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PubMed · 10924087

Determination of EETs using microbore liquid chromatography with fluorescence detection.

K Nithipatikom·P F Pratt·W B Campbell
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Abstract

Epoxyeicosatrienoic acids (EETs) are cytochrome P-450 metabolites of arachidonic acid involved in the regulation of vascular tone. The method of microbore column high-performance liquid chromatography with fluorescence detection was developed to determine 14,15-EET, 11, 12-EET, and the mixture of 8,9-EET and 5,6-EET. Tridecanoic acid (TA) was used as an internal standard. EETs were reacted with 2-(2, 3-naphthalimino)ethyl trifluoromethanesulfonate (NT) to form highly fluorescent derivatives. A C(18) microbore column and a water-acetonitrile mobile phase were used for separation. Samples were excited at 259 nm, and the fluorescence was detected at 395 nm. The overall recoveries were 88% for EETs and 40% for TA. EETs were detected in concentrations as low as 2 pg (signal-to-noise ratio = 3). The method was used to determine the EET production from endothelial cells (ECs). Bradykinin and methacholine (10(-6) M) stimulated an increase in the production of EETs by ECs two- and fivefold, respectively. This sensitive method may be used for determination of EETs at low concentrations normally detected in complex biological samples.

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8,11,14-Eicosatrienoic AcidAnimalsBradykininCattleCells, CulturedChromatography, High Pressure LiquidCoronary VesselsEndothelium, VascularMethacholine ChlorideMicrochemistrySpectrometry, FluorescenceJournal ArticleResearch Support, U.S. Gov't, P.H.S.

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K Nithipatikom, P F Pratt, W B Campbell. 2000. Determination of EETs using microbore liquid chromatography with fluorescence detection.. https://doi.org/10.1152/ajpheart.2000.279.2.h857

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PubMed2007

Mechanisms by which epoxyeicosatrienoic acids (EETs) elicit cardioprotection in rat hearts.

Cytochrome P450 (CYP) epoxygenases and their arachidonic acid (AA) metabolites, the epoxyeicosatrienoic acids (EETs), have been shown to produce reductions in infarct size in canine myocardium following ischemia-reperfusion injury via opening of either the sarcolemmal K(ATP) (sarcK(ATP)) or mitochondrial K(ATP) (mitoK(ATP)) channel. In the present study, we subjected intact rat hearts to 30 min of left coronary artery occlusion and 2 h of reperfusion followed by tetrazolium staining to determine infarct size as a percent of the area at risk (IS/AAR, %). The results demonstrate that the two major regioisomers of the CYP epoxygenase pathway, 11,12-EET (2.5 mg/kg, iv) and 14,15-EET (2.5 mg/kg, iv) significantly reduced myocardial infarct size (IS/AAR, %) in rats as compared with control (41.9+/-2.3%, 40.9+/-1.2% versus 61.5+/-1.6%, respectively), whereas, a third regioisomer, 8,9-EET (2.5 mg/kg, iv) had no effect (55.2+/-1.4). The protective effect of pretreatment with 11,12- and 14,15-EETs was completely abolished (61.9+/-0.7%, 58.6+/-3.1%, HMR; 63.3+/-1.2%, 63.2+/-2.5%, 5-HD) in the presence of the selective sarcK(ATP) channel antagonist, HMR 1098 (6 mg/kg, iv) or the selective mitoK(ATP) channel antagonist, 5-HD (10 mg/kg, iv) given 10 min after 11,12- or 14,15-EET administration but 5 min prior to index ischemia. Furthermore, concomitant pretreatment with 11,12- or 14,15-EET in combination with the free radical scavenger, 2-mercaptopropionyl glycine (2-MPG), at a dose (20 mg/kg, iv) that had no effect on IS/AAR (57.7+/-1.3%), completely abolished the cardioprotective effect of 11,12- and 14,15-EETs (58.2+/-1.6%, 61.4+/-1.0%), respectively. These data suggest that part of the cardioprotective effects of EETs in rat hearts against infarction is the result of an initial burst of reactive oxygen species (ROS) and subsequent activation of both the sarcK(ATP) and mitoK(ATP) channel.

8,11,14-Eicosatrienoic Acid↗
PubMed2006

Beyond vasodilatation: non-vasomotor roles of epoxyeicosatrienoic acids in the cardiovascular system.

Epoxyeicosatrienoic acids (EETs), derived from arachidonic acid by cytochrome P450 epoxygenases, are potent vasodilators that function as endothelium-derived hyperpolarizing factors in some vascular beds. EETs are rapidly metabolized by soluble epoxide hydrolase to form dihydroxyeicosatrienoic acids (DHETs). Recent reports indicate that EETs have several important non-vasomotor regulatory roles in the cardiovascular system. EETs are potent anti-inflammatory agents and might function as endogenous anti-atherogenic compounds. In addition, EETs and DHETs might stimulate lipid metabolism and regulate insulin sensitivity. Thus, pharmacological inhibition of soluble epoxide hydrolase might be useful not only for hypertension but also for abating atherosclerosis, diabetes mellitus and the metabolic syndrome. Finally, although usually protective in the systemic circulation, EETs might adversely affect the pulmonary circulation.

8,11,14-Eicosatrienoic Acid↗
PubMed2006

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Endothelial nitric oxide synthase (eNOS) is a key enzyme in NO-mediated cardiovascular homeostasis and its activity is modulated by a variety of hormonal and mechanical stimuli via phosphorylation modification. Our previous study has demonstrated that epoxyeicosatrienoic acids (EETs), the cytochrome P450 (CYP)-dependent metabolites of arachidonic acid, could robustly up-regulate eNOS expression. However, the molecular mechanism underlying the effects of EETs on eNOS remains elusive. Particularly, whether and how EETs affect eNOS phosphorylation is unknown. In the present study, we investigated the effects of EETs on eNOS phosphorylation with cultured bovine aortic endothelial cells (BAECs). BAECs were either treated with exogenous EETs or infected with recombinant adeno-associated virus (rAAV) carrying CYP2C11-CYPOR, CYP102 F87V mutant and CYP2J2, respectively, to increase endogenous EETs. Both addition of EETs and CYP epoxygenase transfection markedly increased eNOS phosphorylation at its Ser1179 and Thr497 residues. Inhibition of phosphatidylinositol 3-kinase (PI3K) with LY294002 prevented EETs-induced increases of eNOS-Ser(P)1179 but had no effect on the phosphorylation status of Thr497. However, inhibitors of protein kinase B (Akt), mitogen-activated protein kinase (MAPK) and MAPK kinase could block phosphorylation of eNOS at both sites. Inhibition of these kinases also attenuated the up-regulation of eNOS expression by EETs. Finally, administration of viral CYP epoxygenases expression vectors into rats enhanced eNOS phosphorylation and function in vivo. Thus, in addition to up-regulating eNOS expression, EETs also augment eNOS function by enhancing eNOS phosphorylation. EETs-induced up-regulation of eNOS phosphorylation and expression appears to involve in both PI3K/Akt and MAPK pathways.

8,11,14-Eicosatrienoic Acid↗