PHARMACOLOGICAL EFFECTS OF CYTOCHROME P450 4A METABOLISM

描述：（改编自申请人摘要）肾细胞色素 P450介导的类花生酸的产生是一个重要的决定因素， 综合肾功能和肾血管张力。 自发性 高血压大鼠（SHR）， 20-羟基二十碳四烯酸（20-HETE）通过细胞色素P450的酶 CYP 4A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， 将使用高血压大鼠来检验CYP 4A介导的 花生四烯酸的羟基化参与了 高血压 基于肾脏花生四烯酸和omega-1的差异 羟化酶活性，Kroetz博士和她的同事假设一个或多个 CYP 4A基因在高血压大鼠中有更多的差异表达 肾脏与其正常血压对照相比。 基因特异性核糖核酸酶 将使用保护测定来区分 肾皮质和髓质中的CYP 4A 1、CYP 4A 2、CYP 4A 3和CYP 4A 8，以及 将其表达定位于肾单位的特定节段。 表达 模式将与20-HETE形成率相关，以评估 每种CYP 4A亚型在产生这种 降血压类花生酸 这项建议的具体目标是：1） 确定高血压大鼠和血压正常大鼠之间在 mRNA表达的发育模式和肾内分布 在肾皮质和髓质中编码细胞色素P450 4A酶; 2） 建立血压和CYP 4A表达之间的关系， 通过诱导操纵花生四烯酸ω-羟化酶活性 CYP 4A基因或基于机制的CYP 4A蛋白失活; 和3）表征花生四烯酸氧化的代谢概况 通过使用体外蛋白的每种肾脏表达的CYP 4A蛋白 表达系统 了解肾脏CYP 4A mRNA表达水平和 高血压和正常血压大鼠的分布模式， CYP 4A表达对血压的调节，以及关于 给定的CYP 4A基因产物是否代谢生理浓度 花生四烯酸与高血压代谢物的关系将为 CYP 4A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， 原发性高血压的病理生理变化 人类 该计划的长期目标是利用这些知识， 开发针对性的血压调节疗法。

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.