胰岛素对血管平滑肌细胞4E结合蛋白1和核糖体蛋白S6激酶磷酸化的刺激作用

目的研究胰岛素对血管平滑肌细胞(VSMC)蛋白质合成翻译过程的两个调节子,4E-结合蛋白1(4E-BP1)和核糖体蛋白S6激酶,磷酸化的调节作用及其生物学意义.方法培养大鼠胸主动脉VSMC.用3H-亮氨酸和3H-TdR掺入法分别测定蛋白质合成和DNA合成;免疫印迹法检测4E-BP1和核糖体蛋白S6激酶的磷酸化.结果与对照相比,100 nmol/L胰岛素显著增加了VSMC的3H-亮氨酸和3H-TdR掺入.同样浓度的胰岛素作用于VSMC,可以诱导4E-BP1和核糖体蛋白S6激酶发生磷酸化.二者的磷酸化分别于胰岛素刺激后,30 min和10 min达高峰.结论胰岛素可以刺激VSMC的4E-BP1和核糖体蛋白S6激酶发生磷酸化,这可能是胰岛素发挥促进VSMC生长作用的机制.     

核糖体蛋白S6激酶1基因沉默对高糖刺激下肝细胞固醇调节元件结合蛋白1 c表达的影响

目的 探讨核糖体蛋白S6激酶1(S6K1)基因沉默后对高糖刺激下小鼠肝细胞固醇调节元件结合蛋白1c(SREBP1c)表达的影响.方法 将构建的S6K1shRNA重组基因腺病毒(S6K1Ax)注射进db/db小鼠尾静脉,以注射含pU6启动子的腺病毒(pU6Ax)空载体的db/db小鼠作对照组,HE染色观察肝脏病理改变并检测肝脏甘油三酯含量变化.S6K1Ax转染小鼠肝细胞AML12细胞,转染pU6Ax的作为对照组,分别用高糖、高胰岛素,高糖高胰岛素刺激,逆转录聚合酶链式反应分析肝细胞在各种条件刺激后mSREBP1c等的表达,Western blot检测db/db小鼠肝脏及AML12细胞S6K1的蛋白表达.结果 db/db糖尿病小鼠注射S6K1Ax后1周,肝脏S6K1蛋白表达被抑制,对照组与实验组肝脏甘油三酯含量分别为(0.65±0.02)mmol/L和(0.56±0.01)mmol/L,t=4.312,P<0.01,差异有统计学意义.HE染色显示实验组肝细胞胞质含脂肪滴减少,空泡细胞数量减少,脂肪肝得到改善.AML12肝细胞mSREBP1c表达实验组为0.03±0.01,对照组为0.06±0.01,t=5.624,P<0.01,差异有统计学意义.与基础状态相比,给以胰岛素刺激后实验组和对照组mSREBP1c表达均增加,实验组上升至0.06±0.02,t=8.452,P<0.01,对照组上升至0.08±0.02,t=3.591,P<0.05.高糖刺激对实验组和对照组mSREBP1c表达均无明显影响.与高胰岛素刺激组相比,高糖高胰岛素刺激后实验组和对照组mSREBP1c表达均无明显差异.结论 S6K1通过影响脂代谢的关键调控基因mSREBP1c表达参与了脂肪的合成,推测S6K1在脂肪肝的形成中发挥了重要作用.     

核糖体蛋白S6激酶1基因沉默对高脂喂养小鼠肝脏炎性因子和肝脏胰岛素抵抗的影响

目的 研究核糖体蛋白S6激酶1(S6K1)基因沉默对肝脏炎性因子表达的影响,探讨S6K1在肝脏胰岛素抵抗中的作用机制。 方法 将48只体重12.6～14.8 g的6周龄雄性C57BL/6J小鼠按随机数字表法分为4组：普食对照组[普通饲料(ND)＋含U6启动子空载体腺病毒组(pU6Ax组),即ND＋pU6Ax组]、普食实验组[ND＋S6K1短发夹RNA(shRNA)重组基因腺病毒组,即ND＋S6K1Ax组]、高脂对照组[高脂饲料(HFD)＋pU6Ax组,即HFD＋pU6Ax组]、高脂实验组(HFD＋S6K1Ax组),分别喂养16周。实验组和对照组尾静脉分别注射S6K1Ax、pU6Ax(均为普食实验组及对照组90 ml,高脂实验组及对照组120 ml,效价为2.0×10 ¹⁰ pfu/ml)。注射后第6天过夜饥饿后,4组各随机抽取6只行葡萄糖耐量实验,剩余的小鼠处死取肝脏,逆转录-聚合酶链反应(RT-PCR)检测肝脏炎性因子单核细胞趋化蛋白-1(MCP-1)、肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1β、IL-6、IL-10 mRNA的表达,Western blotting观察肝脏蛋白S6K1、S6K1苏氨酸389(S6K1-thr389)、胰岛素受体底物1(IRS1)丝氨酸1101(IRS1 -ser1101)、IRS1丝氨酸636/639(IRS1-ser636/639)、蛋白激酶B丝氨酸473(Akt-ser473)、c-Jun氨基末端激苏氨酸183/酪氨酸185( JNK-thr183/tyr185)表达。两组比较采用 t检验,多组间比较采用单因素方差分析。 结果 肝脏S6K1基因沉默后,与高脂对照组相比,HFD＋S6K1Ax组小鼠炎性因子MCP-1、TNF-α、IL-1β mRNA均表达下降(分别为0.549±0.016比0.871±0.081、0.635±0.079比0.905±0.059、0.642±0.042比1.327±0.025; t＝9.55、6.71、34.07,均 P<0.05)。IL-6 mRNA未表现出组间差异( P>0.05),抗炎因子IL-10 mRNA在HFD＋S6K1Ax组肝脏S6K1基因沉默后升高(0.773±0.076比0.436±0.046, t＝9.27, P<0.05)。Western blotting显示与HFD＋pU6Ax组相比,HFD＋S6K1Ax组肝脏S6K1基因沉默后,IRS1-ser1101、IRS1-ser636/639、S6K1-thr389、JNK-thr183/tyr185表达下降,Akt-ser473表达增强( t＝6.59、8.44、5.37、3.15、6.52,均 P<0.05)。 结论 高脂喂养可引起肝脏低度炎症并介导胰岛素抵抗的发生,肝脏S6K1可能通过促进炎症因子、抑制抗炎因子表达参与了肝脏胰岛素抵抗发生。     

肾细胞癌患者临床病理特征与核糖体S6激酶4表达的相关性

目的探讨肾细胞癌患者核糖体S6激酶4(RSK4)的表达及其与肾细胞癌临床病理特征的相关性。方法应用免疫组化方法对50例肾细胞癌组织中RSK4的表达进行检测,并通过对患者随访,探讨其与肾细胞癌临床病理学参数及患者预后之间的关系。结果肾细胞癌患者中RSK4阳性表达27例,且RSK4阳性表达与Fuhrman分级、TNM分期、集合系统受累、淋巴结转移、病理类型等因素呈正相关;单因素分析显示RSK4(P=0.001)、Fuhrman分级(P=0.001)、TNM分期(P=0.001)、集合系统是否受累(P=0.001)、淋巴结是否转移与患者生存期长短有关;患者性别(P=0.428)、年龄(P=0.060)、肿瘤直径(P=0.621)、病理类型(P=0.335)与生存率无关;Cox回归模型显示,仅RSK4、Fuhrman分级、病理TNM分期、集合系统受累、淋巴结转移与肿瘤生存期相关。结论 RSK4可以成为肾细胞癌的潜在的独立预后因子之一。     

核糖体蛋白S6激酶1基因C19012T多态性与农村超重人群血脂水平相关

目的:研究核糖体蛋白S6激酶1(S6K1)基因C19012T的单核苷酸多态性(SNP)在中国汉族农村人群中与肥胖的相关性,及C19012T与超重人群和正常体重人群血清HDL-C,LDL-C,TC的关系。方法:根据中国成人超重和肥胖症预防与控制指南,以体质指数(BMI)>24为中国成人超重的界限。在1097例超重和699例体重正常研究对象中应用多聚酶链反应,限制性内切酶酶切进行基因分型,测定血清HDL-C,LDL-C,TC,TG,葡萄糖。结果:SNPC19012T的基因型、等位基因频率分布在超重组和体重正常组中差异无统计学意义(P>0.05);超重组中C19012T基因TT,CT,CC基因型的TC,LDL-C水平分别为(5.9±1.2)、(5.8±1.3)、(5.6±1.1)mmol/L和(3.5±1.0)、(3.3±0.9)、(3.2±0.9)mmol/L,基因型组间比较均差异有统计学意义(P=0.0213,P=0.0141),校正年龄,性别,BMI和其他危险因素(吸烟饮酒等)后仍差异有统计学意义(P=0.0150,P=0.0087)。然而,体重正常组中未发现基因型组间血脂水平变化差异有统计学意义。结论:S6K1基因C19012T基因型及等位基因分布在超重人群和体重正常人群之间差异无统计学意义,C19012T分布与中国农村超重人群血脂水平有关。     

核糖体蛋白S6激酶1基因沉默对小鼠肝脏胰岛素抵抗的影响

目的 探讨核糖体蛋白S6激酶1基因沉默对db/db小鼠肝脏胰岛素抵抗的影响及机制。 方法 采用随机数字表法将24只9周龄健康雄性db/db小鼠(体质量44.5～48.2 g)随机分至对照组( n＝12)和实验组( n＝12),实验组小鼠尾静脉注射核糖体蛋白S6激酶1短发夹RNA重组基因腺病毒,对照组尾静脉注射含U6启动子空载体腺病毒。注射病毒后第6天,对照组和实验组采用随机数字表进一步分为进食组( n＝6)及空腹组( n＝6)。在进食和空腹状态处死小鼠,提取肝脏,应用Western blot法观测胰岛素受体底物-1和胰岛素受体底物-2蛋白表达水平。提取肝脏总RNA,应用实时荧光定量逆转录聚合酶链反应检测糖异生基因mRNA表达水平。经尾静脉取血,采用酶联免疫吸附法测定胰岛素水平,采用比色法检测游离脂肪酸、甘油三酯、总胆固醇水平。组间比较采用单因素方差分析。 结果 与对照组相比,进食及空腹状态时,实验组胰岛素受体底物-1、胰岛素受体底物-2蛋白表达均增加。在空腹状态下,实验组与对照组相比,糖异生基因过氧化酶体增殖物受体共激活因子1α(分别为0.072±0.024和0.028±0.012)、磷酸烯醇式丙酮酸羧激酶(分别为23.8±4.0和12.3±2.4)、葡萄糖6磷酸酶(分别为3.0±0.8和1.5±0.4)mRNA表达均下降( F值分别为7.58、5.76、9.15,均 P<0.01)。对照组和实验组胰岛素水平无显著差异( t＝1.26, P>0.05)。空腹及进食状态下,实验组胆固醇水平低于对照组( F＝15.48, P<0.01)。与空腹对照组相比,空腹实验组游离脂肪酸降低( t＝2.56, P<0.05)。 结论 db/db小鼠肝脏核糖体蛋白S6激酶1基因沉默后,空腹糖异生和血清游离脂肪酸水平下降,胰岛素受体底物-1和胰岛素受体底物-2蛋白表达增加,提示营养过剩条件下核糖体蛋白S6激酶1可能在肝脏胰岛素抵抗中发挥重要作用。     

核糖体蛋白S6激酶1基因沉默对小鼠非酒精性脂肪性肝病的影响

目的 探讨核糖体蛋白S6激酶1基因沉默对db/db小鼠非酒精性脂肪性肝病的影响及其作用机制。 方法 根据随机数字表将12只体质量44.5～48.2 g的9周龄雄性db/db小鼠随机分至对照组( n＝6)和实验组( n＝6)。实验组db/db小鼠尾静脉注射制备的核糖体蛋白S6激酶1短发夹RNA重组基因腺病毒,对照组db/db小鼠尾静脉注射含U6启动子空载体腺病毒。在注射病毒后第6天处死小鼠获取肝脏,提取肝脏蛋白,利用Western blot法观察其中胰岛素受体底物1、胰岛素受体底物2、蛋白激酶B及其丝氨酸473蛋白的表达;提取肝脏总RNA用实时荧光定量逆转录聚合酶链反应法检测比较2组参与肝脏脂肪酸合成基因mRNA的表达。处死2组小鼠前1 d禁食16 h后经尾静脉取血,采用比色法检测游离脂肪酸、甘油三酯、总胆固醇水平。组间数据比较采用 t检验。 结果 db/db小鼠注射病毒后6 d,苏木素－伊红染色显示实验组肝细胞胞质含脂肪滴较对照组减少,脂肪肝得到改善。Western blot检测显示实验组核糖体蛋白S6激酶1蛋白表达被抑制,与对照组相比差异有统计学意义(分别为0.12±0.01和0.87±0.06, t＝5.36, P<0.05);实验组胰岛素受体底物1、胰岛素受体底物2、蛋白激酶B丝氨酸473的蛋白表达均较对照组增强(均 P<0.05)。和对照组相比,实验组参与脂肪酸合成的基因固醇调节元件结合蛋白1c(分别为2.33±0.29和1.34±0.39, t＝3.46, P<0.01)、脂肪酸合成酶(分别为7.8±1.2和3.4±0.4, t＝4.67, P<0.01)、硬脂酰辅酶A去饱和酶1(764±116和535±54, t＝6.12, P<0.01)mRNA表达均明显下降。血清生化测定结果显示和对照组相比实验组脂肪酸下降( t＝2.64, P<0.05),胆固醇水平降低( t＝4.25, P<0.01),甘油三酯组间比较差异无统计学意义( P>0.05)。 结论 在营养过剩的条件下,肝脏核糖体蛋白S6激酶1过度激活,可能通过负反馈引起肝脏胰岛素信号传导下降、上调脂代谢关键调控基因固醇调节元件结合蛋白1c表达而参与了肝脏胰岛素抵抗和脂肪肝的发生。     

宫颈癌组织中S6K1和S6K2的表达及其临床意义

目的探讨宫颈癌组织中核糖体蛋白S6激酶(S6K)1和S6K2的表达及其临床意义。方法经手术切除的宫颈癌组织94例、正常宫颈组织52例及宫颈上皮内瘤样病变(CIN)组织103例(CINⅠ33例、CINⅡ29例、CINⅢ41例),采用免疫组化法检测以上组织中S6K1和S6K2的表达情况,分析两者表达与宫颈癌临床病理参数的关系(年龄、淋巴结转移、分化程度、FIGO分期、脉管瘤栓、深肌层浸润及宫旁浸润),采用单因素分析和多因素分析影响宫颈癌预后的因素。结果宫颈癌组织中S6K1和S6K2阳性表达率均高于正常宫颈组织和CIN组织(P<0.05);正常宫颈组织和CIN组织中S6K1、S6K2阳性表达率差异无统计学意义(P>0.05)。S6K1表达与分化程度和FIGO分期均有关(P<0.05),而S6K2表达与淋巴结转移、分化程度、FIGO分期和深肌层浸润均有关(P<0.05);S6K1、S6K2阳性者的5年生存率均低于阴性表达者(P<0.05)。多因素分析发现,淋巴结转移、分化程度、FIGO分期、宫旁浸润及S6K1和S6K2表达为影响宫颈癌预后的独立因素。结论宫颈癌组织中的S6K1和S6K2为高表达,均与分化程度和FIGO分期有关,且S6K1、S6K2阳性者的预后较差,可作为预测预后的影响因素。     

核糖体蛋白S6在肿瘤中作用的研究进展

肿瘤（tumor）是机体在致癌因子的作用下,细胞在基因水平失去对其生长的正常调控,导致克隆性增生而形成的病变。蛋白质合成是基因表达的根本,主要在核糖体内完成。核糖体蛋白s6（ribosomalproteins6,rpS6）是构成核糖体的重要蛋白之一,目前已受到众多学者的关注。现就rpS6在肿瘤中的作用作一综述,旨在探讨其对肿瘤细胞增殖、周期、凋亡和侵袭能力的影响。一、rpS6的结构及其上游调节蛋白核糖体是细胞合成蛋白质的主要场所。根据沉降系数的不同,高等真核生物细胞的核糖体分为40S小亚基和60S大亚基两种。mRNA与40S小亚基的相互作用是蛋白质合成的关键。40S小亚基由一个单一的18SrRNA分子和33种蛋白质组成。     

P85磷酯肌醇-3激酶-蛋白激酶C-ζ复合物对血管紧张素Ⅱ激活平滑肌细胞p70核蛋白体S6激酶的调节作用

作者以前的研究发现蛋白激酶C-ζ介导血管紧张素Ⅱ经Ras-MEK途径激活血管平滑肌细胞丝裂素活化的蛋白激酶MAPK或细胞外信号调节激酶ERK1/2.本文研究了P85磷酯肌醇-3激酶-蛋白激酶C-ζ复合物核蛋白体激酶p70S6激酶活性的调节作用及其信号传递途径. Western Blot分析显示正常培养的血管平滑肌细胞表达p70S6激酶、p85磷酯肌醇-3激酶和蛋白激酶C-ζ.100 nmol血管紧张素Ⅱ和 10 μg/L 血小板源生长因子刺激并不影响p70S6激酶、p85磷酯肌醇-3激酶和蛋白激酶C-ζ表达. p70S6激酶磷酸转移酶活性分析发现血管紧张素Ⅱ与血管平滑肌细胞作用5 min 即开始激活p70S6激酶, 20 min达高峰, 并呈剂量依赖性.磷酯肌醇-3激酶抑制剂wortmannin (10 nmol)、蛋白激酶C-ζ抑制剂Pseudo Z (50 μmol) 和p70S6激酶抑制剂rapamycin (100 μg/L) 均可明显阻断血管紧张素Ⅱ诱导的p70S6激酶激活.有趣的是,我们观察到p85磷酯肌醇-3激酶和蛋白激酶C-ζ受血管紧张素Ⅱ和血小板源生长因子刺激后均向Ras转位并与之结合, 抗Ras抗体可将p85磷酯肌醇-3激酶或蛋白激酶C-ζ混合沉淀, 抗p85磷酯肌醇-3激酶抗体亦可使蛋白激酶C-ζ沉淀下来. 提示Ras、p85磷酯肌醇-3激酶和蛋白激酶C-ζ三种蛋白结合在一起形成一个功能复合体,Wortmannin和Pseudo Z可阻断这个功能复合体的形成.此外,血管紧张素Ⅱ和血小板源生长因子与血管平滑肌细胞孵育24 h 明显促进细胞增殖,氚标胸腺嘧啶脱氧核苷掺入率增加约50%, Wortmannin和非特异性蛋白激酶C抑制剂Chelerythine均抑制血管紧张素Ⅱ和血小板源生长因子对血管平滑肌细胞的促增殖作用.综合上述结果, 说明血管紧张素Ⅱ通过Ras-磷酯肌醇-3激酶-蛋白激酶C-ζ途径激活p70S6激酶和刺激血管平滑肌细胞增殖, p85磷酯肌醇-3激酶-蛋白激酶C-ζ复合物在其中起重要调节作用.     

Differential p70S6k and 4E-BP1 regulation by insulin and amino acids in vascular endothelial and smooth muscle cells

Abstract Differential stimulation of vascular endothelial and smooth muscle cells proliferation is responsible for atherosclerotic lesions. Amino acids and insulin modulate p70S6k and 4E-BP1 activity, regulating cell growth and proliferation. We hypothesised that nutritional (amino acids) and hormonal (insulin) signals differently modulate protein anabolism in human vascular endothelial (HUVEC) and smooth muscle (HVSMC) cells. We evaluated p70S6kinase and 4E-BP1 phosphorylation in the two cell types, grown in amino acid-free medium with or without insulin (INS, 100 nM) or/and amino acids mixture (AA, 3 mM) and with the selective addition or deprivation of branched chain amino acids (BCAA, 0.5 mM). INS stimulated p70S6k and 4E-BP1 phosphorylation transiently in HUVEC and persistently in HVSMC. AA and INS+AA stimulated p70S6k and 4E-BP1 phosphorylation persistently in HUVEC and HVSMC. AA, but not BCAA alone or BCAA-deprived AA, induced p70S6k phosphorylation in HUVEC. BCAA deprivation decreased the p70S6k phosphorylation induced by AA with or without insulin in HVSMC. These results show that anabolic stimuli modulate p70S6k and 4E-BP1 activity differently in the two vascular cell types, suggesting that insulin stimulates protein synthesis for a longer time in HUSMC than in HUVEC. We speculate that hyperinsulinaemia frequently associated with atherosclerosis could induce a selective HVSMC proliferation.     

Phosphorylation of p70S6 kinase predicts overall survival in patients with clear margin‐resected hepatocellular carcinoma

Background/Aims: The mammalian target of rapamycin (mTOR) inhibitors play a key role in regulating signal transduction by blocking the mTOR pathway and combining anticancer and immunosuppressive properties. This study was undertaken to determine the prevalence and clinicopathological relevance of phospho‐p70S6 (p‐p70S6) kinase in hepatocellular carcinoma (HCC) and to investigate the effects of rapamycin on HCC in vitro. Methods: A total of 196 patients with HCCs were treated either with surgical resection (n=106) or liver transplantation (n=90). Tumour tissue was investigated for p‐p70S6, phospho‐AKT, Ki‐67, Cyclin‐D1 and apoptosis, and staining results were correlated with clinicopathologically relevant parameters. Results: Overall, p‐p70S6 was detected in 24.5% (48/196) of HCCs. In the resection group, 26.4% (28/106) of HCC were positive and 22.2% (20/90) in the transplant group. p‐p70S6 was significantly associated with elevated Cyclin‐D1 immunoexpression and was correlated with decreased overall survival (P=0.011) in patients resected with a clear margin. In multivariate COX regression analysis, p‐p70S6 was identified as an independent prognostic parameter in patients resected with a clear margin. Rapamycin induced apoptosis and growth inhibition by G0/G1 cell cycle arrest in vitro. However, in HCC patients p‐p70S6 kinase was not associated with proliferation or apoptosis. Conclusions: Activation of p70S6 kinase indicates aggressive tumour behaviour in patients with clear margin‐resected HCC. Identification of p‐p70S6 kinase in HCC selects high‐risk patients who may benefit from drugs targeting the mTOR pathway.     

Chronic Treatment with FK506 Increases p70 S6 Kinase Activity Associated with Reduced Nitric Oxide Synthase Activity in Rabbit Hearts

FK506, an immunosuppressant, modulates phosphorylation of nitric oxide (NO) synthase, and induces cardiac hypertrophy in clinical settings. Having recently reported that chronic treatment with an inhibitor of NO synthase induces cardiac hypertrophy associated with the activation of 70-kD S6 kinase (p70^S6K), which plays an important role in cardiac hypertrophy by regulating protein synthesis, we investigated the effects of chronic administration of FK506 on NO synthase and p70^S6K activities in hearts. Twenty rabbits were divided into four groups: untreated rabbits, those treated with low-dose FK506 (0.10 mg/kg), those treated with medium-dose FK506 (0.20 mg/kg), and those treated with high-dose FK506 (0.40 mg/kg). FK506 was administered intravenously twice a day. After 4 weeks of treatment with FK506, calcium-dependent NO synthase activity in myocardium in the high-dose FK506 group was lower (P < 0.05) than in the untreated group. p70^S6K activity in myocardium in the high-dose group was higher (P < 0.05) than in the untreated group. There was a significant (P < 0.05) inverse correlation between NO synthase and p70^S6K activities in myocardium. However, the endothelial-dependent vasodilation of aortic rings or plasma levels of NO metabolites during experimental protocols did not differ among the groups studied. These findings suggest that chronic treatment of FK506 activates p70^S6K and reduces NO synthase activity in rabbit hearts. Reduced NO synthase and/or activated p70^S6K activities in hearts might contribute to the cardiac hypertrophy observed in some patients receiving FK506.     

高血压大鼠心脏和血管丝裂素活化蛋白激酶与其磷酸酶-1的表达

目的 :探讨MAPK与MKP 1在高血压心血管重塑中的可能作用。方法 :应用Western blot的方法观察了自发性高血压大鼠(SHR)及WKY大鼠心脏和血管丝裂素活化蛋白激酶及丝裂素活化蛋白激酶磷酸酶表达的改变。结果 :(1)WKY大鼠心脏中未见有丝裂素活化蛋白激酶的表达 ,而SHR心脏中检测到了丝裂素活化蛋白激酶的表达 ;并且SHR血管中丝裂素活化蛋白激酶的表达较WKY大鼠增强90 % (P <0 0 1) ;(2 )SHR心脏及血管中丝裂素活化蛋白激酶磷酸酶 - 1的表达较WKY大鼠分别降低 5 3%及 45 % (P均<0 0 1)。结论 :SHR心脏和血管重塑的发生除与丝裂素活化蛋白激酶表达增强有关外 ,可能还与丝裂素活化蛋白激酶磷酸酶 - 1的表达下降有关。     

NECA at reperfusion limits infarction and inhibits formation of the mitochondrial permeability transition pore by activating p70S6 kinase

The A₁/A₂ adenosine agonist 5′-(N-ethylcarboxamido) adenosine (NECA) limits infarction when administered at reperfusion. The present study investigated whether p70S6 kinase is involved in this anti-infarct effect. Adult rat ventricular myocytes were isolated and incubated in tetramethylrhodamine ethyl ester (TMRE, 100 nM), which causes cells to fluoresce in proportion to their mitochondrial membrane potential. A reduction in TMRE fluorescence serves as an indicator of collapse of the mitochondrial transmembrane potential. Cells were subjected to H₂O₂ (200 μM), which like ischemia induces loss of mitochondrial membrane potential. Fluorescence was measured every 3 min and to facilitate quantification membrane potential was arbitrarily considered as collapsed when fluorescence reached less than 60% of the starting value. Adding NECA (1 mM) to the cells prolonged the time to fluorescence loss (48.0 ± 3.2 min in the NECA group versus 29.5 ± 2.2 min in untreated cells, P < 0.001) and the mTOR/p70S6 kinase inhibitor rapamycin (5 nM) abolished this protection (31.3 ± 3.4 min). Since cyclosporine A offered similar protection, mitochondrial permeability transition pore formation is a likely cause of the H₂O₂-induced loss of potential. The direct GSK-3β inhibitor SB216763 (3 μM) also prolonged the time to fluorescence loss (49.2 ± 2.1 min, P < 0.001 versus control), and its protection could not be blocked by rapamycin (42.2 ± 2.3 min, P < 0.001 versus control). NECA treatment (100 nM) of intact isolated rabbit hearts at reperfusion after 30 min of regional ischemia decreased infarct size from 33.0 ± 3.8% of the risk zone in control hearts to 11.8 ± 2.0% (P < 0.001), and rapamycin blocked this NECA-induced protection (38.3 ± 3.7%). A comparable protective effect was seen for SB216763 (1 μM) with infarct size reduction to 13.5 ± 2.3% (P < 0.001). NECA treatment (200 nM) of intact rabbit hearts at reperfusion also resulted in phosphorylation of p70S6 kinase more than that seen in untreated hearts. This NECA-induced phosphorylation was blocked by rapamycin. These experiments reveal a critical role for p70S6 kinase in the signaling pathway of NECA’s cardioprotection at reperfusion. Returned for 1st revision: 3 November 2005 1st revision received: 3 February 2006 Returned for 2nd revision: 23 February 2006 2nd revision received: 1 March 2006     

DDPH对实验性高血压大鼠血管平滑肌细胞增殖及对热应激蛋白70和抑癌基因p53的影响

目的：观察１－（２，６－二甲基苯氧基）－２－（３，４－二甲氧基苯乙胺基）丙烷盐酸盐（ＤＤＰＨ）对实验性高血压大鼠血管平滑肌细胞增殖的作用及对热应激蛋白７０（ＨＳＰ７０）及其信使核糖核酸和抑癌基因ｐ５３表达的影响。方法：采用主动脉缩窄性高血压模型，用氚－胸腺嘧啶核苷（３Ｈ－ＴｄＲ）参入、电镜、免疫组化、原位杂交等方法观察上述各指标。结果：ＤＤＰＨ在降低高血压模型组血压的同时能减少其肾动脉血管平滑肌细胞的线粒体，粗面内质网和减少肾动脉中膜组织氚－胸腺嘧啶核苷参入量，并能逆转高血压模型组血管平滑肌细胞增殖时ＨＳＰ７０及其信使核糖核酸表达增强，抑癌基因ｐ５３信使核糖核酸表达减弱。结论：ＤＤＰＨ能抑制实验性高血压大鼠的血管平滑肌细胞增殖，与ＨＳＰ７０及抑癌基因ｐ５３的调控有关。     

Protein Kinase C Modulation of Cyclic GMP in Rat Neonatal Pulmonary Vascular Smooth Muscle

Protein kinase C (PKC) has been implicated in the control of vascular tone and mitogenesis in the adult pulmonary vasculature, but little is known about the role of PKC in the neonatal pulmonary vasculature. In addition, the vasodilator nitric oxide (NO) is important in the transition of the pulmonary circulation from fetal to postnatal life, and it is thought that attenuated production of NO and therefore, cGMP may contribute to the pathophysiology of a variety of forms of neonatal pulmonary vascular disease states. Although evidence exists for an interaction between PKC and NO in the adult pulmonary vasculature, the identification of specific roles for PKC in neonatal pulmonary vascular smooth muscle (NPVSM) has not been determined, and no studies have been done on the modulation of cGMP by PKC in NPVSM. Accordingly, immunoblot analysis revealed the expression of the , , , and PKC isozymes in NPVSM. Treatment of NPVSM with 10nM 4- phorbol myristate acetate (PMA), a PKC activator, induced translocation of PKC, and PKC from the soluble to the particulate fraction, while exposure to 10nM endothelin-1 (ET-1), a potent vasoconstrictor and mitogenic substance, caused translocation of PKC and PKC from the soluble to the particulate fraction. Sodium nitroprusside (SNP) significantly increased intracellular cGMP levels, an effect attenuated by PMA but not by ET-1. In addition, pretreatment with the specific PKC isozyme antagonist Go 6983 blocked the effect of PMA on cGMP levels. Collectively, these data demonstrate the expression and activation of multiple PKC isozymes in NPVSM, and indicate that PKC inhibits SNP-stimulated cGMP production in NPVSM. These data also suggest that complex intracellular signaling pathways by specific PKC isozymes may be important in the development of neonatal pulmonary vascular function.     

Effect of Nitric Oxide on Mitogen-Activated Protein Kinases in Neonatal Pulmonary Vascular Smooth Muscle

Mitogen-activated protein kinases (MAPKs) belong to the group of serine/threonine kinases that are rapidly activated in response to growth factor stimulation. In adult mammalian cells, the MAPK family includes extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2 or p44^mapk and p42^mapk), which translocate to the nucleus and integrate signals from second messengers leading to cellular proliferation or differentiation. However, the specific role of MAPKs in neonatal pulmonary vascular smooth muscle is not well understood. Expression of p44^mapk and p42^mapk in primary cultured pulmonary vascular smooth muscle cells from neonatal (1–2 day old) rats was identified by Western immunoblot analysis. Treatment with 10 nM endothelin-1 (ET-1), a potent vasoconstrictor with vascular mitogenic properties, induced phosphorylation of both p44^mapk and p42^mapk , but treatment with the exogenous nitric oxide (NO) donor sodium nitroprusside inhibited both p44^mapk and p42^mapk phosphorylation by ET-1. The specific cGMP-dependent protein kinase (PKG) inhibitor KT5823, the nonspecific nitric oxide synthase (NOS) inhibitor L-NAME, and the specific NOS 1 blocker NPLA all significantly enhanced both p44^mapk and p42^mapk phosphorylation by ET-1. Collectively, these data demonstrate the expression and phosphorylation of specific MAPKs in rat neonatal pulmonary vascular smooth muscle and suggests that the NO signaling pathway modulates MAPK activation by ET-1.     

多巴胺D_1类受体对胰岛素受体介导的血管平滑肌细胞增殖的影响

目的观察多巴胺D1类受体对胰岛素受体介导的血管平滑肌细胞增殖的影响。方法本研究以A10细胞为研究对象,观察刺激D1类受体对胰岛素促增殖作用的影响,并用免疫印迹研究D1类受体影响胰岛素作用的机制。细胞增殖作用采用[3H]胸腺嘧啶核苷(3H-TdR)掺入量表示。结果胰岛素可促进A10细胞的增殖,该作用呈现浓度依赖性的。D1类受体激动剂Fenoldopam本身对A10细胞无增殖影响,但Fenoldopam通过D1类受体可完全阻断胰岛素介导的血管平滑肌细胞增殖作用。免疫印迹显示刺激D1类受体可降低胰岛素受体的蛋白表达,提示该机制可能参与了D1类受体对胰岛素受体作用的过程。结论D1类受体对胰岛素受体介导的血管平滑肌细胞增殖具有抑制作用,该作用可能在高血压的发生发展中发挥一定作用。