Angiotensin AT1 Receptor Research Articles

Selective activation of angiotensin AT2 receptors attenuates progression of pulmonary hypertension and inhibits cardiopulmonary fibrosis.

Pulmonary hypertension (PH) is a devastating disease characterized by increased pulmonary arterial pressure, which progressively leads to right-heart failure and death. A dys-regulated renin angiotensin system (RAS) has been implicated in the development and progression of PH. However, the role of the angiotensin AT2 receptor in PH has not been fully elucidated. We have taken advantage of a recently identified non-peptide AT2 receptor agonist, Compound 21 (C21), to investigate its effects on the well-established monocrotaline (MCT) rat model of PH. A single s.c. injection of MCT (50 mg·kg(-1) ) was used to induce PH in 8-week-old male Sprague Dawley rats. After 2 weeks of MCT administration, a subset of animals began receiving either 0.03 mg·kg(-1) C21, 3 mg·kg(-1) PD-123319 or 0.5 mg·kg(-1) A779 for an additional 2 weeks, after which right ventricular haemodynamic parameters were measured and tissues were collected for gene expression and histological analyses. Initiation of C21 treatment significantly attenuated much of the pathophysiology associated with MCT-induced PH. Most notably, C21 reversed pulmonary fibrosis and prevented right ventricular fibrosis. These beneficial effects were associated with improvement in right heart function, decreased pulmonary vessel wall thickness, reduced pro-inflammatory cytokines and favourable modulation of the lung RAS. Conversely, co-administration of the AT2 receptor antagonist, PD-123319, or the Mas antagonist, A779, abolished the protective actions of C21. Taken together, our results suggest that the AT2 receptor agonist, C21, may hold promise for patients with PH.

Targeting drugs to APJ receptor: the prospect of treatment of hypertension and other cardiovascular diseases.

The APJ is a class A, rhodopsin-like G protein-coupled receptor (GPCR) with high sequence similarity to the angiotensin receptor AT1. APJ has been shown to be widely expressed in humans tissues, including the central nervous system, cardiovascular system, adipocytes and others. APJ plays an important role in the occurrence and development of cardiovascular and metabolic diseases including atherosclerosis (AS), coronary heart disease (CAD), heart failure(HF), pulmonary arterial hypertension (PAH), myocardial hypertrophy and atrial fibrillation, especially hypertension. Previous researchers found that apelin/APJ could induce vasodilation and then reduce blood pressure. Despite APJ is closely associated with many diseases, there are no drugs that can activate or inhibit APJ directly. In the current review, we have summarized recently reported peptides, small molecule agonists and antagonists targeting APJ. Given the role of apelin/APJ in hypertension and other cardiovascular diseases, we believe that the peptides and compounds based on APJ will be developed for treatment of these diseases.

The angiotensin converting enzyme inhibitor, captopril, prevents the hyperactivity and impulsivity of neurokinin-1 receptor gene ‘knockout’ mice: Sex differences and implications for the treatment of attention deficit hyperactivity disorder

Mice lacking functional neurokinin-1 receptors (NK1R−/−) display behavioural abnormalities resembling attention deficit hyperactivity disorder (ADHD): locomotor hyperactivity, impulsivity and inattentiveness. The preferred ligand for NK1R, substance P, is metabolised by angiotensin converting enzyme (ACE), which forms part of the brain renin angiotensin system (BRAS). In view of evidence that the BRAS modulates locomotor activity and cognitive performance, we tested the effects of drugs that target the BRAS on these behaviours in NK1R−/− and wildtype mice. We first tested the effects of the ACE inhibitor, captopril, on locomotor activity. Because there are well-established sex differences in both ADHD and ACE activity, we compared the effects of captopril in both male and female mice. Locomotor hyperactivity was evident in male NK1R−/− mice, only, and this was abolished by treatment with captopril. By contrast, male wildtypes and females of both genotypes were unaffected by ACE inhibition. We then investigated the effects of angiotensin AT1 (losartan) and AT2 (PD 123319) receptor antagonists on the locomotor activity of male NK1R−/− and wildtype mice. Both antagonists increased the locomotor activity of NK1R−/− mice, but neither affected the wildtypes. Finally, we tested the effects of captopril on the performance of male NK1R−/− and wildtype mice in the 5-choice serial reaction-time task (5-CSRTT) and found that ACE inhibition prevented the impulsivity of NK1R−/− mice. These results indicate that certain behaviours, disrupted in ADHD, are influenced by an interaction between the BRAS and NK1R, and suggest that ACE inhibitors could provide a novel treatment for this disorder.

Effect of Combined Treatment with AT1 Receptor Antagonists and Tiagabine on Seizures, Memory and Motor Coordination in Mice.

Losartan and telmisartan, angiotensin AT1 receptor antagonists, are widely used antihypertensive drugs in patients. It is also known that arterial hypertension is often present in people with epilepsy, therefore, drug interactions between AT1 receptor antagonists and antiepileptic drugs can occur in clinical practice. The aim of the current study was to assess the effect of losartan and telmisartan on the anticonvulsant activity of tiagabine, asecond-generation antiepileptic drug, in mice. Additionally, the effect of the combined treatment with AT1 receptor antagonists and TGB on long-term memory and motor coordination has been assessed in animals. The study was performed on male Swiss mice. Convulsions were examined in the maximal electroshock seizure threshold test. Long-term memory was measured in the passive-avoidance task and motor coordination was evaluated in the chimney test. AT1 receptor antagonists and TGB were administered intraperitoneally. Losartan (50 mg/kg) or telmisartan (30 mg/kg) did not influence the anticonvulsant activity of TGB applied at doses of 2, 4and 6mg/kg. However, both AT1 receptor antagonists in combinations with TGB (6mg/kg) impaired motor coordination in the chimney test. The concomitant treatment of the drugs did not decrease retention in the passive avoidance task. It is suggested that losartan and telmisartan should not affect the anticonvulsant action of TGB in people with epilepsy. Because the combined treatment with AT1 receptor antagonists and TGB led to neurotoxic effects in animals, caution is advised during concomitant use of these drugs in patients.

Age-related hypertension and salt sensitivity are associated with unique cortico-medullary distribution of D1R, AT1R, and NADPH-oxidase in FBN rats

We examined effects of normal (NS) and high salt (HS) on blood pressure (BP) and cortico-medullary distribution of dopamine D1 receptor (D1R), angiotensin AT1 receptor (AT1R), NADPH oxidase-gp91phox, and sodium transporters (NHE-3, Na, K ATPase) in adult and aged rats. Aged rats fed with NS diet had higher BP, which further increased with HS. HS increased D1R mRNA and protein levels in cortex and medulla of adult rats. NS or HS fed-aged rats had higher AT1R and gp91phox mRNA levels in cortex and medulla. Aged rats fed with NS diet had higher gp91phox protein levels in cortex. HS diet increased AT1R and gp91phox protein levels in medulla of aged rats. Aged rats fed with NS or HS diet had higher NHE-3 protein levels in medulla. HS increased Na, K ATPase protein levels in medulla of aged rats. HS increased urinary kidney injury molecule-1 (KIM-1) but not protein or albumin levels in aged rats. These results suggest that cortical gp91phox and medullary NHE-3 contribute to age-related hypertension. Whereas D1R (cortical and medullary) together with medullary AT1R, gp91phox and Na, K-ATPase contribute to salt sensitivity in aged rats. And, KIM-1 may be a better marker for kidney damage.

Effect of Combined Treatment with AT1 Receptor Antagonists and Tiagabine on Seizures, Memory and Motor Coordination in Mice*

Background. Losartan and telmisartan, angiotensin AT1 receptor antagonists, are widely used antihypertensive drugs in patients. It is also known that arterial hypertension is often present in people with epilepsy, therefore, drug interactions between AT1 receptor antagonists and antiepileptic drugs can occur in clinical practice.Objectives. The aim of the current study was to assess the effect of losartan and telmisartan on the anticonvulsant activity of tiagabine, a second-generation antiepileptic drug, in mice. Additionally, the effect of the combined treatment with AT1 receptor antagonists and TGB on long-term memory and motor coordination has been assessed in animals.Material and Methods. The study was performed on male Swiss mice. Convulsions were examined in the maximal electroshock seizure threshold test. Long-term memory was measured in the passive-avoidance task and motor coordination was evaluated in the chimney test. AT1 receptor antagonists and TGB were administered intraperitoneally.Results. Losartan (50 mg/kg) or telmisartan (30 mg/kg) did not influence the anticonvulsant activity of TGB applied at doses of 2, 4 and 6 mg/kg. However, both AT1 receptor antagonists in combinations with TGB (6 mg/kg) impaired motor coordination in the chimney test. The concomitant treatment of the drugs did not decrease retention in the passive avoidance task.Conclusion. It is suggested that losartan and telmisartan should not affect the anticonvulsant action of TGB in people with epilepsy. Because the combined treatment with AT1 receptor antagonists and TGB led to neurotoxic effects in animals, caution is advised during concomitant use of these drugs in patients.

Vascular endothelial dysfunction and pharmacological treatment.

The endothelium exerts multiple actions involving regulation of vascular permeability and tone, coagulation and fibrinolysis, inflammatory and immunological reactions and cell growth. Alterations of one or more such actions may cause vascular endothelial dysfunction. Different risk factors such as hypercholesterolemia, homocystinemia, hyperglycemia, hypertension, smoking, inflammation, and aging contribute to the development of endothelial dysfunction. Mechanisms underlying endothelial dysfunction are multiple, including impaired endothelium-derived vasodilators, enhanced endothelium-derived vasoconstrictors, over production of reactive oxygen species and reactive nitrogen species, activation of inflammatory and immune reactions, and imbalance of coagulation and fibrinolysis. Endothelial dysfunction occurs in many cardiovascular diseases, which involves different mechanisms, depending on specific risk factors affecting the disease. Among these mechanisms, a reduction in nitric oxide (NO) bioavailability plays a central role in the development of endothelial dysfunction because NO exerts diverse physiological actions, including vasodilation, anti-inflammation, antiplatelet, antiproliferation and antimigration. Experimental and clinical studies have demonstrated that a variety of currently used or investigational drugs, such as angiotensin-converting enzyme inhibitors, angiotensin AT1 receptors blockers, angiotensin-(1-7), antioxidants, beta-blockers, calcium channel blockers, endothelial NO synthase enhancers, phosphodiesterase 5 inhibitors, sphingosine-1-phosphate and statins, exert endothelial protective effects. Due to the difference in mechanisms of action, these drugs need to be used according to specific mechanisms underlying endothelial dysfunction of the disease.

Alteration of cardiac ACE2/Mas expression and cardiac remodelling in rats with aortic constriction.

The recent discovery of the new components of the renin-angiotensin system (RAS) suggests the importance of the maintenance of cardiovascular structure and functions. To assess the role of the angiotensin-converting enzyme 2 (ACE2)-Mas receptor axis in the regulation of cardiac structure and function, the present work investigated the expression of ACE2 and Mas receptor in the heart in the cardiac remodeling that occurs in aortic constricted rats. Partial abdominal aortic ligation was carried out in Sprague-Dawley rats. Angiotensin AT1 receptor blockade and ACE inhibition were achieved by losartan and enalapril treatment, respectively. Results showed that aortic constriction increased left ventricular hypertrophy, fibrosis, mean arterial pressure (MAP), plasma renin activity (PRA) and cardiac ACE levels, but decreased the expression of cardiac ACE2 and Mas receptor. Losartan treatment significantly decreased MAP, left ventricle hypertrophy (LVH), fibrosis, and increased cardiac ACE2 and Mas expression. Enalapril also improved the cardiac parameters with a rise in cardiac ACE2, but did not change the Mas level. In conclusion, aortic constriction results in cardiac hypertrophy, fibrosis and a rise of cardiac ACE expression. Both AT1 receptor blocker and ACE inhibitor play a cardioprotective role in aortic constriction. However, AT1 receptor blocker particularly promotes cardiac ACE2 and Mas receptor levels. ACE inhibitor is associated with the inhibition of ACE and normalization of cardiac ACE2 activity.

Modulation of Calcium Signaling of Angiotensin AT1, Endothelin ETA, and ETB Receptors by Silibinin, Quercetin, Crocin, Diallyl Sulfides, and Ginsenoside Rb1.

Angiotensin II and endothelin-1 are potent vasoconstrictive peptides that play a central role in blood pressure regulation. Both peptides exert their pleiotropic effects via binding to their respective G-protein-coupled receptors, i.e., angiotensin AT1 and endothelin type A and type B receptors. In the present study, we have selected six structurally different plant-derived compounds with known cardioprotective properties to evaluate their ability to modulate calcium signaling of the above-mentioned receptors. For this purpose, we used and validated a cellular luminescence-based read-out system in which we measured intracellular calcium signaling in Chinese hamster ovary cells that express the calcium sensitive apo-aequorin protein. Firstly, silibinin, a flavanolignan that occurs in milk thistle (Silybum marianum), was investigated and found to be an antagonist for the human angiotensin AT1 receptor with an affinity constant of about 9 µM, while it had no effect on endothelin type A or type B receptor activation. Quercetin and crocin partially impeded intracellular calcium signaling resulting in a non-receptor-related reduction of the responses recorded for the three investigated G-protein-coupled receptors. Two organosulfur compounds, diallyl disulfide and diallyl trisulfide, as well as the triterpene saponin ginsenoside Rb1 did not affect the activation of the angiotensin AT1 and endothelin type A and type B receptors. In conclusion, we were able, by using a nonradioactive cellular read-out system, to identify a novel pharmacological property of the flavanolignan silibinin.

Agonistic autoantibodies against the angiotensin AT1 receptor increase in unstable angina patients after stent implantation.

Agonistic AT1 receptor autoantibodies have been described in patients with hypertension and preeclampsia. These autoantibodies could stimulate proliferation of vascular smooth muscle cells (VSMCs), which are involved in angiotensin II-induced vascular injury in cardiovascular disease. Hence, in this study, we explored the existence of agonistic AT1 receptor autoantibodies in unstable angina (UA) patients and the possible effects of them on the in-stent restenosis of these patients. A total of 95 UA patients and 98 healthy volunteers were enrolled. The serum of each patient was analyzed for the presence of AT1 receptor autoantibodies by enzyme-linked immunosorbent assay. Their effects on VSMC proliferation and c-fos and c-jun expression were studied in vitro. AT1 receptor autoantibodies were detected in 34/95 patients with UA. The incidence was 10.2% in the control group and rose to 47.37% after stent implantation. In vitro, this autoantibody had agonist-like activity, shown as stimulation of VSMC proliferation and upregulation of c-fos and c-jun expression. These effects were similar to that of angiotensin II and could be weakened partly by the AT1-receptor blocker valsartan. Our findings show that the autoantibody from UA patients has similar agonistic activity to angiotensin II and might play a role in the pathogenesis of in-stent restenosis in these patients.

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

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

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

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

The role of local renin-angiotensin system in arterial chemoreceptors in sleep-breathing disorders.

The renin-angiotensin system (RAS) plays pivotal roles in the regulation of cardiovascular and renal functions to maintain the fluid and electrolyte homeostasis. Experimental studies have demonstrated a locally expressed RAS in the carotid body, which is functional significant in the effect of angiotensin peptides on the regulation of the activity of peripheral chemoreceptors and the chemoreflex. The physiological and pathophysiological implications of the RAS in the carotid body have been proposed upon recent studies showing a significant upregulation of the RAS expression under hypoxic conditions relevant to altitude acclimation and sleep apnea and also in animal model of heart failure. Specifically, the increased expression of angiotensinogen, angiotensin-converting enzyme and angiotensin AT1 receptors plays significant roles in the augmented carotid chemoreceptor activity and inflammation of the carotid body. This review aims to summarize these results with highlights on the pathophysiological function of the RAS under hypoxic conditions. It is concluded that the maladaptive changes of the RAS in the carotid body plays a pathogenic role in sleep apnea and heart failure, which could potentially be a therapeutic target for the treatment of the pathophysiological consequence of sleep apnea.

Abstract 022: Evidence for AT2-receptor-MAS Dimerisation from Fluorescence Resonance Energy Transfer (FRET) Imaging and Fluorescence Cross Correlation Spectroscopy (FCCS)

The angiotensin AT2-receptor (AT2R) and the receptor MAS share a strinkingly similar spectrum of signaling mechanisms and protective, physiological actions. Furthermore, cross-inhibition by the respective receptor antagonists has been observed. Therefore we hypothesised that a physical interaction between these two receptors may exist. HEK-293 cells were transfected with vectors encoding MAS or AT2R fused in the C-terminus with the fluorophores CFP or YFP for FRET and GFP or mCherry for FCCS. FRET with photobleaching was used to detect, whether MAS and AT2R are localised in very close proximity (1-10nm) in cell membranes thus indicating dimerisation. FCCS was used to follow simultaneously occurring fluctuations in fluorescence intensity of both labeled molecules. Several controls were applied such as co-transfection of equal amounts of fused and non-fused MAS/AT2R expression vectors for competition, co-tranfection of coding and uncoding pcDNA vectors or co-transfection with an unrelated transmembrane receptor. Experiments were conducted under baseline conditions and in cells treated with AT2R/MAS agonists and antagonists Significant FRET efficiency of 10.8±0.8% was measured for AT2-YFP/MAS-CFP strongly indicating heterodimerisation. FRET efficiency was not altered by AT2R or MAS agonists or antagonists. Non-fluorescent MAS and AT2R competed with fluorescent receptors as indicated by a 50% reduction in FRET efficiency (6.0±0.6%), while empty vectors did not compete (9.6±0.6%). No FRET efficiency was observed with an unrelated transmembrane receptor (0.44±1.44%) indicating specificity of receptor interactions. Both, MAS and AT2R also formed homodimers (7.4±0.8% for MAS, 9.2±0.8% for AT2R). Hetero- and homodimerisations were absent if amino acid C35 of the AT2R was mutated (3,9 ± 1,2%). FCCS corroborated the FRET results and revealed a significantly enhanced cross correlation in cells tranfected with fluorophore-tagged MAS/AT2R when compared to vectors only expressing the fluorophores (8.5±3% vs 11.1±4%; p<0.0001). Our data strongly suggest that MAS and the AT2R form homo- and heterodimers. Studies to investigate the physiological relevance of MAS/AT2R dimerisation are currently being conducted.

Abstract 284: Lower Nrf2 Activity And Decreased Levels Of Pdi And No In The Kidney Contributes To Hypertension In The Aging Fbns

We recently reported that age-associated oxidative stress is causal to higher renal angiotensin AT1 receptor (AT1R) function and blood pressure (BP) in the aging Fischer Brown Norway (FBN) rats. This suggests that redox homeostasis is critical for normal AT1R function and blood pressure (BP). Our objective was to understand the mechanism responsible for the higher oxidative stress in the aging kidneys contributing to impaired AT1R function and high BP in the aged FBNs. Kidneys from adult (3-month) and aged (21-month) FBN rats were isolated. Nrf2 transcription factor, involved in transcriptional activity of diverse antioxidant enzymes genes, was determined in the nuclear and cytosolic fractions of cortical tissues by Western blotting. Nuclear accumulation of Nrf2 is an index of its activation. Protein disulfide isomerase (PDI), an enzyme involved in isomerization reaction, was determined in the cortical homogenates. Moreover, nitrate/nitrite levels were determined in the urine as an index of NO levels in the kidney. We found that the levels of Nrf2 were decreased (adult vs old: 1.86±0.69 vs 0.87±0.18, -53.2%) in the nuclei but increased (adult vs old: 1.20±0.27 vs 1.51±0.36, +25.8%) in the cytosol of aging kidneys. PDI levels also were decreased (adult vs old: 5.38±2.60 vs 3.59±1.76, -33.3%) in the aging kidneys. Furthermore, nitrate/nitrite levels were lower (adult vs old: 372±73 vs 211±30, -43.3%) in the urine from aged than in adult rats. These data suggest that impaired Nrf2 activity in the aging kidneys contributes to increased oxidative stress, renal AT1R function and BP in the aged FNBs. Furthermore, these data also indicate that PDI in an NO-dependent pathway regulates Nrf2 activity contributing to redox-balance in the kidney.

Abstract 643: Unique Cortico-medullary Topology Of D1R, AT1R And NADPH-oxidase Contributes To Hypertension And Salt-sensitivity In Aging Rats

Aging Fisher Brown Norway (FBN) rats exhibit hypertensive phenotype due in part to increased oxidative stress and impaired dopamine D1 receptor (D1R) and angiotensin AT1 receptor (AT1R) functions in the aging kidneys. Here, we tested if these rats develop salt-sensitivity and examined its mechanisms. Adult (2- month) and aged (20- month) FBNs were treated with normal (NS, 0.4%) and high ( HS, 8%) salt for 4-weeks, conscious blood pressure (BP) measured and kidneys harvested for analyses. BP was higher in aged (120 mmHg) than in adult (113 mmHg) rats fed NS, which further increased (126 mmHg) with HS in aged rats. D1R mRNA and protein increased in cortex [NS vs HS: mRNA, 1.00 ± 0.0 vs 1. 21 ± 0.06 AU; Protein, 56.1 ± 2.01 vs 68.8 ± 3.08 DU] and medulla [NS vs HS: mRNA, 1.0 ± 0.0 vs 1.11 ± 0.12 AU; Protein, 54.2 ± 2.89 vs 70.3 ± 1.76 AU] with HS in adult rats. AT1R mRNA in cortex and medulla was higher in aged than in adult rats irrespective of salt treatment [Adult vs Old: Cortex, 1.0 ± 0.00 vs 5.04 ± 0.42; Medulla, 1.0 ± 0.00 vs 5.07 ± 0.09 AU]. However, its protein increased in medulla with HS in only aged rats [NS vs HS: 55.68 ± 4.8 vs 80.68 ± 8.3 DU]. 91phoxgp-NADPH-oxidase mRNA followed a similar pattern as of AT1 mRNA in both cortex and medulla of aged rats. However, its protein was higher in cortex of aged rats irrespective of salt intake [Adult vs Old: NS: 60.3 ± 7.2 vs 112.1 ± 8.6; HS: 61.9 ± 3.7 vs 94.25 ± 15.7 DU], but increased only with HS in medulla [Adult vs Old: 58.3 ± 6.3 vs 86.7 ± 7.2 DU] in these rats. Furthermore, NHE3 protein levels in the medulla were higher irrespective of salt treatment in aged rats [Adult vs Old: NS: 44.0 ± 5.8 vs 62.3 ± 4.6; HS: 45.4 ± 4.7 vs 65.4 ± 2.8 DU]. However, salt treatment increased the levels of Na,K-ATPase protein in the medulla of aged rats (NS vs HS: 11.8 ± 2.2 vs 18.9 ± 2.3 DU). Interestingly, renin levels in cortex were higher than in medulla irrespective of salt intake in aged rats (Cortex vs Medulla: NS: 84. 2 ± 4.5 vs 46. 32 ± 8.18; HS: 82.2 ± 1.18 vs 52.7 ± 3.51 ng/ml/hr). These data suggest that cortical renin and gp91phox-NADPH-oxidase together with medullary NHE-3 contribute to age-related hypertension, while D1R (cortical and medullary) together with medullary AT1R, gp91phox-NADPH-oxidase and Na, K-ATPase contribute to salt-sensitivity in aging FBNs.

Arrestin-dependent Angiotensin AT1 Receptor Signaling Regulates Akt and mTor-mediated Protein Synthesis

Control of protein synthesis is critical to both cell growth and proliferation. The mammalian target of rapamycin (mTOR) integrates upstream growth, proliferation, and survival signals, including those transmitted via ERK1/2 and Akt, to regulate the rate of protein translation. The angiotensin AT1 receptor has been shown to activate both ERK1/2 and Akt in arrestin-based signalsomes. Here, we examine the role of arrestin-dependent regulation of ERK1/2 and Akt in the stimulation of mTOR-dependent protein translation by the AT1 receptor using HEK293 and primary vascular smooth muscle cell models. Nascent protein synthesis stimulated by both the canonical AT1 receptor agonist angiotensin II (AngII), and the arrestin pathway-selective agonist [Sar(1)-Ile(4)-Ile(8)]AngII (SII), is blocked by shRNA silencing of βarrestin1/2 or pharmacological inhibition of Akt, ERK1/2, or mTORC1. In HEK293 cells, SII activates a discrete arrestin-bound pool of Akt and promotes Akt-dependent phosphorylation of mTOR and its downstream effector p70/p85 ribosomal S6 kinase (p70/85S6K). In parallel, SII-activated ERK1/2 helps promote mTOR and p70/85S6K phosphorylation, and is required for phosphorylation of the known ERK1/2 substrate p90 ribosomal S6 kinase (p90RSK). Thus, arrestins coordinate AT1 receptor regulation of ERK1/2 and Akt activity and stimulate protein translation via both Akt-mTOR-p70/85S6K and ERK1/2-p90RSK pathways. These results suggest that in vivo, arrestin pathway-selective AT1 receptor agonists may promote cell growth or hypertrophy through arrestin-mediated mechanisms despite their antagonism of G protein signaling.

Abstract 666: RNAi Therapeutics Targeting Human Angiotensinogen (hAGT) Ameliorate Preeclamptic Sequelae in an Established Transgenic Rodent Model for Preeclampsia

Preeclampsia, a disorder with the hallmark features of new-onset hypertension and proteinuria beginning after 20 weeks of gestation, affects 5% of pregnancies in industrialized nations. It is a major cause of fetal and maternal morbidity/mortality. Several studies have demonstrated that angiotensinogen is involved in the pathogenesis of the disease; however, treatment with ACE Inhibitor or AT1 Receptor blocker is contraindicated due to fetal toxicity. RNAi therapeutics are highly potent mediators of gene-specific silencing. We tested a human angiotensinogen (hAGT)-specifc siRNA, conjugated to triantennary GalNAc, for the ability to ameliorate symptoms of preeclampsia in an established rat model, without affecting the fetus. Transgenic rats expressing hAGT and human renin (hREN) were crossed to produce a model of preeclampsia (PE rat) in the dams. Beginning on day 3 of gestation, transgenic hAGT dams were dosed subcutaneously with 10 mg/kg siRNA every third day through gestation day 15. Mean blood pressure was continuously recorded by radiotelemetry and 24 hour urine samples were collected in metabolic cages at day 18 of gestation. Rats were euthanized at day 21 of gestation. Treatment with the GalNAc-conjugated siRNA reduced the spike in blood pressure seen on gestation day 13 and lasted through study termination (MAP on day 16 of gestation: 155.1 ± 1.4 mmHg untreated vs. 138.4 ± 1.8 mmHg treated). Proteinuria was ameliorated (21.7 ± 3.1 mg/d untreated vs. 2.7 ± 0.6 mg/d treated) and levels of agonistic autoantibodies against the angiotensin receptor AT1 were reduced below the limit of detection. Fetal and uteroplacental unit weights increased with RNAi therapy, demonstrating a reduction in intra-uterine growth restriction (brain to liver ratio (0.95 ± 0.04 untreated vs. 0.73 ± 0.02 treated). mRNA levels of hAGT were reduced to background levels in the liver, but were not affected in the placenta, which is of fetal origin. Our data show that an RNAi therapeutic targeting hAGT ameliorates the clinical sequelae of preeclampsia in a transgenic rat model and improves the outcome of the fetus.

Interactions between levetiracetam and cardiovascular drugs against electroconvulsions in mice

BackgroundHypertension and heart failure belong to common comorbid conditions with epilepsy so drug interactions between antiepileptics and cardiovascular drugs are possible in clinical practice. The aim of this study was to evaluate the effects of angiotensin AT1 receptor antagonists (losartan potassium and candesartan cilexetil), angiotensin-converting enzyme (ACE) inhibitors (captopril and perindopril arginine) and diuretics (hydrochlorothiazide and ethacrynic acid) on the anticonvulsant activity of levetiracetam (LEV) in mice. MethodsThe protective action of LEV was examined in the maximal electroshock seizure threshold test. Drugs were administered intraperitoneally (ip). Additionally, combinations of cardiovascular drugs with LEV were tested for adverse effects in the passive avoidance task and the chimney test. ResultsLosartan potassium (50mg/kg), candesartan cilexetil (8mg/kg), captopril (50mg/kg), hydrochlorothiazide (100mg/kg) and ethacrynic acid (100mg/kg) did not affect the anticonvulsant activity of LEV. Perindopril arginine (10mg/kg) raised the convulsive threshold for LEV administered at doses of 100, 300 and 500mg/kg. This interaction could be pharmacodynamic in nature because the brain concentration of LEV remained unchanged by perindopril. The adverse effects of the combined treatment with LEV and cardiovascular drugs were not observed in the passive avoidance task or the chimney test. ConclusionsAlthough experimental data can be hardly extrapolated to clinical practice, it is suggested that perindopril arginine may positively influence the anticonvulsant action of LEV in epileptic patients. The use of losartan potassium, candesartan cilexetil, captopril, hydrochlorothiazide or ethacrynic acid in patients treated with LEV seems neutral regarding its anticonvulsant activity.

Structural determinants for binding, activation, and functional selectivity of the angiotensin AT1 receptor.

The renin-angiotensin system (RAS) plays an important role in the pathophysiology of cardiovascular disorders. Pharmacologic interventions targeting the RAS cascade have led to the discovery of renin inhibitors, angiotensin-converting enzyme inhibitors, and AT(1) receptor blockers (ARBs) to treat hypertension and some cardiovascular and renal disorders. Mutagenesis and modeling studies have revealed that differential functional outcomes are the results of multiple active states conformed by the AT(1) receptor upon interaction with angiotensin II (Ang II). The binding of agonist is dependent on both extracellular and intramembrane regions of the receptor molecule, and as a consequence occupies more extensive area of the receptor than a non-peptide antagonist. Both agonist and antagonist bind to the same intramembrane regions to interfere with each other's binding to exhibit competitive, surmountable interaction. The nature of interactions with the amino acids in the receptor is different for each of the ARBs given the small differences in the molecular structure between drugs. AT(1) receptors attain different conformation states after binding various Ang II analogues, resulting in variable responses through activation of multiple signaling pathways. These include both classical and non-classical pathways mediated through growth factor receptor transactivations, and provide cross-communication between downstream signaling molecules. The structural requirements for AT(1) receptors to activate extracellular signal-regulated kinases 1 and 2 through G proteins, or G protein-independently through β-arrestin, are different. We review the structural and functional characteristics of Ang II and its analogs and antagonists, and their interaction with amino acid residues in the AT(1) receptor.