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The role of endothelial nitric oxide synthase in cardiovascular regulation. -approach using genetic engineering.

内皮一氧化氮合酶在心血管调节中的作用。

基本信息

批准号：
08457209
负责人：
YOKOYAMA Mitsuhiro
金额：
$ 4.1万
依托单位：
Kobe University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至无数据
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08457209/
关键词：
nitric oxyde synthase endthelial cell transgenic mice genetic engineering

项目摘要

Nitric oxide (NO), constitutively produced by endothelial cell nitric oxide synthase (eNOS), plays a major role in regulation of blood pressure and vascular tone and also acts as an anti-atherogenic molecule.To study chronic effects of lifelong overexpression of eNOS in endothelial cells, we generated transgenic mice overexpressing eNOS by pronuclear microinjection and analyzed their phenotypic changes. We constracted a fusion gene comprising 9.2kb murine preproendothelin-1 gene promoter and 4.1kb bovine eNOS cDNA to direct the expression specifically tp vascular wall. Five founder mice with 9-13 transgene copies out of 104 pups were identified by PCR and subsequent Southem blot analysis. The specific espression of transgene transcript was confirmed in all organs examined by RNase protection assay using bovine eNOS specific riboprobe. In immunohistochemical studies using polyclonal eNOS antibody, eNOS protein was identified predominantly in the vascular endothelial cells in most organs, and also in epithelial cells in the lung and the uterus. Immunoblotting showed significantly increased eNOS protein in particulate fractions of the aorta and the lung, and consistent increase in -calcium-dependent NOS activity, assayd by the conversion of ^3H-arginine to ^3H-citrulline, was identified especially in aorta (53.9 nmol/mg/min, in transgenic to 7.76 nmol/mg/min, in wild-type). General appearance and orgen (heart, lung, and kidney) weights were not significantly different between the transgenic and wild-type mice but blood pressure, measured by direct femoral arterial catheterization, was lower in the transgenic mice. In addition, isotonic tension meserement in aorta revealed the attenuated vasorelaxation to acetylcholine or substance P in transgenic mice compared with wild-type mice.Thus, transgenic mice overexpressing eNOS will be a new experimental model to study the role of NO in cardiovascular system.

一氧化氮(NO)由内皮细胞一氧化氮合酶(ENOS)组成，在血压和血管张力的调节中起重要作用，也是一种抗动脉粥样硬化的分子。为了研究eNOS终生过表达对内皮细胞的慢性影响，我们用原核显微注射的方法建立了过表达eNOS的转基因小鼠，并分析了它们的表型变化。我们构建了9.2kb的小鼠前内皮素1基因启动子与4.1kb的牛enos基因的融合基因，以指导其在血管壁中的特异性表达。通过聚合酶链式反应和随后的Souome印迹分析，从104只仔鼠中鉴定出5只转基因小鼠，其转基因拷贝数为9-13。用牛eNOS特异性核糖核酸探针进行核糖核酸酶保护试验，证实转基因转录产物在所有器官中特异表达。在用多克隆eNOS抗体进行的免疫组织化学研究中，eNOS蛋白主要存在于大多数器官的血管内皮细胞中，也存在于肺和子宫的上皮细胞中。免疫印迹结果显示，大鼠主动脉和肺组织中eNOS蛋白表达显著增加，而~(3 H)-精氨酸转化为~(3 H)-瓜氨酸的结果表明，主动脉中的eNOS活性持续增强(野生型为7.76nmol/mg/min，转基因为53.9nmol/mg/min)。转基因小鼠的一般外观和乌力更(心、肺、肾)重量与野生型小鼠没有显著差异，但通过直接股动脉插管测量的血压在转基因小鼠中较低。此外，主动脉等张张力测定显示，与野生型小鼠相比，转基因小鼠对乙酰胆碱或P物质的血管松弛作用减弱。因此，过表达eNOS的转基因小鼠将成为研究NO在心血管系统中作用的新的实验模型。