arachidonic-acid-omega-9-hydroperoxide and 15-hydroxy-5-8-11-13-eicosatetraenoic-acid

There are considerable data implicating a pancreatic islet 12-lipoxy-genase in glucose-induced insulin secretion. This enzyme traditionally is conceived as converting unesterified arachidonic acid to "free" hydroperoxyeicosatetraenoic acid and metabolites thereof. However, studies employing the provision of exogenous metabolites of arachidonic acid to islet tissue fail to identify convincingly the mediator of insulin release. It is proposed that the islet lipoxygenase directly peroxidizes unsaturated fatty acids esterified within membrane phospholipids, leading to changes in ion flux and enzyme activity (particularly phospholipase A2) at the membrane level. The release of unesterified metabolites of arachidonate, although reflecting islet lipoxygenase activity, may be an epiphenomenon.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 5,8,11,14-Eicosatetraynoic Acid; Arachidonate Lipoxygenases; Arachidonic Acids; Enzyme Activation; Fatty Acids, Unsaturated; Glucose; Hydroxyeicosatetraenoic Acids; Insulin; Islets of Langerhans; Leukotrienes; Lipid Peroxides; Lipoxygenase; Membrane Lipids; Models, Biological; Phospholipases A; Phospholipases A2; Phospholipids

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

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

Arachidonic acid (AA) metabolites derived from both cyclooxygenase (COX) and lipoxygenase (LOX) pathways transduce a variety of signals related to cell growth. Here, we report that the AA LOX pathway also functions as a critical regulator of cell survival and apoptosis. Rat Walker 256 (W256) carcinosarcoma cells express 12-LOX and synthesize 12(S)- and 15(S)-hydroxyeicosatetraenoic acids as their major LOX metabolites. W256 cells transfected with 12-LOX-specific antisense oligonucleotide or antisense oligonucleotides directed to conserved regions of LOXs underwent time- and dose-dependent apoptosis. Likewise, treatment of W256 cells with various LOX but not COX inhibitors induced apoptotic cell death, which could be partially inhibited by exogenous 12(S)- or 15(S)-hydroxyeicosatetraenoic acids. The W256 cell apoptosis induced by antisense oligos and LOX inhibitors was followed by a rapid downregulation of bcl-2 protein, a dramatic decrease in the bcl-2/bax ratio, and could be suppressed by bcl-2 overexpression. In contrast, p53, which is wild type in W256 cells, did not undergo alterations during apoptosis induction. The results suggest that the LOX pathway plays an important physiological role in regulating apoptosis.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Apoptosis; Arachidonate 12-Lipoxygenase; Base Sequence; Cell Division; Cell Line; Cell Survival; Female; Homeostasis; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Leukotriene C4; Leukotrienes; Lipid Peroxides; Lipoxins; Masoprocol; Molecular Sequence Data; Oligonucleotides, Antisense; Plasmids; Rats; Transfection

High extracellular Ca2+ (Ca2+ ec) stimulates the formation of inositol phosphates and diacylglycerol and activates phospholipase A2 in porcine parathyroid cells. Ca2+ ec action is also coupled to the formation of arachidonic acid, the precursor of both the cyclooxygenase and lipoxygenase (LO) pathways. We previously reported that LO pathway products might act as second messengers and play a part in regulating PTH secretion by Ca2+ ec. We have now investigated the effects of hydroxyeicosatetranoic acids (HETEs) on PTH secretion. Collagenase-dispersed porcine parathyroid cells were incubated in low [Ca2+] (0.5 mM, maximal stimulation) with or without HETEs for three 15-min periods. 12- and 15-HETEs inhibited PTH secretion in a dose-dependent manner from 10(-12) to 10(-9) M. Maximal inhibition was with 10(-9) M. Since 12- and 15-HETEs are the metabolic reduction products of 12- and 15-HPETEs, we also examined the effect of those precursors on PTH release. 12- and 15-hydroxyperoxyeicosatetranoic acids (HPETEs) were more potent inhibitors of PTH secretion. The threshold concentrations of both HPETEs that inhibited PTH release were lower than those for HETEs: 10(-9) M suppressed PTH secretion. This effect is comparable to that of high [Ca2+] (2 mM). This provides new evidence that products of 12-LO and 15-LO pathways are potent inhibitors of PTH secretion.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Hydrogen Peroxide; Hydroxyeicosatetraenoic Acids; Leukotrienes; Lipid Peroxides; Lipoxygenase; Parathyroid Hormone; Structure-Activity Relationship; Swine; Time Factors

A modification of a method using high-performance liquid chromatography (HPLC) with chemiluminescence (CL) detection for the measurement of lipid hydroperoxides (LOOH) in human blood plasma has been developed. The system involves separation of different classes of LOOH using reverse-phase HPLC, and post-column detection of CL produced by isoluminol oxidation during the reaction of LOOH with microperoxidase. Complete ultra-violet absorption spectra are collected with an in-line diode-array detector and used to confirm a positive CL response due to LOOH, or other compounds, by the presence or absence, respectively, of the LOOH conjugated diene chromophore. We have used the method to investigate the stability of exogenous 15(S)-HPETE (a hydroperoxide of eicosatetraenoic acid) and conjugated dienes (of both 15(S)-HPETE and its reduced metabolite, 15(S)-HETE) in human plasma stored at various temperatures. A large and rapid loss of the hydroperoxide occurred in plasma incubated at 0 degrees C or 27 degrees C, whereas only a small reduction in the level of conjugated dienes was found. 15(S)-HPETE in PBS was stable under the same conditions, and zero time recovery of the hydroperoxide from denatured plasma and from buffer containing albumin was identical to that of fresh plasma. Our data suggest that the observed temperature-dependent loss of exogenous hydroperoxide from fresh plasma results from a combination of enzymatic degradation to the hydroxy derivative and binding to plasma albumin. 15(S)-HPETE was found to be stable in plasma stored at -70 degrees C for up to 2 weeks and in liquid nitrogen for 3 months in the presence of the antioxidants butylated hydroxytoluene (BHT) and desferal, with no significant loss of conjugated dienes.

Topics: Antioxidants; Blood Preservation; Chromatography, High Pressure Liquid; Humans; Hydroxyeicosatetraenoic Acids; Leukotrienes; Lipid Peroxides; Luminescent Measurements; Spectrophotometry, Ultraviolet; Temperature; Time Factors

Modulation of renin synthesis by lipoxygenase products has been studied in cultured human mesangial cells under basal conditions and in the presence of prostaglandin (PG) E2. Total renin and cyclic AMP productions were stimulated in a dose-dependent manner (0.1-10 microM) by PGE2. The stimulatory effect of PGE2 on renin production was inhibited by 12-hydroxyeicosatetraenoic acid (12-HETE) between 0.1 and 100 nM. Extracellular and intracellular renin were affected similarly. Neither basal and PGE2-dependent cyclic AMP nor basal cyclic GMP productions were modified. 15-Hydroxyeicosatetraenoic acid (15-HPETE), 12-hydroperoxyeicosatetraenoic acid (12-HPETE) and 15-hydroperoxyeicosatetraenoic acid (15-HPETE) had the same effects as 12-HETE. Intracellular calcium concentration was not modified in the presence of 12-HETE. Since oleyl-2-acetylglycerol (OAG), an analog of diacylglycerol, also inhibited PGE2-stimulated renin production, it is hypothesized that the effect of the lipoxygenase products is mediated via protein kinase C stimulation.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Angiotensin II; Calcium; Cyclic AMP; Cyclic GMP; Diglycerides; Dinoprostone; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Kidney Cortex; Leukotrienes; Lipid Peroxides; Lipoxygenase; Renin

Angiotensin II (AII) action on adrenal and smooth muscle cells is mediated via mechanisms that include changes in calcium flux and phosphoinositide hydrolysis. Phosphoinositide metabolism results in the release of arachidonic acid, a precursor of both the cyclooxygenase (CO) and lipoxygenase (LO) pathway. The effects of both LO and CO inhibitors on AII action were studied using both static incubations and perifusions of rat renal cortical slices. 12-Hydroperoxyeicosatetraenoic acid and its stable metabolite 12-hydroxyacid mimicked the inhibitory actions of AII on renin. A specific CO blocker did not alter AII inhibition of renin and a 5-LO blocker U60,257 was also ineffective, whereas the LO blockers BW755c, phenidone, and baicalein all eliminated or interfered with the action of AII on renin. All inhibition in the presence of a LO blocker was restored by adding nanomolar concentrations of 12-hydroperoxyeicosatetraenoic acid. LO inhibitors were specific for blocking AII, as they did not interfere with potassium (K+)-induced renin inhibition. These results imply that 12 and/or 15 products of the LO pathway are involved in AII action.

Topics: 4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyrazol-3-amine; Angiotensin II; Animals; Arachidonate 12-Lipoxygenase; Arachidonate Lipoxygenases; Arachidonic Acids; Cyclooxygenase Inhibitors; Epoprostenol; Flavanones; Flavonoids; Hydroxyeicosatetraenoic Acids; Kidney Cortex; Leukotrienes; Lipoxygenase Inhibitors; Male; Meclofenamic Acid; Potassium; Pyrazoles; Rats; Rats, Inbred Strains; Renin

Release of arachidonic acid from membrane phospholipids is a limiting step in the synthesis of both cyclooxygenase products and lipoxygenase products. The direct effects of prostacyclin and some lipoxygenase products on renin release were studied using rat renal cortical slices. Prostacyclin, at concentrations of 10(-5) M, stimulated renin secretion, but this effect was short-lived. Leukotrienes or their precursor, 5-hydroperoxyeicosatetraenoic acid, did not affect basal renin release. In contrast, 10(-9) M 12-hydroperoxyeicosatetraenoic acid and 10(-8) M 12-hydroxyeicosatetraenoic acid were potent inhibitors of renin secretion. Similarly, 15-hydroperoxyeicosatetraenoic acid and its hydroxy derivative, 15-hydroxyeicosatetraenoic acid, at somewhat higher molar concentrations (10(-6) M) also reduced basal renin. These studies confirm prostacyclin as a potential renin secretagogue; however, its action in vitro is transient, probably because of its rapid degradation. Our studies provide new evidence that products of the 12-lipoxygenase and 15-lipoxygenase pathways, reported to be present in renal vascular tissue, are potent inhibitors of renin secretion and much more active on a molar basis on renin secretion than is prostacyclin. These studies suggest the potential presence of a dual system of stimulation and suppression that may regulate renin secretion in normal and clinical states.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonate 12-Lipoxygenase; Arachidonate 15-Lipoxygenase; Arachidonate Lipoxygenases; Arachidonic Acids; Epoprostenol; Hydroxyeicosatetraenoic Acids; Kidney Cortex; Leukotrienes; Lipid Peroxides; Male; Rats; Rats, Inbred Strains; Renin; SRS-A

Rabbit hydronephrotic cortical interstitial cells in primary culture were labeled with [1-14C]arachidonic acid and the eicosanoids released after stimulation with bradykinin or A23187 were studied by reverse-phase high performance liquid chromatography. The major arachidonic acid metabolite formed was prostaglandin (PG)E2, comprising more than 30% of the total radioactivity released. 12-Hydroxyheptadecatrienoic acid, probably representing spontaneous breakdown of the cyclic endoperoxides PGG2 and/or PGH2, made up 10 to 15% of the radioactivity released. Other cyclooxygenase products that were released included PGF2 alpha, PGD2, 6-keto PGF1 alpha and only minute amounts of thromboxane B2. Small quantities of the lipoxygenase products 15-, 12- and 5-hydroxyeicosatetraenoic acids (HETEs) as well as leukotrienes (LT)B4, LTC4 and LTD4 were also identified. Significantly larger quantities of 15- and 5-HETEs were recovered at 2 to 5 min than after longer incubations with A23187, suggesting esterification of these HETEs into cellular phospholipids. The data indicate that interstitial cells of the hydronephrotic kidney synthesize a variety of cyclooxygenase and lipoxygenase products of arachidonic acid, which may contribute to the pathophysiology of hydronephrosis. Moreover, it is suggested that PGG2 and/or PGH2 that are released from these cells may be metabolized further by adjacent kidney cells or circulating blood elements to other eicosanoid products, thus increasing the diversity of eicosanoids synthesized in the hydronephrotic kidney.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Bradykinin; Calcimycin; Cells, Cultured; Chromatography, High Pressure Liquid; Dinoprostone; Eicosanoic Acids; Hydronephrosis; Hydroxyeicosatetraenoic Acids; Kidney Cortex; Leukotrienes; Male; Prostaglandins E; Prostaglandins G; Rabbits; SRS-A; Strontium

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