PHARMACOLOGICAL EFFECTS OF CYTOCHROME P450 4A METABOLISM

描述：(改编自申请者摘要)肾细胞色素 P450介导的二十烷类化合物的产生是一个重要的决定因素 综合肾功能和肾血管张力。在不由自主的 高血压大鼠(SHR)，形成过程中的变化 细胞色素P450酶催化的20-羟基二十碳四烯酸(20-HETE) 4A(CYP4A)亚家族与血压升高有关 以及肾功能的变化。在拟议的研究中，自发地 高血压大鼠将被用来检验细胞色素P4A介导的假说 花生四烯酸的羟基化参与了血管内皮细胞癌的发生 高血压。基于肾脏花生四烯酸和omega-1的差异 羟基酶活性，Kroetz博士和她的同事假设 更多的细胞色素P4A基因在高血压大鼠中差异表达 与其正常血压对照相比，肾脏。基因特异性核糖核酸酶 保护试验将被用来区分 肾皮质和髓质中的细胞色素P4A1、细胞色素P4A2、细胞色素P4A3和细胞色素P4A8。 将它们的表达定位于肾单位的特定节段。表达式 模式将与20-HETE形成率相关，以评估 各细胞色素P4A亚型在这一过程中的相对贡献 高血压性二十烷基硫酸钠。这项建议的具体目的是：1) 确定高血压大鼠和正常血压大鼠之间的差异 MRNAs表达的发育模式及其在肾内的分布 在肾皮质和髓质编码细胞色素P450 4A酶；2)到 通过以下方式建立血压与细胞色素P4A表达的关系 诱导调控花生四烯酸omega-羟基酶活性 细胞色素P4A基因失活或基于机制的细胞色素4A蛋白失活； 3)花生四烯酸氧化代谢谱的表征 通过使用体外蛋白的每一种肾表达的细胞色素P4A蛋白 表达系统。对肾脏细胞色素P4A基因表达水平的认识 高血压病大鼠和正常血压大鼠的分布模式 细胞色素P4A的表达对血压的调节，以及关于 给定的细胞色素P4A基因产物是否代谢生理浓度 花生四烯酸对高血压代谢物的作用将为 细胞色素P4A家族参与血压调节。 这些数据将为研究类似的机制提供基础 高血压病患者的病理生理改变 人类。该计划的长期目标是利用这些知识 开发靶向调节血压的治疗方法。

DESCRIPTION: (Adapted from the Applicant's Abstract) Renal cytochrome P450-mediated eicosanoid production is an important determinant of integrated kidney function and renal vascular tone. In the spontaneously hypertensive rat (SHR), alterations in the formation of 20-hydroxyeicosatetraenoic acid (20-HETE) by enzymes of the cytochrome P450 4A (CYP4A) subfamily have been associated with increases in blood pressure and changes in renal function. In the proposed studies the spontaneously hypertensive rat will be used to test the hypothesis that CYP4A-mediated hydroxylation of arachidonic acid is involved in the development of hypertension. Based on differences in renal arachidonic acid and omega-1 hydroxylase activity, Dr. Kroetz and her colleagues hypothesize that one or more of the CYP4A genes are differentially expressed in the hypertensive rat kidney as compared to its normotensive control. Gene-specific ribonuclease protection assays will be used to distinguish between the mRNA levels of CYP4A1, CYP4A2, CYP4A3 and CYP4A8 in the renal cortex and medulla, and to localize their expression to specific segments of the nephron. Expression patterns will be correlated with 20-HETE formation rates to assess the relative contribution of each CYP4A isoform in the generation of this prohypertensive eicosanoid. The specific aims of this proposal are: 1) to identify differences between hypertensive and normotensive rats in the developmental pattern of expression and the intrarenal distribution of mRNAs encoding cytochrome P450 4A enzymes in the renal cortex and medulla; 2) to establish the relationship between blood pressure and CYP4A expression by manipulating arachidonic acid omega-hydroxylase activity through induction of the CYP4A genes or mechanism-based inactivation of the CYP4A proteins; and 3) to characterize the metabolic profile for arachidonic acid oxidation by each of the renally expressed CYP4A proteins using an in vitro protein expression system. Knowledge of the renal CYP4A mRNA expression levels and distribution patterns in hypertensive and normotensive rats, the effect of modulation of CYP4A expression on blood pressure, and information about whether a given CYP4A gene product metabolizes physiological concentrations of arachidonic acid to prohypertensive metabolites will provide evidence for the involvement of the CYP4A family in the regulation of blood pressure. These data will provide a basis for investigating a similar mechanism for the pathophysiological changes associated with essential hypertension in humans. The long term goal of this program is to use this knowledge to develop therapies for targeted modification of blood pressure.