The brain metabolizes arachidonic acid into biologically active products via three distinct enzymatic pathways, namely, cyclooxygenase, lipoxygenase, and cytochrome-P450 (cP450). To data, most has focused on the physiology of cyclooxgenase and lipoxygenase products. However, recent work from our laboratory and others has demonstrated that cP450 products of arachidonic acid are potent controllers of biological processes including regulation f cellular ion transport systems and vascular muscle tone. We have preliminary data demonstrating c450 epoxygenase and omega hydroxylase arachidonic acid product formation form cerebral cortex and cortical microvascular tissue of cats which act in nM concentrations to alter resting K+ channel activity in muscle cells form cerebral arterioles. Western blot analysis demonstrates the presence of cP450 4A omega hydroxylase enzymes within cerebral microvascular tissue. Preliminary data demonstrates that cP450 product formation is sensitive to oxygen within the physiological range of tissue PO2 (i.e. between 50 and 30 torr.) the studies outlined in this proposal will isolate cP450 metabolites of arachidonic acid from parenchymal tissue of cat cerebral cortex, the cerebral microvasculature, and endothelial cells by incubation with [14C] arachidonic acid and separation using rpHPLC. Products will be identified via identified via co-elution with known standards and confirmed with GC/MS. We will determine the ability and specificity of inhibitors of cP450 to block product formation in our system. The physiological action of these products will be determined in vivo using laser-doppler flowmetry to measure cerebral blood flow via a cranial window, and in vitro using a myograph to study the response of isolated, pressurized cerebral arteries to cP450 metabolites. Product formation and cP450 enzyme activity will be determined as a function of P02 (between 100 and 20 torr) as will the ability of the cerebral vasculature to respond to hypoxia before and after inhibition of cP450 product formation. We will define the cellular and ionic mechanism of action of cP450 metabolites on arteriolar muscle cells form the cerebral microvasculature by determining the effect of cP450 products on K+ and Ca2+ currents using the patch-clamp technique and measuring [Ca]i with fluorescent probes. These studies are unique in that they incorporate biochemical, cellular, molecular, and functional approaches to determine the importance of cP450 products in brain function and control of """"""""nutritive"""""""" cerebral blood flow. These are vital studies with respect to understanding a relatively unstudied but important biochemical pathway in the brain, and the mechanisms associated with the adaptive and pathological implications of hypoxic insult and stroke.
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