Incorporation and distribution of epoxyeicosatrienoic acids into cellular phospholipids.

The different regioisomers of epoxyeicosatrienoic acids derived from cytochrome P-450 monooxygenase are readily esterified into phospholipids of mastocytoma cells. Incorporation of 14,15-epoxyeicosatrienoic acid was concentration-dependent, with Km = 1.1 microM and Vmax = 36 pmol/min/10(7) cells. Half-maximal incorporation occurred in 30 min, reaching a steady-state concentration of 470 pmol/10(6) cells. This was slightly lower than the values for arachidonic acid (665 pmol/10(6) cells) or 5-hydroxyeicosatetraenoic acid (554 pmol/10(6) cells). The distribution of 14,15-epoxyeicosatrienoic acid was preferential in the order phosphatidylethanolamine greater than phosphatidylcholine greater than phosphatidylinositol greater than phosphatidyl serine much greater than neutral lipids plus fatty acids. This contrasted with 5(S)-hydroxyeicosatetraenoic acid, which was distributed primarily into phosphatidylcholine. Fast atom bombardment/tandem mass spectrometry facilitated identification of molecular species containing epoxyeicosatrienoic acids without relying on radioisotopes. Phosphatidylethanolamine plasmalogens with 16:1 or 18:2 at the sn-1 position, or an 18:0 acyl group, and phosphatidylcholine with 16:0 alkyl ether or an acyl group at the sn-1 position incorporated all possible epoxyeicosatrienoic acid regioisomers. Under basal conditions, cells eliminated 14,15-cis-epoxyeicosatrienoic acid slowly with a half-life of 34.9 +/- 7 h. Cells stimulated with calcium ionophore A23187 eliminated 14,15-epoxyeicosatrienoic acid rapidly. It was notable that its rate of release from phosphatidylcholine and phosphatidylinositol exceeded that for arachidonic acid. A coenzyme A-independent transacylase also catalyzed the transfer of epoxyeicosatrienoic acids from mastocytoma cell membranes into 1-palmitoyl-2-lysophosphatidylcholine. The cellular incorporation, release, and distribution of epoxyeicosatrienoic acids is distinctive and contrasts with most other eicosanoids, suggesting that these compounds may have both autocoid and nonautocoid functions.