11-12-epoxy-5-8-14-eicosatrienoic-acid and 14-15-dihydroxyeicosatrienoic-acid

Cytochrome P450 (P450) eicosanoids regulate vascular tone, renal tubular transport, cellular proliferation, and inflammation. Both the CYP4A omega-hydroxylases, which catalyze 20-hydroxyeicosatetraenoic acid (20-HETE) formation, and soluble epoxide hydrolase (sEH), which catalyzes epoxyeicosatrienoic acid (EET) degradation to the dihydroxyeicosatrienoic acids (DHETs), are induced upon activation of peroxisome proliferator-activated receptor alpha (PPARalpha) by fatty acids and fibrates. In contrast, the CYP2C epoxygenases, which are responsible for EET formation, are repressed after fibrate treatment. We show here that P450 eicosanoids can bind to and activate PPARalpha and result in the modulation of PPARalpha target gene expression. In transactivation assays, 14,15-DHET, 11,2-EET, and 20-HETE were potent activators of PPARalpha. Gel shift assays showed that EETs, DHETs, and 20-HETE induced PPARalpha-specific binding to its cognate response element. Expression of apolipoprotein A-I was decreased 70% by 20-HETE, whereas apolipoprotein A-II expression was increased up to 3-fold by 11,12-EET, 14,15-DHET, and 20-HETE. In addition, P450 eicosanoids induced CYP4A1, sEH, and CYP2C11 expression, suggesting that they can regulate their own levels. Given that P450 eicosanoids have multiple cardiovascular effects, pharmacological modulation of their formation and/or degradation may yield therapeutic benefits.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Apolipoprotein A-I; Apolipoprotein A-II; Aryl Hydrocarbon Hydroxylases; Cell Line, Tumor; Cytochrome P-450 CYP2J2; Cytochrome P-450 CYP4A; Cytochrome P-450 Enzyme System; Cytochrome P450 Family 2; Cytochrome P450 Family 4; Dose-Response Relationship, Drug; Eicosanoids; Epoxide Hydrolases; Gene Expression Regulation, Enzymologic; Hepatocytes; Humans; Hydroxyeicosatetraenoic Acids; Peroxisome Proliferators; PPAR alpha; PPAR gamma; Pyrimidines; Rats; Rats, Sprague-Dawley; Response Elements; Retinoid X Receptors; RNA, Messenger; Steroid 16-alpha-Hydroxylase; Transcriptional Activation; Transfection

Epoxyeicosatrienoic acids (EETs) are potent vasodilators produced by endothelial cells. In many vessels, they are an endothelium-derived hyperpolarizing factor (EDHF). However, it is unknown whether they act as an EDHF on platelets and whether this has functional consequences.. Flow cytometric measurement of platelet membrane potential using the fluorescent dye DiBac4 showed a resting potential of -58+/-9 mV. Different EET regioisomers hyperpolarized platelets down to -69+/-2 mV, which was prevented by the non-specific potassium channel inhibitor charybdotoxin and by use of a blocker of calcium-activated potassium channels of large conductance (BK(Ca) channels), iberiotoxin. EETs inhibited platelet adhesion to endothelial cells under static and flow conditions. Exposure to EETs inhibited platelet P-selectin expression in response to ADP. Stable overexpression of cytochrome P450 2C9 in EA.hy926 cells (EA.hy2C9 cells) resulted in release of EETs and a factor that hyperpolarized platelets and inhibited their adhesion to endothelial cells. These effects were again inhibited by charybdotoxin and iberiotoxin.. EETs hyperpolarize platelets and inactivate them by inhibiting adhesion molecule expression and platelet adhesion to cultured endothelial cells in a membrane potential-dependent manner. They act as an EDHF on platelets and might be important mediators of the anti-adhesive properties of vascular endothelium.

Topics: 8,11,14-Eicosatrienoic Acid; Apamin; Aryl Hydrocarbon Hydroxylases; Biological Factors; Blood Platelets; Cells, Cultured; Charybdotoxin; Cytochrome P-450 CYP2C9; Endothelial Cells; Endothelium, Vascular; Humans; Hydroxyeicosatetraenoic Acids; Ion Channels; Membrane Potentials; Peptides; Platelet Adhesiveness; Platelet Aggregation; Potassium Channels; Recombinant Fusion Proteins; Transfection; Umbilical Veins

Three genes cloned from Fundulus heteroclitus (killifish) define a new P450 subfamily, CYP2P. Structurally, the CYP2Ps are related to fish CYP2Ns and mammalian CYP2Js. CYP2P transcripts are expressed predominantly in liver and intestine. CYP2P3 coexpressed with P450 oxidoreductase in a baculovirus system catalyzed benzphetamine-N-demethylation and arachidonic acid oxidation, forming 14,15-, 11,12-, and 8,9-epoxyeicosatrienoic acids and 19-hydroxyeicosatetraenoic acid. CYP2P3 regio- and enantioselectivities with arachidonic acid were remarkably similar to human CYP2J2 and rat CYP2J3. Epoxyeicosatrienoic acids and their corresponding hydration products, the dihydroxyeicosatrienoic acids, were detected in killifish liver and intestine, indicating metabolism of arachidonic acid by killifish P450s in vivo. Levels of these products in killifish intestine were higher than those in mammalian intestine. 12-O-Tetradecanoyl phorbol 13-acetate suppressed expression of CYP2P2 and CYP2P3 in killifish intestine; fasting itself suppressed expression of CYP2P2/3 but not CYP2P1. In rat intestine fasting similarly depressed the levels of CYP2J proteins. The CYP2Ps and the CYP2Js appear to be derived from a common ancestral gene, likely a fatty acid monooxygenase.

Topics: 8,11,14-Eicosatrienoic Acid; Amino Acid Sequence; Animals; Arachidonic Acid; Benzphetamine; Cloning, Molecular; Conserved Sequence; Cytochrome P-450 Enzyme System; Fasting; Fundulidae; Gene Expression Regulation, Enzymologic; Hydroxyeicosatetraenoic Acids; Male; Molecular Sequence Data; Multigene Family; Organ Specificity; Phylogeny; Rats; Rats, Inbred F344; RNA, Messenger; Sequence Alignment; Tetradecanoylphorbol Acetate; Vertebrates

Epoxyeicosatrienoic acids (EETs) are potent regulators of vascular homeostasis and are bound by cytosolic fatty acid-binding proteins (FABPs) with K(d) values of approximately 0.4 microM. To determine whether FABP binding modulates EET metabolism, we examined the effect of FABPs on the soluble epoxide hydrolase (sEH)-mediated conversion of EETs to dihydroxyeicosatrienoic acids (DHETs). Kinetic analysis of sEH conversion of racemic [(3)H]11,12-EET yielded K(m) = 0.45 +/- 0.08 microM and V(max) = 9.2 +/- 1.4 micromol min(-1) mg(-)(1). Rat heart FABP (H-FABP) and rat liver FABP were potent inhibitors of 11,12-EET and 14,15-EET conversion to DHET. The resultant inhibition curves were best described by a substrate depletion model, with K(d) = 0.17 +/- 0.01 microM for H-FABP binding to 11,12-EET, suggesting that FABP acts by reducing EET availability to sEH. The EET depletion by FABP was antagonized by the co-addition of arachidonic acid, oleic acid, linoleic acid, or 20-hydroxyeicosatetraenoic acid, presumably due to competitive displacement of FABP-bound EET. Collectively, these findings imply that FABP might potentiate the actions of EETs by limiting their conversion to DHET. However, the effectiveness of this process may depend on metabolic conditions that regulate the levels of competing FABP ligands.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acids; Binding, Competitive; Carrier Proteins; Epoxide Hydrolases; Fatty Acid-Binding Protein 7; Fatty Acid-Binding Proteins; Hydroxyeicosatetraenoic Acids; Kinetics; Ligands; Linoleic Acid; Models, Chemical; Myocardium; Neoplasm Proteins; Nerve Tissue Proteins; Oleic Acid; Rats; Recombinant Proteins; Solubility; Water

A modification of the human monooxygenase system have been previously associated with the sudden infant death syndrome (SIDS): the hepatic CYP2C content was markedly enhanced and resulted from an activation of CYP2C gene transcription. To determine the possible consequence of the up-regulation of CYP2C in SIDS, we examined the metabolism of arachidonic acid (AA) an endogenous substrate of CYP2C involved in the physiologic regulation of vascular tone. The overall AA metabolism was extremely low during the fetal period and rose after birth to generate 14,15 epoxyeicosatrienoic acid (EET), 11,12 EET and the sum of 5,6 dihydroxyeicosatrienoic acid (diHETE)+omega/omega-1 hydroxy AA. In SIDS, the accumulation of CYP2C proteins was associated with a significant increase in the formation of 14,15 and 11,12 diHETE, which were shown to be supported by individually expressed CYP2C8 and 2C9 and HETE1 (presumably 15 HETE). This increase was markedly inhibited by addition of sulfaphenazole, a selective inhibitor of CYP2C9. So, we propose that the higher CYP2C content in SIDS stimulates the production of EETs and diHETEs and might have severe pathologic consequences in children.

Topics: 8,11,14-Eicosatrienoic Acid; Adult; Age Factors; Arachidonic Acid; Arachidonic Acids; Aryl Hydrocarbon Hydroxylases; Cytochrome P-450 CYP2C8; Cytochrome P-450 CYP2C9; Cytochrome P-450 Enzyme System; Humans; Hydroxyeicosatetraenoic Acids; Infant; Isoenzymes; Liver; Microsomes, Liver; NADP; Recombinant Proteins; Steroid 16-alpha-Hydroxylase; Steroid Hydroxylases; Sudden Infant Death; Up-Regulation

Cytochrome P450 epoxygenases convert arachidonic acid into 4 epoxyeicosatrienoic acid (EET) regioisomers, which were recently identified as endothelium-derived hyperpolarizing factors in coronary blood vessels. Both EETs and their dihydroxyeicosatrienoic acid (DHET) metabolites have been shown to relax conduit coronary arteries at micromolar concentrations, whereas the plasma concentrations of EETs are in the nanomolar range. However, the effects of EETs and DHETs on coronary resistance arterioles have not been examined. We administered EETs and DHETs to isolated canine coronary arterioles (diameter, 90.0+/-3.4 microm; distending pressure, 20 mm Hg) preconstricted by 30% to 60% of the resting diameter with endothelin. All 4 EET regioisomers produced potent, concentration-dependent vasodilation (EC50 values ranging from -12.7 to -10.1 log [M]) and were approximately 1000 times more potent than reported in conduit coronary arteries. The vasodilation produced by 14,15-EET was not attenuated by removal of the endothelium and indicated a direct action of 14,15-EET on microvascular smooth muscle. Likewise, 14,15-DHET, 11,12-DHET, 8,9-DHET, and the delta-lactone of 5,6-EET produced extremely potent vasodilation (EC50 values ranging from -15.8 to -13.1 log [M]). The vasodilation produced by these eicosanoids was highly potent in comparison to that produced by other vasodilators, including arachidonic acid (EC50=-7.5 log [M]). The epoxide hydrolase inhibitor, 4-phenylchalone oxide, which blocked the conversion of [3H]14,15-EET to [3H]14,15-DHET by canine coronary arteries, did not alter arteriolar dilation to 11,12-EET; thus, the potent vasodilation induced by EETs does not require formation of DHETs. In contrast, charybdotoxin (a KCa channel inhibitor) and KCl (a depolarizing agent) blocked vasodilation by 11,12-EET and 11,12-DHET. We conclude that EETs and DHETs potently dilate canine coronary arterioles via activation of KCa channels. The preferential ability of these compounds to dilate resistance blood vessels suggests that they may be important regulators of coronary circulation.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Calcimycin; Coronary Vessels; Dogs; Dose-Response Relationship, Drug; Female; Hydroxyeicosatetraenoic Acids; Male; Microcirculation; Potassium Channels; Vasodilator Agents