NO SYNTHASE EXPRESSION IN GLOMERULAR ENDOTHELIAL CELLS

该提案的目标是定义一氧化氮的生物学作用 氧化物(N0)在调节肾小球内皮细胞功能中的作用。这个 假设一氧化氮合酶(NOS)和 人肾小球内皮细胞随后释放N0的作用 对它们在健康和疾病中的作用起着关键作用。 最初的目标将集中在研究生理因素上。 它们调节N0酶的激活和N0S的分子表达 HGE细胞的原代培养。由于HGE的有限扩散 细胞和N0在体外的有限表达，是研究的主要推动力 该项目将针对适当的开发和测试 允许HGE有条件永生的载体系统 细胞。有条件的永生将使用一个重组的 编码SV4温度敏感型突变体的逆转录病毒构建 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低 在37摄氏度以上发展HGE细胞系是在 破译N0在这些细胞中的功能作用。刻画 在原代HGE细胞和HGE细胞中都存在哪种特定的亚型 LINES研究将被设计为激活钙依赖 (构成)和细胞因子诱导的NOS亚型。N0活动 将使用以下生化分析方法进行测量(L)瓜氨酸 报告大鼠鸟苷环化酶的形成及激活 成纤维细胞系，形成NO的敏感指标。分子 用Western和Northern印迹方法鉴定N0S基因的表达 分析。生物化学和分子研究都将针对 明确了N0激活的调节机制。随后，我们将 建立一氧化氮合酶过表达或过低表达的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.