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

一氧化氮（NO）由内皮细胞一氧化氮合酶（eNOS）组成，在调节血压和血管张力中起重要作用，同时也是一种抗动脉粥样硬化分子。为了研究eNOS在内皮细胞中终身过表达的慢性效应，我们通过原核微注射制备了过表达eNOS的转基因小鼠，并分析了其表型变化。我们构建了由9.2kb鼠内皮素-1前原基因启动子和4.1kb牛eNOS cDNA组成的融合基因，特异地指导血管壁的表达。通过PCR和southern blot分析，从104只幼鼠中鉴定出5只具有9-13个转基因拷贝的创仔鼠。用牛eNOS特异性核糖探针进行RNase保护实验，证实了转基因转录物在所有器官中的特异性表达。利用多克隆eNOS抗体进行免疫组化研究，发现eNOS蛋白主要存在于大多数器官的血管内皮细胞，以及肺和子宫的上皮细胞。免疫印迹显示，主动脉和肺颗粒部位的eNOS蛋白显著增加，并且通过^ 3h -精氨酸转化为^ 3h -瓜氨酸检测到-钙依赖性NOS活性持续增加，特别是在主动脉（53.9 nmol/mg/min，转基因到7.76 nmol/mg/min，野生型）。转基因小鼠和野生型小鼠的总体外观和器官（心、肺和肾）重量没有显著差异，但通过直接股动脉导管测得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物质的血管舒张作用减弱。因此，过表达eNOS的转基因小鼠将为研究NO在心血管系统中的作用提供新的实验模型。

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.