CHARACTERIZATION OF RENAL NONCYCLOOXYGENASE ARACHIDONATE METABOLISM

肾细胞色素P450依赖的花生四烯酸（AA）单加氧酶 现在被认为是生物医学重要AA的第三个分支 级联。最广泛表征的P450衍生的 代谢物，环氧-和19/20-羟基AA，已经牵连到 诸如控制全身和肾血管反应性的过程， 离子流，激素信号和实验性 高血压因此，我们的研究和其他人的研究表明， 这种酶系统在肾脏和身体控制中的作用 体内平衡然而，由于缺乏适当的分子工具， 方法、肾P450的生理意义及肾P450的定位、 其代谢物的作用模式仍有待明确定义。 项目#2，结合细胞和器官生理学组件 该计划项目，建议利用分子方法， 用于研究的细胞和/或动物模型的开发和使用 P450基因特异性功能表型。我们将应用以下组合 重组DNA和生化技术的设计和构建 高周转率，独立，区域和对映选择性AA 单加氧酶，这些酶在培养细胞中的表达， 插入片段/cDNA依赖性鉴定和机理表征 细胞表型靶向基因破坏将用于 整体动物综合功能和生化分析 肾脏AA表氧化酶的意义和作用， omega/omega-1水解酶。该项目的长期目标是 提供对肾脏P450类花生酸生物学的分子理解 的意义和作用方式。这些重要问题的答案 制定有意义的办法，以便： 明确定义其生理和/或病理生理 显著性，和B）随后药理学和/或临床 干预措施。

The kidney cytochrome P450-dependent arachidonic acid (AA) monooxygenase is now recognized as the third branch of the biomedically important AA cascade. The most extensively characterized of the P450-derived metabolites, the epoxy- and the 19/20-hydroxy AA, have been implicated in processes such as the control of systemic and renal vascular reactivity, ion fluxes, hormonal signaling and the pathophysiology of experimental hypertension. Thus, our studies and that of others indicate significant roles for this enzyme system in the control of kidney and body homeostasis. However, due to paucity of appropriate molecular tools and techniques, the physiological significance of renal P450 and the site and mode of action of its metabolites remains to be unequivocally defined. Project #2, in conjunction with the cell and organ physiology components of this Program Project, proposes to utilize molecular approaches to the development and use of cellular and/or animal models for the study of P450-gene specific functional phenotypes. We will apply a combination of recombinant DNA and biochemical techniques to the design and construction of high turnover, self contained, regio- and enantioselective AA monooxygenases, the expression of these enzymes in cultured cells, and the identification and mechanistic characterization of insert/cDNA-dependent cellular phenotypes. Targeted gene disruption will be employed for the whole animal integrated functional and biochemical analysis of the significance and the role(s) of specific kidney AA epoxygenase and omega/omega-1 hydrolases. The long term goals of this project are to provide a molecular understanding of renal P450 eicosanoid biological significance and mode of action. The answer to these important questions are needed for the development of meaningful approaches to: a) the unequivocal definition of their physiological and/or pathophysiological significance, and b) subsequent pharmacological and/or clinical interventions.