FUNCTION AND REGULATION OF CYTOCHROME P450 4A ISOFORMS

这是一项研究20-羟基二十碳四烯酸(20-HETE)在大鼠肾脏血管和肾小管上皮细胞合成调控机制的建议。我们的研究证实，花生四烯酸(AA)的omega-羟基化产物20-HETE是AA在大鼠肾皮质和外髓的肾小管和血管结构中形成的主要代谢产物。我们和其他人已经证明了20HETE具有强大的生物活性，并提供了证据表明它有助于调节肾血管和肾小管功能，并有助于控制动脉压。包括氨基酸在内的脂肪酸的omega-羟基化是由包括4A1、4A2、4A3和4A8在内的CYP4A家族的酶催化的。我们的研究表明，尽管这些异构体具有很高的同源性，但它们表现出不同的催化性质，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，虽然大鼠的CYP4A酶具有很高的序列同源性和一个共同的独特之处，即在omega碳上将花生四烯酸羟化成20-HETE，但它们显示出不同的催化活性和底物特异性，并显示出不同的局部化模式，很可能处于不同的调节机制下，因此，对内源20-HETE水平有不同贡献的大鼠，表现出不同的催化活性和调节机制，从而显著地对特定组织中的内源20-HETE水平做出贡献。了解调节机制，从而，明确贡献的内源性水平的20-HETE在给定的组织。为了了解肾脏合成这种重要二十烷类化合物的调控机制，拟议的研究旨在进一步在生物化学和分子水平上鉴定这些异构体之间在催化活性和表达调控方面的差异(目标1)，第二，在体外(目标2)和体内(目标3)的细胞和肾功能方面的差异。这些对细胞色素P4A介导的20-HETE产生的细胞、生化和分子研究将加深我们对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.