Differences in positional esterification of 14,15-epoxyeicosatrienoic acid in phosphatidylcholine of porcine coronary artery endothelial and smooth muscle cells
Introduction
Epoxyeicosatrienoic acids (EETs), which are derived from arachidonic acid in a reaction catalyzed by cytochrome P-450 epoxygenases, are important lipid mediators of cardiovascular function. EETs are synthesized by various tissues and cells, including coronary endothelial cells, and produce vasorelaxation in cerebral, renal and coronary circulation [1]. Because the vasodilation produced by EETs is mediated by activation of Ca2+-activated potassium channels, EETs have been proposed to function as endothelium-derived hyperpolarizing factors [2], [3].
EETs are rapidly incorporated into the phospholipids of vascular cells [4], [5], [6], [7], [8], [9]. This may be involved in intracellular regulatory processes such as calcium signaling [10], tyrosine kinase activity [11], and cytokine-induced expression of adhesion molecules [12]. In vascular endothelial and smooth muscle cells, most of the EET is incorporated into phosphatidylcholine (PC) and phosphatidylinositol (PI) [4], [5], [6], [7], [8], [9], phospholipids that are substrates for phospholipases. Activation of phospholipase A2 (PLA2) results in rapid release of fatty acids from the sn-2 position of phospholipids and plays a major role in vascular cell signaling processes.
We recently reported that incorporation of 14,15-EET and 11,12-EET into porcine coronary artery rings resulted in potentiation of endothelium-dependent, but not endothelium-independent, relaxation [7]. Endothelial and smooth muscle cells appear to have similar capacity to incorporate EET [6], [7]. However, the incorporated EETs are rapidly released from endothelial cells with a t1/2 of 2 h [9], but they are only slowly released from smooth muscle cells with a t1/2 of 16 h [5]. One factor which may account for this difference in EET release from smooth muscle versus endothelial cells is differences in the positions of PC and/or PI to which the EETs are esterified. The purpose of the present study was to evaluate this possible mechanism.
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Cell culture and incubations
Porcine coronary artery endothelial cells (PCEC) and smooth muscle cells (PCSMC) were isolated as reported previously [4], [7]. PCEC and PCSMC were grown in M-199 and DMEM supplemented with MEM non-essential amino acids, MEM vitamin solution, 15 mM HEPES, 2 mM l-glutamine and 50 μM gentamicin. Primary cultures were isolated and suspended in the medium described above containing 10% fetal bovine serum. Stocks were subcultured weekly by trypsinization. The cultures were used for experiments between
Uptake and distribution of []14,15-EET
To compare the capacity of PCEC and PCSMC to incorporate EET, the cells were incubated with 1 μM []14,15-EET for various times. Both cell types took up the []14,15-EET rapidly. Uptake was maximum after 60 min of incubation, accounting for about 15% of the added []14,15-EET in both PCSMC and PCEC (PCSMC 970±70 pmol/mg protein, PCEC 891±30 pmol/mg protein, n=3). TLC analysis of the smooth muscle lipids indicated that more than 90% of radioactivity present in the cells was in phospholipids. The
Discussion
EETs can be stored in phospholipids through ester linkages in various tissues and cells [15], [16], [17]. EETs are produced endogenously and released by endothelial cells upon stimulation with bradykinin or methacholine [18]. The endothelial cells also rapidly take up exogenous []14,15-EET, which becomes incorporated primarily into PC and PI [5], [9]. Recent studies indicate that EETs may act through membrane binding sites to produce their biological effects [19], [20]. Thus, free EET
Acknowledgements
This study was supported by American Heart Association research grants 0060413Z and 0230096N (to X.F.), a research award from University of Iowa College of Medicine (to X.F.), and by National Institutes of Health grants HL-49264 and HL-62984 (to A.A.S. and N.L.W.).
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2020, Prostaglandins and Other Lipid MediatorsCitation Excerpt :Certainly, the fish oils EPA and DHA are readily incorporated into phospholipids and the resulting polyunsaturated phospholipids are able to infiltrate lipid rafts as well as form non raft domains [27]. There is also evidence that EETs and DHETs can be incorporated into the sn-2 position of phospholipids, especially phosphatidylcholine and phosphatidylinositol [28–32]. In endothelial cells the incorporation of EETs into a phospholipid pool was reported to be catalyzed by acyl coenzyme synthase [33], and a similar PKC-mediated phenomenon has been described in astroglia [34].
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2016, Vascular PharmacologyCitation Excerpt :Receptor-mediated hydrolysis of phospholipids containing EETs may play a role in stimulus–response coupling in platelets. Certainly, EETs and DHETs can be incorporated into the sn-2 position of phospholipids, especially phosphatidylinositol and phosphatidylcholine [84–88]. In endothelial cells the incorporation of EETs into a phospholipid pool is reported to be catalyzed by acyl coenzyme synthase [89], and a similar PKC-modulated phenomenon has been described in astroglial cells [90].
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2004, Journal of Biological ChemistryCitation Excerpt :Following incubation, the medium and cells were separated, and the lipid-soluble material was extracted and assayed for total radioactivity content. In some experiments, ionophore A23187 was added to determine whether activation of a calcium-mediated signaling pathway increased the efflux of 20-[3H]HETE from the PCEC (25–28). In other experiments, either methyl arachidonoyl fluorophosphonate (MAFP) or bromoenol lactone (BEL), both obtained from Biomol (Plymouth Meeting, PA), was added to investigate the role of phospholipase A2 (PLA2) in mediating the efflux of 20-[3H]HETE from the cells.