Regulation of Na, K-ATPase in renal, cardiac, pulmonaly disease

肾脏、心脏、肺部疾病中 Na、K-ATP 酶的调节

基本信息

批准号：
07044289
负责人：
KAWAKAMI Kiyoshi
金额：
$ 2.75万
依托单位：
Jichi Medical School
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1997
项目状态：
已结题

项目摘要

1.In renal system, we applied cultured distal tubule cells and vascular smooth muscle cells and analyzed the response of Na, K-ATPase genes expression by trearment with corticosteroid hormones, high osmolarity or serum stimulation. Both a and b subunit gene were induced several fold by nortehrn blotting analysis. The induction was also observed when we transfected the reporter luciferase gene fused with the promote region of the Na, K-ATPase a1 and b1 subunit gene, indicating that the regulation was transcriptinal level.2.Na, K-ATPase alpha3 subunit gene is transcribed in a few weeks after birth in rat heart. Using rat cardiocyte as a model system, we identified cis elements and transacting factors responsible for the gene. They are one NF-Y site and two Sp1/Sp3 sites. All three elements act as positive regulatory element and showed synergic activation. Protein occupancy on these elements were observed by in vivo footprinting analysis in cardiocyte specific manner.3.The regulatory element of the beta1 subunit gene in response to hyperoxya was identified at -84 to -44 region. The two Sp1/Sp3 site in the region was not the resonsible element. For the analysis of the induction mechanism of the as subuunit gene during birth period, we established in vitro transcription system of the a1 subuit gene from adult and embyonic lung nuclear extracts. We identified negative regulatory element of the alpha1 subuit gene from -375 to -201. The cis element necessary for the full transcriptin activity was -155 to +31 in embryo while +66 to +92 was also necessary in adult.

1.在肾脏系统中，我们应用了培养的远端小管细胞和血管平滑肌细胞，并通过用皮质类固醇激素，高渗透压或血清刺激来分析Na，K-ATPase基因表达的反应。通过Nortehrn印迹分析，诱导了A和B亚基基因几倍。当我们转染与Na，K-ATPase A1和B1亚基基因的促进区域融合的报告基因荧光素酶基因时，还观察到了诱导。使用大鼠心脏细胞作为模型系统，我们确定了负责该基因的CIS元素和交易因子。它们是一个NF-Y站点和两个SP1/SP3站点。这三个元素充当阳性调节元件，并显示出协同激活。通过以心脏细胞特异性方式通过体内足迹分析观察到这些元素上的蛋白质占用率。3。Beta1亚基基因的调节元件响应于Hyperoxya，在-84至-44区域鉴定出对Hyperoxya的响应。该区域的两个SP1/SP3位点不是可共鸣的元素。为了分析出生期间AS亚征物基因的诱导机制，我们从成人和Embyonic肺核提取物中建立了A1 Subuit基因的体外转录系统。我们从-375至-201确定了Alpha1子女图基因的负调节元件。在胚胎中，完整转录素活性所需的顺式元件为-155至+31，而成人也需要+66至+92。