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Notes: Abstract: We have recently shown that brain slices are capable of metabolizing arachidonic acid by the epoxy-genase pathway. The purpose of this study was to begin to determine the ability of individual brain cell types to form epoxygenase metabolites. We have examined the astrocyte epoxygenase pathway and have also confirmed metabolism by the cyclooxygenase and lipoxygenase enzyme systems. Cultured rat hippocampal astrocyte homogenate, when incubated with radiolabeled [3H]-arachidonic acid, formed products that eluted in four major groups designated as R17–30, R42–50, R51–82, and R83–90 based on their retention times in reverse-phase HPLC. These fractions were further segregated into as many as 13 peaks by normal-phase HPLC and a second reverse-phase HPLC system. The principal components in each peak were structurally characterized by gas chromatography/electron impact-mass spectrometry. Based on HPLC retention times and gas chromatography/electron impactmass spectrometry analysis, the more polar fractions (R17–30) contained prostaglandin D2 as the major cyclooxygenase product. Minor products included 6-keto prostaglandin F1α, prostaglandin E2, prostaglandin F2α, and thromboxane B2. Fractions R42–50, R51–82. and R83–90 contained epoxygenase and lipoxygenase-like products. The major metabolite in fractions R83–90 was 5, 6-epoxyeicosatrienoic acid (EET). Fractions R51–82 contained 14, 15-and 8, 9-EETs, 12-and 5-hydroxyeicosatetraenoic acids, and 8, 9-and 5, 6-dihydroxyeicosatrienoic acids (DHETs). In fractions R42–50, 14, 15-DHET was the major product. When radiolabeled [3H]14, 15-EET was incubated with astrocyte homogenate, it was rapidly metabolized to [3H]14, 15-DHET. The metabolism was inhibited by submicromolar concentration of 4-phenylchalcone oxide, a potent inhibitor of epoxide hydrolase activity. Formation of other polar metabolites such as triols or epoxyalcohols from 14, 15-DHET was not observed. In conclusion, astro-cytes readily metabolize arachidonic acid to 14, 15-EET, 5, 6-EET, and their vicinal-diols. Previous studies suggest these products may affect neuronal function and cerebral blood flow.