AT1 receptor downregulation:A mechanism for improving glucose homeostasis

There is a pathophysiological correlation between arterial hypertension and diabetes mellitus, established since the pre-diabetic state in the entity known as insulin resistance. It is known that high concentrations of angiotensin-Ⅱ enable chronic activation of the AT1 receptor, promoting sustained vasoconstriction and the consequent development of high blood pressure. Furthermore, the chronic activation of the AT1 receptor has been associated with the development of insulin resistance. From a molecular outlook, the AT1 receptor signaling pathway can activate the JNK kinase. Once activated, this kinase can block the insulin signaling pathway, favoring the resistance to this hormone. In accordance with the previously mentioned mechanisms, the negative regulation of the AT1receptor could have beneficial effects in treating metabolic syndrome and type 2diabetes mellitus. This review explains the clinical correlation of the metabolic response that diabetic patients present when receiving negatively regulatory drugs of the AT1 receptor.

Potential protective role of ACE-inhibitors and AT1 receptor blockers against levodopa-induced dyskinesias:a retrospective case-control study

Growing evidence has highlighted that angiotensin-converting enzyme(ACE)-inhibitors(ACEi)/AT1 receptor blockers(ARBs)may influence the complex interplay between dopamine and the renin-angiotensin system in the nigrostriatal pathway,thus affecting the development of levodopa-induced dyskinesia in Parkinson’s disease(PD).In the present study,we analyzed whether the use of this class of medication was associated with a reduced occurrence of levodopa-induced dyskinesia,using electronically-stored information of idiopathic PD patients enrolled at Novara University Hospital“Maggiore della Carità”.We conducted a retrospective case-control study identifying PD patients with dyskinesias(PwD;n=47)as cases.For each PwD we selected a non-dyskinetic control(NoD),nearly perfectly matched according to sex,Unified Parkinson’s Disease Rating Scale(UPDRS)part III score,and duration of antiparkinsonian treatment.Binary logistic regression was used to evaluate whether dyskinesias were associated with ACEi/ARBs use.Ninety-four PD patients were included,aged 72.18±9 years,with an average disease duration of 10.20±4.8 years and 9.04±4.9 years of antiparkinsonian treatment.The mean UPDRS part III score was 18.87±7.6 and the median HY stage was 2.In the NoD group,25(53.2%)were users and 22(46.8%)non-users of ACEi/ARBs.Conversely,in the PwD group,11(23.4%)were users and 36 non-users(76.6%)of this drug class(Pearson chi-square=8.824,P=0.003).Concerning general medication,there were no other statistically significant differences between groups.After controlling for tremor dominant phenotype,levodopa equivalent daily dose,HY 3-4,and disease duration,ACEi/ARBs use was a significant predictor of a lower occurrence of dyskinesia(OR=0.226,95%CI:0.080-0.636,P=0.005).Therefore,our study suggests that ACEi/ARBs may reduce levodopa-induced dyskinesia occurrence and,thanks to good tolerability and easy management,represent a feasible choice when dealing with the treatment of hypertension in PD patients.The study was approved by the Ethics Committee of Novara University Hospital“Maggiore della Carità”(CE 65/16)on July 27,2016.

Design and synthesis of 2-alkylbenzimidazole derivatives as novel non-peptide angiotensin Ⅱ AT1 receptor antagonists

A series of 2-alkylbenzimidazole derivatives 9a-n have been designed and synthesized as a novel class of non-peptide angiotensin H AT1 receptor antagonists. The synthesized compounds were evaluated for their antagonism of angiotensin H, induced contraction in the rabbit thoracic aortic ring and the results showed that compounds 9a, 9g and 9j exhibited potent antagonistic activity of AT1 receptor.

Effects of autoantibodies against AT1-receptor and angiotensin Ⅱ on refractory hypertension

Objective The study will explore effects of the autoantibodies against AT1 receptor and angiotensin Ⅱ on the refractory hypertension. Methods Seventy-seven patients (46 men and 31 women) with essential hypertension were divided into groups of refractory hypertension (RH) and hypertension (HT) according to the 1999 WHO-ISH Guidelines for the Management of Hypertension. Forty normotensives (22 men) were recruited as controls. The mean age was 54. 3±13 years old in RH group, 53. 5±9 years old in HT group and 51. 2±11. 9 years old in normotensives (NT) group. The mean blood pressure was 154. 2±9. 4/98. 4± 8. 2 mmHg in RH group and 130. 1±7. 6/80. 5±6. 7 mmHg in HT group after combination drug therapy of hypertension for 4 weeks. Blood pressure in NT group was 120. 8±11. 7/76. 4 ± 7. 2 mmHg. The epitope of the 2nd extracellular loops of AT1 receptor was synthesized and used as antigens to screen the autoantibodies by ELISA. Plasma angiotensin (Ang) II were examined by a radioimmunoassay. Results The autoantibodies against AT1 receptor were positive in 18 (46. 15 %) patients with RH, in 4 (10. 5 % ) hypertension and in 3 (7. 5 % ) normotensives, P < 0. 01. Ang Ⅱwas 57. 01±52. 63 pmol/L in patients with RH. Both the autoantibodies positive and the Ang Ⅱ increasing were 4 (10. 3 % ) cases, both normal were 7 (17. 9 % ) cases, the autoantibodies positive or Ang II increasing was all of 14 (35. 9 % ) cases (x2 = 0. 09, P>0. 05) . There was no relationship between the autoantibodies against AT1 receptor and the angiotensin Ⅱ in refractory hypertension. Conclusion The autoantibodies against AT1 receptor and Ang Ⅱ might be two independent factors in developing of refractory hypertension. The findings suggest that AT1 receptor an-tagnist used in the treatment of refractory hypertension might have an important value.

Activation of angiotensin Ⅱ type 1 receptors in the median preoptic nucleus induces a diuretic and natriuretic response in rats

Objective： To investigate the effect of activation of angiotensin Ⅱ（AngⅡ） type 1 （AT1） receptors in the median preoptic nucleus （MnPO） of rats on renal sodium excretion. Methods： After anesthesia, the rats were injected into the MnPO via an implanted cannula. Urine samples were collected via a bladder cannula, and the urine sodium concentration was assayed with flame spectrophotometry. The serum level of endogenous digitalis-like factor （EDLF） and Na^＋,K^＋-ATPase activity in the renal cortex tissue were assayed respectively with a radioimmunoassay and with an ammonium molybdophosphate-based kit. Results： Both the urinary volume and the sodium excretion peaked 60 min after AngⅡ was administered into the MnPO. The responses were accompanied by an increase in serum EDLF and a decrease in Na^＋,K^＋-ATPase activity in the renal cortex. The responses of diuresis and natriuresis, as well as an increase in serum EDLF and a decrease in Na^＋,K^＋-ATPase activity in the renal cortex induced by MnPO adminstration with AngⅡ were inhibited by pior treatment with the AngⅡ receptor blocking agent losartan into the MnPO. Conclusion： These results suggest that activation of AT1 receptors in the MnPO of rat induces diuretic and natriuretic responses. The responses are associated with an increase release of EDLF and with the inhibition of Na^＋,K^＋-ATPase activity in renal cortex tissue.

The dopamine receptor D4 regulates the proliferation of pulmonary arteries smooth muscle in broilers by downregulating AT1R

The major cause of pulmonary vascular remodeling in broilers is abnormal proliferation of vascular smooth muscle cells(VSMCs),and one of the main causes of pulmonary hypertension syndrome(PHS)in broilers is pulmonary artery vascular remodeling.Forty Arbor Acres(AA)broilers were randomly divided into four groups(n=10):a control group(deionized water,Og/L NaCl),a freshwater group(FW,deionized water+1 g/L NaCl),highly salinized freshwater group 1(H-SFW-1,deionized water+2.5 g/L NaCl)and highly salinized freshwater group 2(H-SFW-2,deionized water+5 g/L NaCl).The results of in vivo experiments showed that vascular smooth muscle of the broilers could be significantly proliferated by intake of high-salinity fresh water(H-SFW-1&H-SFW-2),which significantly increased the content of angiotensin II(Ang II)and the expression of angiotensin II type 1(AT1)receptor protein.Meanwhile,it significantly decreased the expression of dopamine receptor D4(DRD4)protein.The results of in vitro experiments showed that exogenous Ang II induced the proliferation of primary VSMCs in broilers,which could be significantly inhibited by DRD4 agonists(D4A,HY-101384A)and enhanced by DRD4 inhibitors(D4I;HY-B0965).In addition,the results of immunoblotting and fluorescence quantitative PCR showed that AT1 receptors could be negatively regulated by DRD4 in VSMCs of broilers,either at the transcriptional or translational level.At the same time,the expression of AT1 receptor could be increased by DRD4 inhibition by D4I and decreased by DRD4 activation by D4A.The negative regulatory effect of DRD4 on AT1 receptor occurred in a dose-dependent manner.These results indicate that long-term intake of highly salinized fresh water can cause PHS in broilers,accompanied by varying degrees of proliferation of pulmonary artery smooth muscle.This mechanism may involve response of its receptor being induced by increased Ang II,while DRD4 can negatively regulate it.

EFFECT OF ANGIOTENSIN Ⅱ RECEPTOR SUBTYPE ⅠON THE FIRING RATE IN CATECHOLAMINERGIC TUMOR CELLS

Objective To study the action of brain angiotensin Ⅱ(Ang Ⅱ) receptors and underlying intracellular mechanism in the catecholaminergic system Methods Action potentials (APs) of the primary co cultured catecholaminergic tumor (CATH.a) cells were recorded with the whole cell patch clamp configuration in current clamp mode. Expression of Ang Ⅱ receptors subtypes (AT 1 and AT 2 ) was detected by RT PCR technique. Results The differentiated CATH.a cells represented a neuron like characterization. All CATH.a cells expressed mRNA encoding both Ang Ⅱ AT 1 and AT 2 receptor subtypes. Ang Ⅱ increased the firing rate in the CATH.a cells, which was inhibited completely by addition administration of the AT 1 but not AT 2 receptor antagonist, and partially by using the inhibitors of signal molecules, U73122 (10 μmol·L -1 ), or KN 93 (10 μmol·L -1 ), or calphostin C (10 μmol·L -1 ). Conclusion Ang Ⅱ increases firing rate in CATH.a cells via AT 1 receptor. The CATH.a cells expressing functional AT 1 and AT 2 receptor subtypes may be of general utility for the study of the Ang Ⅱ receptor induced modulation of brain catecholaminergic system.

Involvement of Ca^(2+)-activated K^+ Channels in Receptor-Regulated Sperm Motility in Rats

Previous voltage clamp studies have demonstrated the modulation of sperm Ca 2+ activated K + (KCa) channels expressed in Xenopus oocytes by angiotensin II (Ang II) and extracellular ATP via AT 1 receptor and P 2U receptor, respectively. In the present study, we investigated the involvement of KCa channels in receptor regulated sperm motility of the rat using a computer aided sperm analysis system, HTM IVOS, in conjunction with Ca 2+ mobilizing agents, receptor agonists/antagonists and KCa channels blockers. The percentage of motile sperm was increased by ionomycin (0.5 μmol/L), which could be inhibited by K + channel blockers, tetraethylammonium (TEA 1 μmol/L ) or charybdotoxin (ChTX, 300 nmol/L) indicating the presence of KCa channels. Ang II, at low concentration, 10 nmol/L, was found to increase motility, however, at higher concentration, 1 μmol/L, percentage of motility was found to be suppressed. Both stimulatory and inhibitory effects of Ang II could be reversed by losartan, a specific antagonist of AT 1 receptors, but not AT 2 antagonist PD123177, indicating the involvement of AT 1 but not AT2 receptor in mediating both effects. ChTX also abolished both stimulatory and inhibitory effects of Ang II, suggesting the involvement of KCa channels. The percentage of motility was also enhanced by extracellular ATP, a factor known to be involved in sperm activation. The ATP enhanced sperm motility was mimicked by UTP, and inhibited by ChTX and reactive blue, an antagonist of P 2 receptor, indicating the involvement of both P 2U and KCa channels. RT PCR study was also conducted to confirm the expression of KCa channels, AT 1 receptors and P 2U receptor, but not AT 2 receptor, in rat caudal epididymal sperm. The present findings suggest an important role of KCa channels in the regulation of sperm motility by AT 1 and P 2U receptors.

Effects on Cell Viability and on Apoptosis in Tumoral(MCF-7)and in Normal(MCF10A)Epithelial Breast Cells after Human Chorionic Gonadotropin and Derivated-Angiotensin Peptides Treatments

Angiotensin-(1 - 7) [Ang-(1 - 7)] is an endogenous heptapeptide hormone of the renin-angiotensin system that has antiproliferative properties. The aim of this work was to evaluate the anti-proliferative and pro-apoptotic properties of Ang-(1 - 7) and of Ang-(1 - 7)-substituents 9-fluorenylmethyloxycarbonyl (Fmoc) e Ang II-derivatives containing the TOAC (2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid) in normal (MCF10A) and in tumoral (MCF7) epithelial mammary cell lines. Both cell lines received an hCG and angiotensin peptides 24-hour treatment, in combination or alone followed by cell viability, apoptosis and cell cycle assays performed by flow cytometer (GUAVA). After hCG, Ang-(1 - 7), hCG + Ang-(1 - 7) and hCG + Ang-(1 - 7)-Fmoc treatments, MCF7 displayed cell viability decrease and mid-apoptosis increase. We also observed cell viability decrease in MCF10A after Ang-(1 - 7), Ang-(1 - 7) Fmoc and hCG + AngII Toac treatments. These cells had an increase in late apoptosis and necrosis after AngII Toac, hCG + Ang-(1 - 7) and hCG + Ang-(1 - 7)-Fmoc treatments. Regarding the cell cycle analysis, we did not observed any changes in cell cycle phases. In summary, cell viability was decreased and apoptosis (initial, mid and late) was increased after hCG and/or Ang-(1 - 7) peptides treatments. These results point out hCG and Ang-(1 - 7) as effective compounds to inhibit cell proliferation, since they decrease cell viability and increase apoptosis in both normal and in tumoral breast cells, being the effect more pronounced in the tumoral cell line. Our results support the idea of investigating more closely the putative use of these compounds as novel therapeutic agents for breast cancer.

腺苷A_1受体阻断剂对学习记忆的影响及机制分析

目的 探讨腺苷A1 受体阻断剂对学习记忆的影响及其与胆碱能、氨基酸能神经的关系。方法 采用避暗实验、分光光度法和HPLC法 ,观察腺苷A1 受体特异性阻断剂 8 环戊 1 ,3 二丙基黄嘌呤 (DPCPX)对东莨菪碱(Scop)、2 氨基 5 磷戊酸 (AP5)致小鼠记忆障碍及脑胆碱酯酶 (AChE)活性、氨基酸水平的影响。结果 DPCPX可显著改善Scop致记忆障碍 ,但对AP5致记忆障碍无影响 ;在体内外高剂量DPCPX可显著抑制小鼠脑AChE活性 ;DPCPXicv可显著升高小鼠脑Glu和Asp含量 ,降低GABA含量 ,使脑内Glu GABA比值显著升高。结论 腺苷A1 受体特异性阻断剂DPCPX可显著改善Scop而不能改善AP5致记忆障碍 ,在高剂量时可影响脑AChE活性和脑氨基酸水平、升高脑内Glu GABA比值。

Interferon Regulatory Factor 5 and Renin-Angiotensin-Aldosterone System Polymorphisms in Coronary Artery Disease: An Overview of Experimental and Clinical Studies

Heart diseases are the main cause of mortality in Mexico, being coronary heart disease the most frequent in the country. Its high prevalence makes important the study of the pathophysiology and the search for prognostic factors. Different genes and polymorphisms promote atherogenesis and coronary artery disease, they affect inflammatory and vascular pathological processes. Interferon regulatory factor 5 (IRF5) is associated with coronary heart disease, it promotes chronic inflammation and cytokines release;it could trigger immune reactions and its activating receptors express in the vascular endothelium. Besides, polymorphisms in the renin-angiotensin-aldosterone system (RAAS) are implied with coronary disease, they are found in angiotensinogen (AGT), angiotensin II type 1 receptor (AT1R), angiotensin II type 2 receptor (AT2R), and angiotensin-converting enzyme (ACE) genes. These genetic polymorphisms are associated with a prothrombotic state, endothelial dysfunction, and immune activation. Multiple experimental studies showed that chronic activation of RAAS and chronic expression of IRF5 generates an environment prone to the development of atherosclerosis, and autoimmune and cardiovascular diseases. Studying these specific genes and their relationship with coronary heart disease will allow a better understanding of the pathological process and possibly the quest for new treatments.

The mechanism of signal transduction during vascular smooth muscle cell proliferation induced by autoantibodies against angiotensin AT1 receptor from hypertension

Background Autoantibodies against angiotensin AT1 receptor have been discovered in patients with preeclampsia or malignant hypertension. Some studies have demonstrated that the autoantibodies are involved in the immunopathogenesis of hypertension and have an agonist effect similar to angiotensin II. Methods Autoantibodies against AT1 receptor were purified from sera of patients with primary hypertension by affinity chromatography. Proliferation of cultured rat vascular smooth muscle cells was detected by bromodeoxyuridine incorporation and activation of signalling molecules detected by Western blotting and electrophoretic mobility shift assay. Results The AT1-RAb caused a significant proliferation similar to the Ang II during first 24 hours. The levels of nuclear factor-KB （NF-KB）, phosphorylated JAK2., phosphorylated STAT1 （pSTAT1） and phosphorylated STAT3 （pSTAT3） molecules were increased in response to the autoantibodies. In contrast, the activations of NF-KB and JAK-STAT were blocked by Iosartan, pyrrolidinedithiocarbamate （a specific inhibitor of NF-KB） and AG490 （a specific inhibitor of the JAK2. tyrosine kinase）. The expressions of NF-KB, pSTAT1 and pSTAT3 reached peak levels at different times. Moreover, the relative densities of electrophoretic bands showed that activation of pSTAT3 was more significant than STAT1 induced by AT1 -RAb. Conclusions These results suggest that the autoantibodies against AT1 receptor have an agonist effect similar to Ang II in proliferation of VSMCs and the NF-KB and JAK-STAT proteins play essential roles. The effect is different from Angll in that STAT3 is the main downstream activating molecule in JAK-STAT signalling pathway.

Macrophage exosomes transfer angiotensin Ⅱ type 1 receptor to lung fibroblasts mediating bleomycin-induced pulmonary fibrosis

Background:Macrophages are involved in the pathogenesis of idiopathic pulmonary fibrosis,partially by activating lung fibroblasts.However,how macrophages communicate with lung fibroblasts is largely unexplored.Exosomes can mediate intercellular communication,whereas its role in lung fibrogenesis is unclear.Here we aim to investigate whether exosomes can mediate the crosstalk between macrophages and lung fibroblasts and subsequently induce fibrosis.Methods:In vivo,bleomycin(BLM)-induced lung fibrosis model was established and macrophages infiltration was examined.The effects of GW4869,an exosomes inhibitor,on lung fibrosis were assessed.Moreover,macrophage exosomes were injected into mice to observe its pro-fibrotic effects.In vitro,exosomes derived from angiotensin Ⅱ(Ang Ⅱ)-stimulated macrophages were collected.Then,lung fibroblasts were treated with the exosomes.Twenty-four hours later,protein levels ofα-collagen I,angiotensin Ⅱ type 1 receptor(AT1R),transforming growth factor-β(TGF-β),and phospho-Smad2/3(p-Smad2/3)in lung fibroblasts were examined.The Student's t test or analysis of variance were used for statistical analysis.Results:In vivo,BLM-treated mice showed enhanced infiltration of macrophages,increased fibrotic alterations,and higher levels of Ang Ⅱ and AT1R.GW4869 attenuated BLM-induced pulmonary fibrosis.Mice with exosomes injection showed fibrotic features with higher levels of Ang Ⅱ and AT1R,which was reversed by irbesartan.In vitro,we found that macrophages secreted a great number of exosomes.The exosomes were taken by fibroblasts and resulted in higher levels of AT1R(0.22±0.02 vs.0.07±0.02,t=8.66,P=0.001),TGF-β(0.54±0.05 vs.0.09±0.06,t=10.00,P<0.001),p-Smad2/3(0.58±0.06 vs.0.07±0.03,t=12.86,P<0.001)andα-collagen I(0.27±0.02 vs.0.16±0.01,t=7.01,P=0.002),and increased Ang Ⅱ secretion(62.27±7.32 vs.9.56±1.68,t=12.16,P<0.001).Interestingly,Ang Ⅱ increased the number of macrophage exosomes,and the protein levels of Alix(1.45±0.15 vs.1.00±0.10,t=4.32,P=0.012),AT1R(4.05±0.64 vs.1.00±0.09,t=8.17,P=0.001),and glyceraldehyde-3-phosphate dehydrogenase(2.13±0.36 vs.1.00±0.10,t=5.28,P=0.006)were increased in exosomes secreted by the same number of macrophages,indicating a positive loop between Ang Ⅱ and exosomes production.Conclusions:Exosomes mediate intercellular communication between macrophages and fibroblasts plays an important role in BLM-induced pulmonary fibrosis.

白细胞介素-1受体相关激酶-4在血吸虫病门脉高压小鼠肠系膜静脉的表达变化及其意义

目的探讨血吸虫病门脉高压小鼠肠系膜静脉组织白细胞介素-1受体相关激酶-4（IRAK-4）的表达变化及意义。方法将55只雄性昆明小鼠随机分为对照组（n=15）和感染组（n=40）。感染组采用腹部敷贴法感染日本血吸虫尾蚴，分别于感染后12、20周随机分为12周组（n=20）、20周组（n=20）；采用逆转录-聚合酶链反应（RT—PCR）法检测肠系膜静脉IRAK-4mRNA表达与肿瘤坏死因子（TNF）-α mRNA表达，Westernblot法检测肠系膜静脉IRAK-4蛋白表达。结果感染组小鼠肠系膜静脉IRAK-4mRNA表达强度值（12周组0．734±0．081，20周组0．682±0．069）和IRAK-4蛋白表达水平（12周组：1034．52±89．72，20周组：989．56±69．35）较对照组（IRAK-4mRNA表达强度值0．163±0．031，IRAK4蛋白表达水平667．45±72．22）明显升高（P〈0．01，P〈0．05）；20周组IRAK-4mRNA和蛋白表达与12周组比较差异无统计学意义（P〉0．05）；各组IRAK-4mRNA的表达与其效应分子TNF-αmRNA的表达呈显著正相关（r=5．56，P〈0．01）。结论门静脉高压症小鼠内脏血管IRAK-4在转录和翻译水平的表达升高，并激活下游炎性反应，是参与门静脉高压性血管病变的重要因素。