arachidonic-acid-omega-9-hydroperoxide and 5-hydroxy-6-8-11-14-eicosatetraenoic-acid

Capsaicin, a pungent ingredient of hot peppers, causes excitation of small sensory neurons, and thereby produces severe pain. A nonselective cation channel activated by capsaicin has been identified in sensory neurons and a cDNA encoding the channel has been cloned recently. However, an endogenous activator of the receptor has not yet been found. In this study, we show that several products of lipoxygenases directly activate the capsaicin-activated channel in isolated membrane patches of sensory neurons. Among them, 12- and 15-(S)-hydroperoxyeicosatetraenoic acids, 5- and 15-(S)-hydroxyeicosatetraenoic acids, and leukotriene B(4) possessed the highest potency. The eicosanoids also activated the cloned capsaicin receptor (VR1) expressed in HEK cells. Prostaglandins and unsaturated fatty acids failed to activate the channel. These results suggest a novel signaling mechanism underlying the pain sensory transduction.

Topics: Animals; Capsaicin; Cell Line; Cells, Cultured; Dinoprostone; Eicosanoids; Ganglia, Spinal; Humans; Hydroxyeicosatetraenoic Acids; Inflammation; Ion Channel Gating; Leukotriene B4; Leukotrienes; Ligands; Lipid Peroxides; Lipoxygenase; Molecular Structure; Neurons, Afferent; Prostaglandin D2; Prostaglandin H2; Prostaglandins H; Rats; Receptors, Drug; Structure-Activity Relationship

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

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonate 12-Lipoxygenase; Arachidonate 5-Lipoxygenase; Arachidonate Lipoxygenases; Arachidonic Acids; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Leukocytes; Leukotrienes; Lipid Peroxides; Substrate Specificity; Swine

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

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonate Lipoxygenases; Arachidonic Acids; Calcimycin; Humans; Hydroxyeicosatetraenoic Acids; Leukocytes; Leukotrienes; Lipoxygenase; SRS-A