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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS032321-04
Application #
2460565
Study Section
Neurology A Study Section (NEUA)
Program Officer
Jacobs, Tom P
Project Start
1994-09-01
Project End
1998-07-31
Budget Start
1997-08-01
Budget End
1998-07-31
Support Year
4
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Medical College of Wisconsin
Department
Type
Organized Research Units
DUNS #
073134603
City
Milwaukee
State
WI
Country
United States
Zip Code
53226
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Bylund, Johan; Harder, Adam G; Maier, Kristopher G et al. (2003) Leukotriene B4 omega-side chain hydroxylation by CYP4F5 and CYP4F6. Arch Biochem Biophys 412:34-41
Medhora, Meetha; Bousamra 2nd, Michael; Zhu, Daling et al. (2002) Upregulation of collagens detected by gene array in a model of flow-induced pulmonary vascular remodeling. Am J Physiol Heart Circ Physiol 282:H414-22
Liu, Yanping; Harder, David R; Lombard, Julian H (2002) Interaction of myogenic mechanisms and hypoxic dilation in rat middle cerebral arteries. Am J Physiol Heart Circ Physiol 283:H2276-81
Bylund, Johan; Zhang, Chenyang; Harder, David R (2002) Identification of a novel cytochrome P450, CYP4X1, with unique localization specific to the brain. Biochem Biophys Res Commun 296:677-84
Nithipatikom, K; Grall, A J; Holmes, B B et al. (2001) Liquid chromatographic-electrospray ionization-mass spectrometric analysis of cytochrome P450 metabolites of arachidonic acid. Anal Biochem 298:327-36
Medhora, M; Narayanan, J; Harder, D (2001) Dual regulation of the cerebral microvasculature by epoxyeicosatrienoic acids. Trends Cardiovasc Med 11:38-42
Medhora, M; Narayanan, J; Harder, D et al. (2001) Identifying endothelium-derived hyperpolarizing factor: recent approaches to assay the role of epoxyeicosatrienoic acids. Jpn J Pharmacol 86:369-75
Gebremedhin, D; Lange, A R; Lowry, T F et al. (2000) Production of 20-HETE and its role in autoregulation of cerebral blood flow. Circ Res 87:60-5
Medhora, M; Harder, D (1998) Functional role of epoxyeicosatrienoic acids and their production in astrocytes: approaches for gene transfer and therapy (review). Int J Mol Med 2:661-9

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