arachidonic-acid-omega-9-hydroperoxide and arachidonic-acid-5-hydroperoxide

Electrospray ionization ion trap mass spectra of 5-, 12-, and 15-hydroperoxyeicosatetraenoic (HPETE), hydroxyeicosatetraenoic (HETE), and ketoeicosatetraenoic (KETE) acids were recorded. The HPETE were partly dehydrated to the corresponding KETE in the heated capillary of the mass spectrometer. 12-HPETE and 15-HPETE were also converted to KETE by collision-induced dissociation (CID) in the ion trap, whereas CID of 5-HPETE yielded little formation of 5-KETE. Subcellular fractions of bovine corneal epithelium were incubated with arachidonic acid (AA) and the metabolites were analyzed. 15-HETE and 12-HETE were consistently formed, whereas significant accumulation of HPETE and KETE was not detected. Biosynthesis of 12- and 15-HETE was quantified with octadeuterated 12-HETE and 15-HETE as internal standards. The average biosynthesis of 15-HETE and 12-HETE from 30 microM AA by the cytosol was 38 +/- 8 and below 3 ng/mg protein/30 min, respectively, which increased to 78 +/- 21 and 10 +/- 4 ng/mg protein/30 min in the presence of 1 mM free Ca2+. The microsomal biosynthesis was unaffected by Ca2+. The microsomes metabolized AA to 15-HETE as the main metabolite at a low protein concentration (0.3 mg/mL), whereas 12-HETE and 15-HETE were formed in a 2:1 ratio at a combined rate of 0.7 +/- 0.2 microg/mg protein/30 min at a high protein concentration (1.8 mg/mL). The level of 12-HETE in corneal epithelial cells was 50 +/- 13 pg/mg tissue, whereas the endogenous amount of 15-HETE was low or undetectable (<3 pg/mg tissue). Incubation of corneas for 20 min at 37 degrees C before processing selectively increased the amounts of 12-HETE in the epithelium fourfold to approximately 0.2 ng/mg tissue. We conclude that 12-HETE is the main endogenously formed lipoxygenase product of bovine corneal epithelium.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Cattle; Chromatography, Liquid; Epithelium, Corneal; Hydroxyeicosatetraenoic Acids; Leukotrienes; Lipid Peroxides; Lipoxygenase; Mass Spectrometry; Subcellular Fractions

The overexpression of phospholipid hydroperoxide glutathione peroxidase (PHGPx) by RBL-2H3 cells was used as the basis for an investigation of the effects of PHGPx on the formation of leukotrienes. The rates of production of leukotriene C4 (LTC4) and leukotriene B4 (LTB4) in cells that overexpressed PHGPx were 8 times lower than those in a control line of cells. The reduction in rates of production of leukotrienes apparently resulted from the increase in the PHGPx activity since control rates of formation of leukotrienes could be achieved in PHGPx-overexpressing cells upon inhibition of PHGPx activity by diethyl malate. The conversion of radioactively labeled arachidonic acid to intermediates in the lipoxygenase pathway, such as 5-hydroxyeicosatetraenoic acid (5-HETE), LTC4, and LTB4, was strongly inhibited in PHGPx-overexpressing cells that had been prelabeled with [14C]arachidonic acid. PHGPx apparently inactivated the 5-lipoxygenase that catalyzed the conversion of arachidonic acid to 5-hydroperoxyeicosatetraenoic acid (5-HPETE) since 5-HPETE is a common precursor of 5-HETE, LTC4, and LTB4. The rates of formation of LTC4 and LTB4 in PHGPx-overexpressing cells returned to control rates upon the addition of a small amount of 12-HPETE. Flow cytometric analysis revealed that the rapid burst of formation of lipid hydroperoxides induced by A23187 was suppressed in PHGPx-overexpressing cells as compared with the control lines of cells. Subcellular fractionation analysis showed that the amount of PHGPx associated with nuclear fractions from PHGPx-overexpressing cells was 3.5 times higher than that from the control line of cells. These results indicate that PHGPx might be involved in inactivation of 5-lipoxygenase via reductions in levels of the fatty acid hydroperoxides that are required for the full activation of 5-lipoxygenase. Thus, in addition to its role as an antioxidant enzyme, PHGPx appears to have a novel function as a modulator of the production of leukotrienes.

Topics: Animals; Antioxidants; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Calcimycin; Cell Line; Cell Separation; Chromatography, High Pressure Liquid; Flow Cytometry; Glutathione Peroxidase; Hydroxyeicosatetraenoic Acids; Ionophores; Leukotrienes; Phospholipid Hydroperoxide Glutathione Peroxidase; Proteins; Rats; Selenoproteins

The effects of 1 microM concentrations of arachidonic acid hydroperoxide (HPETES) products of 5-, 12- and 15-lipoxygenase on Na+, K(+)-ATPase activity were investigated in synaptosomal membrane preparations from rat cerebral cortex. 5-HPETE inhibited Na+, K(+)-ATPase activity by up to 67 %. In contrast, 12-HPETE and 15-HPETE did not inhibit Na+, K(+)-ATPase activity. In addition, neither 5-HETE or LTA4 inhibited Na+, K(+)-ATPase activity. Dose-response studies indicated that 5-HPETE was a potent (IC25 = 10(-8) M) inhibitor of Na+, K(+)-ATPase activity. These findings indicate that 5-HPETE inhibits Na+, K(+)-ATPase activity by a mechanism that is dependent on the hydroperoxide position and independent of further metabolism by 5-lipoxygenase. It is proposed that 5-HPETE production by 5-lipoxygenase and subsequent inhibition of neuronal Na+, K(+)-ATPase activity may be a mechansim for modulating synaptic transmission.

Topics: Animals; Arachidonate Lipoxygenases; Cerebral Cortex; Enzyme Inhibitors; Hydroxyeicosatetraenoic Acids; Leukotriene A4; Leukotrienes; Lipid Peroxides; Male; Neurons; Rats; Rats, Sprague-Dawley; Sodium-Potassium-Exchanging ATPase; Synaptic Transmission; Synaptosomes

The acute activation of adrenal glucocorticoid synthesis by ACTH has long been believed to be mediated by cAMP as the major second messenger, although increases in cellular cAMP concentration have not been observed at low concentrations of ACTH. We found that steroidogenesis in bovine adrenal fasciculata-reticularis cells was activated by the addition of arachidonic acid or its 15-lipoxygenase metabolite, 15-hydroperoxyeicosatetraenoic acid. The cellular 15-lipoxygenase pathway was significantly activated by 1 pM ACTH, at which concentration no increase in cellular cAMP synthesis was observed. The 1 pM ACTH-induced stimulation of steroidogenesis was completely suppressed by a lipoxygenase inhibitor, AA-861. The stimulation was independent of the increase in cellular cAMP. These results show that the action of 1 pM ACTH on steroidogenesis may be mediated by the 15-lipoxygenase metabolite(s) as a solo second messenger. The addition of ACTH at concentrations higher than 10 pM increased both the 15-lipoxygenase activity and cellular cAMP synthesis. Under these conditions, the 15-lipoxygenase metabolite(s) and cAMP were shown to mediate the activation of steroidogenesis synergistically. The presence of a dual second messenger system could explain the stimulation of steroidogenesis by ACTH at physiological concentrations.

Topics: Animals; Arachidonate 15-Lipoxygenase; Arachidonic Acids; Benzoquinones; Bucladesine; Cattle; Cells, Cultured; Cosyntropin; Cyclic AMP; Dihydrotestosterone; Enzyme Inhibitors; Indomethacin; Isoquinolines; Kinetics; Leukotrienes; Lipid Peroxides; Lipoxygenase Inhibitors; Masoprocol; Pregnenolone; Sulfonamides; Tetrahydronaphthalenes; Zona Fasciculata; Zona Reticularis

Intracellular loading of nonhydrolyzable GTP analogs into innervated muscle cells in Xenopus cultures led to a marked increase in the frequency of spontaneous synaptic currents (SSCs), while extracellular application of the drugs at the same concentration was without effect. The increase in SSC frequency appeared to be unrelated to changes in the muscle membrane sensitivity toward acetylcholine (ACh), but resulted from an elevated spontaneous ACh secretion from the presynaptic nerve terminal. Postsynaptic loading of arachidonic acid (AA) produced a similar effect as the GTP analogs, and the potentiation effect of both GTP analogs and AA was reversed by an inhibitor of AA metabolism, AA861. Further studies indicate that a lipoxygenase metabolite, 5-HPETE, appears to be a likely candidate for the retrograde factor involved in modulating ACh secretion. These results suggest that G protein activation of the AA cascade in the postsynaptic cell could produce a retrograde signal to modulate transmitter secretion from the presynaptic nerve terminal at developing synapses.

Topics: Acetylcholine; Animals; Arachidonic Acid; Benzoquinones; Cyclic AMP; Diglycerides; Electric Conductivity; Embryo, Nonmammalian; Guanosine Triphosphate; Leukotrienes; Neuromuscular Junction; Organ Culture Techniques; Synapses; Xenopus

Oxygen radical-induced genetic damage may be mediated by products of lipid peroxidation, in particular, arachidonic acid. Several isomeric hydroxy- and hydroperoxy-6,8,11,14-eicosatetraenoic acids (HETEs and HPETEs), intermediates of arachidonic acid metabolism, were evaluated for their ability to cause sister chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells. Both HETEs and HPETEs induced SCEs in a dose-dependent fashion at concentrations of 5, 10, and 20 microM. At each concentration, HETEs were more effective in producing SCEs than the corresponding HPETEs. Each of the isomeric forms used were equally effective in producing genetic damage. Antioxidants (superoxide dismutase, catalase and mannitol) were protective suggesting an intermediate role for the hydroxyl radical. Iron chelation by desferrioxamine suppressed SCE induction by 45% and an additional 33% inhibition was observed upon the addition of the calcium chelator EGTA.

Topics: Animals; Catalase; Cell Line; Cricetinae; Deferoxamine; Egtazic Acid; Free Radicals; Hydroxyeicosatetraenoic Acids; Leukotrienes; Lipid Peroxidation; Lipid Peroxides; Mannitol; Sister Chromatid Exchange; Superoxide Dismutase

Sulfhydryl reagents such as dithiothreitol stabilized human leukocyte 5-lipoxygenase (5-LO) during purification. During enzyme assay, however, these reagents led to irreproducible or unexpectedly low activity. This inconsistency in the assay was eliminated by inclusion of hydroperoxyeicosatetraenoic acids (1-5 microM) during the reaction which effected a 10-20-fold stimulation of 5-LO activity. Structural studies indicated that an intact hydroperoxy function, and a long-chain fatty acyl moiety were required for 5-LO stimulation. These data suggest that human leukocyte 5-LO is activated by hydroperoxy fatty acids, and that this results in a requirement for exogenous hydroperoxide in the presence of sulfhydryl reagents.

Topics: Arachidonate Lipoxygenases; Arachidonic Acids; Enzyme Activation; Humans; Leukocytes; Leukotrienes; Lipid Peroxides; Lipoxygenase; Sulfhydryl Reagents

Isolated pancreatic islets from the rat have been demonstrated by stable isotope dilution-mass spectrometric methods to synthesize the 12-lipoxygenase product 12-hydroxyeicosatetraenoic acid (12-HETE) in amounts of 1.7 to 2.8 ng per 10(3) islets. No detectable amounts of 5-HETE and only trace amounts of 15-HETE could be demonstrated by these methods. Nordihydroguaiaretic acid (NDGA) and BW755C have been demonstrated to inhibit islet 12-HETE synthesis and also to inhibit glucose-induced insulin secretion. Inhibition of insulin secretion and of 12-HETE synthesis exhibited similar dependence on the concentration of these compounds. Eicosa-5,8,11,14-tetrynoic acid (ETYA) also inhibited glucose-induced insulin secretion, as previously reported, at concentrations which inhibit islet 12-HETE synthesis. Exogenous 12-HETE partially reversed the suppression of glucose-induced insulin secretion by lipoxygenase inhibitors, but exogenous 12-hydroperoxyeicosatetraenoic acid (12-HPETE), 15-HPETE, 5-HPETE, 15-HETE, or 5-HETE did not reverse this suppression. These observations argue against the recently suggested hypothesis that islet synthesis of 5-HETE modulates insulin secretion. Suppression of glucose-induced insulin secretion by ETYA, BW755C and NDGA may be due to inhibition of the islet 12-lipoxygenase by these compounds. The possibility that other processes involved in glucose-induced insulin secretion are inhibited by ETYA, BW755C and NDGA cannot yet be excluded.

Topics: 4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyrazol-3-amine; 5,8,11,14-Eicosatetraynoic Acid; Animals; Arachidonate Lipoxygenases; Arachidonic Acid; Arachidonic Acids; Gas Chromatography-Mass Spectrometry; Hydroxyeicosatetraenoic Acids; Insulin; Insulin Secretion; Islets of Langerhans; Leukotrienes; Lipoxygenase; Male; Pyrazoles; Rats; Rats, Inbred Strains

Previous studies in a line of rat basophilic leukemia (RBL 1) cells have indicated that the slow reacting substance (SRS) made during stimulation with the divalent cation ionophore, A23187, is derived from arachidonic acid (AA). In the present report, various inhibitors of AA metabolism were compared with regard to their effects on SRS formation and incorporation of radioactivity from [1-14C]-AA into known metabolites of the lipoxygenase and cyclooxygenase pathways. An apparently close parallel between lipoxygenase product formation and SRS synthesis is demonstrated. In addition, exogenous 5-hydroperoxy-eicosatetraenoic acid (5-HPETE) has been shown to markedly enhance SRS synthesis, even when A23187 is absent. The data provide very strong evidence that SRS is produced through the lipoxygenase pathway.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 5,8,11,14-Eicosatetraynoic Acid; Animals; Arachidonic Acids; Autacoids; Basophils; Leukemia; Leukotrienes; Linoleic Acids; Linolenic Acids; Lipoxygenase Inhibitors; Muscle Contraction; Peroxides; Pyrogallol; Rats