Role of Novel CYP Isoforms in Regulation of Cerebral Blood Flow

新型 CYP 亚型在脑血流调节中的作用

• 批准号: 6967913
• 负责人: David Rae Harder
• 金额: $ 48.57万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-12-01 至 2009-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6967913
• 关键词: animal tissue; arachidonate; brain circulation; brain metabolism; clinical research; cytochrome P450; eicosanoid metabolism; eicosanoids; enzyme mechanism; fatty acid metabolism; hemodynamics; human tissue; inflammation; isozymes; laboratory rat; leukotrienes; omega 3 fatty acid; oxidation; unsaturated fatty acids; vasoactive agent

This project is based on the hypothesis that oxygenation of polyunsaturated fatty acids (PUFAs) in the brain by cytochrome P450 (CYP) enzymes plays an important role in the regulation of cerebral blood flow and inflammation. We have previously focused on the NADPH-dependent metabolism of arachidonate (AA) to epoxyeicosatrienoates (EETs) and 20-hydroxyeicosatetraenoate (20-HETE), and the involvement of these metabolites in control of cerebral vascular tone through their action on Ca2+activated K+ channels. In this renewal, we will continue to study the functional significance of NADPH-dependent CYP metabolism of AA, however, we will extend these studies to also include other PUFAs, such as docosahexaenoate (DHA) and eicosapentaenoate (EPA), as well as the potent inflammatory mediator leukotriene B4 (LTB4). In our preliminary studies, we have identified CYP enzymes that are expressed in the brain and are capable of metabolizing PUFAs into vasoactive metabolites. These enzymes belong to two different subfamilies, i.e. the CYP4F and CYP4X. Initial data suggests that enzymes of the CYP4F subfamily can oxygenate AA and LTB4 into co-side chain hydroxy metabolites, and DHA and EPA into epoxy metabolites. CYP4X1 is a novel rat enzyme, cloned in our laboratory, which is highly and predominantly expressed in specific neurons and cerebral vascular endothelial cells. Preliminary results show that the CYP4X1 protein is highly conserved between different species and that recombinant CYP4X1 is capable of converting AA into EETs. However, the functional consequences of CYP4F and CYP4X1 catalyzed oxygenation of PUFAs in the brain are unknown. Specific Aim 1: will characterize the expression and catalytic properties of CYP4F isoforms in the brain, and to characterize the biological action of CYP4F oxygenated metabolites of PUFAs. Specific Aim 2: will determine the functional significance of LTB4 metabolism by CYP4F enzymes in the brain. Specific Aim 3: will characterize the expression and catalytic properties of human, mouse and rat CYP4X1 and define the biological activities of CYP4Xl-generated metabolites. This project will increase our understanding about the function of newly identified CYP isoforms in the brain and the actions of metabolites of PUFAs other than AA in the control of inflammation and cerebral blood flow.

该项目基于以下假设：通过细胞色素P450（CYP）酶对大脑中多不饱和脂肪酸（PUFAS）的氧合作用在调节大脑血流和炎症的调节中起重要作用。 以前，我们曾关注蛛网膜酸（AA）的NADPH依赖性代谢（aa）对环氧树叶酸酯（EET）（EET）和20-羟基乙酸酯（20-HETE），以及这些代谢物通过控制Ca2+ Actived Aivic Actived k+ Channels的脑血管张力的参与。在此续约中，我们将继续研究AA NADPH依赖性CYP代谢的功能意义，但是，我们将把这些研究扩展到 包括其他PUFA，例如Docosahexaenoate（DHA）和Eicosapentaenoate（EPA），以及有效的炎症介质白细胞B4（LTB4）。在我们的初步研究中，我们确定了在大脑中表达并能够将PUFA代谢为血管活性代谢物的CYP酶。这些酶属于两个不同的亚家族，即CYP4F和CYP4X。初始数据表明，CYP4F亚家族的酶可以将AA和LTB4氧化为共侧链羟基代谢物，而DHA和EPA成为环氧代谢物。 CYP4X1 是一种新型的大鼠酶，克隆在我们的实验室中，它在特定的神经元和脑血管内皮细胞中高度表达。初步结果表明，CYP4X1蛋白在不同物种之间高度保守，重组CYP4X1能够将AA转化为EET。然而，CYP4F和CYP4X1催化PUFAS中PUFA的功能后果尚不清楚。具体目标1：将表征CYP4F同工型在大脑中的表达和催化特性，并表征PUFAS的CYP4F氧化代谢产物的生物学作用。具体目标2：将确定CYP4F酶在大脑中的LTB4代谢的功能意义。具体目标3：将表征人，小鼠和大鼠CYP4X1的表达和催化特性，并定义CYP4XL生成的代谢物的生物学活性。 该项目将提高我们对大脑中新鉴定的CYP同工型功能的理解以及AA以外的PUFAS代谢物在控制炎症和脑血流动方面的作用。