Abstract
Epoxyeicosatrienoic acids (EETs) are synthesized by cytochrome P-450 monooxygenases and released into the blood. When taken up by vascular endothelial and smooth muscle cells, the EETs are primarily esterified to phospholipids or converted to dihydroxyeicosatetraenoic acids (DHETs) and released. In the present studies, radiolabeled 8,9-, 11,12-, and 14,15-DHETs released into the medium from vascular smooth muscle cells were isolated and incubated for 4-16 h with cultured bovine aortic endothelial cells. The uptake ranged from 2 to 50% for the three regioisomers. Hydrolysis of the endothelial lipids and gas chromatographic-mass spectral analyses of the products indicated that all three DHET regioisomers were incorporated intact into phosphatidylcholine and phosphatidylinositol. Similar incubations with EETs confirmed that small amounts of DHETs were also esterified to endothelial phospholipids. These studies indicate that DHETs are incorporated into phospholipids either at the time of EET conversion to DHET or upon release and re-uptake of DHETs. Beside demonstrating for the first time that fatty acid diols are incorporated intact into endothelial lipids, these studies raise the possibility that both EETs and DHETs remain long enough in the vascular wall to produce chronic vasoactive effects.
Highlights
In contrast to EET vasoactivity, very little is known about the disposition of circulating EETs and their dihydroxyeicosatetraenoic acids (DHETs) products
Fitzpatrick and colleagues [28] suggested that small amounts of the vicinal diol, 14,15-DHET, are taken up by mastocytoma cells; they were unable to find any 14,15-DHET esterified to phospholipids when they were analyzed by tandem mass spectrometry
The present study demonstrates that DHET regioisomers are taken up by vascular endothelial cells and incorporated intact into membrane phospholipids
Summary
Synthesis of EET and DHET Standards—EET and DHET regioisomers were synthesized from unlabeled, tritiated, and deuterated arachidonic acids [3, 17]. The upper, aqueous phase was removed, mixed vigorously for 5 min with 5 volumes of a chloroform/methanol/phosphate buffer solution (86:14:1), and recentrifuged. The products were transferred to a silylated glass centrifuge tube, and the pH was adjusted to 7.8 – 8.0 with 0.5 M phosphate buffer (pH 6.6) This mixture was shaken with 10 volumes of ice-cold, water-saturated ethyl acetate and centrifuged (450 ϫ g for 10 min at 4 °C). After being dried under N2 and mixed with 1.0 g of unlabeled EET and DHET for retention time markers, 8000 dpm of the saponified phospholipids was dissolved in 50 l of methanol and injected onto a guard and analytical column positioned in series (50 ϫ 4.6 mm (inner diameter) plus 250 ϫ 4.6 mm (inner diameter)) containing 5-m C18 particles Individual equivalent chain length values were determined from the interpolated log (retention times)
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