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.