Activation of the lipoxygenase pathway in the methionine enkephalin induced respiratory burst in human polymorphonuclear leukocytes

In comparative studies of f-met-Leu-Phe (FMLP) and methionine enkephalin (ME) induced polymorphonuclear leukocyte (PMNL) stimulation the following results were obtained: (i) both FMLP and ME increased the intracellular killing (IK) capability of human PMNLs probably through NADPH oxidase activation, (ii) the ME-induced respiratory burst (RB) differed from the chemotactic peptide FMLP-triggered superoxide generation because the former was not accompanied by the activation of the glutathione system and the duration of the superoxide production was prolonged. The reaction was dependent on lipoxygenation, was potentiated by indomethacin (IM) and was inhibited by nordihidro-guairetic acid (NDGA), (iii) both 14C-arachidonic acid (14C-AA) release and leukotriene B4 (LTB4) synthesis of ME-treated PMNLs were elevated as compared to those of FMLP triggered cells. Our results suggest that lipoxygenation and even an increased LTB4 synthesis are involved in the ME-induced RB of leukocytes.     

Recent investigations into the lipoxygenase pathway of plants

The plant lipoxygenase (LOX) pathway is in many respects the equivalent of the 'arachidonic acid cascade' in animals. The LOX-catalyzed dioxygenation of the plant fatty acids, linoleic and linolenic acids, is followed by metabolism of the resulting fatty acid hydroperoxides by other enzymes. Although the physiological functions of the end-products do not appear to be fully defined at this time, hormonal and anti-fungal activities have been reported.     

Effect of inhibitors of the lipoxygenase pathway on mouse myoblast fusion

In this study we examined the effects of inhibitors of the lipoxygenase and cyclooxygenase pathways on mouse myoblast fusion. The fusion of cloned mouse myoblasts was markedly inhibited, in a dose-dependent manner, when cells were cultured in medium supplemented with either phenidone (1-phenyl-3-pyrazolidione) or BW755c (3-amino-1-(3-tri-fluoromethylphenyl)-2-pyrazoline), drugs which have been reported to inhibit lipoxygenase and cyclo-oxygenase activities. Fusion was also inhibited when these cells were cultured in medium supplemented with esculetin (6,7-dihydroxycoumarin) which has been reported to inhibit lipoxygenase activity. Removal of the above inhibitors resulted in a return to control levels of fusion. Fusion was not demonstrably inhibited with aspirin (acetylsalicylic acid) and only inhibited to a minor extent with indomethacin (1-(p-chlorobenzoyl)-5-methoxy-2-methylindole-3-acetic acid); both of these drugs are inhibitors of cyclo-exygenase activity.     

Mechanism of mosaic attenuation of the lungs on computed tomography in induced bronchospasm

The purpose of this study was to investigatewhether hypoxic pulmonary vasoconstriction is the major determinant ofthe computed tomography (CT) pattern of mosaic attenuation in asthmaticpatients with induced bronchoconstriction. Thin-section CT wasperformed at suspended full inspiration immediately and 30 min aftermethacholine bronchoprovocation in 22 asthmatic subjects, who wererandomly assigned to breathe room air (group A,n = 8), oxygen via nasal prongs at 5 l/min (group B,n = 8), and oxygen via face mask at 12 l/min (group C,n = 6). CT changes were quantified interms of global lung density and density in hypodense and hyperdense areas. Lung parenchymal density increases were greatest ingroup C and greater ingroup B than in groupA, globally (P = 0.03) and in hypodense regions (P = 0.01).On bivariate analysis, the only change in cross-sectional area wasrelated to change in global density. In hypodense regions, densitychange was related both to reduction in cross-sectional area(P < 0.0005) and to oxygen administration (P = 0.01). Aftercorrection for changes in global lung density, only oxygen wasindependently related to density increase in hypodense areas(P = 0.02). In inducedbronchoconstriction, the CT appearance of mosaic attenuation can belargely ascribed to hypoxic vasoconstriction rather than to changes inlung inflation.     

Stimulation of the lipoxygenase pathway is associated with systemic resistance induced in bean by a nonpathogenic Pseudomonas strain

Systemic defense reactions induced in bean by the non-pathogenic Pseudomonas putida BTP1 strain reduced disease caused by Botrytis cinerea. Phenylalanine ammonialyase activity and the level of endogenous free salicylic acid were compared in plant growth-promoting rhizobacteria-treated versus control plants, but no significant differences were detected. Furthermore, no enhanced fungitoxicity was detected in methanolic leaf extracts, suggesting that accumulation of bean phytoalexins was not part of the stimulated defense mechanisms. However, BTP1-inoculated plants showed increased levels of both linoleic and linolenic acids. On this basis, we further investigated whether the lipoxygenase pathway, leading to antifungal phytooxylipins, could have been stimulated. Two key enzymatic activities of this metabolic route, namely lipoxygenase and hydroperoxide lyase, were significantly stimulated during the first four days after challenging BTP1-treated plants with the pathogen. This was observed in parallel with a more rapid consumption of the respective substrates of these enzymes, as revealed by measurements of endogenous concentrations of linolenic acid and their hydroperoxide derivatives. Moreover, headspace-gas chromatography analyses showed significantly higher concentrations of the fungitoxic final product Z-3-hexenal in leaves from BTP1-inoculated beans as compared with control plants. Taken together, these results strongly suggest that the oxylipin pathway can be associated with enhanced disease resistance induced in bean plants by nonpathogenic rhizobacteria.     

The role of arachidonate lipoxygenase in the release of SRS-A from guinea-pig chopped lung

The immunological release of SRS-A was investigated in guinea-pig chopped lung. A number of unsaturated fatty acids, all of which are substrates for arachidonate lipoxygenase were found to potentiate the release of SRS-A. This potentiation was enhanced by indomethacin, a cyclo-oxygenase inhibitor, and completely reversed by nordihydroguaiaretic acid (NDGA) and eicosatetraynoic acid (ETA) which inhibit lipoxygenase. This suggests that some aspect of arachidonate lipoxygenase action stimulates release of SRS-A and that release of SRS-A is increased by redirection of arachidonic acid (AA) metabolism via the lipoxygenase pathway (Hamberg, 1976). However, although exogenous ¹⁴C-AA increased SRS-A output it was not incorporated into SRS-A.     

The role of arachidonate lipoxygenase in the release of SRS-A from guinea-pig chopped lung

The immunological release of SRS-A was investigated in guinea-pig chopped lung. A number of unsaturated fatty acids, all of which are substrates for arachidonate lipoxygenase were found to potentiate the release of SRS-A. This potentiation was enhanced by indomethacin, a cyclo-oxygenase inhibitor, and completely reversed by nordihydroguaiaretic acid (NDGA) and eicosatetraynoic acid (ETA) which inhibit lipoxygenase. This suggests that some aspect of arachidonate lipoxygenase action stimulates release of SRS-A and that release of SRS-A is increased by redirection of arachidonic acid (AA) metabolism via the lipoxygenase pathway (Hamberg, 1976). However, although exogenous 14C-AA increased SRS-A output it was not incorporated into SRS-A.     

Products of the lipoxygenase pathway in human natural killer cell cytotoxicity

As earlier data suggested the importance of lipoxygenase activation for expression of human NK cell cytotoxicity, four different lipoxygenase inhibitors were tested for suppression of natural killer (NK) cell lysis. All inhibitors were found active at nontoxic concentrations with 50% inhibition at approximately 15 microM for nordihydroguaiaretic acid (NDGA). NK cell lysis could be reconstituted to NDGA-suppressed cells with leukotriene B4 (LTB4), the all-trans isomers 6-trans-LTB4 and 12-epi-6-trans-LTB4, and 20-COOH-LTB4. LTB4 reconstitution was best in the concentration range 1-100 pM and near control levels at both higher and lower concentrations. Herpesvirus Ateles-transformed killer T cells could also be inhibited by NDGA. These data indicate that lipoxygenase activity is required for human NK cell lysis and that several different LTB4-related products can restore NK activity in inhibited cells; they also suggest that the lipoxygenase pathway is present in the killer cell population.     

Differential distribution of the lipoxygenase pathway enzymes within potato chloroplasts

The lipoxygenase pathway is responsible for the production of oxylipins, which are important compounds for plant defence responses. Jasmonic acid, the final product of the allene oxide synthase/allene oxide cyclase branch of the pathway, regulates wound-induced gene expression. In contrast, C6 aliphatic aldehydes produced via an alternative branch catalysed by hydroperoxide lyase, are themselves toxic to pests and pathogens. Current evidence on the subcellular localization of the lipoxygenase pathway is conflicting, and the regulation of metabolic channelling between the two branches of the pathway is largely unknown. It is shown here that while a 13-lipoxygenase (LOX H3), allene oxide synthase and allene oxide cyclase proteins accumulate upon wounding in potato, a second 13-lipoxygenase (LOX H1) and hydroperoxide lyase are present at constant levels in both non-wounded and wounded tissues. Wound-induced accumulation of the jasmonic acid biosynthetic enzymes may thus commit the lipoxygenase pathway to jasmonic acid production in damaged plants. It is shown that all enzymes of the lipoxygenase pathway differentially localize within chloroplasts, and are largely found associated to thylakoid membranes. This differential localization is consistently observed using confocal microscopy of GFP-tagged proteins, chloroplast fractionation, and western blotting, and immunodetection by electron microscopy. While LOX H1 and LOX H3 are localized both in stroma and thylakoids, both allene oxide synthase and hydroperoxide lyase protein localize almost exclusively to thylakoids and are strongly bound to membranes. Allene oxide cyclase is weakly associated with the thylakoid membrane and is also detected in the stroma. Moreover, allene oxide synthase and hydroperoxide lyase are differentially distributed in thylakoids, with hydroperoxide lyase localized almost exclusively to the stromal part, thus closely resembling the localization pattern of LOX H1. It is suggested that, in addition to their differential expression pattern, this segregation underlies the regulation of metabolic fluxes through the alternative branches of the lipoxygenase pathway.     

Metabolic profile of linoleic acid in porcine leukocytes through the lipoxygenase pathway

Porcine neutrophilic leukocytes were found to contain a lipoxygenase which converted linoleic acid into 13-hydroxy-9,11-octadecadienoic acid (n-6 specificity), arachidonic acid into 12-hydroxy-5,8,10,14-eicosatetraenoic acid (n - 9 specificity) and 5-hydroxy-6,8,11,14-eicosatetraenoic acid into 5,12-dihydroxy-6,8,10,14-eicosatetraenoic acid. This lipoxygenase was partially purified and it appeared that its substrate specificity and other properties were quite different from the 12-lipoxygenase of blood platelets. Incubations of intact or broken porcine leukocytes with added linoleic acid revealed the formation of not only 13-hydroxy-9,11-octadecadienoic acid but also of substantial amounts of epoxyhydroxy and trihydroxy isomers. These products from linoleate, collectively described by the name 'octadecanoids' were characterized in detail by a combination of chemical, chromatographic and mass spectrometric techniques. The phospholipids of porcine leukocytes contain more than twice as much linoleate than arachidonate (22 vs. 8%). In accordance with this fatty acid composition we found that in the stimulated neutrophil the endogenous production of octadecanoids often surpassed that of the eicosanoids. Lipoxygenation of endogenously liberated linoleic acid was especially pronounced when a suspension of leukocytes in citrated plasma was recalcified and allowed to clot.     

The lipoxygenase pathway and chemiluminescence in horse eosinophilic leukocytes

It was shown in several cell types that the dual lipoxygenase and cyclooxygenase inhibitor eicosatetraynoic acid but not the cyclooxygenase inhibitor acetylsalicylic acid suppressed luminol-dependent chemiluminescence. Since lipoxygenase is known to generate chemiluminescence in vitro, these observations were interpreted as evidence for a direct contribution of the lipoxygenase pathway to light emission in intact cells. We have investigated a possible contribution of the lipoxygenase to the chemiluminescence of horse eosinophils by directly comparing the formation of the byproduct chemiluminescence with the formation of stable end-products of the lipoxygenase pathway, leukotrienes and HETEs. Azide as well as eicosatetraynoic acid almost completely inhibited chemiluminescence stimulated by the calcium ionophore A23187 but had less effect on the formation of leukotrienes. The tumour-promoting ester, phorbol myristate acetate, stimulated chemiluminescence in an azide- and eicosatetraynoic acid-sensitive manner and failed to evoke the production of leukotrienes. Azide, but also eicosatetraynoic acid inhibited the luminol-dependent chemiluminescence generated by isolated eosinophil peroxidase in the presence of H₂O₂. Our results argue against a direct role of the lipoxygenase pathway in the generation of light in horse eosinophilic leukocytes but do not exclude that product(s) of this pathway may be involved in stimulus-response coupling.     

Evidence for lipoxygenase pathway involvement in allergic tracheal contraction

Challenge of actively sensitized guinea-pig trachea in vitro led to a contraction which was enhanced by the cyclo-oxygenase inhibitors, indomethacin and sodium meclofenamate. Cyclo-oxygenase inhibitors eliminated the release of PGE-like material induced by arachidonic acid (AA), histamine, and antigen challenge. AA (10 microgram./ml.) and PGE2 (100 ng./ml.) usually relaxed the trachea, whereas in the presence of cyclo-oxygenase inhibitors a contraction occurred. Phenidone and ETYA, which also blocked the lipoxygenase pathway of AA metabolism inhibited the enhancement of allergic tracheal contraction induced by cyclo-oxygenase inhibitors, decreased the time that the trachea remained contracted, and also eliminated the contraction induced by AA and PGE2. Thus, cyclo-oxygenase inhibitors may enhance allergic tracheal contraction by diverting AA metabolism into the lipoxygenase pathway and product of the latter pathway, possibly SRS-A, may be responsible for the enhancement and for the prolonged phase of allergic tracheal contraction. An analogous mechanism may account for aspirin-induced asthma in man.     

Evidence for lipoxygenase pathway involvement in allergic tracheal contraction

Challenge of actively sensitized guinea-pig trachea in vitro led to a contraction which was enhanced by the cyclo-oxygenase inhibitors, indomethacin and sodium meclofenamate. Cyclo-oxygenase inhibitors eliminated the release of PGE-like material induced by arachidonic acid (AA), histamine, and antigen challenge. AA (10 μg./ml.) and PGE₂ (100 ng./ml.) usually relaxed the trachea, whereas in the presence of cyclo-oxygenase inhibitors a contraction occurred. Phenidone and ETYA, which also blocked the lipoxygenase pathway of AA metabolism inhibited the enhancement of allergic tracheal contraction induced by cyclo-oxygenase inhibitors, decreased the time that the trachea remained contracted, and also eliminated the contraction induced by AA and PGE₂. Thus, cyclo-oxygenase inhibitors may enhance allergic tracheal contraction by diverting AA metabolism into the lipoxygenase pathway and a product of the latter pathway, possibly SRS-A, may be responsible for the enhancement and for the prolonged phase of allergic tracheal contraction. An analogous mechanism may account for aspirin-induced asthma in man.     

Heat shock induces alterations of the lipoxygenase pathway in human polymorphonuclear granulocytes

We have investigated the effect of the heat shock response on the leukotriene generation, chemotaxis, and generation of oxygen radicals of human polymorphonuclear granulocytes (PMNs) by preincubating the PMNs at 42 degrees C. Subsequently, the different test systems were performed at 37 degrees C. As we confirmed by the release of lactate dehydrogenase and beta-glucuronidase the elevated temperatures did not result in cytotoxic or degranulating processes. After heat shock treatment the generation of leukotrienes induced by the Ca(++)-ionophore A23187, fMLP or opsonized zymosan was inhibited in a time and temperature dependent manner (preincubation phase) as was measured by HPLC-analysis. In contrast, the conversion of 14C-arachidonic acid revealed the generation of LTB4, 5-HPETE and 5-HETE solely as a result of the preincubation at 42 degrees C without any further stimulation. In addition, the chemiluminescence response induced by opsonized zymosan and the chemotaxis against C5a and LTB4 was clearly inhibited after heat shock treatment. With regard to enzyme activities of the heat treated PMNs the protein kinase C activities were enhanced whereas the LTD4-dipeptidase and the LTB4-omega-hydroxylase were not affected.     

Arachidonate metabolism in the mouse thyroid implication of the lipoxygenase pathway in thyrotropin action

The present study of compares the effects of various inhibitors of arachidonate metabolism on mouse thyroid cyclo-oxygenase and lipoxygenase activities and thyrotropin-augmented cyclic-AMP accumulation. Mouse thyroid homogenate converts [1-14C]- arachidonate to several products of the cyclo-oxygenase pathway as well as one major product of the lipoxygenase pathway, 12-L-hydroxyeicosatetraenoic acid (12-Hete). Prostaglandin (PG) formation in thyroid homogenates is inhibited by 1-10 microM indomethacin and etya. 12-HETE accumulation is reduced by 91%, 83% and 20% by 5 microM ETYA, 15-HETE, and indomethacin, respectively. Thyrotropin-stimulated cyclic-AMP accumulation, measured in whole thyroid lobes by radioimmunoassay, is reduced by 45% and 73% by 50 microM and 100 microM ETYA, respectively; indomethacin is without effect at these concentrations. 15-HETE reduces thyrotropin-augmented cyclic-AMP accumulation by 57% and 100 microM. In product inhibition studies, 10 microM 12-HETE reduced the formation of radiolabeled 12-HETE by 20%. 10 microM PGE2, PGF2 alpha or PGD2 had no effect on [1-14C]-PG formation. 12-HETE, however, reduced PG synthesis by 76% at 10 microM. This is the first report implicating the arachidonate lipoxygenase pathway in thyrotropin action at the level of cyclic-AMP regulation. Additionally, our finding that 12-HETE inhibits prostaglandin synthesis suggests that the cyclo-oxygenase and lipoxygenase pathways in the mouse thyroid may be highly integrated.     

Arachidonate lipoxygenase inhibitors in guinea-pig isolated trachea. Effects on contractions to antigen and various agonists

We compared the effects of the leukotriene (LT) D₄ receptor antagonist FPL55712 and some lipoxygenase inhibitors on contractions of isolated guinea-pig trachea induced by antigen (ovalbumin, OA) and calcium ionophore A23187 in the presence of the cyclooxygenase inhibitor indomethacin (5 μM), and by arachidonic acid (AA), melittin and LTD₄. FPL55712 (0.1 and 1 μM) inhibited contractions induced by AA (100 μM) and the phospholipase A₂ activator melittin (3 μg/ml), while the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA, 10 μM) was a more effective inhibitor of the melittin response than the response. FPL55712 inhibited contractions induced by OA (100 μg/ml) more than by A23187 (1 μg/ml), and these inhibitory effects of FPL55712 were much less in the presence of l-serine-borate complex (45 mM), an inhibitor of LTC₄ conversion to LTD₄. NDGA (10 μM) had no significant effect on the OA response, whereas the lipoxygenase inhibitors 1-phenyl-3-pyrazolidone (phenidone, 10 μM) and 5,8,11,14-eicosatetraynoic acid (ETYA, 10 μM) clearly inhibited it. In contrast, NDGA and phenidone inhibited the A23187 response, but ETYA had no effect on it. FPL55712, phenidone and ETYA, but not NDGA, had a large inhibitory effect on LTD₄-induced contractions, but these inhibitors had no effect on histamine-induced contractions. These results suggest that in the guinea-pig trachea inhibitors of LTD₄-induced contractions decrease antigen-induced contractions, whereas lipoxygenase inhibitors reduce the contraction to A23187.     

Arachidonate lipoxygenase inhibitors in guinea-pig isolated trachea. Effects on contractions to antigen and various agonists

We compared the effects of the leukotriene (LT) D4 receptor antagonist FPL55712 and some lipoxygenase inhibitors on contractions of isolated guinea-pig trachea induced by antigen (ovalbumin, OA) and calcium ionophore A23187 in the presence of the cyclooxygenase inhibitor indomethacin (5 microM), and by arachidonic acid (AA), melittin and LTD4. FPL55712 (0.1 and 1 microM) inhibited contractions induced by AA (100 microM) and the phospholipase A2 activator melittin (3 micrograms/ml), while the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA, 10 microM) was a more effective inhibitor of the melittin response than the AA response. FPL55712 inhibited contractions induced by OA (100 micrograms/ml) more than by A23187 (1 microgram/ml), and these inhibitory effects of FPL55712 were much less in the presence of l-serine-borate complex (45 mM), an inhibitor of LTC4 conversion to LTD4. NDGA (10 microM) had no significant effect on the OA response, whereas the lipoxygenase inhibitors 1-phenyl-3-pyrazolidone (phenidone, 10 microM) and 5,8,11,14-eicosatetraynoic acid (ETYA, 10 microM) clearly inhibited it. In contrast, NDGA and phenidone inhibited the A23187 response, but ETYA had no effect on it. FPL55712, phenidone and ETYA, but not NDGA, had a large inhibitory effect on LTD4-induced contractions, but these inhibitors had no effect on histamine-induced contractions. These results suggest that in the guinea-pig trachea inhibitors of LTD4-induced contractions decrease antigen-induced contractions, whereas lipoxygenase inhibitors reduce the contraction to A23187.