5-6-epoxy-8-11-14-eicosatrienoic-acid and 20-hydroxy-5-8-11-14-eicosatetraenoic-acid

Arachidonic acid is metabolized to biologically active substances by three major enzyme systems including cyclooxygenases, lipoxygenases and cytochrome P450s. The third pathway, P450-dependent pathway, includes allylic oxidation, omega-hydroxylation, and epoxidation of arachidonic acid. Of these metabolites, the physiological role of 20-hydroxyeicosatetraenoic acid (20-HETE) produced by CYP4A isoforms has been extensively studied. 20-HETE affects ion transport, constricts blood vessels and participates in tubuloglomerular feed back. Increased production of 20-HETE is a major factor in elevating blood pressure in spontaneously hypertensive rats (SHR). We have found that CYP4A2 level in SHR is much higher than that of normotensive rat. Recently, factors of endothelial origin other than nitric oxide and prostaglandins were reported. Inhibitors of P450-dependent arachidonic acid metabolism greatly reduce the vasodilator effect and this factor is speculated to be an epoxide of arachidonic acid. We have isolated CYP2C23 from rat kidney and have found that it produces arachidonic acid epoxides. We have investigated changes in the CYP2C23 levels in physiological and pathophysiological conditions. Multiple pathways of arachidonic acid metabolism by P450 have been reported and the diverse properties of these metabolites and the wide distribution of the P450 system make them prime candidates for participation in regulatory mechanisms of the circulation and transporting epithelia.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Blood Pressure; Cytochrome P-450 CYP2J2; Cytochrome P-450 CYP4A; Cytochrome P-450 Enzyme System; Hydroxyeicosatetraenoic Acids; Ion Transport; Mixed Function Oxygenases; Rats; Rats, Inbred SHR; Vasoconstriction

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Blood Pressure; Cells, Cultured; Cytochrome P-450 Enzyme System; Hydroxyeicosatetraenoic Acids; Kidney; Sodium-Potassium-Exchanging ATPase; Vasodilator Agents

Cytochrome P450 metabolites of arachidonic acid (AA) belong to eicosanoids and are potent lipid mediators of inflammation. It is well-known that eicosanoids play an important role in numerous pathophysiological processes. Therefore, quantitative analysis of cytochrome P450 metabolites of AA, including hydroxyeicosatetraenoic acids (HETEs), epoxyeicosatreinoic acids (EETs), and dihydroxyeicosatrienoic acids (DHETs) can provide crucial information to uncover underlying mechanisms of cytochrome P450 metabolites of AA related diseases. Herein, we developed a highly sensitive method to identify and quantify HETEs, EETs, and DHETs in lipid extracts of biological samples based on stable isotope probe labeling coupled with ultra high-performance liquid chromatography/mass spectrometry. To this end, a pair of stable isotope probes, 2-dimethylaminoethylamine (DMED) and d4-2-dimethylaminoethylamine (d4-DMED), were utilized to facilely label eicosanoids. The heavy labeled eicosanoid standards were prepared and used as internal standards for quantification to minimize the matrix and ion suppression effects in mass spectrometry analysis. In addition, the detection sensitivities of DMED labeled eicosanoids improved by 3-104 folds in standard solution and 5-138 folds in serum matrix compared with unlabeled analytes. Moreover, a good separation of eicosanoids isomers was achieved upon DMED labeling. The established method provided substantial sensitivity (limit of quantification at sub-picogram), high specificity, and broad linear dynamics range (3 orders of magnitude). We further quantified cytochrome P450 metabolites of AA in rat liver, heart, brain tissues and human serum using the developed method. The results showed that 19 eicosanoids could be distinctly detected and the contents of 11-, 15-, 16-, 20-HETE, 5,6-EET, and 14,15-EET in type 2 diabetes mellitus patients and 5-, 11-, 12-, 15-, 16-, 20-HETE, 8,9-EET, and 5,6-DHET in myeloid leukemia patients had significant changes, demonstrating that these eicosanoids may have important roles on the pathogenesis of type 2 diabetes mellitus and myeloid leukemia.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Brain; Case-Control Studies; Chromatography, High Pressure Liquid; Cytochrome P-450 Enzyme System; Deuterium; Diabetes Mellitus, Type 2; Eicosanoids; Humans; Hydroxyeicosatetraenoic Acids; Isotope Labeling; Leukemia, Myeloid; Liver; Male; Myocardium; Organ Specificity; Rats, Sprague-Dawley; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry

The endocannabinoid anandamide is an arachidonic acid derivative that is found in most tissues where it acts as an important signaling mediator in neurological, immune, cardiovascular, and other functions. Cytochromes P450 (P450s) are known to oxidize arachidonic acid to the physiologically active molecules hydroxyeicosatetraenoic acids (HETEs) and epoxyeicosatrienoic acids (EETs), which play important roles in blood pressure regulation and inflammation. To determine whether anandamide can also be oxidized by P450s, its metabolism by human liver and kidney microsomes was investigated. The kidney microsomes metabolized anandamide to a single mono-oxygenated product, which was identified as 20-HETE-ethanolamide (EA). Human liver microsomal incubations with anandamide also produced 20-HETE-EA in addition to 5,6-, 8,9-, 11-12, and 14,15-EET-EA. The EET-EAs produced by the liver microsomal P450s were converted to their corresponding dihydroxy derivatives by microsomal epoxide hydrolase. P450 4F2 was identified as the isoform that is most probably responsible for the formation of 20-HETE-EA in both human kidney and human liver, with an apparent Km of 0.7 microM. The apparent Km values of the human liver microsomes for the formation of the EET-EAs were between 4 and 5 microM, and P450 3A4 was identified as the primary P450 in the liver responsible for epoxidation of anandamide. The in vivo formation and biological relevance of the P450-derived HETE and EET ethanolamides remains to be determined.

Topics: 8,11,14-Eicosatrienoic Acid; Arachidonic Acids; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System; Endocannabinoids; Epoxy Compounds; Humans; Hydrogen-Ion Concentration; Hydroxyeicosatetraenoic Acids; Kidney; Kinetics; Microsomes, Liver; Polyunsaturated Alkamides; Spectrometry, Mass, Electrospray Ionization

Neuronal activity evokes localized changes in blood flow. Although this response, termed neurovascular coupling, is widely used to monitor human brain function and diagnose pathology, the cellular mechanisms that mediate the response remain unclear. We investigated the contribution of glial cells to neurovascular coupling in the acutely isolated mammalian retina. We found that light stimulation and glial cell stimulation can both evoke dilation or constriction of arterioles. Light-evoked and glial-evoked vasodilations were blocked by inhibitors of cytochrome P450 epoxygenase, the synthetic enzyme for epoxyeicosatrienoic acids. Vasoconstrictions, in contrast, were blocked by an inhibitor of omega-hydroxylase, which synthesizes 20-hydroxyeicosatetraenoic acid. Nitric oxide influenced whether vasodilations or vasoconstrictions were produced in response to light and glial stimulation. Light-evoked vasoactivity was blocked when neuron-to-glia signaling was interrupted by a purinergic antagonist. These results indicate that glial cells contribute to neurovascular coupling and suggest that regulation of blood flow may involve both vasodilating and vasoconstricting components.

Topics: 8,11,14-Eicosatrienoic Acid; Adenosine Triphosphate; Amidines; Animals; Arterioles; Calcium Signaling; Caproates; Cyclic N-Oxides; Cytochrome P-450 CYP2J2; Cytochrome P-450 CYP4A; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Eye Proteins; Hydrazines; Hydroxyeicosatetraenoic Acids; Imidazoles; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Male; Miconazole; Neuroglia; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Photolysis; Proadifen; Purinergic Antagonists; Rats; Rats, Long-Evans; Retinal Vessels; Signal Transduction; Vasoconstriction; Vasodilation; Vasomotor System

The 5,6 epoxyeicosatrienoic acid (5,6-EET) exhibits a range of biological activities but the functional significance of this labile eicosanoid is unknown due, in part, to difficulties of quantitation in biological samples. We have developed a sensitive and specific method to measure 5,6-EET utilizing its selective capacity to form a lactone. The initial conversion of 5,6-EET and 5,6-dihydroxyeicosatrienoic acid (5,6-DHT) to 5,6-delta-lactone is followed by selective purification using reverse phase high performance liquid chromatography (HPLC), reconversion to 5,6-DHT and quantitation by gas chromatography-mass spectrometry (GCMS). In oxygenated Krebs' buffer, 5,6-EET degrades to 5,6-delta-lactone and 5,6-DHT with a t1/2 approximately 8 min. In the presence of camphorsulfonic acid, 5,6-EET and 5,6-DHT convert to a single HPLC peak (lambda = 205) comigrating with 5,6-delta-lactone. Incubation of 5,6-delta-lactone with triethylamine resulted in a single HPLC peak with the retention time of 5,6-DHT. In the perfusate from the isolated kidney, release of 5,6-EET (20 +/- 5 pg/ml), measured indirectly via conversion to 5,6-DHT, was approx. 6-fold less than that reported for prostaglandin E2 (PGE2) and 20-HETE. The coronary perfusate concentration of 5,6 EET was 9 +/- 2 pg/ml. 5,6-EET recovered from renal and coronary perfusates was increased 2-fold to 45.5 +/- 5.5 pg/ml and 21.6 +/- 6.3 pg/ml, respectively, by arachidonic acid.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Chromatography, High Pressure Liquid; Dinoprostone; Gas Chromatography-Mass Spectrometry; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Kidney; Male; Myocardium; Perfusion; Rats; Rats, Wistar; Reference Standards; Sensitivity and Specificity

Recent studies from this laboratory demonstrated that bradykinin transiently elevates intracellular Ca2+ and inhibits Cl-reabsorption in the in vitro microperfused medullary thick ascending limb (mTAL) of the rat. The present study was designed to identify the intracellular signaling mechanism(s) that mediate this response. Preincubation with the intracellular calcium chelator BAPTA (10(-5) M) completely eliminated the bradykinin-dependent increase in intracellular Ca2+ and the suppression of Cl- transport. Preincubation with the cGMP-dependent protein kinase inhibitor H-89 (10(-5) M) had no effect on the transport response to bradykinin. In contrast, 17-octadecynoic acid (17-ODYA; 10(-5) M), a suicide-substrate inhibitor of renal cytochrome P450 omega-hydroxylase, completely blocked the transport response to bradykinin, while the cyclooxygenase inhibitor sodium meclofenamate (10(-5) M) had no effect. Finally, addition of the cytochrome P450 omega-hydroxylase metabolite 20-hydroxyeicosatetraenoic acid (20-HETE; 10(-8) M) to the bathing medium significantly inhibited Cl- transport in the mTAL (delta -39 +/- 6.0%; p < 0.05), while the epoxygenase metabolite 5,6-epoxyeicosatrienoic acid (5,6-EET; 10(-8) M) had no effect. These data suggest that the bradykinin-dependent inhibition of Cl- transport in the mTAL of the rat is mediated by cytochrome P450 dependent metabolite(s) of arachidonic acid.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Bradykinin; Calcium; Cyclic GMP; Cyclooxygenase Inhibitors; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Enzyme Inhibitors; Fatty Acids, Unsaturated; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Kidney Tubules, Distal; Male; Rats; Rats, Sprague-Dawley; Sodium Chloride

The renovascular effects of cytochrome P450-dependent arachidonic acid (P450-AA) metabolites synthesized by rat and rabbit kidneys were studied in the rabbit isolated kidney under conditions of constant flow and examined for their dependency on cyclooxygenase relative to their expression of vasoactivity. Kidneys were perfused with Krebs-Henseleit solution, and perfusion pressure was raised to levels of 90 to 110 mm Hg with the addition of 2 to 3 microM phenylephrine to the perfusate. Close arterial injection of 1 to 20 micrograms of 5,6-, 8,9- and 11,12-epoxyeicosatrienoic acid (EET) dose-dependently decreased perfusion pressure. The 5,6-EET was the most potent and the only epoxide dependent on cyclooxygenase for expression of vasoactivity, being inhibited by indomethacin (2.8 microM). In contrast, 14,15-EET resulted in dose-dependent increases in perfusion pressure. The vasodilator effects of the omega- and omega-1 oxidation products, 20-hydroxyeicosatetraenoic acid (HETE) and the stereoisomers of 19-HETE, were also inhibited by indomethacin. Furthermore, the renal vasodilator responses to 5,6-EET were not inhibited by either superoxide dismutase (10 U) or catalase (40 U) and, therefore, were unrelated to the formation of oxygen radicals generated during transformation of the epoxide by cyclooxygenase. As 5,6-EET and 19- and 20-HETE are synthesized by the renal tubules and can affect movement of salt and water, expression of vasoactivity by P450-dependent arachidonic acid metabolites, and after release from a nephron segment, may represent a mechanism that couples altered renal tubular function to appropriate changes in local blood flow.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Blood Pressure; Cyclooxygenase Inhibitors; Cytochrome P-450 Enzyme System; Eicosanoids; Free Radicals; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Kidney; Male; Prostaglandin-Endoperoxide Synthases; Rabbits; Renal Circulation; Vascular Resistance