EFFECT OF PROTEIN-KINASE-C MODULATORS ON 14,15-EPOXYEICOSATRIENOIC ACID INCORPORATION INTO ASTROGLIAL PHOSPHOLIPIDS

Our previous studies have shown that 14,15-epoxyeicosatrienoic acid (1 4,15-EET) is a major product of arachidonic acid metabolism in astrocy tes. The purpose of this study was to investigate cellular regulation of 14,15-EET incorporation, distribution, and metabolism in primary cu ltures of rat brain cortical astrocytes. Incorporation of 14,15-EET in to astrocytes was lower (93,390 +/- 11,121 dpm/5 x 10(6) cells) than i ncorporation of 8,9-EET (226,500 +/- 5,567 dpm/5 x 10(6) cells) and ar achidonic acid (321,600 +/- 1,200 dpm/5 x 10(6) cells). 14,15-EET was distributed in the order neutral lipids and free fatty acids (solvent front) >> phosphatidylcholine (PC) > phosphatidylinositol (PI) > phosp hatidylethanolamine. In contrast, 8,9-EET and arachidonic acid were ex clusively incorporated into PC. During incubation, astroglial epoxide hydrolase selectively metabolized 14,15-EET, but not 8,9-EET, to its v ic-diol. Although 4-phenylchalcone oxide, a potent inhibitor of epoxid e hydrolase, completely inhibited 14,15-EET metabolism, a large amount of cell-incorporated radioactivity remained as free 14,15-EET. Long-t erm exposure of astrocytes to 4 beta-phorbol 12-myristate 13-acetate ( 4 beta-PMA) resulted in a time-dependent incorporation of 14,15-EET in to PI but not in control cells exposed to 4 alpha-phorbol 12,13-dideca noate. PKC down-regulation completely inhibited epoxide hydrolase meta bolism of 14,15-EET. Following recovery of downregulated PKC, 1 week a fter treatment with 4 beta-PMA, astrocytes regained their normal patte rn of low incorporation of 14,15-EET. Protein kinase C (PKC) inhibitio n by staurosporine enhanced 14,15-EET incorporation without affecting its metabolism to 14,15-dihydroxyeicosatrienoic acid. Incorporation of 14,15-EET by PKC-down-regulated cells was inhibited by thimerosal, a known inhibitor of fatty acyl-CoA synthase. Our results suggest that t he lower incorporation of 14,15-EET into astroglial cells may be due t o modulation of PKC-mediated cellular mechanism(s).