Arachidonic acid metabolism in isolated pancreatic islets. IV. Negative ion-mass spectrometric quantitation of monooxygenase product synthesis by liver and islets.
Deuterium-labelled standards of four regionally isomeric epoxyeicosatrienoic acids (EETs) and their hydrolysis products, the dihydroxyeicosatrienoic acids (DHETs), have been prepared and analyzed by capillary column gas chromatography (GC)-negative ion (NI)-methane chemical ionization (MCI)-mass spectrometry (MS) as the pentafluorobenzyl esters. As little as 40 pg of these compounds were readily visualized by these methods, and the deuterium-labelled standards were used in a stable isotope dilution mass spectrometric assay which was linear from near the detection limit over several orders of magnitude. NADPH-dependent synthesis of both EETs and DHETs from arachidonate by hepatic microsomal cytochrome P-450-mono-oxygenase activity was demonstrable with these methods and was significantly suppressed by the compound BW755C (500 microM), but not by eicosa-5,8,11,14-tetraynoic acid (ETYA, 20 microM) or by nordihydroguaiaretic acid (NDGA, 50 microM). All three compounds suppress glucose-induced insulin secretion and 12-hydroxyeicosatetraenoic acid (12-HETE) synthesis by isolated pancreatic islets with similar concentration dependence. Microsomes derived from isolated pancreatic islets synthesized less than 3% of the EET and DHET compounds as a comparable amount of hepatic microsomes. Intact islets synthesized less than 3% by mass of the EET and DHET compounds compared to the mass of 12-HETE produced by the islets. Islets also failed to convert 3H-labelled arachidonate to 3H-labelled EETs or DHETs under conditions where conversion to [3H]12-HETE and to [3H]prostaglandin E2 (but not to [3H]leukotriene C4, D4, or E4) was clearly demonstrable. Neither exogenous EETs nor leukotriene C4 stimulated insulin secretion from the isolated islets or reversed the suppression of glucose-induced secretion by the lipoxygenase inhibitor BW755C. The cytochrome P-450-monooxygenase inhibitor, metyrapone (50 microM), did not influence insulin secretion from the isolated islets under conditions where the lipoxygenase inhibitor, NDGA, suppressed glucose-induced secretion. These observations argue against the recently suggested hypothesis that EETs derived from arachidonate by monooxygenase action participate in glucose-induced insulin secretion by isolated pancreatic islets.
D015772:4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyrazol-3-amine; D015117:5,8,11,14-Eicosatetraynoic Acid / Q000378:metabolism; D015126:8,11,14-Eicosatrienoic Acid / Q000378:metabolism; D000818:Animals; D001091:Arachidonate Lipoxygenases; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000378:metabolism*; D002396:Catechols / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System; D008401:Gas Chromatography-Mass Spectrometry; D006893:Hydroxyeicosatetraenoic Acids / Q000378:metabolism; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D008084:Lipoxygenase / Q000378:metabolism; D009637:Masoprocol; D008797:Metyrapone / Q000494:pharmacology; D008861:Microsomes / Q000201:enzymology; D008862:Microsomes, Liver / Q000201:enzymology*; D009249:NADP / Q000378:metabolism; D010105:Oxygenases / Q000378:metabolism; D011720:Pyrazoles / Q000494:pharmacology; D051381:Rats; D013189:SRS-A / Q000494:pharmacology
D001095:Arachidonic Acids; D002396:Catechols; D006893:Hydroxyeicosatetraenoic Acids; D007328:Insulin; D011720:Pyrazoles; D013189:SRS-A; D015117:5,8,11,14-Eicosatetraynoic Acid; D016718:Arachidonic Acid; D009249:NADP; D015772:4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyrazol-3-amine; D009637:Masoprocol; D003577:Cytochrome P-450 Enzyme System; D010105:Oxygenases; D001091:Arachidonate Lipoxygenases; D008084:Lipoxygenase; D015126:8,11,14-Eicosatrienoic Acid; D008797:Metyrapone