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 (14,15-EET) is a major product of arachidonic acid metabolism in astrocytes. The purpose of this study was to investigate cellular regulation of 14,15-EET incorporation, distribution, and metabolism in primary cultures of rat brain cortical astrocytes. Incorporation of 14,15-EET into astrocytes was lower (93,390 +/- 11,121 dpm/5 x 10(6) cells) than incorporation of 8,9-EET (226,500 +/- 5,567 dpm/5 x 10(6) cells) and arachidonic 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) > phosphatidylethanolamine. In contrast, 8,9-EET and arachidonic acid were exclusively incorporated into PC. During incubation, astroglial epoxide hydrolase selectively metabolized 14,15-EET, but not 8,9-EET, to its vic-diol. Although 4-phenylchalcone oxide, a potent inhibitor of epoxide hydrolase, completely inhibited 14,15-EET metabolism, a large amount of cell-incorporated radioactivity remained as free 14,15-EET. Long-term exposure of astrocytes to 4 beta-phorbol 12-myristate 13-acetate (4 beta-PMA) resulted in a time-dependent incorporation of 14,15-EET into PI but not in control cells exposed to 4 alpha-phorbol 12,13-didecanoate. PKC down-regulation completely inhibited epoxide hydrolase metabolism of 14,15-EET. Following recovery of down-regulated PKC, 1 week after treatment with 4 beta-PMA, astrocytes regained their normal pattern of low incorporation of 14,15-EET. Protein kinase C (PKC) inhibition by staurosporine enhanced 14,15-EET incorporation without affecting its metabolism to 14,15-dihydroxyeicosatrienoic acid.(ABSTRACT TRUNCATED AT 250 WORDS)
Shivachar
A C
AC
Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298-0613, USA.
Willoughby
K A
KA
Ellis
E F
EF
D015126:8,11,14-Eicosatrienoic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D001253:Astrocytes / Q000378:metabolism*; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D010716:Phosphatidylinositols / Q000378:metabolism; D010740:Phospholipases / Q000037:antagonists & inhibitors; D010741:Phospholipases A / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
D010716:Phosphatidylinositols; D010743:Phospholipids; D016718:Arachidonic Acid; C046782:14,15-epoxy-5,8,11-eicosatrienoic acid; D011493:Protein Kinase C; D010740:Phospholipases; D010741:Phospholipases A; D015126:8,11,14-Eicosatrienoic Acid