Incorporation and distribution of epoxyeicosatrienoic acids into cellular phospholipids.

K Bernstrom,K Kayganich,R.C Murphy,F.A Fitzpatrick

doi:10.1016/s0021-9258(19)50579-8

K Bernstrom, K Kayganich + Show 2 more

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https://doi.org/10.1016/s0021-9258(19)50579-8

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Abstract

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.

Highlights

From the $Department of Pharmacology C236, University of Colorado Health Sciences Center, Denver, Colorado80262 and the §National Jewish Center for Immunology and Respiratory Medicine, Denuer, Colorado80206
Phosphatidylethament, followed bynegativeionchemicalionization mass nolamine plasmalogens with 16:l or 182 at the sn-1 spectrometry of their pentafluorobenzyl estedrerivatives, inposition, or an 18:0 acyl group, and phosphatidylcho- dicate that kidney and liver contain appreciable amounts of line with16:O alkyl ether or an acyl groautpthe sn-1 epoxyeicosatrienoic acids (EETs) esterified at thesn-2 position of phospholipid subclasses position incorporated allpossible epoxyeicosatrienoic [13,14,15,16,17]
Classes and Molecular Species-Mast cells were incubated with 5,6, 8,9, ll,lZ, or 14,15-EET for 2 h, and their phospholipids were analyzed by FAB/triple quadrupole mass spectrometry to generate (M-H)- ions for phosphatidylethanolamine and phosphatidylinositol or (M-CH3)- ions for phose, phatidylcholine species [303,3, 34]

Summary

RESULTS

Cellular Incorporation of 14,15-EET-Murine mast cells rapidly incorporated 14,15-EET5, -HETE,or arachidonic acid into theirphospholipids. Steady-state incorporation of [3H]14,15-EET (470 pmol/106 cells) waslower than ['HHIarachidonic acid (665 pmol/106 cells) or [3H]5HETE (554 pmol/106 cells) (Fig. 1, lower panel). Hydrolytic instability of 14,15-EET might partly account for this difference, since 14,15-diHETE, the hydration product of 14,15EET, was not readily incorporated, reaching a steady-state content of 151pmol/106cells. After a 2-h incubation [3H]14,15-EET was distributed among phospholipids in the order PE > PC > PI (Fig. 1, top panel). Phosphatidylserine, neutral lipids, and free fatty acid pools in mast cells contained insignificant amounts of labeled material. 90% of maximal incorporation required 1.9 h for arachidonic acid (AA), 2.6 h for 5-HETE, and 4.5 h was distributed among PE.

Effect of albumin on EETstability and cellular incorporation

Identification of Separate EET Isomers within Phospholipid

PC rnin"

DISCUSSION