METABOLISM OF ARACHIDONIC-ACID TO EPOXYEICOSATRIENOIC ACIDS, HYDROXYEICOSATETRAENOIC ACIDS, AND PROSTAGLANDINS IN CULTURED RAT HIPPOCAMPAL ASTROCYTES

We have recently shown that brain slices are capable of metabolizing a rachidonic acid by the epoxygenase pathway. The purpose of this study was to begin to determine the ability of individual brain cell types t o form epoxygenase metabolites. We have examined the astrocyte epoxyge nase pathway and have also confirmed metabolism by the cyclooxygenase and lipoxygenase enzyme systems. Cultured rat hippocampal astrocyte ho mogenate, when incubated with radiolabeled [H-3]-arachidonic acid, for med products that eluted in four major groups designated as R17-30. R4 2-50, R51-82, and R83-90 based on their retention times in reverse-pha se HPLC. These fractions were further segregated into as many as 13 pe aks by normal-phase HPLC and a second reverse-phase HPLC system. The p rincipal components in each peak were structurally characterized by ga s chromatography/electron impact-mass spectrometry. Based on HPLC rete ntion times and gas chromatography/electron impact-mass spectrometry a nalysis, the more polar fractions (R17-30) contained prostaglandin D2 as the major cyclooxygenase product. Minor products included 6-keto pr ostaglandin F1alpha, prostaglandin E2, prostaglandin F2alpha, and thro mboxane 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 [H-3]l 4,15-EET wa s incubated with astrocyte homogenate, it was rapidly metabolized to [ H-3] 1 4,15-DHET. The metabolism was inhibited by submicromolar concen tration of 4-phenylchalcone oxide, a potent inhibitor of epoxide hydro lase activity. Formation of other polar metabolites such as triols or epoxyalcohols from 14,15-DHET was not observed. In conclusion, astrocy tes readily metabolize arachidonic acid to 14,15-EET, 5,6-EET, and the ir vicinal-diols. Previous studies suggest these products may affect n euronal function and cerebral blood flow.