This Program Project Grant proposal aims at investigating the functions and mechanisms of regulation of vascular and renal eicosanoids in relation to the activity of vasoactive hormonal systems, sodium intake and blood pressure in normotensive and hypertensive animal models. The proposal focuses on eicosanoids arising form arachidonic acid via metabolism by cyclooxygenase(s), lipoxygenase(s), cytochrome P450 epoxygenase(s) and cytochrome P450 omega/omega hydroxylase(s). The experimental strategies combine molecular, cellular, isolated organ and whole animal-approaches. The proposed research activities are organized into five projects supported by two core units. Project l will define the mechanisms by which vasoactive hormones, oxygenases and cytokines interact within the kidney- at organ and cellular levels- to regulate renal function through arachidonate products generated by cytochrome P450-dependent monoxygenases. Project 2 will test the hypothesis that lipoxygenase products formed in excess by arterial vessels of hypertensive rats contribute to increased blood pressure by fostering the expression of PGH2-mediated vasoconstriction and by limiting the activity of vasodilatory mechanisms mediated by PGI2; it also will test the hypothesis that excessive production of 20-HETE promotes renal and systemic vasoconstriction and contributes to the mechanisms of hypertension in SHR. Project 3 will examine whether arachidonic acid epoxides (EETs) derived via renal metabolism by cytochrome P450 epoxygenase(s) are involved in the regulation of renal vascular function by activating K+ channels and promoting vasodilation, and by counteracting the renal vasoconstrictor actions of pressor hormones; it will also investigate the mechanism underlying hyperexpression of renal vasodilatory responses to arachidonic acid in SHR. Project 4 will characterize the mechanisms regulating 2O-HETE synthesis by tubular and vascular structures in kidneys of SHR and normotensive rats. This will include relating the expression of cytochrome P450 4A isoforms, protein and mRNA, to 2O-HETE synthesis, examining the effect of dietary sodium on renal cytochrome P450 4A expression and 20- HETE synthesis, and characterizing the catalytic activity of the various cytochrome P450 4A gene products expressed in an in vitro system. Project 5 will study the mechanism and the eicosanoid mediator(s) of the inhibitory effect of arachidonic acid on apical K+ channel activity in the thick ascending limb, the second messengers involved, and the role of such eicosanoids as modulators of the actions of angiotensin II and vasopressin on K+ channel activity in the loop of Henle. Core A will serve the administrative needs of the Program Project. Core B will serve the needs of Program Project Investigators for analysis of eicosanoids by gas chromatography-mass spectrometry.
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