CHARACTERIZATION OF RENAL NONCYCLOOXYGENASE ARACHIDONATE METABOLISM

肾细胞色素 P450 依赖性花生四烯酸 (AA) 单加氧酶 现已被公认为具有生物医学重要性的 AA 的第三个分支 级联。 P450 衍生的最广泛特征 代谢物，环氧-和 19/20-羟基 AA，与 控制全身和肾血管反应性等过程， 离子通量、激素信号传导和实验的病理生理学 高血压。因此，我们的研究和其他人的研究表明， 该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和 技术、肾 P450 的生理意义及其部位和 其代谢物的作用方式仍有待明确定义。 项目#2，与细胞和器官生理学成分相结合 该计划项目的建议利用分子方法 开发和使用细胞和/或动物模型来研究 P450基因特异性功能表型。我们将应用组合 重组DNA和生化技术的设计和构建 高周转、独立、区域选择性和对映选择性 AA 单加氧酶，这些酶在培养细胞中的表达，以及 插入/cDNA依赖的鉴定和机制表征 细胞表型。靶向基因破坏将用于 整个动物的综合功能和生化分析 特定肾脏 AA 环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 类二十烷酸生物活性的分子了解 意义和作用方式。这些重要问题的答案 需要制定有意义的方法来： 其生理和/或病理生理学的明确定义 意义，和 b) 随后的药理学和/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.