomerular endothelial cells (hPGECs) and evaluating this expression in the kidney tissues of a 5/6 nephrectomy mouse model as well as patients with hypertension. Hypertension-mimicking devices and KLF2 siRNA were used to downregulate KLF2 expression, while the expression of KLF2 was upregulated by administering simvastatin. After 4 mmHg of pressure was applied on hPGECs for 48 h, KLF2 mRNA expression decreased, while alpha-smooth muscle actin (αSMA) mRNA expression increased. Apoptosis and fibrosis rates were increased under pressure, and these phenomena were aggravated following KLF2 knockdown, but were alleviated after simvastatin treatment; additionally, these changes were observed in angiotensin II, angiotensin type-1 receptor (AT1R) mRNA, and interleukin-18 (IL-18), but not in angiotensin type-2 receptor mRNA. Reduced expression of KLF2 in glomerular endothelial cells due to hypertension was found in both 5/6 nephrectomy mice and patients with hypertensive nephropathy. Thus, our study demonstrates that the pressure-induced apoptosis and fibrosis of glomerular endothelial cells result from angiotensin II, AT1R activation, and KLF2 inhibition, and are associated with IL-18.

We investigated using immunohistochemistry the possible protective effects of ascorbic acid, α-tocopherol and selenium during chemotherapy treatment with cyclophosphamide. Thirty female Wistar rats were divided into five groups of six: group 1, untreated control; group 2, 75 µg/kg cyclophosphamide; group 3, 75 µg/kg cyclophosphamide + 150 µg/kg/day α-tocopherol; group 4, 75 µg/kg cyclophosphamide + 200 µg/kg/day ascorbic acid and group 5, 75 µg/kg cyclophosphamide + 40 ppm/kg/day selenium. Proliferating cell nuclear antigen (PCNA) staining was used to detect cell proliferation and AT1 was used to evaluate structural damage. Caspase-8, caspase-9 and caspase-3 signal molecules were used to investigate apoptosis. In group 2, epithelium, alveolar macrophages, infiltrated lymphocytes and connective tissue were immunostained moderately to strongly with PCNA. Bronchus, alveolar wall and infiltrated lymphocytes were immunostained moderately to strongly with AT1 and diffuse strong caspase immunoreactions were observed throughout the lung tissue. AT1 and caspase immunoreactions in groups 4 and 5 were similar to group 2. In group 3, PCNA immunoreactivity was strong in the bronchiolus epithelium, endothelial cell nuclei and in stacks of infiltrated lymphocyte cell nuclei. In group 3, AT1 and caspase immunoreactions were identical to group 1. It appears that α-tocopherol inhibits lung tissue damage in rats during chemotherapy. [ABSTRACT FROM AUTHOR]

Background The renin-angiotensin system plays a crucial role in human physiology, and its main hormone, angiotensin, activates 2 G-protein-coupled receptors, the angiotensin type-1 and type-2 receptors, in almost every organ. However, controversy exists about the location, distribution, and expression levels of these receptors. Concerns have been raised over the low sensitivity, low specificity, and large variability between lots of commercially available antibodies for angiotensin type-1 and type-2 receptors, which makes it difficult to reconciliate results of different studies. Here, we describe the first non-antibody-based sensitive and specific targeted quantitative mass spectrometry assay for angiotensin receptors. Methods and Results Using a technique that allows targeted analysis of multiple peptides across multiple samples in a single mass spectrometry analysis, known as TOMAHAQ (triggered by offset, multiplexed, accurate mass, high resolution, and absolute quantification), we have identified and validated specific human tryptic peptides that permit identification and quantification of angiotensin type-1 and type-2 receptors in biological samples. Several peptide sequences are conserved in rodents, making these mass spectrometry assays amenable to both preclinical and clinical studies. We have used this method to quantify angiotensin type-1 and type-2 receptors in postmortem frontal cortex samples of older adults (n=28) with Alzheimer dementia. We correlated levels of angiotensin receptors to biomarkers classically linked to renin-angiotensin system activation, including oxidative stress, inflammation, amyloid-β load, and paired helical filament-tau tangle burden. Conclusions These robust high-throughput assays will not only catalyze novel mechanistic studies in the angiotensin research field but may also help to identify patients with an unbalanced angiotensin receptor distribution who would benefit from angiotensin receptor blocker treatment.

Scorpion stings are mainly associated with cardiovascular disturbances that may be the cause of death. In this study, the involvement of angiotensin II (Ang II) in cardiac and aortic inflammatory response was studied. Mice were injected with Androctonus australis hector ( Aah ) scorpion venom (0.5 mg/kg, subcutaneously), in the presence or absence of an angiotensin converting enzyme (ACE) inhibitor, captopril (15 mg/kg/day/1 day intraperitoneally) or an angiotensin type-1 receptor (AT1R) antagonist, valsartan (15 mg/kg/day/15 days, orally). In the envenomed group, results revealed severe tissue alterations with a concomitant increase of metabolic enzymes (CK and CK-MB) in sera. An important inflammatory cell (neutrophil and eosinophil) infiltration into the heart and aorta were observed, accompanied by imbalanced redox status (NO, MDA, catalase and GSH) and high cytokine levels (IL-6 and TNF-α) in sera with the expression of MMP-2 and MMP-9 metalloproteinases. However, the blockade of the actions of AngII by the ACE inhibitor or by the AT1R antagonist prevented cardiac and aortic tissue alterations, inflammatory cell infiltration, as well as the oxidative stress generation and cytokine and metalloproteinase expression. These results suggest the involvement of AngII, through its AT1R in the inflammation induced by Aah venom, in the heart and the aorta. [ABSTRACT FROM AUTHOR]

The blockade of the central nucleus of the amygdala (CeA) with the GABA A receptor agonist muscimol significantly reduces hypertonic NaCl and water intake by sodium-depleted rats. In the present study we investigated the effects of previous injection of losartan, an angiotensin II type-1 (AT1) receptor antagonist, into the CeA on 0.3 M NaCl and water intake reduced by muscimol bilaterally injected into the same areas in rats submitted to water deprivation–partial rehydration (WD–PR) and in rats treated with the diuretic furosemide (FURO). Male Sprague–Dawley rats with stainless steel cannulas bilaterally implanted into the CeA were used. Bilateral injections of muscimol (0.2 nmol/0.5 μl, n = 8 rats/group) into the CeA in WD–PR-treated rats reduced 0.3 M NaCl intake and water intake, and pre-treatment of the CeA with losartan (50 μg/0.5 μl) reversed the inhibitory effect of muscimol. The negative effect of muscimol on sodium and water intake could also be blocked by pretreatment with losartan microinjected into the CeA in rats given FURO ( n = 8 rats/group). However, bilateral injections of losartan (50 μg/0.5 μl) alone into the CeA did not affect the NaCl or water intake. These results suggest that the deactivation of CeA facilitatory mechanisms by muscimol injection into the CeA is promoted by endogenous angiotensin II acting on AT1 receptors in the CeA, which prevents rats from ingesting large amounts of hypertonic NaCl and water. [ABSTRACT FROM AUTHOR]

Kruppel-like factor 2 (KLF2) regulates endothelial cell metabolism; endothelial dysfunction is associated with hypertension and is a predictor of atherosclerosis development and cardiovascular events. Here, we investigated the role of KLF2 in hypertensive nephropathy by regulating KLF2 expression in human primary glomerular endothelial cells (hPGECs) and evaluating this expression in the kidney tissues of a 5/6 nephrectomy mouse model as well as patients with hypertension. Hypertension-mimicking devices and KLF2 siRNA were used to downregulate KLF2 expression, while the expression of KLF2 was upregulated by administering simvastatin. After 4 mmHg of pressure was applied on hPGECs for 48 h, KLF2 mRNA expression decreased, while alpha-smooth muscle actin (αSMA) mRNA expression increased. Apoptosis and fibrosis rates were increased under pressure, and these phenomena were aggravated following KLF2 knockdown, but were alleviated after simvastatin treatment; additionally, these changes were observed in angiotensin II, angiotensin type-1 receptor (AT1R) mRNA, and interleukin-18 (IL-18), but not in angiotensin type-2 receptor mRNA. Reduced expression of KLF2 in glomerular endothelial cells due to hypertension was found in both 5/6 nephrectomy mice and patients with hypertensive nephropathy. Thus, our study demonstrates that the pressure-induced apoptosis and fibrosis of glomerular endothelial cells result from angiotensin II, AT1R activation, and KLF2 inhibition, and are associated with IL-18. [ABSTRACT FROM AUTHOR]

Obesity is a widespread health concern that is associated with an increased prevalence of hypertension and cardiovascular disease. Both obesity and hypertension have independently been associated with increased levels of inflammatory cytokines and immune cells within specific brain regions, as well as increased activity of the renin–angiotensin system (RAS). To test the hypothesis that high-fat diet (HFD) induced obesity leads to an angiotensin-II (Ang-II)-dependent increase in inflammatory cells within specific forebrain regions that are important for cardiovascular regulation, we first assessed microglial activation, astrocyte activation, inflammation and RAS component gene expression within selected metabolic and cardiovascular control centers of the forebrain in adult male C57BL/6 mice given either a HFD or a low-fat diet (LFD) for 8 weeks. Subsequently, we assessed the necessity of the paraventricular nucleus of the hypothalamus (PVN) angiotensin type-1a (AT1a) receptor for these responses by using the Cre/lox system in mice to selectively delete the AT1a receptor from the PVN. These studies reveal that in addition to the arcuate nucleus of the hypothalamus (ARC), the PVN and the subfornical organ (SFO), two brain regions that are known to regulate blood pressure and energy balance, also initiate proinflammatory responses after the consumption of a diet high in fat. They further indicate that some, but not all, of these responses are reversed upon deletion of AT1a specifically within the PVN. [ABSTRACT FROM AUTHOR]

BACKGROUND AND PURPOSE The renin-angiotensin system (RAS) is critical for the control of blood pressure by the CNS. Recently, direct renin inhibitors were approved as antihypertensive agents. However, the signalling mechanism of renin, which regulates blood pressure in the nucleus tractus solitarii (NTS) remains unclear. Here we have investigated the signalling pathways involved in renin-mediated blood pressure regulation, at the NTS. EXPERIMENTAL APPROACH Depressor responses to renin microinjected into the NTS of Wistar-Kyoto rats were elicited in the absence and presence of the endothelial nitric oxide synthase (eNOS)-specific inhibitor, N(5)-(-iminoethyl)-L-ornithine, Akt inhibitor IV and LY294002, a PI3K inhibitor and GP antagonist-2A [Gq inhibitor]. Lisinopril (angiotensin converting enzyme inhibitor), losartan, valsartan (angiotensin AT1 receptor antagonists), D-Ala7-Ang-(1-7) (angiotensin-(1-7) receptor antagonist) were used to study the involvement of RAS on renin-induced depressor effects. KEY RESULTS Microinjection of renin into the NTS produced a prominent depressor effect and increased NO production. Pretreatment with Gq-PI3K-Akt-eNOS pathway-specific inhibitors significantly attenuated the depressor response evoked by renin. Immunoblotting and immunohistochemical studies further showed that inhibition of PI3K significantly blocked renin-induced eNOS-Ser117 and Akt-Ser473 phosphorylation in situ. In addition, pre-treatment of the NTS with RAS inhibitors attenuated the vasodepressor effects evoked by renin. Microinjection of renin also increased Ras activation in the NTS. CONCLUSIONS AND IMPLICATIONS Taken together, these results suggest renin modulated blood pressure at the NTS by AT1 and Mas receptor-mediated activation of Gq and Ras to evoke PI3K-Akt-eNOS signalling. [ABSTRACT FROM AUTHOR]

Abstract: Angiotensin II (Ang II) stimulates oral water intake by causing thirst in all terrestrial vertebrates except anurans. Anuran amphibians do not drink orally but absorb water osmotically through ventral skin. In this study, we examined the role of Ang II on the regulation of water-absorption behavior in the Japanese tree frog (Hyla japonica). In fully hydrated frogs, intracerebroventricular (ICV) and intralymphatic sac (ILS) injection of Ang II significantly extended the residence time of water in a dose-dependent manner. Ang II-dependent water uptake was inhibited by ICV pretreatment with an angiotensin II type-1 (AT1) receptor antagonist but not a type-2 (AT2) receptor antagonist. These results suggest that Ang II stimulates water-absorption behavior in the tree frog via an AT1-like but not AT2-like receptor. We then cloned and characterized cDNA of the tree frog AT1 receptor from the brain. The tree frog AT1 receptor cDNA encodes a 361 amino acid residue protein, which is 87% identical to the toad (Bufo marinus) AT1 receptor and exhibits the functional characteristics of an Ang II receptor. AT1 receptor mRNAs were found to be present in a number of tissues including brain (especially in the diencephalon), lung, large intestine, kidney and ventral pelvic skin. When tree frogs were exposed to dehydrating conditions, AT1 receptor mRNA significantly increased in the diencephalon and the rhombencephalon. These data suggest that central Ang II may control water intake behavior via an AT1 receptor on the diencephalon and rhombencephalon in anuran amphibians and may have implications for water consumption in vertebrates. [ABSTRACT FROM AUTHOR]

Suicide has been suggested to involve disturbances in the stress response system and to be related to genetics. The renin-angiotensin system (RAS) has been shown to affect the stress response, and several functional polymorphisms in RAS-related genes have been predicted to alter protein function. We hypothesized that the dysregulation of RAS was involved in suicide, and examined the association between completed suicides and four functional polymorphisms of RAS-related genes: the angiotensinogen M235T, angiotensin-converting enzyme (ACE) insertion(I)/deletion(D), angiotensin type-1 receptor A1166C, and G-protein-β3 C825T gene polymorphisms. The I allele of the ACE I/D polymorphism was found to be more frequent in completed suicides than in controls ( P = 0.014). The I allele was also found to be more frequent in male completed suicides ( P = 0.022) than in male controls, while this was not the case in females. These results suggest that the alteration of RAS function caused by the genetic polymorphism is involved in the susceptibility to suicide in males. [ABSTRACT FROM AUTHOR]

Summary Background Angiotensin receptors are the specific receptors of angiotensin II of the renin–angiotensin system. However, expression of the receptors in hair follicles has not been determined. Objects To clarify the expression and localization of angiotensin receptors in human anagen hair follicles and basal cell carcinomas. Methods We studied immunohistochemically the expression of angiotensin type 1(AT1) and type 2 (AT2) receptors in human anagen hair follicles and in 16 cases of basal cell carcinoma (BCC) (nine of solid BCC of the circumscribed type, two of adenoid BCC, five of BCC with follicular differentiation). Results Our experiments demonstrated the localization of AT1 in the inner root sheath and the inner layers of the outer root sheath. In BCC, positive staining with AT1 was revealed in the tumour cells of basal cell carcinoma with follicular differentiation. Conclusions AT1 may have a role in association with follicular keratinization. Studying AT1 distribution may be useful in understanding the pathophysiology of human hair follicles and the hair follicle-associated tumours. [ABSTRACT FROM AUTHOR]

Beneficial effects of angiotensin type-1 receptor (AT1) inhibition have been observed in a number of brain processes mediated by oxidative stress and neuroinflammation, including Parkinson's disease. However, important counterregulatory interactions between dopamine and angiotensin systems have recently been demonstrated in several peripheral tissues, and it is possible that a decrease in dopamine levels due to AT1 inhibition may interfere with neuroprotective strategies. The present experiments involving rats with normal dopaminergic innervation indicate that chronic treatment with the AT1 antagonist candesartan does not significantly affect striatal levels of dopamine, serotonin or metabolites, as does not significantly affect motor behavior, as evaluated by the rotarod test. Interestingly, chronic administration of candesartan to normal rats induced a marked increase in dopamine D1 and a decrease in dopamine D2 receptor expression. In a rat model of Parkinson's disease treated with L-DOPA, no differences in striatal dopamine and serotonin levels were observed between candesartan-treated rats and untreated, which suggests that chronic treatment with candesartan does not significantly affect the process of L-DOPA decarboxylation and dopamine release in Parkinson's disease patients. Candesartan did not induce any differences in the striatal expression of dopamine D1 and D2 and serotonin 5-HT1B receptors in 6ydroxydopamine-lesioned rats treated with L-DOPA. The results suggest that chronic treatment with AT1 antagonists as a neuroprotective strategy does not significantly affect striatal dopamine release or motor behavior. This article is part of the Special Issue entitled 'The Synaptic Basis of Neurodegenerative Disorders'., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The mechanism by which estrogen protects dopaminergic neurons has not yet been clarified. It is not known if changes in RhoA/Rho kinase activity are involved in the enhanced vulnerability of dopaminergic neurons observed after estrogen depletion. The present study shows that the MPTP-induced loss of dopaminergic neurons is increased by estrogen depletion and inhibited by estrogen replacement, the Rho kinase inhibitor Y27632 and deletion of the angiotensin type-1 receptor. In ovariectomized mice, treatment with MPTP induced a marked increase in Rho kinase activity, and RhoA and RhocK II mRNA and protein expression, which were significantly higher than in ovariectomized mice treated with MPTP and estrogen replacement or type-1 receptor deletion. Estrogen depletion increased Rho kinase activity, via enhancement of the angiotensin type-1 receptor pathway, and Rho kinase activation increased type-1 receptor expression suggesting a vicious cycle in which Rho kinase and type-1 receptor activate each other and promote the degenerative process. The results suggest that type-1 receptor antagonists and Rho kinase inhibitors may provide a new neuroprotective strategy, which may circumvent the potential risks of estrogen replacement therapy and be particularly useful in elderly women or women affected by long-term lack of estrogen., (Copyright © 2013 Elsevier Inc. All rights reserved.)