NO SYNTHASE EXPRESSION IN GLOMERULAR ENDOTHELIAL CELLS

该提案的目标是确定氮的生物作用 氧化物（N0）在肾小球内皮细胞功能的调节。 的 假设是一氧化氮合酶（NOS）表达， 人肾小球内皮细胞（HGE）随后释放N0 在健康和疾病中发挥关键作用。 最初的目标将集中在调查生理因素 其调节N0酶的活化和N0S的分子表达， HGE细胞的原代培养物。 由于HGE的有限增殖， 细胞和N0在体外的有限表达， 该项目将针对开发和测试适当的 允许HGE有条件永生化的载体系统 细胞 条件性永生化将使用重组的 编码SV4温度敏感突变体的逆转录病毒构建体 大T抗原。 这种不耐热的转化蛋白在 允许温度（33摄氏度），但在更高的温度下会降解 超过37摄氏度。开发HGE细胞系是关键的第一步， 破译N0在这些细胞中的功能作用。 表征 所述特定同种型存在于原代HGE细胞和HGE细胞中 线研究将被设计为激活钙依赖性 （组成型）和细胞因子诱导型NOS同种型。 NO活性 将使用以下生化测定进行测量1）瓜氨酸 报告大鼠鸟苷酸环化酶的活化 成纤维细胞系，NO形成的敏感指标。分子 通过Western和北方印迹表征NOS的表达 分析. 生物化学和分子研究都将针对 定义N0激活的调节机制。 后续我们将 建立过表达或低表达NOS的HGE细胞系， 对HGE细胞功能的生理影响。

The goals of the proposal are to define the biological role of nitric oxide (N0) in the regulation of glomerular endothelial cell function. The hypothesis is that expression of nitric oxide synthase (N0S) and subsequent release of N0 by human glomerular endothelial (HGE) cells play a key role in their function in both health and disease. The initial aims will focus on investigating the physiological factors which regulate N0 enzyme activation and molecular expression of N0S in primary cultures of HGE cells. Due to the finite proliferation of HGE cells and limited expression of N0 in vitro, the major thrust of the project will be directed at the development and testing of appropriate vector systems which will allow for conditional immortalization of HGE cells. Conditional immortalization will be achieved using a recombinant retroviral construct encoding the temperature - sensitive mutant of SV4 large T antigen. This thermolabile transforming protein is active at permissive temperature (33 degrees C) but degrades at temperatures greater than 37 degrees C. Developing HGE cell lines is a crucial first step in deciphering the functional role of N0 in these cells. To characterize which particular isoform exist in both primary HGE cells and in HGE cell lines studies will be designed to activate the calcium dependent (constitutive) and the cytokine inducible isoforms of N0S. N0 activity will be measured utilizing the following biochemical assays l) citrulline forming assay and 2) activation of guanylate cyclase in reporter rat fibroblast cell line, sensitive index of the formation of NO. Molecular expression of N0S will be characterized by Western and Northern blot analysis. Both biochemical and molecular studies will be directed at defining the regulator mechanisms of N0 activation. Subsequently, we will develop HGE cell line which over or under - express NOS and examine the physiological effects on HGE cell function.