Role of Novel CYP Isoforms in Regulation of Cerebral Blood Flow

该项目基于这样的假设，即细胞色素P450(CYP)酶对脑内多不饱和脂肪酸(PUFAs)的氧化在调节脑血流和炎症过程中起着重要作用。 我们以前主要关注花生四烯酸(AA)依赖NADPH代谢成环氧二十碳三烯酸酯(EETs)和20-羟基二十碳四烯酸(20-HETE)，以及这些代谢物通过作用于钙激活的K+通道参与脑血管张力的控制。在这次更新中，我们将继续研究AA依赖NADPH的CYP代谢的功能意义，但我们将把这些研究扩展到 包括其他多不饱和脂肪酸，如二十二碳六烯酸酯(DHA)和二十碳五烯酸酯(EPA)，以及有效的炎症介质白三烯B4(LTB4)。在我们的初步研究中，我们已经确定了在大脑中表达的能够将多不饱和脂肪酸代谢成血管活性代谢物的CYP酶。这些酶属于两个不同的亚家族，即细胞色素P4F和细胞色素P4X。初步数据表明，CYP4F亚家族的酶可以将AA和LTB4氧化成共侧链羟基代谢物，将DHA和EPA氧化成环氧代谢物。细胞色素P4X1 是本实验室克隆的一种新型大鼠酶，在特定的神经元和脑血管内皮细胞中高水平表达。初步结果表明，不同物种间的细胞色素P4X1蛋白高度保守，重组的细胞色素P4X1能够将氨基酸转化为内毒素。然而，CYP4F和CYP4X1催化的多不饱和脂肪酸在大脑中氧化的功能后果尚不清楚。具体目的1：将表征细胞色素P4F亚型在脑中的表达和催化性质，以及多不饱和脂肪酸的细胞色素P4F氧化代谢产物的生物学作用。具体目标2：将通过大脑中的细胞色素P4F酶来确定LTB4代谢的功能意义。具体目标3：将表征人、小鼠和大鼠细胞色素P4X1的表达和催化性质，并确定细胞色素P4X1产生的代谢物的生物学活性。 这个项目将增加我们对新发现的CYP亚型在脑中的功能以及AA以外的PUFAs代谢物在控制炎症和脑血流中的作用的了解。

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.