Role of Novel CYP Isoforms in Regulation of Cerebral Blood Flow

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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.