This Program Project Grant proposal aims at investigating the function and regulation of cytochrome P450 (CYP)-derived eicosanoids in relation to vascular and renal mechanism of blood pressure control. The proposal focuses on vascular and/or renal mechanisms involving 20-hydroxyeicosatetraenoic acid (20-HETE), CYP2E1-derived 19(R)- and 18(R)-HETE, and epoxyeicosatrienoic acids (EETs) of both the cis-EET and trans-EET configuration. The experimental strategies combine molecular, cellular, isolated organ and whole animal approaches. The proposed research activities are organized into four projects supported by three cores. Project 1 investigates the role of CYP2E1-derived eicosanoids in the regulation of vascular reactivity and blood pressure. Blood pressure and vascular reactivity to constrictor stimuli will be studied in relation to vascular synthesis of CYP2E1-derived eicosanoids and 20-HETE in animals subjected to interventions that increase (CYP2E1 gene transfer) or decrease (CYP2E1 inhibition;CYP2E1-null mice) the synthesis of CYP2E1-derived eicosanoids. Project 2 investigates the role of 20-HETE as a determinant of endothelial cell dysfunction and activation in rats made hypertensive by in vivo CYP4A2 gene transfer. Relationships will be examined between vascular 20-HETE production, indices of endothelial dysfunction, and blood pressure. The contribution of endothelial versus smooth muscle 20-HETE will be assessed. The molecular mechanisms of 20-HEETE-induced endothelial dysfunction will be addressed. Project 3 will investigate the role of EETs in regulation of Na transport in the cortical collecting duct (CCD). The EETs-dependent effects of arachidonic acid and adenosine on ENaC will be studied as a function of Na intake and epoxygenase expression. The signaling mechanisms underlying the inhibitory action of EETs on ENaC will be defined. Project 4 will study the renal synthesis and release of cis- and trans-EETs in relation to pressure and oxidative stress, examine their vasoregulatory actions, and determine the relative contribution of cis and trans-EETs to the exaggerated renal vasodilatory effect of arachidonic acid in SHR. Core A provides administrative support. Core B is responsible for measurement of eicosanoids by mass spectrometry. Core C provides viral vectors to transfer CYP genes in the sense and antisense orientation.
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