Evidence for inhibition of leukotriene A4 synthesis by 5,8,11,14-eicosatetraynoic acid in guinea pig polymorphonuclear leukocytes

The sensitivity of the 5-lipoxygenase to inhibition by 5,8,11,14-eicosatetraynoic acid (ETYA) is species- and/or tissue-dependent. Guinea pig peritoneal polymorphonuclear leukocytes prelabeled with [3H]arachidonic acid and stimulated with ionophore A23187 formed 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE), as well as several dihydroxy fatty acids, including 5(S),12(R)-dihydroxy-6,8,10-(cis/trans/trans)-14-(cis)-eicosatetraenoic acid. ETYA (40 microM) did not inhibit, but, rather, increased the incorporation of 3H label into 5-HETE. In contrast, ETYA markedly inhibited the formation of radiolabeled dihydroxy acid metabolites by the A23187-stimulated cells. Assay of products from polymorphonuclear leukocytes incubated with exogenous arachidonic acid plus A23187, by reverse phase high performance liquid chromatography combined with ultraviolet absorption, showed a concentration-dependent inhibition of the formation of dihydroxy acid metabolite by ETYA (1-50 microM) and an increase in 5-HETE levels (maximum of 2- to 3-fold). The latter finding was verified by stable isotope dilution assay with deuterated 5-HETE as the internal standard. Another lipoxygenase inhibitor, nordihydroguaiaretic acid, potently inhibited the formation of both 5-HETE and dihydroxy acids, with an IC50 of 2 microM. The data suggest that ETYA can inhibit the enzymatic step whereby 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid is converted to leukotriene A4 in guinea pig polymorphonuclear leukocytes.     

Effects of protein deficiency on the metabolism of arachidonic acid by rat pleural polymorphonuclear leukocytes

The effects of protein deficiency on the biosynthesis of metabolites of arachidonic acid by rat pleural polymorphonuclear leukocytes stimulated with calcium ionophore were investigated. The major products of metabolism by lipoxygenase in these cells were leukotriene B4 and 5-hydroxy-6,8,11,14-eicosatetraenoic acid, whereas the major cyclooxygenase products were thromboxane B2 and 12-hydroxy-5,8,10-heptadecatrienoic acid. At high substrate concentrations (100 microM), the formation of all products by polymorphonuclear leukocytes was lower for protein-deficient rats than for controls. Similar results were obtained when products synthesized from endogenous substrate were measured, except that there was no change in the amount of 5-hydroxy-6,8,11,14-eicosatetraenoic acid formed. The biosynthesis of prostaglandins E2 and F2 alpha by homogenates of rat kidney medulla was reduced as a result of protein deficiency. Acetylsalicylic acid inhibited the formation of cyclooxygenase products and stimulated the formation of lipoxygenase products by polymorphonuclear leukocytes. Protein deficiency did not alter the effects of acetylsalicylic acid on the biosynthesis of these products, although at any given concentration the amounts of products formed were less with protein-deficient rats than with rats fed control diets.     

Modulation of insulin secretion by lipoxygenase products of arachidonic acid. Relation to lipoxygenase activity of pancreatic islets

Glucose (16.7 mM)-induced insulin secretion from isolated pancreatic islets of rats was inhibited by nordihydroguaiaretic acid (NDGA), 1-phenyl-3-pyrazolidinone (phenidone), 3-amino-1-(3-trifluoromethylphenyl)-2-pyrazoline (BW755C), 2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone (AA861), and 2,6-di-tert-butyl-4-methylphenol (BHT). Indomethacin and aspirin, however, failed to inhibit the glucose-induced insulin secretion but rather tended to enhance it. The glucose-induced insulin secretion was inhibited by 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) (50 microM), 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE) (100 microM), and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) (100 microM), but not by 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) (100 microM). Exogenous 5-HETE (10 microM) induced significant insulin secretion in a low glucose (3.3 mM) medium. Racemic 5-HETE also showed insulinotropic effect in a concentration-dependent manner with the concentrations 20 microM or above, whereas 12-HETE, 15-HETE, 15-HPETE, 5,12-dihydroxy-6,8,10,14-eicosatetraenoic acid, 5-hydroxy-6-glutathionyl-7,9,11,14-eicosatetraenoic acid, 5-hydroxy-6-cysteinylglycinyl-7,9,11,14-eicosatetraenoic acid, prostaglandin E2, and prostaglandin F2 alpha failed to induce insulin secretion. Although significant insulin release was observed with arachidonic acid (greater than or equal to 100 microM), reduce cell viability was evident at 200 microM. When the 10,000 X g supernatant of isolated pancreatic islet homogenate was incubated with [3H]arachidonic acid at 37 degrees C in the presence of GSH and Ca2+, and the labeled metabolites then extracted with ethyl acetate and subjected to reverse phase high pressure liquid chromatography, several radioactive peaks, coeluted with authentic 15-, 12-, and 5-HETE, were observed. The radioactive peaks were completely suppressed by the addition of either NDGA, BW755C, or phenidone into the medium. The results support our contention i.e. the involvement of lipoxygenase product(s) in the secretory mechanism of insulin, and further suggest that 5-lipoxygenase system may play a role.     

Ability of 15-hydroxyeicosatrienoic acid (15-OH-20:3) to modulate macrophage arachidonic acid metabolism

Mouse peritoneal macrophages metabolize dihomogammalinolenic acid (20:3n-6) primarily to 15-hydroxy-8,11,13-eicosatrienoic acid (15-OH-20:3). Since the biological properties of this novel trienoic eicosanoid remain poorly defined, the effects of increasing concentrations of 15-OH-20:3 and its arachidonic acid (20:4n-6) derived analogue. 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE), on mouse macrophage 20:4n-6 metabolism were investigated. Resident peritoneal macrophages were prelabeled with [3H]-20:4n-6 and subsequently stimulated with zymosan in the presence of either 15-OH-20:3 or 15-HETE (1-30 microM). After 1 hr, the radiolabeled soluble metabolites were analyzed by reverse phase high performance liquid chromatography. 15-OH-20:3 inhibited zymosan-induced leukotriene C4 (IC50 = 2.4 microM) and 5-HETE (IC50 = 3.1 microM) synthesis. In contrast to the inhibition of macrophage 5-lipoxygenase, 15-OH-20:3 enhanced 12-HETE synthesis (5-30 microM) and had no measurable effect on cyclooxygenase metabolism (1-10 microM) i.e., 6-keto-prostaglandin F1 alpha and prostaglandin E2 synthesis. Addition of exogenous 15-HETE produced similar effects. These results suggest that the manipulation of macrophage 15-OH-20:3n-6 levels may provide a measure of cellular control over 20:4n-6 metabolism, specifically, leukotriene production.     

Inhibition of leukotriene formation in human leukocytes by halothane

The effects of an inhalation anesthetic, halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) on the formation of 5-lipoxygenase metabolites such as leukotriene B4, 5(S)-hydroxyeicosatetraenoic acid (5-HETE), 6-trans-isomers of leukotriene B4 and leukotriene C4 were studied in human leukocytes stimulated with calcium ionophore A23187. Halothane inhibited the formation of all these metabolites dose dependently and the formation was restored by removal of the drug. The anesthetic also reversibly inhibited the release of [3H]arachidonic acid from neutrophils with a half-inhibition concentration of less than 0.19 mM. The formation of 5-lipoxygenase metabolites was not inhibited by the anesthetic when leukocytes were stimulated with the ionophore in the presence of exogenous arachidonic acid. These observations indicate that the inhibitory effect of halothane on the formation of 5-lipoxygenase metabolites in leukocytes is mainly due to the inhibition of arachidonic acid release.     

Activation of a 15-lipoxygenase/leukotriene pathway in human polymorphonuclear leukocytes by the anti-inflammatory agent ibuprofen

Human peripheral blood polymorphonuclear leukocytes (PMNs) metabolized [14C]arachidonic acid predominantly by lipoxygenase pathways. The major products were 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) and 15-HETE. These and other lipoxygenase products, including their derived leukotrienes, have been implicated as mediators of inflammatory and allergic reactions. In human platelets, the nonsteroidal anti-inflammatory drug ibuprofen inhibited production of the cyclooxygenase product thromboxane B2 (I50 = 65 microM), whereas the lipoxygenase product 12-HETE was not appreciably affected even at 5 mM ibuprofen. The 5-lipoxygenase of human PMNs (measured by 5-HETE formation) was inhibited by ibuprofen but was about six times less sensitive (I50 = 420 microM) than the platelet cyclooxygenase. The unexpected observation was made that the human PMN 15-lipoxygenase/leukotriene pathway was selectively activated by 1-5 mM ibuprofen. Metabolites were identified by ultraviolet spectroscopy, by radioimmunoassay, or by retention times on high pressure liquid chromatography in comparison with authentic standards. The major product was 15-HETE; and in all of 19 donors tested, 15-HETE formation was stimulated up to 20-fold by 5 mM ibuprofen. Other identified products included 12-HETE and 15- and 12-hydroperoxyeicosatetraenoic acid. Activation of the 15-lipoxygenase by ibuprofen occurred within 1 min and was readily reversible. The effects of aspirin, indomethacin, and ibuprofen on the PMN 15-lipoxygenase were compared in six donors. Ibuprofen produced an average 9-fold stimulation of the enzyme, whereas aspirin and indomethacin resulted in an average 1.5- and 2-fold enhancement, respectively.     

Action of novel eicosanoids lipoxin A and B on human natural killer cell cytotoxicity: effects on intracellular cAMP and target cell binding

Lipoxin A (5,6,15L-trihydroxy-7,9,11,13-eicosatetraenoic acid) and lipoxin B (5D,14,15-trihydroxy-6,8,10,12-eicosatetraenoic acid), two newly isolated compounds derived from the oxygenation of arachidonic acid in human leukocytes, inhibit the cytotoxic activity of human natural killer (NK) cells. Dose-response studies showed that both lipoxin A and lipoxin B inhibit, at submicromolar concentrations (ID50 10(-7) M), NK cell activity assayed against K562 target cells. Prostaglandin E2 (PGE2) also inhibited cytotoxicity, whereas both 15-HETE (5(S)-hydroxy-5,8,11,13-eicosatetraenoic acid) and leukotriene B4 (synthetic and biologically derived) were ineffective. PGE2 stimulated a time- and dose-dependent increase in intracellular cAMP, which was accompanied by a decrease in NK target cell binding. Lipoxin A and lipoxin B did not elevate intracellular cAMP, nor did they inhibit target cell binding. Together these findings suggest that lipoxin A and lipoxin B abrogate NK cell cytotoxicity at a step distal to target effector cell recognition. In contrast, PGE2 appears to exert its effect, at least in part, on cytotoxicity indirectly by decreasing the binding between target and effector cells (in vitro). Moreover, they suggest that novel oxygenated derivatives of arachidonic acid (i.e., lipoxin A, lipoxin B) may regulate the activities of NK cells.     

Platelet activating factor. Stimulation of the lipoxygenase pathway in polymorphonuclear leukocytes by 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine

1-O-Alkyl-2-O-acetyl-sn-glycero-3-phosphocholine (AAGPC) triggered the release of [3H]arachidonate but not [14C]stearate from cellular phospholipids in cytochalasin B-treated rabbit polymorphonuclear leukocytes. Concentrations of AAGPC up to 20 nM caused a dose-dependent release and subsequent metabolism of the released [3H]arachidonic acid. Most of the release of the [3H]arachidonate had taken place within the first 2 min of stimulation. Phosphatidylinositol and phosphatidylcholine served as the sources of [3H]arachidonate with about 50% of the label coming from each pool. Challenge of cytochalasin B-treated polymorphonuclear leukocytes with AAPGC led to the production of [3H]hydroxyeicosatetraenoic acids and [3H]dihydroxyeicosatetraenoic acids. No significant production of [3H]prostaglandins or [3H]thromboxanes was detected. AAGPC also caused a dose-dependent degranulation of cytochalasin B-treated rabbit polymorphonuclear leukocytes as shown by the release of beta-glucuronidase and lysozyme. Both the AAGPC-stimulated production of arachidonate metabolites and the degranulation response were blocked by eicosatetraynoic acid and non-dihydroguaiaretic acid at similar inhibitor concentrations. These findings suggest the bioactions of AAGPC on polymorphonuclear leukocytes may be mediated by the release of arachidonic acid and the production of mono- and dihydroxyeicosatetraenoic acids.     

Relationship of cyclic-AMP levels in leukotriene B4-stimulated leukocytes to lysosomal enzyme release and the generation of superoxide anions

Leukotriene B4 stimulated the formation of cyclic AMP, the release of lysosomal enzyme and generation of superoxide anions by human leukocytes. Dose-response curves have shown that the enzyme release proceeded in parallel with increments in cyclic AMP, suggesting a linkage between cyclic AMP and leukotriene B4-induced leukocyte activation. However, preincubation of the cells with (5S,12S)-dihydroxy-6,8,10,14-eicosatetraenoic acid or leukotriene B4 resulted in a dose-dependent inhibition of leukotriene B4-induced degranulation, without causing parallel changes in the levels of cyclic AMP. Both dihydroxy acids also blocked leukotriene B4-induced superoxide anion generation. These results suggest that the leukocyte responses to leukotriene B4 and the concomitant cyclic-AMP increments may be merely coincidental. In addition, the present study further supports the suggestion that (5S,12S)-dihydroxy-6,8,10,14-eicosatetraenoic acid may modulate the action of leukotriene B4 in the leukocyte.     

Characterization and separation of the arachidonic acid 5-lipoxygenase and linoleic acid omega-6 lipoxygenase (arachidonic acid 15-lipoxygenase) of human polymorphonuclear leukocytes

The cytosolic fraction of human polymorphonuclear leukocytes precipitated with 60% ammonium sulfate produced 5-lipoxygenase products from [14C]arachidonic acid and omega-6 lipoxygenase products from both [14C]linoleic acid and, to a lesser extent, [14C]- and [3H]arachidonic acid. The arachidonyl 5-lipoxygenase products 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE) and 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) derived from [14C]arachidonic acid, and the omega-6 lipoxygenase products 13-hydroperoxy-9,11-octadecadienoic acid (13-OOH linoleic acid) and 13-hydroxy-9,11-octadecadienoic acid (13-OH linoleic acid) derived from [14C]linoleic acid and 15-hydroxyperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE), and 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) derived from [14C]- and [3H]arachidonic acid were identified by TLC-autoradiography and by reverse-phase high-performance liquid chromatography (RP-HPLC). Products were quantitated by counting samples that had been scraped from replicate TLC plates and by determination of the integrated optical density during RP-HPLC. The arachidonyl 5-lipoxygenase had a pH optimum of 7.5 and was 50% maximally active at a Ca2+ concentration of 0.05 mM; the Km for production of 5-HPETE/5-HETE from arachidonic acid was 12.2 +/- 4.5 microM (mean +/- S.D., n = 3), and the Vmax was 2.8 +/- 0.9 nmol/min X mg protein (mean +/- S.D., n = 3). The omega-6 linoleic lipoxygenase had a pH optimum of 6.5 and was 50% maximally active at a Ca2+ concentration of 0.1 mM in the presence of 5 mM EGTA. When the arachidonyl 5-lipoxygenase and the omega-6 lipoxygenase were separated by DEAE-Sephadex ion exchange chromatography, the omega-6 lipoxygenase exhibited a Km of 77.2 microM and a Vmax of 9.5 nmol/min X mg protein (mean, n = 2) for conversion of linoleic acid to 13-OOH/13-OH linoleic acid and a Km of 63.1 microM and a Vmax of 5.3 nmol/min X mg protein (mean, n = 2) for formation of 15-HPETE/15-HETE from arachidonic acid.     

LTA(4)-derived 5-oxo-eicosatetraenoic acid: pH-dependent formation and interaction with the LTB(4) receptor of human polymorphonuclear leukocytes

5-oxo-(7E,9E,11Z,14Z)-eicosatetraenoic acid (5-oxo-ETE) has been identified as a non-enzymatic hydrolysis product of leukotriene A(4) (LTA(4)) in addition to 5,12-dihydroxy-(6E,8E,10E, 14Z)-eicosatetraenoic acids (5,12-diHETEs) and 5,6-dihydroxy-(7E,9E, 11Z,14Z)-eicosatetraenoic acids (5,6-diHETEs). The amount of 5-oxo-ETE detected in the mixture of the hydrolysis products of LTA(4) was found to be pH-dependent. After incubation of LTA(4) in aqueous medium, the ratio of 5-oxo-ETE to 5,12-diHETE was 1:6 at pH 7.5, and 1:1 at pH 9.5. 5-Oxo-ETE was isolated from the alkaline hydrolysis products of LTA(4) in order to evaluate its effects on human polymorphonuclear (PMN) leukocytes. 5-Oxo-ETE induced a rapid and dose-dependent mobilization of calcium in PMN leukocytes with an EC(50) of 250 nM, as compared to values of 3.5 nM for leukotriene B(4) (LTB(4)500 nM for 5(S)-hydroxy-(6E,8Z,11Z,14Z)-eicosatetraenoic acid (5-HETE). Pretreatment of the cells with LTB(4) totally abolished the calcium response induced by 5-oxo-ETE. In contrast, the preincubation with 5-oxo-ETE did not affect the calcium mobilization induced by LTB(4). The calcium response induced by 5-oxo-ETE was totally inhibited by the specific LTB(4) receptor antagonist LY223982. These data demonstrate that 5-oxo-ETE can induce calcium mobilization in PMN leukocyte via the LTB(4) receptor in contrast to the closely related analog 5-oxo-(6E,8Z,11Z, 14Z)-eicosatetraenoic acid which is known to activate human neutrophils by a mechanism independent of the receptor for LTB(4).     

Metabolism of 5-hydroxyicosatetraenoate by human neutrophils: production of a novel omega-oxidized derivative

Human neutrophils incorporated 5-hydroxy-E,Z,Z,Z-6,8,11,14-eicosatetraenoic acid (5-HETE) into cellular triglyceride and phospholipid. They also metabolized 5-HETE into a novel, extracellularly released derivative, 5,20-dihydroxy-E,Z,Z,Z-6,8,11,14-eicosatetraenoic acid (5,20-diHETE). 5,20-diHETE formation predominated at higher substrate concentrations and longer incubation intervals. In the absence of added 5-HETE, 1 X 10(8) neutrophils stimulated with 20 microM ionophore A23187 produced up to 243 ng of 5,20-diHETE, indicating that both endogenously formed and exogenously added substrate could be oxidized at carbon 20. 5,20-diHETE was about 10- to 100-fold weaker than 5-HETE in enhancing human neutrophil degranulation responses to platelet-activating factor. omega-Oxidation appears to be a general enzymatic mechanism for inactivation of arachidonic acid metabolites.     

Transformation of arachidonic acid by 5- and 15-lipoxygenase pathways in bovine adrenal fasciculata cells

[1-14C]Arachidonic acid was incubated with isolated bovine adrenal fasciculata cells for 15 min at 37gC. The metabolites were separated and purified by reverse- and straight-phase high performance liquid chromatography, and identified by gas chromatography-mass spectrometry or radioimmunoassay. Identified metabolites were 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE), 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE), leukotriene B4 and 11,14,15-trihydroxy-5,8,12-eicosatrienoic acid (11,14,15-THET). Addition of 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE), an intermediate metabolite of 15-lipoxygenase pathway to microsomes of bovine adrenal fasciculata cells resulted in the formation of 11,14,15-THET. The formation of 11,14,15-THET by microsomes was not dependent on the presence of NADPH, while it was dose-dependently suppressed by ketoconazole, a potent inhibitor of cytochrome P-450 dependent enzymes. These results indicate that 5- and 15-lipoxygenase pathways of arachidonic acid may exist in bovine adrenal fasciculata cells and that 15-HPETE is further metabolized to 11,14,15-THET by adrenal microsomal cytochrome P-450.     

Quantitation of arachidonic acid metabolites in small tissue biopsies by reversed-phase high-performance liquid chromatography

Arachidonic acid metabolites exert a variety of distinct biological effects on the initiation and resolution of inflammatory diseases and their measurements in tissue can be critical to evaluate their regulatory function during the course of inflammation and to supplement in vitro experiments. The aim of this study was the detection and quantitative analysis of four arachidonic acid metabolites in small-sized biopsies of human periodontal tissues. The biopsies were homogenized and injected directly into a single analytical column of a RP-HPLC system. Detection was performed by a photodiode array detector. Calibration was established by dilutions of authentic standards of prostaglandin E2 (PGE2), leukotriene B4 (LTB4), 12(R)-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), and 15(S)-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE). A total of 38 specimens weighing between 19 and 191 mg (wet tissue) were analyzed (mean = 59.9 +/- 30.2 mg). The detection limits were 1 pg for LTB4 and 12-HETE, 0.5 pg for 15-HETE, and 10 ng for PGE2. The concentrations of PGE2 and LTB4 were significantly higher in inflamed than in healthy periodontal tissues (P = 0.0079; P = 0. 0114). 12-HETE was detected in one biopsy (30 pg/g); 15-HETE was not detected. This method of homogenization, extraction, and analysis of arachidonic acid metabolites by RP-HPLC appears to be well suited for studies of human oral biopsies. Only small tissue samples and minimal laboratory equipment were required for a sensitive analysis.     

Lipoxygenase products of arachidonic acid modulate biosynthesis of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by human neutrophils via phospholipase A2

When human neutrophils, previously labeled in their phospholipids with [14C]arachidonate, were stimulated with the Ca2+-ionophore, A23187, plus Ca2+ in the presence of [3H]acetate, these cells released [14C]arachidonate from membrane phospholipids, produced 5-hydroxy-6,8,11,14-[14C]eicosatetraenoic acid (5-HETE) and 14C-labeled 5S,12R-dihydroxy-6-cis,8,10-trans, 14-cis-eicosatetraenoic acid ([14C]leukotriene B4), and incorporated [3H]acetate into platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine). Ionophore A23187-induced formation of these radiolabeled products was greatly augmented by submicromolar concentrations of exogenous 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE), 5-HETE, and leukotriene B4. In the absence of ionophore A23187, these arachidonic acid metabolites were virtually ineffective. Nordihydroguaiaretic acid (NDGA) and several other lipoxygenase/cyclooxygenase inhibitors (butylated hydroxyanisole, 3-amino-1-(3-trifluoromethylphenyl)-2-pyrazoline and 1-phenyl-2-pyrazolidinone) caused parallel inhibition of [14C]arachidonate release and [3H]PAF formation in a dose-dependent manner. Specific cyclooxygenase inhibitors, such as indomethacin and naproxen, did not inhibit but rather slightly augmented the formation of these products. Furthermore, addition of 5-HPETE, 5-HETE, or leukotriene B4 (but not 8-HETE or 15-HETE) to neutrophils caused substantial relief of NDGA inhibition of [3H]PAF formation and [14C]arachidonate release. As opposed to [3H]acetate incorporation into PAF, [3H]lyso-PAF incorporation into PAF by activated neutrophils was little affected by NDGA. In addition, NDGA had no effect on lyso-PAF:acetyl-CoA acetyltransferase as measured in neutrophil homogenate preparations. It is concluded that in activated human neutrophils 5-lipoxygenase products can modulate PAF formation by enhancing the expression of phospholipase A2.     

Synthesis of two hydroxy fatty acids from 7,10,13,16,19-docosapentaenoic acid by human platelets

Platelets metabolize 7,10,13,16,19-docosapentaenoic acid (22:5(n-3] into 11-hydroxy-7,9,13,16,19- and 14-hydroxy-7,10,12,16,19-docosapentaenoic acid via an indomethacin-insensitive pathway. Time-dependent studies with 20 microM substrate show a lag in the synthesis of both the 11- and 14-isomers which was not observed for the synthesis of thromboxane B2 (TXB2), 5,8,10-heptadecatrienoic acid, and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) from arachidonic acid. When platelets were incubated with increasing concentrations of 22:5(n-3), the 11- and 14-isomers were not produced until the substrate concentration exceeded 5 microM unless arachidonic acid was also added to the incubations. The stimulatory effect of arachidonic acid was not blocked by indomethacin thus suggesting that 12-hydroperoxyeicosatetraenoic acid or 12-HETE derived from arachidonic acid may activate the platelet lipoxygenase(s) which metabolize 22:5(n-3). Incubations containing 20 microM 22:5(n-3) and increasing levels of [1-14C]arachidonic acid show that the (n-3) acid inhibits the synthesis of both 5,8,10-heptadecatrienoic acid and TXB2 from arachidonic acid. At the same time, 12-HETE synthesis increased due to substrate shunting to the lipoxygenase pathway.     

Stimulation of gonadotropin release by arachidonic acid and its lipoxygenase metabolites in superfused pituitary cells

Luteinizing hormone and follicle stimulating hormone secretion was stimulated by 4 min pulses of arachidonic acid (3 X 10(-5) to 10(-4)M) in superfused rat pituitary cells. The effect of its lipoxygenase metabolites, 5-hydroxy-6,8,11,14-eicosatetranoic acid (5-HETE) and 15-hydroxy-5,8,10,14-eicosatetranoic acid (15-HETE) was more potent on hormone release when added in the same dose. Using 3 X 10(-5)M 5-HETE, its releasing activity on gonadotropins was comparable to that of GnRH (10(-9)M). 15-HETE (3 X 10(-5)M) was even more potent on LH and FSH secretion than 5-HETE. The secretory profile induced by 5-HETE and 15-HETE was also similar to that shown for GnRH, resulting in a rapid increase and a more prolonged decline of the hormone release. The addition of these fatty acids to superfused pituitary cells did not alter the response of the cells to their physiological ligand. These findings give further support to the proposal that metabolites of arachidonic acid may be involved in receptor-mediated mechanisms of gonadotropin release in pituitary cells.     

5-oxo-ETE is a major oxidative stress-induced arachidonate metabolite in B lymphocytes

B lymphocytes convert arachidonic acid (AA) to the 5-lipoxygenase products leukotriene B4 (LTB4) and 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) when subjected to oxidative stress. 5-HETE has little biological activity, but can be oxidized by a selective dehydrogenase in some cells to 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE), a potent eosinophil chemoattractant. We found that CESS cells, a B lymphocyte cell line, convert AA to 5-oxo-ETE and this is selectively stimulated by oxidative stress. In the presence of H2O2, 5-oxo-ETE is a major AA metabolite in these cells (5-oxo-ETE≈5-HETE>LTB4). The cyclooxygenase product 12-hydroxy-5,8,10-heptadecatrienoic acid is also formed, but is not affected by H2O2. Diamide had effects similar to those of H2O2 and both substances had similar effects on human tonsillar B cells. H2O2 also stimulated 5-oxo-ETE formation from its direct precursor 5-HETE in tonsillar B and CESS cells, and this was inhibited by the glutathione reductase inhibitor carmustine. H2O2 concomitantly induced rapid increases in GSSG and NADP+ and reductions in GSH and NADPH. We conclude that oxidative stress stimulates 5-oxo-ETE synthesis in B lymphocytes by two mechanisms: activation of 5-lipoxygenase and increased oxidation of 5-HETE by NADP+-dependent 5-hydroxyeicosanoid dehydrogenase. B lymphocyte-derived 5-oxo-ETE could contribute to eosinophilic inflammation in asthma and other allergic diseases.     

Effects of stilbene derivatives on arachidonate metabolism in leukocytes

The effects of various alpha-phenylcinnamic acid derivatives (i.e., alpha-(3,4-dihydroxyphenyl)cinnamic acid, alpha-(3,4-dihydroxyphenyl)-3-hydroxycinnamic acid, alpha-(3,4-dihydroxyphenyl)-4-hydroxycinnamic acid and alpha-(3,4-dihydroxyphenyl)-3, 4-dihydroxycinnamic acid) synthesized from 3,4-dihydroxyphenyl acetic acid and hydroxy-benzaldehyde, and 3,3',4-trihydroxystilbene obtained by decarboxylation of alpha-(3,4-dihydroxyphenyl)-3-hydroxycinnamic acid on rat peritoneal polymorphonuclear leukocyte lipoxygenase and cyclooxygenase activities were studied. 3,3',4-Trihydroxystilbene was found to inhibit the 5-lipoxygenase product, 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HETE), and cyclooxygenase products, 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and thromboxane B2; its concentrations for 50% inhibition (IC50) were 0.885 +/- 0.016 microM for the leukocyte lipoxygenase product, 5-HETE, 7.70 +/- 0.104 microM for the formations of HHT and 7.96 +/- 0.143 microM for the formation of thromboxane B2. Alpha-(3,4-Dihydroxyphenyl)cinnamic acid, alpha-(3,4-dihydroxyphenyl)-3-hydroxycinnamic acid and alpha-(3,4-dihydroxyphenyl)-3,4-dihydroxycinnamic acid also inhibited the formations of 5-HETE, HHT and thromboxane B2, although less strongly. Their IC50 values were, respectively, 91.3 +/- 3.62 microM, 947.5 +/- 28.7 microM, 453.3 +/- 229.3 microM and 148.8 +/- 50.6 microM for the formation of 5-HETE, 894.0 +/- 5.57 microM, 792.5 +/- 15.9 microM, greater than 1000 microM and 925.0 +/- 7.64 microM for the formation of HHT and 941.0 +/- 18.0 microM, 825 +/- 14.4 microM, greater than 1000 microM and 932.7 +/- 3.93 microM for the formation of thromboxane B2.     

Correlation of intracellular and extracellular calcium ion concentrations with synergy between 1,2-dioctanoyl-sn-glycerol and ionomycin in platelet arachidonic acid mobilization

The potentiation by 1,2-dioctanoyl-sn-glycerol (DiC8) of ionomycin-induced platelet production of 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) was investigated in correlation with extracellular Ca2+ concentrations and increases in [Ca2+]i, as detected with aequorin and fura-2. Extracellular Ca2+ concentrations greatly influenced the production of arachidonic acid metabolites induced by DiC8 and ionomycin, while that induced by ionomycin alone was minimally affected by variation of the extracellular Ca2+ concentration. In the synergy between ionomycin and 20 microM DiC8, the optimal concentrations of ionomycin shifted from high to low with increasing concentrations of extracellular Ca2+, suggesting that there might be a range of optimal [Ca2+]i for the production of the arachidonic acid metabolites. This hypothesis was confirmed by simultaneous measurements of [Ca2+]i increases, and the production of the arachidonic acid metabolites. With the aequorin method, the optimal concentrations of [Ca2+]i fell to between 10 microM and 20 microM, and with the fura-2 method, it fell to between 800 nM and 1800 nM. Direct measurements of [14C]arachidonic acid release suggested that the DiC8-potentiated production of arachidonic acid metabolites induced by ionomycin was attributable to increased arachidonic acid release. Since ionomycin and DiC8 induced relatively low levels of phosphatidic acid production, an indicator of phospholipase C activation, it was suggested that the increased arachidonic acid release was largely dependent upon phospholipase A2. Synergy between DiC8 and ionomycin was also observed with aggregation and serotonin release. Aggregation was induced by lower concentrations of ionomycin, and appeared to be more dependent upon extracellular Ca2+, while serotonin release required higher concentrations of ionomycin, and variations in extracellular Ca2+ affected the response minimally. These findings suggest that the mechanisms underlying the synergy between protein kinase C activation and Ca2+ mobilization differ among the three functions evaluated in this study.