Differences in positional esterification of 14,15-epoxyeicosatrienoic acid in phosphatidylcholine of porcine coronary artery endothelial and smooth muscle cells

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Abstract

Epoxyeicosatrienoic acids (EETs) are readily incorporated into phospholipids of smooth muscle cells (SMC) and endothelial cells (EC). Incorporation of EETs into intact porcine coronary arteries potentiates EC-dependent relaxation, but not vasorelaxation induced by agents that act solely on SMC. To explore the potential mechanisms responsible for this difference, porcine coronary artery SMC and EC preloaded with [3H]14,15-EET were treated with calcium ionophore A23187. Although the amount of EET incorporated into EC and SMC was similar, A23187 stimulated a five-fold increase in release of radioactivity from EC, but only a 21% increase in release from SMC. Thin layer chromatography (TLC) examination of cell lipids demonstrated that >70% of the incorporated radioactivity was present in phosphatidylcholine (PC) in both SMC and EC. After treatment of EC PC with PLA2, TLC analysis indicated that ≅75% of radioactivity was present as free EET, and 25% of radioactivity was present as lyso-PC. Therefore, most of the 14,15-EET was esterified into the sn-2 position of PC in EC. However, in SMC, ≅70% of radioactivity was present as lyso-PC after PLA2 treatment, indicating that the EET was predominately esterified into the sn-1 position. In contrast, all of the 14,15-EET was esterified into the sn-2 position of PI in both EC and SMC. These results suggest that the preferential incorporation of 14,15-EET into the sn-1 position of PC in SMC may help to explain the greater retention of the compound in SMC, while incorporation into the sn-2 position of PC in EC may facilitate agonist-induced 14,15-EET release and potentiation of EC-dependent porcine coronary artery relaxation.

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.

Section snippets

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 [3H]14,15-EET

To compare the capacity of PCEC and PCSMC to incorporate EET, the cells were incubated with 1 μM [3H]14,15-EET for various times. Both cell types took up the [3H]14,15-EET rapidly. Uptake was maximum after 60 min of incubation, accounting for about 15% of the added [3H]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 [3H]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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