FUNCTION AND REGULATION OF CYTOCHROME P450 4A ISOFORMS

本研究旨在探讨大鼠肾脏血管和肾小管细胞20-羟基二十碳四烯酸（20-HETE）合成的调控机制。我们的研究已经确定，20- HETE，花生四烯酸（AA）的ω-羟基化产物，是在大鼠肾皮质和外髓质的管状和血管结构中形成的主要AA代谢物。我们和其他人已经证明，20 HETE具有有效的生物活性，并提供了证据表明，它有助于调节肾血管和肾小管功能，并控制动脉压。脂肪酸（包括AA）的ω-羟基化由CYP 4A家族的酶（包括4A 1、4A 2、4A 3和4A 8）催化。我们的研究表明，尽管同源性高，这些异构体显示不同的催化性能，包括动力学参数，产物分布和抑制剂敏感性的差异。我们假设大鼠CYP 4A酶，尽管具有非常高的序列同源性和共同的独特性，即，- 花生四烯酸在ω碳上羟基化形成20-HETE，表现出不同的催化活性和底物特异性，并表现出不同的局部模式，最可能的是，处于不同的调节机制下，从而对20-HETE的内源性水平有不同的贡献，表现出不同的催化活性和调节机制，从而在给定组织中显著地促进20-HETE的内源性水平。为了了解调节机制，从而在给定的组织中显著地促进内源性20-HETE水平。为了了解这种重要的类二十烷酸的肾脏合成的调控机制，提出的研究旨在进一步确定这些异构体之间的差异，在生化和分子水平上的催化活性和表达调控（目的1），其次，在体外（目的2）和体内（目的3）的细胞和肾功能方面的这种差异。对CYP 4A介导的20-HETE产生的这些细胞、生化和分子研究将增加我们对20-HETE的生理功能及其可能参与高血压和心血管疾病发病机制的理解。

This is a proposal to investigate the mechanisms underlying the regulation of 20-hydroxyeicosatetraenoic acid (20-HETE) synthesis in vascular and tubular cells of the rat kidney. Our studies have established that 20- HETE, the omega-hydroxylation product of arachidonic acid (AA), is the principal AA metabolite formed in tubular and vascular structures of the rat renal cortex and outer medulla. We and others have demonstrated that 20 HETE is endowed with potent biological activities and have provided evidence that it contributes to the regulation of renal vascular and tubular functions and to the control of arterial pressures. The omega- hydroxylation of fatty acids, including AA, is catalyzed by enzymes of the CYP4A family including 4A1, 4A2, 4A3 and 4A8. Our studies indicated that despite the high homology, these isoforms display distinct catalytic properties including differences in kinetic parameters, product profile and inhibitor sensitivity. We hypothesize that the rat CYP4A enzymes, although sharing very high sequence homology and a common unique, i.e., hydroxylation of arachidonic acid at the omega carbon to form 20-HETE, exhibit different catalytic activities and substrate specificities, and display distinct patterns of localized and ,most likely, are under different regulatory mechanisms, thereby, distinct contributing to the endogenous level of 20-HETE, exhibit different catalytic activities and regulatory mechanisms, thereby distinctively contributing to the endogenous level of 20-HETE in a given tissue. To understand the regulatory mechanisms, thereby, distinctively contributing to the endogenous level of 20-HETE in a given tissue. To understand the regulatory mechanisms underlying the renal synthesis of this important eicosanoid, the proposed studies are designed to further identify differences among these isoforms at the biochemical and molecular levels with respect to catalytic activity and regulation of expression (Aim 1) and, secondly, to implicate such differences in terms of cell and renal function in vitro (Aim 2) and in vivo (Aim 3). These cellular, biochemical, and molecular studies of CYP4A-mediated, 20-HETE production will increase our understanding of the physiological function of 20-HETE and its possible involvement in the pathogenesis of hypertension and cardiovascular diseases.