二酰甘油－蛋白激酶C信使系统在LA90细胞转化中的作用

以ＬＡ９０细胞（ＲＳＶ温度敏感突变株ＬＡ９０转化的小鼠成纤维细胞〕为模型,研究了二酰甘油（ＤＧ）－蛋白激酶Ｃ（ＰＫＣ）信使系统在细胞转化中的作用。通过免疫沉淀法观察到ＬＡ９０细胞在允许温度（３３℃）时具有很高的ｐｐ６０ｖ－ｓｒｃ激酶活性,远高于非允许温度（３９℃）,当从３９℃转至３３℃１０分钟,激酶活性就已显著升高。同时运用３Ｈ－甘油掺入并结合板层析分离方法和酶活性测定,发现ＬＡ９０细胞中ＤＧ含量和ＰＫＣ活性在３３℃条件下高于３９℃,当由３９℃转至３３℃１０分钟,细胞内ＤＧ含量和ＰＫＣ活性均明显增加。我们进一步探讨了ＰＫＣ活性与癌基因ｖ－ｓｅｒ、ｐ５３基因表达的相关性,实验表明,ＰＫＣ的激活剂ＴＰＡ可刺激３９℃条件下的ＬＡ９０细胞中ｖ－ｓｒｅ和ｐ５３基因表达,ＰＫＣ选择性抑制剂Ｈ７可抑制３３℃条件下ＬＡ９０细胞中ｖ－ｅｒｃ和ｐ５３基因表达。看来,ｐｐ６０ｖ－ｓｒｃ可通过刺激磷脂酰肌醇（ＰＩ）代谢激活ＤＧ－ＰＫＣ（ｄｉａｃｙｌｇｌｙｃｅｒｏｌ－ｐｒｏｔｅｉｎｋｉｎａｓｅＣ）信使系统,后者通过某种途径调控ｖ－ｓｒｃ和ｐ５３等癌基因之表达。因此ＤＧ－ＰＫＣ信使系统可能是ｐｐ６０ｖ－ｓｒｃ转化细胞及被转化细胞维持转     

血管平滑肌细胞增殖与Cdk抑制蛋白p27的表达

ｐ２７蛋白是细胞周期素依赖性激酶（Ｃｄｋ）抑制蛋白家族中的一种,主要对外部促进或抑制细胞增殖的信号起反应。本研究应用流式细胞仪（ＦＣＭ）双标记的方法观察血管紧张素Ⅱ（ＡｎｇⅡ）、血管加压素（ＡＶＰ）和血小板源生长因子（ＰＤＧＦ）对血管平滑肌细胞（ＶＳＭＣｓ）细胞周期百分比和ｐ２７蛋白表达量的影响。静止状态培养的ＶＳＭＣｓ加入ＡｎｇⅡ,ＡＶＰ,ＰＤＧＦＢＢ后,在不同时间收集细胞,用碘化丙啶（ＰＩ）标记细胞ＤＮＡ,以确定细胞所处的周期。用ｐ２７蛋白的单抗和标记了ＦＩＴＣ的二抗标记细胞,通过流式细胞仪测定被激发出的荧光量来确定细胞ｐ２７蛋白表达的相对量。结果显示,ＡｎｇⅡ刺激ＶＳＭＣｓ增生,其蛋白含量增加了４３６％（Ｐ＜００１）,但不抑制ｐ２７蛋白的表达;ＡＶＰ可轻度抑制ｐ２７的表达,有轻度促进ＶＳＭＣｓ增殖和增生的作用（Ｐ＜００５）;ＰＤＧＦ明显抑制ｐ２７的表达,引起细胞增殖。本研究结果提示,ｐ２７蛋白抑制ＶＳＭＣｓ通过Ｇ１期进入Ｓ期,是抑制ＶＳＭＣｓ增殖的重要调节因子。     

低氧、血管紧张素Ⅱ对肺内小动脉平滑肌细胞膜Ca2 ┐ATPase活力的影响

本课题观察了低氧及血管紧张素Ⅱ（ａｎｇｉｏｔｅｎｓｉｎⅡ,ＡｎｇⅡ）对分离培养家兔肺内小动脉平滑肌细胞（ＰＡＳＭ－Ｃｓ）膜Ｃａ２＋－ＡＴＰａｓｅ活力的影响,同时用钙通道阻断剂维拉帕米（ｖｅｒａｐａｍｉｌ,ＶＰ）进行干预,进一步了解细胞内钙与Ｃａ２＋－ＡＴＰａｓｅ活力的关系。结果表明：ＰＡＳＭＣｓ膜Ｃａ２＋－ＡＴＰａｓｅ活力对低氧具有短暂的耐受性,随低氧时间延长,Ｃａ２＋－ＡＴＰａｓｅ活力呈时间依赖性抑制;低氧、ＡＮＧⅡ均能抑制Ｃａ２＋－ＡＴＰａｓｅ活力（Ｐ＜０．０１）低氧＋ＡⅡ对Ｃａ２＋－ＡＴＰａｓｅ活力的抑制具叠加效应（Ｐ＜０．０５）;ＶＰ可逆转低氧、ＡｎｇⅡ、低氧＋ＡｎｇⅡ对Ｃａ２＋－ＡＴＰａｓｅ活力的抑制（Ｐ＜０．０１）。结果提示：低氧,ＡＮＧⅡ可通过抑制肺血管平滑肌细胞膜Ｃａ２＋－ＡＴＰａｓｅ活力而可能削弱肺血管平滑肌舒张功能也可能是低氧性肺动脉高压（ＨＰＨ）形成的原因之一。     

血管紧张素Ⅱ诱导培养的成年大鼠心肌细胞凋亡及其细胞机制

研究血管紧张素Ⅱ（ＡｎｇｉｏｔｅｎｓｉｎⅡ,ＡｎｇⅡ）诱导培养的成年大鼠心肌细胞（Ａｄｕｌｔｒａｔｖｅｎｔｒｉｃｕｌａｒｍｙ－ｏｃｙｔｅｓ,ＡＲＶＭ）凋亡。酶灌流消化法分离培养ＡＲＶＭ,不同处理后,光镜观察形态改变,琼脂糖凝胶电泳定性分析ＤＮＡ降解程度。结果发现培养的ＡＲＶＭ经１０μｍｏｌ／ＬＡｎｇⅡ处理４８ｈ后,大部分细胞变圆,胞浆浓缩;电泳显示核酸断裂片段“梯形”结构,上述改变在７２ｈ更为明显。上述作用可被氯沙坦、维拉帕米和ｓｔａｕｒｏｓｐｏｒｉｎｅ所取消。结果表明,ＡｎｇⅡ诱导培养的ＡＲＶＭ凋亡由ＡＴ１受体介导、细胞内钙升高和ＰＫＣ激活起重要作用     

丝裂素活化蛋白激酶参与内皮素刺激的兔主动脉平滑肌细胞增生

内皮素（ｅｎｄｏｔｈｅｌｉｎ,ＥＴ）是已知的体内活性最强的缩血管物质,其缩血管作用由Ｇ蛋白偶联受体所介导。但ＥＴ强大的促血管平滑肌细胞（ＶＳＭＣ）增生效应的机理尚未完全阐明。本研究选用培养的兔胸主动脉ＶＳＭＣ,探讨丝裂素活化蛋白激酶（ＭＡＰＫ）在ＥＴ促细胞增生中的作用。结果表明：ＥＴ－１呈时间和浓度依赖性地促进细胞摄取￣３Ｈ－ＴｄＲ和激活ＭＡＰＫ,此作用可被蛋白激酶Ｃ（ｐｒｏｔｅｉｎｋｉｎａｓｅＣ,ＰＫＣ）抑制剂Ｓｔａｕｒｏｓｐｏｒｉｎｅ（ＳＴＰ）,Ｈ－７和ＥＴ＿Ａ受体拮抗剂ＢＱ１２３所抑制,但不被酪氨酸激酶抑制剂ＨｅｒｂｉｍｙｃｉｎＡ（Ｈｅｒｂ）所抑制,用ＰＫＣ激动剂ＰＭＡ（Ｐｈｏｒｂｏｌｍｙｒｉｓｔａｔｅａｃｅｔａｔｅ）预处理ＶＳＭＣ,使其ＰＫＣ活性下调,可显著减弱ＥＴ－１对ＭＡＰＫ的激活能力。本结果提示：（１）ＭＡＰＫ参与ＥＴ－１所致的ＶＳＭＣ增生;（２）ＥＴ－１促细胞增生与激活ＭＡＰＫ的作用是由ＥＴ＿Ａ受体和ＰＫＣ介导的。     

人主动脉硫酸乙酰肝素蛋白聚糖对培养的人血管壁细胞生长的作用

通过培养的人主动脉平滑肌细胞（ｈＡＳＭＣ）及脐静脉内皮细胞（ｈＵＶＥＣ）,应用３Ｈ－ＴｄＲ参入、Ｎｏｒｔｈｅｒｎｂｌｏｔ分析、逆转录多聚酶链反应（ＲＴ－ＰＣＲ）、放射免疫分析（ＲＩＡ）、和紫外比色法等技术观察了人主动脉中硫酸乙酰肝素蛋白聚糖（ＨＳＰＧ）对ｈＡＳＭＣ和ｈＵＶＥＣＤＮＡ合成的作用及对血小板源生长因子（ＰＤＧＦ）、ＰＤＧＦ受体、转化生长因子β（ＴＧＦ－β）、内皮素－１（ＥＴ－１）或碱性成纤维细胞生长因子（ｂＦＧＦ）基因表达和肾素－血管紧张系统（ＲＡＳ）的影响,结果显示,ＨＳＰＧ明显抑制培养的ｈＡＳＭＣ基础的ＤＮＡ合成（ｃｐｍ值为：１０３８５±３２６３ｖｓ,２５５４１±６４２１,Ｐ＜０．０１）及外源性ＰＤＧＦ诱导的ＤＮＡ合成（ｃｐｍ值为：９８７８±１９４７ｖｓ．１３４８１±４４ｌ０,Ｐ＜０．０５）;抑制ＰＤＧＦＡ链、ＴＧＦ－Ｂｐ和ＥＴ－１ｍＲＮＡ表达,提高ＰＤＧＦａ和β受体ｍＲＮＡ的表达;显著降低ｈＡＳＭＣ培养液中血管紧张素Ⅱ（ＡｎｇⅡ）的浓度和血管紧张素转换酶（ＡＣＥ）的活性,推测ＨＳＰＧ抑制ＰＤＧＦＡ链、ＴＧＦ－β及ＥＴ－１ｍＲＮＡ表达,降低ＡＣＥ活性及ＡｎｇⅡ浓度是其抑制ｈＡＳＭＣ增殖的重要机     

同型半胱氨酸对大鼠血管平滑肌细胞增殖的作用

血中同型半胱氨酸（ｈｏｍｏｃｙｓｔｅｉｎｅ,ＨＣＹ）浓度的升高已成为动脉粥样硬化发生的一个独立危险因子．为进一步阐明ＨＣＹ促进血管平滑肌细胞（ｖａｓｃｕｌａｒｓｍｏｏｔｈｍｕｓｃｌｅｃｅｌｓ,ＶＳＭＣｓ）增殖,从而引起动脉粥样硬化发生的机理．本实验采用细胞计数、３Ｈ－ＴｄＲ参入、细胞周期分析、Ｎｏｒｔｈｅｒｎ杂交方法证明,一定剂量的ＨＣＹ可促进离体培养的ＷＫＹ大鼠血管平滑肌细胞增殖,使其ＤＮＡ合成增加,细胞周期中Ｓ期细胞所占比例增加４３％,并促进ｃ－ｍｙｃ与ｃ－ｆｏｓ原癌基因ｍＲＮＡ表达增加．提示ＨＣＹ可能通过促进ＶＳＭＣｓ增殖而诱发动脉粥样硬化     

系列腺病毒伴随病毒载体的构建及表达β-半乳糖苷酶的研究

为了便于开展用重组腺病毒伴随病毒（ｒｅｃｏｍｂｉｎａｎｔ ａｄｅｎｏ－ａｓｓｏｃｉａｔｅｄ ｖｉｒｕｓ,ｒＡＡＶ）载体进行基因治疗的研究,构建了三种通用型ＡＡＶ载体ｐＷＡＶ－１、ｐＷＡＶ－２和ｐＳＮＡＶ,每种载体均含有ＡＡＶ－２两端的倒转末端重复序列（ｉｎｖｅｒｔｅｄ ｔｅｒｍｉｎａｌ ｒｅｐｅａｔｓ,ＩＴＲ）,中间依次为巨细胞病毒（ＣＭＶ）立早增强子和启动子、多克隆位点和ｐｏｌｙＡ信号。研究只     

血管紧张素Ⅱ对大鼠心肌肌浆网Ca~(2+),Mg~(2+)-ATPase基因转录调节的影响

观察血管紧张素Ⅱ（ＡｎｇⅡ）对心肌肌浆网Ｃａ２＋,Ｍｇ２＋－ＡＴＰａｓｅ基因（ＳＥＲＣＡ２ａ）转录调节的影响,评价ＤＭＰ８１１对此效应的干预作用．６周龄雄性ＳＤ大鼠随机分为３组,每组６只．组１：生理盐水输注;组２：ＡｎｇⅡ输注＋ＤＭＰ８１１管饲（３ｍｇ·ｄ－１·ｋｇ－１）;组３：ＡｎｇⅡ输注（２００ｎｇ·ｍｉｎ－１·ｋｇ－１．１周后称其体重,取心脏并称重,提取心脏总ＲＮＡ后采用Ｎｏｒｔｈｅｒｎｂｌｏｔ的方法检测ＳＥＲ－ＣＡ２ａ的转录水平,采用ＲＴ－ＰＣＲ检测ＡｎｇⅡ１型受体（ＡＴ１）ｍＲＮＡ水平．实验后,组３心重（ＣＷ）、心重／体重（Ｃ／Ｂ）、ＡＴ１受体转录水平均高于组１（分别增加４．７±０．４％,４．９±０．９％和２４．７±３．５％;Ｐ＜０．０１）,而ＳＥＲＣＡ２ａ基因转录水平显著低于组１（降低２０．１±３．０％,Ｐ＜０．０１）,并且ＳＥＲＣＡ２ａｍＲＮＡ水平与ＡＴ１受体ｍＲＮＡ水平呈负相关（ｒ＝－０．７４,Ｐ＜０．０１）．ＡｎｇⅡ导致的上述改变能被ＤＭＰ８１１完全阻断．ＡｎｇⅡ通过其Ⅰ型受体的介导,诱导了ＳＥＲＣＡ２ａ的转录下调     

表达强毒pp38决定簇的马立克病病毒疫苗毒CVI988点突变株的构建和特性

用鸡马立克病病毒（ＭＤＶ）强毒ＧＡ株的３８ｋＤ磷蛋白（ｐｐ３８）基因克隆ＤＮＡ转染Ｉ型弱毒疫苗ＣＡＩ９８８／Ｒｉｓｐｅｎｓ株ＭＤＶ感染的鸡胚成纤维细胞,再用能识别Ｉ型强毒ｐｐ３８的单克隆抗体Ｈ１９做免疫荧光试验,筛选到能在ｐｐ３８基因上表达强毒株特异性抗原决定簇的定向点突变弱毒株ＣＶＩ／ｒｐｐ３８。用＾３５Ｓ－蛋氨酸标记的细胞裂解物做免疫沉淀反应表明,单抗Ｈ１９不能识别天然ＣＶＩ９８８株ＭＤＶ中的     

Role of Ras in ethanol modulation of angiotensin II activated p42/p44 MAP kinase in rat hepatocytes

Angiotensin II plays a role in both liver cell proliferation and liver injury but the effects of ethanol on angiotensin II signaling in liver are not clearly understood. We have investigated the role of Ras in ethanol modulation of p42/p44 mitogen-activated protein kinase (MAPK) stimulated by angiotensin II (Ang II) in primary cultures of rat hepatocytes. Hepatocytes were incubated with ethanol (100 mM) for 24 h, then stimulated with Ang II (100 nM). The level of p42/p44 MAPK phosphorylation was measured by Western blot analysis and Ras activation was assessed by specific binding of Ras-GTP (activated form) to a GST-RBD fusion protein containing Ras-binding domain (RBD) of Raf-1. Ethanol potentiated p42/p44 MAPK activation by Ang II, whereas ethanol alone did not significantly affect phosphorylation of p42/p44 MAPK. Ang II increased Ras activity by about 2 fold. Ethanol exposure increased Ang II stimulated Ras activity by an additional about 2 fold. Ethanol alone elicited a small increase in basal Ras activity. Pretreatment with manumycin A (10 microM), a Ras farnesylation inhibitor, partially blocked both Ang II-activated and ethanol-potentiated MAPK activities. These data provided the first evidence that ethanol potentiation of Ang II stimulated p42/p44 MAPK is mediated, in part, by Ras in hepatocytes.     

Role of the actin cytoskeleton in angiotensin II signaling in human vascular smooth muscle cells

Angiotensin II (Ang II) regulates vascular smooth muscle cell (VSMC) function by activating signaling cascades that promote vasoconstriction, growth, and inflammation. Subcellular mechanisms coordinating these processes are unclear. In the present study, we questioned the role of the actin cytoskeleton in Ang II mediated signaling through mitogen-activated protein (MAP) kinases and reactive oxygen species (ROS) in VSMCs. Human VSMCs were studied. Cells were exposed to Ang II (10-7 mol/L) in the absence and presence of cytochalasin B (10-6 mol/L, 60 min), which disrupts the actin cytoskeleton. Phosphorylation of p38MAP kinase, JNK, and ERK1/2 was assessed by immuno blotting. ROS generation was measured using the fluoroprobe chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (4 micromol/L). Interaction between the cytoskeleton and NADPH oxidase was determined by evaluating the presence of p47phox in the Triton X-100 insoluble membrane fraction. Ang II significantly increased phosphorylation of p38MAP kinase, JNK, and ERK1/2 (two- to threefold above control, p < 0.05). Cytochalasin B pretreatment attenuated p38MAP kinase and JNK effects (p < 0.05) without altering ERK1/2 phosphorylation. ROS formation, which was increased in Ang II stimulated cells, was significantly reduced by cytochalasin B (p < 0.01). p47phox, critically involved in NADPH oxidase activation, colocalized with the actin cytoskeleton in Ang II stimulated cells. Our data demonstrate that Ang II mediated ROS formation and activation of p38MAP kinase and JNK, but not ERK1/2, involves the actin cytoskeleton in VSMCs. In addition, Ang II promotes interaction between actin and p47phox. These data indicate that the cytoskeleton is involved in differential MAP kinase signaling and ROS generation by Ang II in VSMCs. Together, these studies suggest that the cytoskeleton may be a central point of crosstalk in growth- and redox-signaling pathways by Ang II, which may be important in the regulation of VSMC function.     

Dephosphorylation or antibody binding to the carboxy terminus stimulates pp60c-src.

Phosphorylation of pp60c-src at Tyr-527, six residues from the carboxy terminus, has been implicated in regulation of the protein-tyrosine kinase activity of pp60c-src. Here we show that dephosphorylation of pp60c-src by phosphatase treatment in vitro caused a 10- to 20-fold increase in pp60c-src protein-tyrosine kinase activity. Binding of specific antibody to the region of pp60c-src which contains phosphotyrosine-527 also increased kinase activity. Each treatment increased phosphorylation of added substrates and of Tyr-416 within pp60c-src by a similar mechanism that involved altered interactions with ATP and increased catalytic rate. We suggest that the phosphorylated carboxy terminus acts as an inhibitor of the protein kinase domain of pp60c-src, unless its conformation is altered by either dephosphorylation or antibody binding. The antibody additionally stimulated the phosphorylation of forms of pp60c-src that had reduced gel mobility, much like those phosphorylated in kinase reactions containing pp60c-src activated by polyomavirus medium tumor antigen. These in vitro experiments provide models for the activation of pp60c-src in cells transformed by polyomavirus. We also show that autophosphorylation of pp60c-src at Tyr-527 occurs only to a very limited extent in vitro, even when Tyr-527 is made available for phosphorylation by treatment with phosphatase. This suggests that other protein-tyrosine kinases may normally phosphorylate Tyr-527 and regulate pp60c-src in the cell.     

Angiotensin II activation of focal adhesion kinase and pp60c-Src in relation to mitogen-activated protein kinases in hepatocytes

We have investigated signaling pathways leading to angiotensin II (Ang II) activation of mitogen-activated protein kinase (MAPK) in hepatocytes. MAPK activation by Ang II was abolished by the Ang II type 1 (AT1) receptor antagonist losartan, but not by the Ang II type 2 (AT2) receptor antagonist PD123319. Ang II (100 nM) induced a rapid phosphorylation of Src (peak approximately 2 min) and focal adhesion kinase (FAK, peak approximately 5 min) followed by a decrease to basal levels in 30 min. An increased association between FAK and Src in response to Ang II was detected after 1 min, which declined to basal levels after 30 min. Treatment with the Src kinase inhibitor PP-1 inhibited FAK phosphorylation. Downregulation of PKC, intracellular Ca2+ chelator BAPTA or inhibitors of PKC, Src kinase, MAPK kinase (MEK), Ca2+/calmodulin dependent protein kinase, phosphatidylinositol 3-kinase all blocked Ang II-induced MAPK phosphorylation. In contrast to other cells, there was no evidence for the role of EGF receptor transactivation in the activation of MAPK by Ang II. However, PDGF receptor phosphorylation is involved in the Ang II stimulated MAPK activation. Furthermore, Src/FAK and Ca/CaM kinase activation serve as potential links between the Ang II receptor and MAPK activation. These studies offer insight into the signaling network upstream of MAPK activation by AT1 receptor in hepatocytes.     

川芎嗪抑制血管紧张素Ⅱ诱导的平滑肌细胞NF-κB激活和骨形成蛋白-2表达降低

本文旨在观察血管紧张素Ⅱ（angiotensinⅡ,AngⅡ）对血管平滑肌细胞核转录因子-κB（nuclear factor-κB,NF-κB）的活性及骨形成蛋白-2（bone morphogenetic protein-2,BMP-2）表达的影响,以探讨AngⅡ参与动脉粥样硬化的机制,并探讨川芎嗪是否能抑制AngⅡ的促动脉粥样硬化作用。采用Western blot、免疫组化和原位杂交等方法分别检测AngⅡ刺激和川芎嗪干预后NF-κB活性、BMP-2蛋白和mRNA表达的变化。结果显示：（1）AngⅡ刺激激活NF-κB。AngⅡ刺激15min即有NF-κB p65核转移,30min达高峰（P〈0．01）,1h后减退。川芎嗪抑制AngⅡ诱导的NF-κB激活,与AngⅡ组比较,川芎嗪＋AngⅡ组NF-κB活性显著降低（P〈0．01）。（2）AngⅡ刺激6h时BMP-2表达增强（P〈0．05）,12h时减弱（P〈0．01）,24h时更弱（P〈0．01）。川芎嗪＋AngⅡ组中,川芎嗪干预6h时BMP-2表达亦增强,12与24h时保持正常水平。（3）川芎嗪对正常细胞的NF-κB活性和BMP-2表达无影响。以上结果表明,AngⅡ刺激后激活NF-κB并最终使生长抑制因子BMP-2表达下降,这可能是其参与动脉粥样硬化发生的机制之一。BMP-2一过性增高可能不依赖NF-κB通路的激活。川芎嗪可抑制AngⅡ诱导的NF-κB激活与BMP-2表达降低,提示它在抗动脉粥样硬化形成中起重要作用。     

Mass spectral identification of the blocked N-terminal tryptic peptide of the ATPase inhibitor from beef heart mitochondria

The endogenous cellular oncogene products, pp60c-src, exhibits a protein kinase activity, but is itself a phosphoprotein. Based on the assumption that pp60c-src might play a role in the control of cell proliferation, we have studied its behaviour as a substrate for phosphorylation known to occur when quiescent, serum-deprived cells are stimulated to enter cell cycle following addition of either serum, platelet-derived growth factor or the phorbol ester derivative, 12-O-tetradecanoyl-phorbol-13-acetate. For this purpose a partial purification of pp60c-src on DEAE ion-exchange chromatography was combined with immune precipitation. A 2-4-fold increase in serine phosphorylation of pp60c-src was consistently observed after stimulation of quiescent cells to growth.     

Dimethylarginine dimethylaminohydrolase-1 mediates inhibitory effect of interleukin-10 on angiotensin II-induced hypertensive effects in vascular smooth muscle cells of spontaneously hypertensive rats

In hypertension studies, anti-inflammatory cytokine interleukin-10 (IL-10) has been shown to prevent angiotensin II (Ang II)-induced vasoconstriction and regulate vascular function by down-regulating pro-inflammatory cytokine and superoxide production in vascular cells. However, little is known about the mechanism behind the down-regulatory effect of IL-10 on Ang II-induced hypertensive mediators. In this study, we demonstrated the effects of IL-10 on expression of dimethylarginine dimethylaminohydrolase (DDAH)-1, a regulator of NO bioavailability, as well as the down-regulatory mechanism of action of IL-10 in relation to Ang II-induced hypertensive mediator expression and cell proliferation in vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR). IL-10 increased DDAH-1 but not DDAH-2 expression and increased DDAH activity. Additionally, IL-10 attenuated Ang II-induced DDAH-1 inhibition in SHR VSMCs. Increased DDAH activity due to IL-10 was mediated mainly through Ang II subtype II receptor (AT₂ R) and AMP-activated protein kinase (AMPK) activation. DDAH-1 induced by IL-10 partially mediated the inhibitory action of IL-10 on Ang II-induced 12-lipoxygenase (LO) and endothelin (ET)-1 expression in SHR VSMCs. In addition, the inhibitory effect of IL-10 on proliferation of Ang II-induced VSMCs was mediated partially via DDAH-1 activity. These results suggest that DDAH-1 plays a potentially important role in the anti-hypertensive activity of IL-10 during Ang II-induced hypertension.     

pp60c-src tyrosine kinase, myristylation, and modulatory domains are required for enhanced mitogenic responsiveness to epidermal growth factor seen in cells overexpressing c-src.

In previous studies examining the potential role of pp60c-src in cellular proliferation, we demonstrated that C3H10T1/2 murine embryo fibroblasts overexpressing transfected chicken genomic c-src displayed an epidermal growth factor (EGF)-induced mitogenic response which was 200 to 500% of the response exhibited by parental control cells (Luttrell et al., Mol. Cell. Biol. 8:497-501, 1988). In order to examine specific structural and functional requirements for pp60c-src in this event, 10T1/2 cells were transfected with chicken c-src genes encoding pp60c-src deficient in tyrosine kinase activity (pm430), myristylation, (pm2A), or a domain hypothesized to modulate the interaction with substrates or regulatory components (dl155). Neomycin-resistant clonal cell lines overexpressing each of the mutated c-src genes were assayed for EGF mitogenic responsiveness by measuring [3H]thymidine incorporation into acid-precipitable material or into labeled nuclei. The results were compared with those obtained with lines overexpressing the cDNA form of wild-type (wt) c-src or control cells transfected with the neomycin resistance gene only. As previously described for cells overexpressing wt genomic c-src (Luttrell et al., 1988), clones overexpressing wt cDNA c-src also exhibited enhanced EGF mitogenic responses ranging from approximately 300 to 400% of the control cell response. In contrast, clones overexpressing unmyristylated, modulation-defective, or kinase-deficient c-src not only failed to support an augmented response to EGF but also exhibited EGF responses lower than that of the control cells. Furthermore, there were no significant differences in the mitogenic responses to 10% fetal calf serum among any of the cells tested. These results indicate that pp60(c-scr) can potentiate mitogenic signaling generated by EGF but not all growth factors. This potentiation requires the utilization of pp60(c-scr) myristylation, and modulatory and tyrosine kinase domains and can me mediated by cDNA-encoded as well as by genome-encoded wt pp60(c-scr).     

Ethanol-induced mitogen activated protein kinase activity mediated through protein kinase C.

The aim of this study was to determine the pathway(s) by which ethanol activates mitogen-activated protein kinase (MAPK) signaling and to determine the role of Ca2+ in the signaling process. MAPK signaling was determined by assessing MAPK activity, measuring phosphorylated extracellular signaling-regulated kinase (pp 44 ERK-1 and pp 42 ERK-2) expression and ERK activity by measuring ERK-2-dependent phosphorylation of a synthetic peptide as a MAPK substrate in rat vascular smooth muscle cells. Ethanol activated extracellular signal-regulated kinase expression (ERK 1 and 2) could be observed when vascular smooth muscle cells (VSMCs) were stimulated for 5 min or less, but was inhibited when cells are treated for 10 min or more with 1-16 mM of ethanol. Maximum ethanol-induced MAPK activity was observed within 5 min with 4 or 8 mM. Ethanol stimulated MAPK activity was blocked by the protein kinase C (PKC) inhibitor (GF109203X) and epidermal growth factor (EGF) receptor antagonist (PD153035) by 41 +/- 24 and 34 +/- 12.3%, respectively. The calcium channel blocker, diltiazem and the chelating agent, BAPTA, reduced the activation of MAPK activity by ethanol, significantly. The data demonstrate that ethanol-stimulated MAPK expression is mediated partially through both the EGF-receptor and PKC intermediates and that activation through the PKC intermediate is calcium-dependent.     

血管紧张素Ⅱ上调自发性高血压大鼠和Wistar-Kyoto大鼠血管平滑肌细胞外信号调节激酶的信号转导

本文研究血管紧张素Ⅱ(angiotensin Ⅱ,Ang Ⅱ)对自发性高血压大鼠(spontaneously hypertensive rat,SHR)和Wistar- Kyoto(WKY)大鼠血管平滑肌细胞(vascular smooth muscle cells．VSMCs)细胞外信号调节激酶(extracellular signal-regulated pro- tein kinases,ERKs)信号途径的影响。体外培养SHR和WKY大鼠的VSMCs,先在培养基中加入终浓度为1×105mmol／L 的缬沙坦或1×105mmol／L的PD98059或不加药物,再给予1×107mmol／L的Ang Ⅱ刺激24 h后收集细胞,以无血清培养基 培养的VSMCs作对照。用免疫沉淀法测定ERK活性;用Western-blot方法检测总ERK(total ERK,t-ERK)、磷酸化ERK (phosphorylated-ERK,p-ERK)及丝裂素活化蛋白激酶磷酸酶-1(mitogen-activated protem kinases phosphatase-1,MKP-1)水 平;用RT-PCR法半定量测定MKP-1 mRNA的含量。结果显示:(1)SHR和WKY大鼠Ang Ⅱ刺激组VSMCs中ERK活 性、p-ERK、MKP-1及MKP-1 mRNA水平均明显高于对照组(P<0．05);SHR和WKY大鼠Ang Ⅱ+缬沙坦组和Ang Ⅱ +PD98059组的上述指标与对照组比较均无显著性差异。(2)SHR大鼠VSMCs中ERK活性、P-ERK、MKP-1及MKP-1 mRNA均显著高于相同干预的WKY大鼠(P<0．01)。(3)SHR和WKY大鼠之间以及对照组、Ang Ⅱ刺激组、Ang Ⅱ+缬沙 坦组和Ang Ⅱ+PD98059组间VSMCs中t-ERK水平均无显著性差异。以上结果表明,Ang Ⅱ可能主要通过其1型(Ang Ⅱ type 1,AT)受体激活SHR和WKY大鼠VSMCs中ERK途径,增加ERK活性和p-ERK蛋白水平,继而引起MKP-1及 MKP-1 mRNA水平升高。