The production of 5-HETE and leukotriene B in rat neutrophils from carrageenan pleural exudates

Rat neutrophils isolated from three-hour carrageenan pleural exudates actively metabolize arachidonic acid into three major metabolites, HHT, 11-HETE and 15-HETE. However, in the presence of the calcium ionophore, A23187, or the non-ionic detergent, BRIJ 56, these cells also produce 5-HETE and LTB. The production of these lipoxygenase products is calcium dependent. While non-steroidal anti-inflammatory drugs do not affect 5-HETE or LTB production, BW 755C and ETYA inhibit formation of these metabolites from exogenously added arachidonic acid.     

The production of 5-HETE and leukotriene B in rat neutrophils from carrageenan pleural exudates

Rat neutrophils isolated from three-hour carrageenan pleural exudates actively metabolize arachidonic acid into three major metabolites, HHT, 11-HETE and 15-HETE. However, in the presence of the calcium ionophore, A₂₃₁₈₇, or the non-ionic detergent, BRIJ 56, these cells also produce 5-HETE and LTB. The production of these lipoxygenase products is calcium dependent. While non-steroidal anti-inflammatory drugs do not affect 5-HETE or LTB production, BW 755C and ETYA inhibit formation of these metabolites from exogenously added arachidonic acid.     

Neutrophil degranulation: evidence pertaining to its mediation by the combined effects of leukotriene B4, platelet-activating factor, and 5-HETE

Stimulus-activated polymorphonuclear neutrophils (PMN) produce leukotriene B4 (LTB4), 5-hydroxyeicosatetraenoate (5-HETE), and platelet-activating factor (PAF). Each of these lipids promotes PMN degranulation; in combination they have additive and potentiating effects that result in prominent degranulation responses at relatively low concentrations. Thus, the combined interactions of LTB4, 5-HETE, and PAF may mediate responses in PMN activated by other stimuli. This possibility was examined by measuring the responses of PMN made insensitive to one or more of these lipids. Cells were pretreated with LTB4, 5-HETE, and/or PAF for 8 min; exposed for 2 min to cytochalasin B (which is required for lipid-induced degranulation); and then challenged. PMN challenged with only buffer released minimal amounts of granule-bound enzymes. Furthermore, the lipid-pretreated cells were hyporesponsive to challenge with 1) various combinations of these same lipids or 2) ionophore A23187. The relative potencies of the lipids in producing hyporesponsiveness to themselves or A23187 were: 5-HETE less than PAF less than or equal to LTB4 less than PAF + LTB4 less than PAF + LTB4 + 5-HETE. For both types of challenge, reduced responsiveness occurred in cells pretreated with greater than 0.1 nM LTB4 and/or greater than 0.2 nM PAF, persisted in cells washed after lipid pretreatment, and did not develop in cells pretreated with various combinations of bioinactive structural analogues of the lipids. Thus, PAF, LTB4, and 5-HETE interacted to desensitize PMN, and the degranulating actions of A23187 required cells that were fully responsive to each of the three lipids. This supports the concept that the lipids act together in mediating certain of the ionophore's effects. However, lipid-desensitized PMN degranulated fully when challenged with C5a, a formylated oligopeptide, or phorbol myristate acetate. Degranulation responses, therefore, may proceed through various pathways, only some of which involve the lipid products studied here.     

Comparative effect of leukotriene B4 and leukotriene B5 on calcium mobilization in human neutrophils

Leukotriene B4 (LTB4) is reported to exert its biological activity in neutrophils through the increase in cytosolic free calcium that follows binding to its specific receptor. Leukotriene B5 has been shown to be far less active than LTB4. Therefore we compared the capacity of LTB4 and LTB5 to stimulate the rise in cytosolic free calcium using fura-2-loaded human neutrophils, to assess the relationship between the calcium mobilizing activity and biological potency of LTB4 and LTB5. At any concentration tested, LTB5 was less active than LTB4 in increasing cytosolic free calcium. ED50 for LTB4 and LTB5 were 5 X 10(-10) M and 5 X 10(-9) M, respectively. The difference in the binding affinities of LTB4 and LTB5 to the LTB4 receptor has been reported to explain the difference in their biological activities. In the present study we further demonstrated that the calcium mobilizing activity of LTB4 and LTB5 also correlates the different biological activity of the two compounds.     

Na+/H+ exchange modulates the production of leukotriene B4 by human neutrophils.

Human neutrophils produce various compounds of the 5-lipoxygenase pathway, including (5S)-hydroxyeicosatetraenoic acid, leukotriene B4, its 6-trans isomers and omega-oxidation metabolites of LTB4, when the cells are stimulated with the Ca2+ ionophore A23187. The elevation in the extracellular pH (pHo) facilitated the cytoplasmic alkalinization induced by the ionophore as determined fluorometrically using 2',7'-bis(carboxyethyl)carboxyfluorescein and enhanced the production of all the 5-lipoxygenase metabolites. The production decreased when the alkalinization was blocked by the decrease in the pHo, the removal of the extracellular Na+ or the addition of specific inhibitors of the Na+/H+ exchange, such as 5-(NN-hexamethylene)amiloride, 5-(N-methyl-N-isobutyl)amiloride and 5-(N-ethyl-N-isopropyl)amiloride. The alkalinization of the cytoplasm with methylamine completely restored the production suppressed by the removal of Na+ from the medium. These findings suggest that the change in the cytoplasmic pH (pHi) mediated by the Na+/H+ exchange regulates the production of the lipoxygenase metabolites. The site of the metabolism controlled by the pHi change seemed to be the 5-lipoxygenase, because the production of all the metabolites decreased in parallel and the release of [3H]arachidonic acid from the neutrophils in response to the ionophore was not affected by the pHi change. Furthermore, the production of the 5-lipoxygenase metabolites stimulated by A23187 with or without exogenous arachidonic acid showed a similar pHo-dependence and the production induced by N-formylmethionyl-leucylphenylalanine (chemotactic peptide) with exogenous arachidonic acid also decreased when the cytoplasmic alkalinization was inhibited.     

Binding and metabolism of leukotriene B4 by neutrophils and their subcellular organelles

The subcellular distribution of leukotriene (LT)B4 binding and metabolizing sites was investigated in human neutrophils. Cells were disrupted by nitrogen cavitation and fractionated by Percoll density gradient centrifugation to yield cytoplasm, membranes, azurophilic granules, and specific granules. Only membrane fractions contained high affinity [3H]LTB4 binding sites. Binding of radiolabeled ligand to membranes was rapid, reversible, and saturable; it was blocked by a series of LTB4 analogues at concentrations corresponding to their respective potencies in 1) blocking [3H]LTB4 binding to whole cells and 2) stimulating neutrophil degranulation responses. In contrast, [3H]LTB4 was metabolized by fractions enriched with markers for cytoplasm plus endoplasmic reticulum. The metabolic activity was sedimented by ultracentrifugation, enhanced by NADPH, and inhibited at 4 degrees C. The cell-free system, like intact cells, metabolized [3H]LTB4 to omega-oxidized product rapidly and quantitatively at 37 degrees C but was inactive at 4 degrees C. Whole cells converted radiolabel to 20-hydroxy (approximately 30% of product) and 20-carboxy (approximately 70% of product) derivatives; the cell-free system formed principally 20-hydroxy-[3H]LTB4. These products were less bioactive than LTB4. Nevertheless, metabolism of LTB4 played little role in limiting the cells' response to the ligand: neutrophils completed degranulation and became desensitized to LTB4 within 3-5 min of exposure. Within this time frame, they oxidized less than 30% of the stimulus, and the extracellular fluid of these neutrophil suspensions was fully capable of activating fresh cells. We conclude that neutrophils transmit bioactions of LTB4 via a specific receptor integrally associated with their plasmalemma and/or endoplasmic reticulum. They inactivate the stimulus via a particulate omega-oxidase. At the level of the individual cell, receptor down-regulation, rather than ligand metabolism, appears to limit functional responses such as degranulation.     

Perturbation of beta-glucan receptors on human neutrophils initiates phagocytosis and leukotriene B4 production

Normal human neutrophils were stimulated with the yeast cell wall product, zymosan, and examined for two biologic responses, ingestion of particles and production of leukotriene B4 (LTB4), under conditions that were comparable and optimal for the quantitation of each response. Monolayers of adherent neutrophils ingested unopsonized zymosan particles, at particle-to-cell ratios of 12.5:1 to 125:1, in a dose- and time-related manner. At a ratio of 125:1, the percentages of neutrophils ingesting greater than or equal to 1 and greater than or equal to 3 zymosan particles reached plateau levels of 55 +/- 6 and 32 +/- 9% (mean +/- SD, n = 8), respectively, within 30 min. At this same ratio, neutrophils during gravity sedimentation with zymosan particles synthesized LTB4 in a time-dependent manner for at least 45 min. The maximum amount of immunoreactive LTB4 released into supernatants was 3.8 +/- 1.2 ng per 10(6) neutrophils (mean +/- SD, n = 5) and the corresponding total immunoreactive LTB4 was 6.2 +/- 1.9 ng per 10(6) neutrophils. Treatment of 2 x 10(7) suspended neutrophils with 250 micrograms of trypsin for 20 min before concurrent assessment of neutrophil phagocytosis and LTB4 production reduced both of these responses by about 50%. Pretreatment of neutrophils with 800 micrograms/ml of soluble yeast beta-glucan inhibited their ingestion of zymosan by 84% (mean +/- SD, n = 3), with 50% inhibition occurring with 100 micrograms/ml of soluble beta-glucan; 800 micrograms/ml of soluble yeast alpha-mannan had no inhibitory effect. Pretreatment of neutrophils with 400 micrograms/ml of soluble yeast beta-glucan inhibited neutrophil synthesis of LTB4 by 90%, with 50% occurring with 200 micrograms/ml; 400 micrograms/ml of soluble yeast alpha-mannan had no inhibitory effect. The presence of 1.25 micrograms/ml of cytochalasin B during incubation with zymosan particles reduced neutrophil phagocytosis from 65 to 6%, and neutrophil synthesis of LTB4 from total levels of 6.0 +/- 0.3 ng/10(6) cells to zero (mean +/- SD, n = 3). Pretreatment with either cytochalasin B or vinblastine did not alter neutrophil generation of LTB4 induced by calcium ionophore. Neutrophils pretreated with vinblastine, at 4 x 10(-6) to 4 x 10(-4) M, and then maintained at one-half these concentrations during incubation with unopsonized zymosan particles exhibited no diminution in particle ingestion, but were markedly reduced in zymosan-induced synthesis of LTB4.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evaluation of the process for superoxide production by NADPH oxidase in human neutrophils: evidence for cytoplasmic origin of superoxide

We present an up-to-date insight into the function of NADPH oxidase in human neutrophils, the signalling pathways involved in activation of this enzyme and the process of association of its components with the cytoskeleton. We also discuss the functional implications of morphological studies revealing localization of the sites of NADPH oxidase activity. An original model of the process of superoxide (O2*-) production in human neutrophils is shown. Organization of NADPH oxidase is associated with several components. Upon stimulation, tri-phox cytosolic components of NADPH oxidase (p40-phox, p47-phox and p67-phox) bind to actin filaments. This process involves other actin-binding proteins, such as cofilin and coronin. Activated protein kinase C, translocated from the plasma membrane, phosphorylates cytosolic components at a scaffold of cytoskeleton. Subsequently, p40-phox, responsible for maintaining the resting state of NADPH oxidase, is separated from other two cytosolic phox proteins following an attachment of the active form of small GTP-binding protein Rac to p67-phox. Cytosolic duo-phox proteins (p47-phox and p67-phox) conjugate with membrane components (gp91-phox, p22-phox and Rapla) of NADPH oxidase residing within membranes of intracellular compartments. This chain of events triggers production of O2*-. Then, oxidant-producing intracellular compartments associate with the plasma membrane. Eventually, intracellularly produced O2*- is released to the extracellular environment through the orifice formed by fusion of oxidant-producing compartments with the plasma membrane. Intracellular movement of the oxidant-producing compartments may be regulated by myosin light chain kinase. The review emphasizes that functional assembly of NADPH oxidase and, therefore, generation of O2*- is accomplished essentially within the intracellular compartments. Upon neutrophil stimulation, intracellularly generated O2*- is transported to the plasma membrane to be released and to ensure host defense against infection.     

Evaluation of the process for superoxide production by NADPH oxidase in human neutrophils: evidence for cytoplasmic origin of superoxide

Abstract

We present an up-to-date insight into the function of NADPH oxidase in human neutrophils, the signalling pathways involved in activation of this enzyme and the process of association of its components with the cytoskeleton. We also discuss the functional implications of morphological studies revealing localization of the sites of NADPH oxidase activity. An original model of the process of superoxide (O2) production in human neutrophils is shown. Organization of NADPH oxidase is associated with several components. Upon stimulation, tri-phox cytosolic components of NADPH oxidase (p40-phox, p47-phox and p67-phox) bind to actin filaments. This process involves other actin-binding proteins, such as cofilin and coronin. Activated protein kinase C, translocated from the plasma membrane, phosphorylates cytosolic components at a scaffold of cytoskeleton. Subsequently, p40-phox, responsible for maintaining the resting state of NADPH oxidase, is separated from other two cytosolic phox proteins following an attachment of the active form of small GTP-binding protein Rac to p67-phox. Cytosolic duo-phox proteins (p47-phox and p67-phox) conjugate with membrane components (gp91-phox, p22-phox and Rap1a) of NADPH oxidase residing within membranes of intracellular compartments. This chain of events triggers production of O2. Then, oxidant-producing intracellular compartments associate with the plasma membrane. Eventually, intracellularly produced O2 is released to the extracellular environment through the orifice formed by fusion of oxidant-producing compartments with the plasma membrane. Intracellular movement of the oxidant-producing compartments may be regulated by myosin light chain kinase. The review emphasizes that functional assembly of NADPH oxidase and, therefore, generation of O2 is accomplished essentially within the intracellular compartments. Upon neutrophil stimulation, intracellularly generated O2 is transported to the plasma membrane to be released and to ensure host defense against infection.     

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.     

Effect of the 5-lipoxygenase inhibitor piriprost on superoxide production by human neutrophils

The effect of 6,9-deepoxy-6,9-(phenylimino)-delta 6,8-prostaglandin I1 (Piriprost) on the oxidative response was studied in human neutrophils stimulated by N-formyl-methionyl-leucyl-phenylalanine (fMLP), phorbol 12-myristate, 13-acetate (PMA) or opsonized zymosan. Piriprost inhibited the stimulatory effect of fMLP on superoxide anion (O2-) generation, at concentrations higher than those which depress leukotriene B4 (LTB4) formation. This inhibition was overcome by increasing the concentration of fMLP. Neither exogenous LTB4 nor indomethacin were able to reverse the inhibitory effect of piriprost on fMLP action. In contrast, piriprost did not inhibit the stimulation of O2- production induced by PMA or zymosan. Piriprost behaves thus as a specific and apparently competitive antagonist of fMLP: this action does not seem to involve lipoxygenase inhibition and might be exerted at the level of the fMLP receptor or its associated mechanisms of transduction.     

Stereospecificity of leukotriene B4 and structure-function relationships for chemotaxis of human neutrophils

The chemotactic activity of leukotriene B4 (5S, 12R Dihydroxy 6, 14 cis, 8, 10 trans eicosatetraenoic acid) (LTB4) was examined by using a sensitive Boyden-chamber assay. The activity of LTB4 was compared to other biosynthetic stereoisomers: 5S, 12R Dihydroxy 6, 8, 10 trans 14 cis eicosatetraenoic acid (6-trans LTB4); 5S, 12S Dihydroxy 6, 8, 10 trans 14 cis eicosatetraenoic acid (12-epi-6-trans LTB4), 5S, 12S DiHETE; the metabolic product 20-Hydroxy LTB4 (20-OH LTB4); methylated LTB4 (Methyl-LTB4), and the related monoHETE 5-HETE and 12-HETE. The compounds were purified by several steps of reverse phase and straight phase HPLC. The LTB4 exhibits measurable chemotactic activity at 10(-9) M with maximal activity at 10(-7) M and an ED50 of 10(-8) M. The LTB4 isomers and monoHETE were less chemotactic than previously reported. The monoHETE (5-HETE and 12-HETE), the isomer 12-epi-6-trans LTB4, and 5S, 12S DiHETE fail to attract neutrophils at levels between 10(-6) and 10(-5) M. If these compounds are chemotactic, then activity is at least four orders of magnitude less than that of LTB4. The isomer 6-trans LTB4 at 10(-6) to 10(-5) M induced chemotaxis with an extrapolated ED50 value of 10(-5) M, indicating that a trans for cis change in configuration at position 6 reduces the chemotactic activity of LTB4 by 1000-fold. Conversely, the metabolic product 20-OH LTB4 is at least as active as the native compound LTB4. Methylation of the carboxyl group of LTB4 reduces its chemotactic activity by two orders of magnitude. These results indicate a high degree of stereospecificity for the LTB4 receptor with strict dependence on hydroxyl group, and triene configuration and considerable dependence on the carboxyl group. Modification at the aliphatic omega end of the LTB4 molecule has a minimal effect on function, suggesting that the hydrophobicity of this portion of the molecule is not important for optimal activity. Furthermore, we propose that metabolic products of LTB4 may be of greater importance than LTB4 as physiologic inflammatory mediators in vivo.     

Intracellular retention of the 5-lipoxygenase pathway product, leukotriene B4, by human neutrophils activated with unopsonized zymosan

The cellular and extracellular distribution of leukotriene B4 (LTB4) generated in human neutrophilic polymorphonuclear leukocytes (PMN) stimulated with unopsonized zymosan has been compared with that generated in PMN activated by the calcium ionophore. The amounts of extracellular and intracellular LTB4 were quantitated by radioimmunoassay. The authenticity of the immunoreactive LTB4 was confirmed by the elution of a single immunoreactive peak after reverse phase-high performance liquid chromatography (RP-HPLC) at the retention time of synthetic LTB4, by the identical elution time of a peak of radiolabeled product derived from [3H]arachidonic acid-labeled PMN with the immunoreactive product, and by the comparable chemotactic activity on a weight basis of immunoreactive LTB4 and synthetic LTB4 standard. Under optimal conditions of stimulation by unopsonized zymosan, more than 78% of the generated immunoreactive LTB4 remained intracellular, whereas with optimal activation by the ionophore, less than 8.6% of immunoreactive LTB4 was retained. Resolution by RP-HPLC of the products from the supernatants and cell extracts of [3H]arachidonic acid-labeled PMN stimulated with unopsonized zymosan and those stimulated with calcium ionophore allowed identification and measurement of 5-hydroxyeicosatetraenoic acid (5-HETE), 6-trans-LTB4, LTB4, and omega oxidation products of LTB4 by radioactivity. With zymosan stimulation of PMN, 5-HETE and the 6-trans-LTB4 diastereoisomers were not released, LTB4 was partially released, and the omega oxidation products of LTB4 were preferentially extracellular in distribution. In contrast, with ionophore stimulation, only 5-HETE had any duration of intracellular residence being equally distributed intra- and extracellularly throughout the 30-min period of observation; 6-trans-LTB4, LTB4, and the omega oxidation products of LTB4 were retained at less than 19%. The respective distributions of 5-HETE after zymosan and ionophore stimulation were not altered by the introduction of albumin to the reaction mixtures to prevent reacylation, or by hydrolysis of the cell extract to uncover any product that had been reacylated. The finding that stimulation of PMN with unopsonized zymosan results in the cellular retention of 5-lipoxygenase products suggests that release of these metabolites may be an event that is regulated separately from their generation.     

The adherence of human neutrophils and eosinophils to schistosomula: evidence for membrane fusion between cells and parasites

Human neutrophils and eosinophils adhere to the surface of schistosomula of Schistosoma mansoni that have been preincubated with antischistosomular sera with or without complement. Neutrophils are seen to form small (< 0.5 micrometer), heptalaminar and large (5-8 micrometer), pentalaminar fusions with the normal pentalaminar parasite surface membrane. By freeze-fracture techniques, attachment areas 5-8 micrometer in diameter are seen to form between neutrophils and schistosomula. These areas have three zones--an edge and two centrally located areas, one of which is rich and one of which is poor in intramembrane particles (IMPs). The edge zone is continuous around the attachment areas and is usually composed of a skip-fracture that passes out of the schistosomular outer membrane into the inner membrane. In some cases, the edge zone is made up of a string of IMPs. The IMP-rich central areas have an IMP concentration similar to that of unattached neutrophil membranes, are raised off of the surface of the schistosomulum, and have two normal schistosomular membranes underneath indicating that they are indeed unattached. the IMP-poor central areas are composed of a fused or hybrid membrane that is continuous with the neutrophil plasma membrane but that bears the same spatial relationship to the schistosomular inner membrane that the normal outer membrane does. Similar changes are seen in samples prepared with glycerination. Eosinophils generally do not fuse with the schistosomular outer membrane but, instead, discharge their granular contents onto the surface of the schistosomula and appear to adhere to the parasite through this discharged material. It is suggested that schistosomula have a capability to fuse with mammalian cells and that this fusion proceeds from a fusion of the outer leaflets to a fusion of the bilayers, as appears also to be the case in other systems.     

Modulation of the heterogeneous membrane potential response of neutrophils to N-formyl-methionyl-leucyl-phenylalanine (FMLP) by leukotriene B4: evidence for cell recruitment

Individual human neutrophils (PMN) isolated by Hypaque-Ficoll gradient sedimentation, dextran sedimentation, or buffy coat preparation were assessed for the effects of leukotriene B4 (5S,12R dihydroxy 6,14-cis-8, 10 trans eicosatetraenoic acid (LTB4)-pretreatment on N-formylmethionyl-leucyl-phenylalanine (FMLP)-mediated membrane potential or oxidative responses by using flow cytometry and a lipophilic probe of membrane potential (di-pentyl-oxacarbocyanine, di-O-C(5)3), or the nitroblue tetrazolium dye (NBT) reduction test, respectively. Although exposure to LTB4 (10(-7) M) had no effect on the membrane potential of resting PMN and little effect on oxidant production, pretreating PMN with LTB4 followed by FMLP (10(-6) M) demonstrated a significant enhancement in the proportion of depolarizing PMN over that seen with FMLP alone (p = 0.0014, N = 9). This recruitment of previously unresponsive cells by LTB4 was dose and time dependent, with the maximal relative increase in the proportion of depolarizing cells occurring at LTB4 concentrations of 10(-8) to 10(-7) M and within 1 min of LTB4 addition. The recruitment effect persisted despite vigorous washing of the cells. LTB4 also increased the proportion of NBT-positive PMN in response to FMLP. Although LTB4 alone did not depolarize PMN it did induce a light scatter shift indicative of cell activation. 3H-FMLP binding studied at 0 degree C comparing buffer and LTB4-treated PMN indicated no significant change in the number or affinity of FMLP binding. The data provide evidence for the recruitment of a greater proportion of cells into a FMLP-responsive state as a mechanism for the enhanced functional response of PMN pretreated with LTB4, as well as for a dissociation of the membrane potential and light scattering responses of cells to this pro-inflammatory LT. The mechanism of recruitment remains unclear, but it most likely involves the modulation of a post-FMLP binding step.     

Transformation of 15-hydroperoxyeicosapentaenoic acid to lipoxin A5 and B5, mono- and dihydroxyeicosapentaenoic acids by porcine leukocytes

15-Hydroperoxy[1-14C]eicosapentaenoic acid derived from eicosapentaenoic acid (EPA) was incubated with suspensions of porcine leukocytes. Incubation with porcine leukocytes resulted in the formation of seven dihydroxy compounds, one monohydroxy and one hydroxyepoxy compound. After separation by reverse-phase and straight-phase HPLC, GC/MS analysis revealed that these metabolites were four isomers of 8,15-diHEPEs, two isomers of 14,15-diHEPEs, one isomer of 5,15-diHEPE, 15-HEPE and an epoxyalcohol: 13-hydroxy-14,15-epoxyeicosatetraenoic acid. In addition to the above metabolites, two trihydroxytetraene derivatives were also isolated. GC/MS and ultraviolet spectroscopy identified the two trihydroxypentaene derivatives as 5,6,15-trihydroxy-7,9,11,13,17-eicosapentaenoic acid (lipoxin A5) and 5,14,15-trihydroxy-6,8,10,12,17-eicosapentaenoic acid (lipoxin B5). This study demonstrated that the 15-hydroperoxide of EPA can be actively converted to various hydroxylated products via the 5-, 12- and 15-lipoxygenase as well as epoxyisomerase pathways in the porcine leukocytes.     

Effects of heavy metal ions on phospholipid metabolism in human neutrophils: relationship to ionophore-mediated cytotoxicity

This study examined the effects of 0.1mM heavy metal ions (Au3+, Zn2+, Cr3+, Mn2+, and Cu2+) on ionophore-treated human neutrophils. Treatment of human neutrophils with 5-10 microM ionophore A23187 resulted in phospholipid deacylation and eicosanoid release within 5 min. After approximately 20 min, viability decreased significantly with near total cell death by 50 min. Heavy metal ions altered phospholipid metabolism, eicosanoid synthesis, and cytotoxicity in parallel fashion. Radioimmunoassays for 5-HETE and LTB4 demonstrated that Au3+ and Zn2+ stimulated, Cr3+ had little effect on, and Mn2+ and Cu2+ inhibited eicosanoid release from ionophore-treated neutrophils. Cells prelabelled with [3H]arachidonic acid exhibited similar metal-mediated effects on lipid metabolism. Strong negative correlations between metal effects on viability and the metabolism of arachidonic acid suggest that eicosanoids participate in ionophore-induced cytotoxicity.     

Comparison of leukotriene A4 metabolism into leukotriene C4 by human platelets and endothelial cells.

Leukotriene (LT) A4 metabolism was studied in human platelets and endothelial cells, since both cells could be involved in transcellular formation of LTC4. Upon addition of exogenous LTA4, both cells produced LTC4 as a major metabolite at various incubation times, and no LTB4, LTD4, or LTE4 was detected. Kinetic studies revealed a higher apparent Km for LTA4 in endothelial cells as compared to platelets (5.8 microM for human umbilical vein endothelial cells (HUVEC) versus 1.3 microM for platelets); platelets were more efficient in this reaction with a higher Vmax (174 pmol/mg protein/min) versus 15 pmol/mg protein/min in HUVEC. The formation of LTC4 and corresponding kinetic parameters were not modified when platelets or endothelial cells were stimulated by thrombin prior to or simultaneously with the addition of LTA4. In both cells LTC4 synthase activity was not modified by repeated addition of LTA4 showing that it is not a suicide-inactivated enzyme. Furthermore, in platelets and endothelial cells, the enzyme activity was localized in the membrane fraction and was distinct from cytosolic glutathione-S-transferases. Platelet membrane fractions showed apparent Km values of 31 microM and 1.2 mM for LTA4 and GSH, respectively. Inhibition of LTC4 formation from platelets and endothelial cells preparations by S-substituted glutathione derivatives was correlated to the length of the S-alkyl chain. The same substances inhibited cytosolic glutathione-S-transferases with significantly lower IC50, confirming the distinct nature of the two enzymes. These results show that platelets and HUVEC possess similar enzymes for the production of LTC4 from LTA4; however, platelets seem to have a higher efficiency than HUVEC in performing this reaction.