Association of Polymorphisms in Angiotensin-converting Enzyme and Type 1 Angiotensin Ⅱ Receptor Genes with Coronary Heart Disease and the Severity of Coronary Artery Stenosis

To explore the relation of angiotensin-converting enzyme （ACE） and angiotensin Ⅱ type 1 receptor （AT1R） gene polymorphism with coronary heart disease （CHD） and the severity of coronary artery stenosis, 130 CHD patients who underwent coronary angiography were examined for the number of affected coronary vessels （≥75% stenosis） and coronary Jeopardy score. The insertion/deletion of ACE gene polymorphism and AT1R gene polymorphism （an A→C transversion at nucleotide position 1166） were detected by using polymerase chain reaction （PCR） and restriction fragment length polymorphism （RFLP） in CHD patients and 90 healthy serving as controls. The resuits showed that DD genotype and of ACE were more frequent in CHD patients than that in control group （38.5% vs 14.4%, P〈0.001）. The frequency of the ATIR A/C genotypes did not differ between the patients and the controls （10% vs 13.1%, P〉0.05）. The relative risk associated with the ACE-DD was increased by AT1R-AC genotype. Neither the number of affected coronary vessels nor the coronary score differed among the ACE I/D genotypes （P〉0.05）. But the number of affected coronary vessels and the coronary score were significantly greater in the patients with the AT1R-AC genotype than in those with the AA genotype （P〈0.05）. In conclusion, DD genotype may be risk factor for CHD and MI in Chinese people, and is not responsible for the development of the coronary artery stenosis. The AT1R-C allele may increase the relative risk associated with the ACE-DD genotype, and may be involved in the development of the stenosis of coronary artery.

Construction of shRNA Targeted to the Rat Angiotensin Ⅱ Type 1 Receptors and Its RNAi in Cytoplasma

The expression vector of shRNA targeted to the rat angiotensin Ⅱ receptor gene was constructed and the efficacy of siRNAs to modulate the expression of target gene in the in vitro cultured mammalian cells was investigated for antihypertensive therapy in spontaneous hypertensive rat （SHR） at post-transcriptional level. The sense and antisense RNA oligonucleotides strands targeting angiotensin Ⅱ receptor mRNA were synthesized individually according to the sequence of the rat angiotensin Ⅱ receptor. For preparation of duplexes, sense- and antisense-stranded oligonucleotides were mixed and annealed, and the annealed duplexes were cloned into the pGenesil-1 vector. The rat glioma cells were transfected with constructed pGenesil-1-shRNA plasmid and scrambled plasmid. The cultured cells were collected at different phases. RT-PCR and Western blot were performed. The AT1 mRNA and protein levels behaved ultimately same. Compared to control after 48 h, AT1 mRNA levels were decreased to 35.5%±3.0 %, and the levels reached their lowest point after 72 h （20.7% ±4 % of control）. At 24 and 48 h, AT1 protein was reduced to 46.9%±4.2% and 36.98%±3.7% respectively compared to control and a maximum reduction was observed after 72 h of incubation （28.1%± 4% compared to controls）. Plasmid-based shRNA expression systems targeted against the rat angiotensin Ⅱ receptor gene were generated successfully. The shRNAs with a 22-nt stem and a short loop were cleaved into small interfering dsRNA （siRNA） by the Dicer. The in vitro transcribed siRNA enables the effective silencing of gene expression to the target mRNA and leads to effective inhibition of translation of proteins and will be lay the foundation of application of gene silencing technology to hypertensive rats.

Expression of Angiotensin Ⅱ Receptors in Aldosterone-producing Adenoma of the Adrenal Gland and Their Clinical Significance

The expression of angiotensin Ⅱ type 1 receptor (AT1R) and angiotensin Ⅱ type 2 receptor (AT2R) in aldosterone-producing adenoma (APA) of the adrenal gland was detected, and their relationship with clinical indexes of APA was analyzed. The mRNA expression of AT1R and AT2R in 50 cases of APA and tissues adjacent to tumors and 12 cases of normal adrenal tissues was detected by using reverse transcriptase polymerase chain reaction (RT-PCR). The expression of AT1R and AT2R proteins in paraffin-embedded slices of tissue was detected by immunohistochemistry. The expression of AT1R in adenoma, tissues adjacent to tumor, and normal tissues of the adrenal gland showed no significant differences. The expression of AT2R in APA tissue was lower than that in normal adrenal gland tissues (P<0.05). Correlation analysis of the mRNA expression level of AT2R and clinical data from patients demonstrated that AT2R expression was negatively related to plasma aldosterone concentration (PAC) (r=-0.467, P<0.05), but positively related with plasma renin activity (PRA) (r=0.604, P<0.05). It is concluded that down-regulation of the AT2R expression is possibly related with the tumorigenesis of APA.

Effect of nuclear factor-κB and angiotensin Ⅱ receptor type 1 on the pathogenesis of rat non-alcoholic fatty liver disease

AIM: To investigate the roles of nuclear factor(NF)-κB and angiotensin Ⅱ receptor type 1(AT1R) in the pathogenesis of non-alcoholic fatty liver disease(NAFLD).METHODS: Forty-two healthy adult male SpragueDawley rats were randomly divided into three groups:the control group(normal diet), the model group,and the intervention group(10 wk of a high-fat diet feeding, followed by an intraperitoneal injection of PDTC); 6 rats in each group were sacrificed at 6, 10,and 14 wk. After sacrifice, liver tissue was taken,paraffin sections of liver tissue specimens were prepared, hematoxylin and eosin(HE) staining was performed, and pathological changes in liver tissue(i.e., liver fibrosis) were observed by light microscopy.NF-κB expression in liver tissue was detected by immunohistochemistry, and the expression of AT1 R in the liver tissue was detected by reverse transcriptionpolymerase chain reaction(RT-PCR). The data are expressed as mean ± SD. A two-sample t test was used to compare the control group and the model group at different time points, paired t tests were used to compare the differences between the intervention group and the model group, and analysis of variance was used to compare the model group with the control group. Homogeneity of variance was analyzed with single factor analysis of variance. H variance analysis was used to compare the variance. P < 0.05 wasconsidered statistically significant.RESULTS: The NAFLD model was successful after 6wk and 10 wk. Liver fibrosis was found in four rats in the model group, but in only one rat in the intervention group at 14 wk. Liver steatosis, inflammation, and fibrosis were gradually increased throughout the model. In the intervention group, the body mass,rat liver index, serum lipid, and transaminase levels were not increased compared to the model group.In the model group, the degree of liver steatosis was increased at 6, 10, and 14 wk, and was significantly higher than in the control group(P < 0.01). In the model group, different degrees of liver cell necrosis were visible and small leaves, punctated inflammation,focal necrosis, and obvious ballooning degeneration were observed. Partial necrosis and confluent necrosis were observed. In the model group, liver inflammatory activity scores at 6, 10, and 14 wk were higher than in the control group(P < 0.01). Active inflammation in liver tissue in the intervention group was lower than in the model group(P < 0.05). HE staining showed liver fibrosis only at 14 wk in 4/6 rats in the model group and in 1/6 rats in the intervention group. NF-κB positive cells were stained yellow or ensemble yellow,and NF-κB was localized in the cytoplasm and/or nucleus. The model group showed NF-κB activation at6, 10, and 14 wk in liver cells; at the same time points,there were statistically significant differences in the control group(P < 0.01). Over time, NF-κB expression increased; this was statistically lower(P < 0.05) at14 weeks in the intervention group compared to the model group, but significantly increased(P < 0.05)compared with the control group; RT-PCR showed that AT1 R mRNA expression increased gradually in the model group; at 14 wk, the expression was significantly different compared with expression at 10 weeks as well as at 6 weeks(P < 0.05). In the model group, AT1 R mRNA expression was significantly higher than at the same time point in the control group(P <0.01).CONCLUSION: With increasing severity of NAFLD,NF-κB activity is enhanced, and the inhibition of NF-κB activity may reduce AT1 R mRNA expression in NAFLD.

Effects of angiotensin Ⅱ receptor antagonist, Losartan on the apoptosis, proliferation and migration of the human pancreatic stellate cells

AIM： To investigate the effects of AT, （Type 1 angiotensin Ⅱ receptor） antagonist （Losartan） on the apoptosis, proliferation and migration of the human pancreatic stellate cells （hPSCs）. METHODS： hPSCs were isolated from pancreatic sample of patients with pancreatic carcinoma using radioimmunoassay （RIA） technique to detect the concentration of AngⅡ in culture media and cell homogenate. Immunocytochemistry （ICC） and in situ hybridization （ISH） methods were utilized to test AT1 expression in hPSCs. Effects of Losartan on hPSCs proliferation, apoptosis and migration were investigated using BrdU incorporation, TUNEL, flow cytometry （FCM）, and phase-contrast microscope separately when cells treated with Losartan. Immunofluorescence and Western blot were applied to quantify the expression of type Ⅰ collagen in hPSCs. RESULTS： There exists AT1 expression in hPSCs, while no AngⅡ was detected in culture media and cell homogenate. Losartan induces cell apoptosis in a doseand time-dependent manner （apparently at 10^-5 mol/L）, no pro-proliferative effect was observed in the same condition. Corresponding dosage of Losartan can also alleviate the motion capability and type Ⅰ collagen content of hPSCs compared with AngⅡ treatment and non-treatment control groups. CONCLUSION： These findings suggest that paracrine not autocrine functions of AngⅡ may have effects on hPSCs, which was mediated by AT1 expressed on cells, while Losartan may exert anti-fibrotic effects by inhibiting hPSCs motion and partly by inducing apoptosis.

DNA methylation of angiotensin Ⅱ receptor gene in nonalcoholic steatohepatitis-related liver fibrosis

AIM To clarify whether Agtr1 a methylation is involved in the development of nonalcoholic steatohepatitis(NASH)-related liver fibrosis in adult rats.METHODS A choline-deficient amino acid(CDAA) diet model was employed for methylation analysis of NASH-related liver fibrosis.Agtr1 a methylation levels were measured in the livers of CDAA- and control choline-sufficient amino acid(CSAA)-fed rats for 8 and 12 wk using quantitative methylation-specific PCR.Hepatic stellate cells(HSCs) were isolated by collagenase digestion of the liver,followed by centrifugation of the crude cell suspension through a density gradient.Agtr1 a methylation and its gene expression were also analyzed during the activation of HSCs.RESULTS The mean levels of Agtr1 a methylation in the livers of CDAA-fed rats(11.5% and 18.6% at 8 and 12 wk,respectively) tended to be higher(P = 0.06 and 0.09,respectively) than those in the livers of CSAA-fed rats(2.1% and 5.3% at 8 and 12 wk,respectively).Agtr1 a was not methylated at all in quiescent HSCs,but was clearly methylated in activated HSCs(13.8%,P < 0.01).Interestingly,although Agtr1 a was hypermethylated,the Agtr1 a m RNA level increased up to 2.2-fold(P < 0.05) in activated HSCs compared with that in quiescent HSCs,suggesting that Agtr1 a methylation did not silence its expression but instead had the potential to upregulate its expression.These findings indicate that Agtr1 a methylation and its upregulation of gene expression are associated with the development of NASH-related liver fibrosis.CONCLUSION This is the first study to show that DNA methylation is potential y involved in the regulation of a renin-angiotensin system-related gene expression during liver fibrosis.

Effect of Microinfusion of Angiotensin Ⅱ into the RVLM in Rats on the Baroreceptor Reflex Sensitivity

Objective: The present study was designed to investigate the effect of microinfusion angiotensin Ⅱ(Ang Ⅱ),Ang Ⅱ type 1(AT_1)receptor antagonist losartan into the rostral ventrolateral medulla(RVLM)on the baroreceptor reflex sensitivity(BRS)in urethane-anesthetized rats. Methods: Reflex changes in heart rate(HR)were elicited by bolus intravenous injection of phenylephrine before and during RVLM microinfusion of saline(0.5 μl/h),Ang Ⅱ (1.5 nmol/h),losartan(250 nmol/h),and Ang Ⅱ(1.5 nmol/h)pretreated with microinjection of losartan (50 nmol/0.51 μl)into the RVLM.The average ratio between changes in HR in beats per minute(beats·min -1)and changes in mean arterial pressure [MAP,mmHg(1 mmHg=0.133 kPa)] was used as an index of BRS. Results: Ang Ⅱ resulted in a significant decrease in the BRS for reflex bradycardia compared with control(-2.1±0.1 vs-3.9±0.4 beats·min -1·mmHg -1).Microinfusion of losartan had no significant effect on BRS for reflex bradycardia.The effect of Ang Ⅱ was almost completely abolished by pretreatment with microinjection of losartan. Conclusion:These results showed that the exogenous Ang Ⅱ in the RVLM produces inhibitory modulation of BRS,which is mediated by AT_1 receptor.However,AT_1 receptor in the RVLM is not involved in the tonic control of BRS.

Isolation and Characterization of Proteins Interacting with Activin Type Ⅱ Receptors

Regulation of the number of aetivin receptors that are present in the cell membrane plays a key role in the modulation of cellular responses to activin. In order to find the regulators, a novel protein ARIPzip, interacting with activin type II receptors, was searched and identified by using yeast two-hybrid screening. ARIPzip is a splicing variant of ARIP2. This has been discussed previously. ARIPzip can specifically interact with ActR Ⅱ A, and is widely distributed in mouse tissues. Overexpression of ARIPzip can cause the activin-induced transcriptional activities to increase in a dose-dependent manner while the overexpression of ARIV2 can decrease these activities. These data suggest that the C-terminal rezions of ARIP2 and ARIPzip are involved in the regulation of activin signaling.

Association of Polymorphisms in Angiotensin Ⅱ Receptor Genes with Aldosterone-producing Adenoma

This study examined the association of polymorphisms in angiotensinⅡreceptor genes（AT1R and AT2R） with the risk for aldosterone-producing adenoma（APA） in a Chinese Han population.Four polymorphisms including rs5182（573T/C） in exon 4,rs5186（1166A/C） in 3＇-untranslated region（3＇-UTR） in AT1R gene and rs5194（2274G/A） in 3＇-UTR,rs1403543（1675G/A） in intron 1 in AT2R gene were detected in 148 APA patients and 192 normal subjects（serving as control） by using a MGB-Taqman probe.The distribution of genotypes of each locus was in accordance with Hardy-Weinberg Equilibrium（HWE） in the APA and control groups（P0.05）.The allele A frequency at rs5194 was significantly higher in the APA group（0.49） than in the control group（0.35）（χ2=12.08,P=0.001）.Subjects with homozygotic genotype AA and heterozygotic genotype GA were at an increased risk for APA as compared to those with GG genotype（OR=2.66,95% CI=1.45-4.87;OR=1.67,95% CI=1.02-2.74）.Furthermore,rs5194 single-nucleotide polymorphism（SNP） at AT2R gene was significantly associated with APA in additive（OR=1.64,95% CI=1.21-2.20,P=0.001）,dominant（OR=1.94,95% CI=1.23-3.06,P=0.003）,and recessive model（OR=2.01,95% CI=1.17-3.45,P=0.01）.It was concluded that rs5194 polymorphism at AT2R gene was associated with the risk for APA,which may constitute a genetic marker of APA.

Effect of Atorvastatin on Expression of Peroxisome Proliferator-activated Receptor Beta/delta in Angiotensin Ⅱ-induced Hypertrophic Myocardial Cells In Vitro

Objective To explore the effect of atorvastatin on cardiac hypertrophy and to determine the potential mechanism involved. Methods An in vitro cardiomyocyte hypertrophy from neonatal rats was induced with angiotensinⅡ(Ang Ⅱ) stimulation. Before AngⅡ stimulation, the cultured rat cardiac myocytes were pretreated with atorvastatin at different concentrations(0.1, 1, and 10 μmol/L). The following parameters were evaluated: the myocyte surface area, 3H-leucine incorporation into myocytes, m RNA expressions of atrial natriuretic peptide, brain natriuretic peptide, matrix metalloproteinase 9, matrix metalloproteinase 2, and interleukin-1β, m RNA and protein expressions of the δ/β peroxisome proliferator-activated receptor(PPAR) subtypes. Results It was shown that atorvastatin could ameliorate Ang Ⅱ-induced neonatal cardiomyocyte hypertrophy in the area of cardiomyocytes, 3H-leucine incorporation, and the expression of atrial natriuretic peptide and brain natriuretic peptide markedly. Meanwhile, atorvastatin also inhibited the augmented m RNA level of several cytokines in hypertrophic myocytes. Furthermore, the down-regulated expression of PPAR-δ/β at both the m RNA and protein levels in hypertrophic myocytes could be significantly reversed by atorvastatin treatment. Conclusions Atorvastatin could improve AngⅡ-induced cardiac hypertrophy and inhibit the expression of cytokines. Such effect might be partly achieved through activation of the PPAR-δ/β pathway.

妊娠高血压患者血管紧张素Ⅱ及AT1R、AT2R的表达及意义

目的:探讨妊娠高血压(HDCP)患者血管紧张素Ⅱ(AngⅡ)及AngⅡ受体-1(AT1R)和AngⅡ受体-2(AT2R)的表达及意义。方法:选取2021年1月—2022月年9月孝感市中心医院收治的90例HDCP患者作为观察组,并将观察组根据病情程度分为HDCP组、轻度子痫前期组和重度子痫前期组3个亚组,将同期产检的90例健康孕妇作为对照组。检测并比较观察组与对照组、观察组不同亚组AngⅡ水平、AT1R和AT2R阳性表达情况。结果:观察组产前母血、产后脐血AngⅡ水平低于对照组,产后母血AngⅡ水平、AT1R、AT2R总阳性率高于对照组,差异有统计学意义(P<0.05)。不同病情程度HDCP患者产前母血、产后脐血AngⅡ水平比较,HDCP组>轻度子痫前期组>重度子痫前期组;不同病情程度HDCP患者产后母血AngⅡ水平比较,HDCP组<轻度子痫前期组<重度子痫前期组;不同病情程度HDCP患者AT1R、AT2R阳性情况比较,HDCP组<轻度子痫前期组<重度子痫前期组,差异有统计学意义(P<0.05)。结论:HDCP患者母血、脐血AngⅡ存在异常表达,其AT1R、AT2R阳性率随病情加重而升高,检测上述指标有助于为HDCP发病机制、早期诊断与治疗提供参考。

A Quantitative Study on Vascular Angiotensin Ⅱ Receptors in Rats with Portal Hypertension

Angiotension-Ⅱ(A-Ⅱ) receptor maximal binding capacity (Bmax) and dissociation constants (Kd) of different blood vessels in rats with prehepatic portal hypertension were studied by radioligand binding analysis. The results showed that the A-Ⅱreceptor Bmax. in the thoracic aorta, superior mesenteric artery and portal vein of portal hypertensive animals (113. 7±19. 4 fmol/mg protein, 206.9 ±39. 3 fmol/mg protein and 31. 5±9. 2 fmol/mg protein respectively ) was all significantly lower than that of controls (146. 8±24. 5 fmol/mg protein, 297. 2±44. 7 fmol/mg protein and 53. 4±12.1 fmol/mg protein respectively, P＜0. 01).The A-Ⅱ receptor Kd in the superior mesenteric artery was markedly increased in portal hypertensive animals (1. 03±0.11 nmol/L) compared with that in controls (0. 88±0. 08 nmol/L, P＜0. 05). In the thoracic aorta and portal vein, the A-Ⅱ receptor Kd in portal hypertensive animals was slightly higher than that in controls, but no significant difference was observed between the two groups. The results suggested that the vascular hyporesponsiveness to A-Ⅱ in portal hypertension was caused partially by a reduction in number and a decrease in affinity of vascular A- Ⅱ receptors, and these changes might possibly lead to the formation of hyperdynamic circulation.

子痫前期患者血清CTRP6、sTNFR-Ⅱ水平及其诊断价值

目的:探究子痫前期(PE)患者血清C1q/肿瘤坏死因子相关蛋白6(CTRP6)、可溶性肿瘤坏死因子受体(sTNFR)-Ⅱ水平及其诊断价值。方法:选取2021年6月-2022年6月在本院确诊为PE的患者148例临床资料为PE组,根据病情程度分为轻度PE组(85例)和重度PE组(63例),常规产前检查健康孕妇75例为对照组。酶联免疫吸附测定法测定各组血清CTRP6、sTNFR-Ⅱ水平;Pearson法分析PE患者血清CTRP6、sTNFR-Ⅱ的相关性;受试者工作特征曲线(ROC)分析CTRP6、sTNFR-Ⅱ对PE发生发展的诊断价值。结果:重度PE组、轻度PE组、对照组尿蛋白定量、ALT、Fib、Cr、收缩压、舒张压水平依次降低,PT值依次增加,血清CTRP6、sTNFR-Ⅱ水平依次降低(均P<0.05)。相关性分析,PE患者血清CTRP6与sTNFR-Ⅱ的表达呈正相关(r=0.354,P<0.001)。ROC曲线分析,CTRP6与sTNFR-Ⅱ联合诊断PE发生的曲线下面积(AUC)(0.975)高于CTRP6、sTNFR-Ⅱ单独诊断,其敏感度、特异性分别为79.7%和92.3%;诊断重度PE的AUC为0.925,敏感度、特异性分别为39.7%和98.8%,高于CTRP6和sTNFR-Ⅱ单独诊断(均P<0.001)。结论:PE患者血清CTRP6、sTNFR-Ⅱ水平异常升高,二者与疾病严重程度有关,且联合诊断可提高PE的诊断效能。

The role of angiotensinⅡtype 1 receptor pathway in cerebral ischemia-reperfusion injury:Implications for the neuroprotective effectof ARBs

Cerebral ischemia-reperfusion(I/R)injury is a crucial factor that impacts the prognosis of recanalization therapy for acute ischemic stroke(AIS).It has been found that the brain renin-angiotensin system,especially the angiotensinⅡtype 1 receptor(AT1R)pathway,plays a significant role in cerebral I/R injury.This pathway is involved in processes such as oxidative stress,neuroinflammation,apoptosis,and it affects cerebrovascular autoregulation and the maintenance of blood-brain barrier.AT1R blocker(ARB),widely used as an antihypertensive agent,has demonstrated stroke prevention capabilities in numerous prospective studies,independent of its antihypertensive characteristics.Studies focusing on neurological diseases like Alzheimer's disease,Parkinson's disease,and cognitive impairment have confirmed that ARBs exhibit neuroprotective effects and aid in improving neurological functions.Preclinical studies have shown that ARBs can reduce infarct volume and brain edema,inhibit multiple signaling pathways associated with I/R injury,restore energy levels in damaged brain regions,and rescue the penumbra by promoting neovascularization in cerebral I/R models.These findings suggest that ARBs have potential to become a novel category of neuroprotecting agents for clinical treatment of Als.Therefore,this review primarily provides a theoretical foundation and practical evidence for the future clinical utilization of ARBs as neuroprotective agents following reperfusion therapy for Als.It outlines the role of cerebral I/R injury through the AT1R pathway and highlights the research progressmadeonARBs in I/Rmodels.

ANGⅡ-AT_1 Receptor Pathway Is Involved in the Anti-fibrotic Effect of β-elemene

To investigate the effects of β-elemene on the ANG Ⅱ-ATI receptor pathway in rats with liver fibrosis, a model of hepatic fibrosis was induced by hypodermical injection of carbon tetrachloride （CC14） into Wistar male rats. D-elemene was intraperitonealy administered into the rats for 8 weeks （0.1 mL/100 g body weight per day）. Masson staining was used to observe the liver fibrosis of rats and liver functions were measured by enzymatic kinetic analysis. The content of hydroxyproline in liver tissues was detected by specimen alkaline hydrolysis. The level of plasma ANG Ⅱ in blood plasma was detected by radioimmunoassay. The expression of AT1R in rat liver were measured using reverse transcriptional-polymerase chain reaction and immunohistochemistry respectively. The results showed that β-elemene could reduce the collagen disposition in liver and inhibit the progression of liver fibrosis. In addition, the levels of plasma ANG Ⅱ and the expression of hepatic AT1R in rats with liver fibrosis were also suppressed by β-elemene. It is concluded that the ANG Ⅱ-AT1 receptor pathway plays an important role in the development of hepatic fibrosis and D-elemene could down-regulate the levels of plasma ANG Ⅱ and the expression of hepatic ATIR in rats with liver fibrosis.

Hydrogen peroxide impairs angiotensin Ⅱ contraction of afferent arteriole in mice with renal ischemia-reperfusion injury

Renal ischemia reperfusion injury increases renal generation of angiotensin II （Ang Ⅱ） which could wors- en renal vasocontraction. Thus, we investigated the hypothesis that renal ischemia reperfusion injury alters renal af- ferent arteriolar responses to Ang II via production of hydrogen peroxide （ H202 ）, or superoxide （ O2 ） or via al- tered angiotensin type 1 receptor （AT1R） expression. Afferent arterioles of mouse kidneys 24h after renal ischemia repeffusion or sham procedures were isolated and perfused. Responses to Ang II or norepinephrine （NE） were as- sessed by measurement of arteriolar luminal diameter. The mRNA expressions of AT1 receptor （ AT1 R） and AT2 re- ceptor （ATzR） were evaluated by quantificational real-time polymerase chain reaction. Compared to sham group, afferent arterioles from mouse kidneys after renal ischemia reperfusion had impaired contractions to Ang II （ -4.63 ± 3.06） % versus （ - 29.95 ± 1.31 ） % at 10 -9 tool · ^-1, p 〈 0.05 , （ - 27.07 ± 1 50） % versus （ - 41 74 ± 0.60） % at 10^-7 tool · L^-1, P 〈 0.05 ） that were normalized by incubation with PEG-catalase , but unaffected by PEG-SOD. However, the NE responses of afferent arterioles after renal ischemia reperfusion were unchanged. Com- pared to the sham group, renal ischemia reperfusion significantly increased the renal cortical H202 （0. 123 ± -1 0. 006） versus （0. 087 ± 0. 003） mmol·mg protein, P 〈 0.01 ）, reduced catalase activity [ （ 14.81 ± 3.22） ver- sus （28.49 ± 1.62） units · mg^-1 protein, P 〈 0.01 ] and downregulated mRNA for AT1R （0.27 ± 0.02 versus 0.95 ± 0.02, P 〈 0.01 ）. We conclude that afferent arteriolar responses to Ang II are impaired selectively in mice after renal ischemia reperfusion by accumulation of H202 and reduced expression of AT1R.

Suppression of angiotensin Ⅱ stimulated responses in aortic vascular smooth muscle cells of experimental cirrhotic rats

Functional responses to angiotensin Ⅱ (AT-Ⅱ) were determined in aortic vascular smooth muscle cells (VSMCs) from experimental cirrhotic rats. Our data showed that AT-Ⅱ-stimulated extracellular acidification rate (ECAR), which was measured by Cytosensor microphysiometry, was significantly reduced in the aortic VSMCs from the cirrhotic rats as compared to those from the control animals. The ability of AT-Ⅱ to promote formation of inositol phosphates, the second messenger produced by the activation of Gq-coupled receptors, was also considerably suppressed in the cirrhotic VSMCs. Furthermore, the maximal p42/44 MAPK phosphorylation stimulated by AT-Ⅱ was significantly reduced in the cirrhotic VSMCs in contrast to that in the normal VSMCs. Taken together, our data clearly demonstrated that the functional responses to AT-Ⅱ was severely suppressed in aortic VSMCs in cirrhosis, indicating the impairment of general Gq-coupled receptor signaling and subsequent biological function in the cirrhotic VSMCs.

POLYMORPHISM OF ANGIOTENSIN I TYPE 1 RECEPTOR GENE IN ELDERLY PATIENTS WITH ESSENTIAL HYPERTENSION

Objective To detect the A/C1165 polymorphism of angiotensin Ⅱ type Ⅰ receptor (AT1-R)gene in essential hypertensive elderly. Methods The A/C1166 polymorphism of AT1-R gene was assessed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in a case-control study of 87 essential hypertensive elders (EH) and 55 normolensive elders (NT). Results The genotype frequencies of AA, AC, CC were 0 .805 , 0.161, 0 .034 in EH group and 0 .927 ,0 .073 ,0 .000 in NT group respectively. The frequency of C61166 allele was higher in EH group (0.115) than in NT group (0 .036 )(P<0 .05 ). Conclusion The resultsindicate that A/C1166 polymorphism of AT1-R gene may be associated with essential hypertension in elderly.

磷酸弗林酸性簇分选蛋白2对血管紧张素Ⅱ诱导的心肌肥大的影响及其机制

目的探究磷酸弗林酸性簇分选蛋白2(PACS-2)对血管紧张素Ⅱ(AngⅡ)诱导的心肌肥大的影响及其机制。方法剪取并消化出生1~2 d的SD大鼠乳鼠的心脏组织,采用差速贴壁法获取乳鼠心肌细胞(NRCMs),原代培养24 h。以浓度为1.5μmol/L的AngⅡ处理NRCMs 0、3、6、12、24 h,采用Western blot方法检测细胞中FUN14域蛋白1(FUNDC1)、PACS-2、三磷酸肌醇受体蛋白(IP3R)的表达水平,采用实时荧光定量PCR(RT-qPCR)方法检测细胞中心钠肽(ANP)、脑钠肽(BNP)、β-肌球蛋白重链(β-MHC)mRNA的表达水平。将NRCMs分为A~C组,A组使用无血清培养基培养,B、C组分别转染si-NC和si-PACS-2,采用Western blot方法检测各组细胞中PACS-2蛋白的表达水平。将NRCMs分为D~G组,D组使用无血清培养基培养,E组以浓度0.15μmol/L的AngⅡ培养,F、G组分别转染si-NC、si-PACS-2后再以浓度0.15μmol/L的AngⅡ培养,采用TRITC-鬼笔环肽染色技术检测各组心肌细胞表面积,RT-qPCR检测各组细胞ANP、BNP、β-MHC mRNA的表达水平,以Fluo-4,AM探针检测各组细胞胞质Ca^(2+)的水平。将NRCMs分为H~K组,H组使用无血清培养基培养,I、J组分别转染si-NC、si-PACS-2,K组转染si-PACS-2并且以浓度1μmol/L的钙调蛋白(CaM)拮抗剂处理后,采用RT-qPCR方法检测各组细胞ANP、BNP、β-MHC mRNA的表达水平。结果以浓度1.5μmol/L的AngⅡ处理NRCMs 0、3、6、12、24 h,NRCMs中ANP、BNP、β-MHC mRNA的表达水平呈时间依赖性上调(F=25.73~58.30,P<0.05),并且处理第24小时时相较于第0小时时均显著上调(t=5.35~37.50,P<0.05)。以浓度1.5μmol/L的AngⅡ处理NRCMs 0、3、6、12、24 h,NRCMs中IP3R、PACS-2以及FUNDC1蛋白的表达水平均呈时间依赖性下调(F=5.37~9.07,P<0.05),并且处理第24小时时相较于第0小时时显著下调(t=6.55~7.42,P<0.05)。与B组相比,C组NRCMs中PACS-2蛋白的表达水平显著下调(t=5.92,P<0.05)。与F组相比,G组NRCMs中心肌细胞表面积增大,ANP、BNP、β-MHC mRNA的表达水平及胞质Ca^(2+)水平均上调(t=3.50~26.60,P<0.05)。与J组相比,K组NRCMs中ANP、BNP、β-MHC mRNA表达水平均显著下调(t=3.27~5.13,P<0.05)。结论敲低PACS-2可增加NRCMs中胞质Ca^(^(2+))水平,并且可能以Ca^(^(2+))-CaM依赖的方式加重AngⅡ诱导的心肌肥大的发生。