E. coli hemolysin-induced lipid mediator metabolism in alveolar macrophages: impact of eicosapentaenoic acid

Escherichia coli hemolysin (HlyA) is a prototype of a large family of pore-forming proteinaceous exotoxins that have been implicated in the pathogenetic sequelae of severe infection and sepsis, including development of acute lung injury. In the present study in rabbit alveolar macrophages (AMs), subcytolytic concentrations of purified HlyA evoked rapid synthesis of platelet-activating factor, with quantities approaching those in response to maximum calcium ionophore challenge. In parallel, large quantities of leukotriene (LT) B(4) and 5-, 8-, 9-, 12-, and 15-hydroxyeicosatetraenoic acid (HETE) were liberated from HlyA-exposed AMs depending on exogenous arachidonic acid (AA) supply. Coadministration of eicosapentaenoic acid (EPA) dose dependently suppressed generation of the proinflammatory lipoxygenase products LTB(4) and 5-, 8-, 9-, and 12-HETE in parallel with the appearance of the corresponding EPA-derived metabolites LTB(5) and 5-, 8-, 9-, and 12-hydroxyeicosapentaenoic acid (HEPE). At equimolar concentrations, EPA turned out to be the preferred substrate over AA for these AM lipoxygenase pathways, with the sum of LTB(5) and 5-, 8-, 9-, and 12-HEPE surpassing the sum of LTB(4) and 5-, 8-, 9-, and 12-HETE by >80-fold. In contrast, coadminstration of EPA did not significantly reduce HlyA-elicited generation of the anti-inflammatory AA lipoxygenase product 15-HETE. We conclude that AMs are sensitive target cells for HlyA attack, resulting in marked proinflammatory lipid mediator synthesis. In the presence of EPA, lipoxygenase product formation is shifted from a pro- to an anti-inflammatory profile.     

Guinea pig alveolar eosinophils and macrophages produce leukotriene B4 but no peptido-leukotriene

The metabolism of arachidonic acid (AA) was investigated in purified guinea pig alveolar eosinophils and macrophages. Alveolar eosinophils produced 12S-hydroxy-5,8,10-heptadecatraenoic acid (HHT) and small amounts only of 5-lipoxygenase products when stimulated by AA (10 microM) or ionophore A23187 (2 microM). However, when the cell suspensions were stimulated with both AA and A23187, the cells produced HHT, leukotriene (LT) B4, and 5S-hydroxy-6,8,11,14-eicosatetraenoic acid, whereas LTC4, D4, and E4 were undetectable. Similarly, alveolar macrophages stimulated with A23187 produced HHT, 5-hydroxy-6,8,11,14-eicosatetraenoic acid, and LTB4 but no peptido-leukotrienes. When LTA4 was added to suspensions of eosinophils and macrophages, only LTB4 was formed, whereas in parallel experiments, intact human platelets incubated with LTA4 produced LTC4. These data suggest that guinea pig alveolar eosinophils and macrophages contain both cyclooxygenase and 5-lipoxygenase, but do not produce peptido-leukotrienes, probably lacking LTA4 glutathione transferase activity. These studies demonstrate that guinea pig eosinophils differ from eosinophils of other animal species which have been shown to be major sources of leukotriene C4. The present data imply that eosinophils and macrophages are not the source of peptido-leukotrienes in anaphylactic guinea pig lungs.     

Metabolism of arachidonic acid through the 5-lipoxygenase pathway in normal human peritoneal macrophages

Peritoneal macrophages (PM), obtained from 39 healthy women with normal laparoscopy findings, were stimulated with the ionophore A23187 or/and arachidonic acid (AA) both in adherence and in suspension. AA lipoxygenase metabolites were determined by reversed-phase HPLC. The major metabolites identified were 5-hydroxyeicosatetraenoic acid (5-HETE), leukotriene (LT)B4 and LTC4. The 20-hydroxy-LTB4, 20-carboxy-LTB4, and 15-HETE were not detected. Incubations of adherent PM with 2 microM A23187 induced the formation of LTB4, 110 +/- 19 pmol/10(6) cells, 5-HETE, 264 +/- 53 pmol/10(6) cells and LTC4, 192 +/- 37 pmol/10(6) cells. When incubated with 30 microM exogenous AA, adherent PM released similar amounts of 5-HETE (217 +/- 67 pmol/10(6) cells), but sevenfold less LTC4 (27 +/- 12 pmol/10(6) cells) (p less than 0.01). In these conditions LTB4 was not detectable. These results indicate that efficient LT synthesis in PM requires activation of the 5-lipoxygenase/LTA4 synthase, as demonstrated previously for blood phagocytes. When stimulated with ionophore, suspensions of Ficoll-Paque-purified PM produced the same lipoxygenase metabolites. The kinetics of accumulation of the 5-lipoxygenase/LTA4 synthase products in A23187-stimulated adherent cells varied for the various metabolites. LTB4 reached a plateau by 5 min, whereas LTC4 levels increased up to 60 min, the longest incubation time studied. Levels of 5-HETE were maximal at 5 min, and then slowly decreased with time. Thus, normal PM, in suspension or adherence, have the capacity to produce significant amounts of 5-HETE, LTB4, and LTC4. The profile of lipoxygenase products formed by the PM and the reactivity of this cell to AA and ionophore A23187 are similar to those of the human blood monocyte, but different from those of the human alveolar macrophage.     

Inhibition of production of LTB4 and chemotactic agent from rat alveolar macrophages treated with t-butyl hydroperoxide is independent of ATP depletion

In rat alveolar macrophages treated with 100 microM t-butyl hydroperoxide (tBOOH), leukotriene B4 (LTB4) synthesis was significantly lower than the basal level while levels of cyclooxygenase pathway products were increased. LTB4, 5,6-dihydroxyeicosatetraenoic acid (5,6-DiHETEs), and 5-hydroxyeicosatetraenoic acid (5-HETE) production in macrophages was significantly stimulated by 2 microM A23187, but this was suppressed 40% by simultaneous addition of 10 microM tBOOH and completely abolished by 100 microM tBOOH. Basal and A23187-stimulated macrophage production of chemotactic agents were similarly suppressed by addition of tBOOH; this effect paralleled depression of cellular LTB4 synthesis. In contrast to the significant depression of A23187-stimulated formation of 5-lipoxygenase products by 10 microM tBOOH, cellular adenosine triphosphate (ATP) was unchanged. Macrophages pretreated with KCN led to a 42% decline in ATP levels; however, LTB4, 5,6-DiHETEs, and 5-HETE production in response to A23187 was not suppressed. The results indicate that inhibition of 5-lipoxygenase pathway products in macrophages treated with tBOOH did not occur by depletion of cellular ATP levels.     

Comparison of the effect of lipoxygenase metabolites of arachidonic acid and eicosapentaenoic acid on human natural killer cell cytotoxicity

We compared in vitro effect of lipoxygenase (LO) products derived from arachidonic acid (AA) and eicosapentaenoic acid (EPA) on cytotoxic activity of human natural killer (NK) cell against human erythroleukemia cell line K-562. Leukotriene B4 (LTB4) derived from AA was found to significantly augment NK cell activity compared to the control level (in the absence of LTB). LTB5 showed a weak, but not significant, enhancing effect on NK cell activity. LTB4 was significantly more potent than LTB5 in the enhancement of NK cell activity. On the other hand, both 5- and 15-hydroperoxy fatty acids derived from AA and EPA significantly enhanced NK cell activity compared to the control level with similar potencies.     

Age-dependent change in arachidonic acid metabolic capacity in rat alveolar macrophages

Arachidonic acid (AA) metabolism was assessed in cultured alveolar macrophages (AM) obtained from newborn (10 days old) and adult (2 months and 4 months old) rats. The AMs were stimulated with the calcium ionophore, A23187 (10 microM). The released radiolabelled AA metabolites were measured by thin layer chromatography. The results showed that among different aged rats, the synthesis of 5-lipoxygenase (5-LO) metabolites, LTB4, LTC4, LTD4 and 5-HETE were increased with age inspite of similar levels of [14C]AA release. In response to A23187, 5-LO metabolic capacity of 2 and 4 months old adult rat AMs were increased 21-fold and 34-fold, respectively, compared with 10 days old rat AMs. As the metabolic capacity increased, the release of prostaglandins and thromboxane B2 tended to decrease markedly. Newborn rats (10 days old) AM, at the initial developmental stage, did not produce a noticeable amount of 5-LO metabolites which, conceivably, contribute to high susceptibility of neonatal lung to infection.     

Modulation of alveolar macrophage-derived 5-lipoxygenase products by the sulfhydryl reactant, N-ethylmaleimide

The sulfhydryl reactant N-ethylmaleimide (NEM) stimulates the release and cyclooxygenase metabolism of arachidonic acid in rat alveolar macrophages. Because both 5-lipoxygenation and leukotriene (LT) C4 synthesis represent sulfhydryl-dependent steps in the 5-lipoxygenase pathway, we examined the effect of NEM on 5-lipoxygenase, as well as cyclooxygenase, metabolism in resting and agonist-stimulated cells by reverse-phase high performance liquid chromatography and radioimmunoassay. NEM at 5-10 microM stimulated the synthesis of thromboxane, but not prostaglandin E2 or the 5-lipoxygenase products LTC4, LTB4, or 5-hydroxyeicosatetraenoic acid from endogenously released arachidonate. In the presence of exogenous fatty acid, however, NEM stimulated the synthesis of large quantities of LTB4. The effect of NEM on arachidonate metabolism stimulated by the calcium ionophore A23187 and the particulate zymosan was also investigated. NEM augmented arachidonate release and thromboxane synthesis stimulated by A23187 but inhibited A23187-induced LTC4 synthesis with an IC50 of approximately 4.3 microM. This inhibitory effect closely paralleled the ability of NEM to deplete intracellular glutathione (IC50 approximately 4.3 microM). Preincubation with the intracellular cysteine delivery agent L-2-oxothiazolidine-4-carboxylate augmented intracellular glutathione concentration and A23187-stimulated LTC4 synthesis and attenuated the capacity of NEM to deplete glutathione and inhibit LTC4 synthesis. While LTB4 and 5-hydroxyeicosatetraenoic synthesis were unaffected at these low NEM concentrations, LTB4 synthesis was inhibited at high concentrations (IC50 approximately 210 microM). Zymosan-induced eicosanoid synthesis was modulated by NEM in a similar fashion. Thus, NEM is an agonist of arachidonate metabolism with the capacity to modulate the spectrum of macrophage-derived eicosanoids by virtue of specific biochemical interactions with substrates and enzymes of the 5-lipoxygenase pathway.     

Effect of dietary oils on the production of n-3 and n-6 metabolites of leukocyte 5-lipoxygenase in five rat strains

We examined the influence of various dietary oils, including linseed and fish oil on the relative rates of leukotriene B4 (LTB4) and LTB5 production by rat peritoneal exudate cells in five rat strains. While there was an association between the membrane phospholipid levels of the fatty acid precursors (arachidonic acid (AA) and eicosapentaenoic acid (EPA)) and the rate of synthesis of their respective 5-lipoxygenase products (LTB4 and LTB5), the rate of LTB4 synthesis was a combined function of both AA and EPA levels. We observed a strong linear relationship (correlation coefficient = 0.99) between the ratio of EPA/AA in the cell membrane phospholipids and the ratio of LTB5/LTB4 produced by these cells in vitro; this association was independent of genetic (strain) variability and was independent of the source of EPA (dietary EPA or EPA endogenously synthesized from dietary alpha-linolenic acid).     

15-Hydroperoxyeicosapentaenoic acid, but not eicosapentaenoic acid, shifts arachidonic acid away from cyclooxygenase pathway into acyl-CoA synthetase pathway in rabbit kidney medulla microsomes

Under physiological conditions, small amounts of free arachidonic acid (AA) are released from membrane phospholipids, and cyclooxygenase (COX) and acyl-CoA synthetase (ACS) competitively act on this fatty acid to form prostaglandins (PGs) and arachidonoyl-CoA (AA-CoA). In the present study, we investigated the effects of eicosapentaenoic acid (EPA) and 15-hydroperoxyeicosapentaenoic acid (15-HPEPE) on the PG and AA-CoA formations from high and low concentrations of AA (60 and 5 microM) in rabbit kidney medulla microsomes. The kidney medulla microsomes were incubated with 60 or 5 microM [(14)C]-AA in 0.1M Tris/HCl buffer (pH 8.0) containing cofactors of COX (reduced glutathione and hydroquinone) and cofactors of ACS (ATP, MgCl(2) and CoA). After incubation, PG (as total PGs), AA-CoA and residual AA were separated by selective extraction using petroleum ether and ethyl acetate. EPA reduced the PG and AA-CoA formations from both 60 and 5 microM AA. In contrast, 15-HPEPE decreased the PG formation without affecting the AA-CoA formation from 60 microM AA, and increased the AA-CoA formation at the expense of PG formation when 5 microM AA was used as substrate concentration. The experiments utilizing Fe(2+) and an electron spin resonance (ESR) revealed that 15-HPEPE elicits these effects in the form of hydroperoxy adduct. These results suggest that 15-HPEPE, but not EPA, has the potential to shift AA away from COX pathway into ACS pathway at low substrate concentration (close to the physiological concentration of AA).     

Arachidonic acid metabolism and casein secretion in lactating rabbit mammary epithelial cells: effects of inhibitors of prostaglandins and leukotrienes synthesis

The metabolism of arachidonic acid (AA) in fragments of lactating rabbit mammary glands in vitro was studied by considering the distribution of 13-[14C]AA in the cells, and the effects of inhibitors of cyclooxygenase and lipoxygenase pathway on the basal and prolactin (PRL)-stimulated casein secretion. 13-[14C]AA was incorporated in all classes of lipids and PRL increased transiently the percentage of free fatty acid after 1 and 5 min. Ten microM ETYA (5,8,11,14-Eicosatetraynoic acid), a tetrayne analogue of AA inhibited prostaglandins F2 alpha (PGF2 alpha) production but not leukotrienes B4 and C4 (LTB4 and LTC4) production and increased basal casein secretion. 10(-4) M DCHA (Docosahexaenoic acid) a competitive inhibitor of prostaglandin-synthetase inhibited PGF2 alpha production but did not affect basal nor PRL-stimulated casein secretion. Fourteen microM indomethacin inhibited PGF2 alpha and LTC4 production and PRL-stimulated casein secretion. Ten microM NdgA (nordihydroguaiaretic acid) an inhibitor of lipoxygenase pathway, inhibited LTB4 and LTC4 production, increased basal level of casein secretion and inhibited PRL-stimulated casein secretion. Hundred microM caffeic acid, an inhibitor of glutathione-S-transferase (GST), a class of enzymes implied in the transformation of LTA4 into LTC4, had the same effect that NDGA on basal and PRL-stimulated casein secretion. These findings show that inhibitors of AA metabolites alter casein secretion.     

Arachidonic acid cytotoxicity in leukocytes: implications of oxidative stress and eicosanoid synthesis

Arachidonic acid (AA)-induced cytotoxicity was evaluated in leukocytes: the human leukemia cell lines HL-60, Jurkat and Raji and in rat lymphocytes. Such cytotoxicity was dose- and time-dependent. At concentrations below 5 microM, AA was not toxic; at 10-400 microM, AA induced apoptosis and at concentrations beyond 400 microM, necrosis. The minimum exposure time to trigger cell death was of around 1 h, but the effect was increased by longer exposure times until 6-24 h. Apoptosis was morphologically characterized by a decrease in cell and nuclear volume, chromatin condensation and DNA fragmentation and the presence of lipid bodies, without changes in organelle integrity. Biochemically, AA-induced apoptosis was associated with internucleosomal fragmentation and caspase activation, evaluated by PARP cleavage and the use of a caspase inhibitor. Necrosis was characterized by increased cell volume, presence of loose chromatin, appearance of vacuoles, loss of membrane integrity and of the definition of organelles. The apoptotic effect of AA was studied as to oxidative-reductive imbalance and the participation of eicosanoids. Apoptotic AA treatment was accompanied by an increase in the quantity of thiobarbituric acid reactive substances (TBARS), low-level chemiluminescence and in the glutathione disulfide/reduced glutathione ratio, indicating oxidative stress. The addition of tocopherol, ascorbate, prostaglandin E2 and lipoxygenase inhibitors delayed cell death, whereas the inhibition of cyclooxygenase promoted AA-induced cell death. Cell treatment with AA was accompanied by increased cellular production of LTB4. AA, therefore, is cytotoxic at physiological and supraphysiological concentrations, causing apoptosis and necrosis. Cell treatment with apoptotic concentrations of AA involves oxidative stress and changes in eicosanoid biosynthesis.     

The effect of calcium ionophore A23187 on the metabolism of arachidonic and eicosapentaenoic acids in neutrophils from a marine teleost fish rich in (n-3) polyunsaturated fatty acids

1. [1-14C]AA and [1-14C]EPA were incorporated equally into plaice neutrophil phospholipids. 2. Incubation with A23187 resulted in the loss of label from total phospholipids and increased label released from the neutrophils. 3. Labelled LTB4 and LTB5 production was increased 2.5- and 3-fold, respectively, by A23187 treatment. 4. However, the incorporated AA was generally more metabolically active than the incorporated EPA with approx. 3-4 times as much LTB4 produced than LTB5 by cells in the presence or absence of A23187, respectively. 5. The results obtained in this (n-3) PUFA-rich species were discussed in comparison with current knowledge of AA and EPA metabolism in mammalian neutrophils.     

Dietary effects of eicosapentaenoic and docosahexaenoic acid esters on lipid metabolism and immune parameters in Sprague-Dawley rats

Sprague-Dawley rats were fed eicosapentaenoic (EPA) and docosahexaenoic acid (DHA) ethyl esters at the 2% level for 3 weeks to clarify their effects on immune functions. In the rats fed EPA or DHA, serum cholesterol, triglyceride, and phospholipid (PL) levels were significantly lower than those in the rats fed safflower oil. In PL fractions of serum, liver, lung, splenocytes, and peritoneal exudate cells (PEC), increases in linoleic and dihomo-gamma-linolenic acid contents and a decrease in arachidonic acid (AA) content were observed in the rats fed EPA or DHA. In addition, the EPA content increased in the rats fed EPA and DHA. In the rats fed EPA or DHA, a decrease of LTB4 productivity and an increase of LTBs productivity were observed in the PEC, in response to the treatment with 5 microM calcium ionophore A23187 for 20 min. The changes in leukotriene production were more marked in EPA-fed rats than in DHA-fed rats. These results suggest that dietary EPA affects lipid metabolism and leukotriene synthesis more strongly than DHA.     

Phospholipase A2 activation in human neutrophils. Differential actions of diacylglycerols and alkylacylglycerols in priming cells for stimulation by N-formyl-Met-Leu-Phe

Both 1,2-diacyl- and 1-O-alkyl-2-acylglycerols are formed during stimulation of human neutrophils (PMN), and both can prime respiratory burst responses for stimulation by the chemotactic peptide, N-formyl-Met-Leu-Phe (fMLP); however, mechanisms of priming are unknown. Arachidonic acid (AA) release through phospholipase A2 activation and metabolism by 5-lipoxygenase are important activities of PMN during inflammation and could be involved in the process of primed stimulation. Therefore, we have examined the ability of diacyl- and alkylacylglycerols to act as priming agents for AA release and metabolism in human neutrophils. After prelabeling PMN phospholipids with [3H]AA, priming was tested by incubating human PMN with the diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG), or its alkylacyl analog, 1-O-delta 9-octadecenyl-2-acetylglycerol (EAG) before stimulating with fMLP. fMLP (1 microM), OAG (20 microM), or EAG (20 microM) individually caused little or no release of labeled AA. However, after priming PMN with the same concentrations of either OAG or EAG, stimulation with 1 microM fMLP caused rapid (peak after 1 min) release of 6-8% of [3H]AA from cellular phospholipids; total release was similar with either diglyceride. Priming cells with OAG also enhanced conversion of released AA to leukotriene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE) upon subsequent fMLP stimulation, but AA metabolites were not increased in EAG-primed PMN. If fMLP was replaced with the calcium ionophore A23187 (which directly causes release of AA and production of LTB4 and 5-HETE), priming by both diglycerides again enhanced release of [3H]AA, but only OAG priming increased lipoxygenase activity. Indeed, EAG pretreatment markedly reduced LTB4 and 5-HETE production. Thus, both diglycerides prime release of AA from membrane phospholipids but have opposite actions on the subsequent metabolism of AA.     

Analysis of leukotrienes, prostaglandins, and other oxygenated metabolites of arachidonic acid by high-performance liquid chromatography

High-performance liquid chromatography procedures were developed which separate leukotrienes (LTs), hydroxy-fatty acids (HETEs), prostaglandins (PGs), the stable metabolite of prostacyclin (6-keto-PGF1 alpha), the stable metabolite of thromboxane A2 (TXB2), 12-hydroxyheptadecatrienoic acid (HHT), and arachidonic acid (AA). Two methods employing reverse-phase columns are described. One method uses a radial compression system, the other a conventional steel column. Both systems employ methanol and buffered water as solvents. The radial compression system requires 60 min for separation of the AA metabolites, while the conventional system requires 100 min. Both methods provide good separation and recovery of 6-keto-PGF1 alpha, TXB2, PGE2, PGF2 alpha, PGD2, LTC4, LTB4, LTD4, LTE4, HHT, 15-, 12-, and 5-HETE; and AA. The 5S,12S-dihydroxy-6-trans, 8-cis, 10-trans, 14-cis-eicosatetraenoic acid (5S,12S-diHETE), a stereoisomer of LTB4, coelutes with LTB4. To determine the applicability of the methods to biologic systems, AA metabolism was studied in two models, guinea pig lung microsomes and rat alveolar macrophages. Both HPLC systems demonstrated good recovery and resolution of eicosanoids from the two biological systems. A simple evaporation technique for HPLC sample preparation, which avoids the use of chromatographic and other time-consuming methodology, is also described.     

Modulation of lymphocyte proliferation by macrophages and macrophages loaded with arachidonic acid

Arachidonic acid (AA) is incorporated and exported by macrophages. This fatty acid is also transferred from macrophages (Mphi) to lymphocytes (LY) in co-culture. This observation led us to investigate the effect of macrophages pre-loaded with AA on concanavalin A (Con A)-stimulated lymphocyte proliferation. The experiments were performed in co-culture. This condition reproduces the in vivo microenvironment in which the modulation of lymphocyte proliferation is dependent on the interaction with macrophages. Lymphocytes obtained from untreated rats or from intraperitoneally thioglycolate-injected rats (THIO-treated) were co-cultured with macrophages from the same rats. Firstly, macrophages were co-cultured for 48 h with Con A-stimulated lymphocytes in different proportions: 0.5, 1, 2.5, 5, 10, 20 and 30% of 5 x 10(5) lymphocytes per well. At 1% proportion, macrophages caused maximum stimulation of lymphocyte proliferation; a four- to five-fold increase, for cells from both thioglycolate-treated and untreated rats, respectively, whereas at 20% it caused maximum inhibition. In addition, 1 or 20% macrophages were pre-loaded with several AA concentrations during a period of 6 h and co-cultured with lymphocytes. At 180 microM AA and 1% macrophages, lymphocyte proliferation was inhibited (by 25%), whereas at 20% macrophages, proliferation was increased, by 25- and three-fold, respectively, for cells from untreated and THIO-treated rats. AA added directly to the medium reduced lymphocyte proliferation, also being toxic to these cells at 100 microM. No toxic effects of AA were observed on macrophages. Additional evidence suggests that nitric oxide production is involved in the modulation of lymphocyte proliferation by AA-pre-loaded macrophages. These findings support the proposition that AA can directly modulate lymphocyte proliferation and the interaction between macrophages and lymphocytes.     

Cell-cell interaction of macrophages and vascular smooth muscle cells in the synthesis of leukotriene b(4)

Biosynthesis of LTB(4) during cell-cell interaction between vascular smooth muscle cells (SMC) and alveolar macrophages (AM) has been investigated by use of both high-pressure Hquid chromatography (HPLC) and radtoimmunoassay (RIA). Both interleukin-beta (IL-beta) and tumour necrosis factor-alpha (TNFalpha) induced a time- and dose-dependent synthesis of 15-, and 5-hydroxyeicosatetraenoic acids (HETEs) from cultured SMC. However, neither TNFalpha nor IL-1beta induced a significant LTB(4) production in SMC alone or AM alone after 24 h of incubation. Addition of IL-1beta and TNFalpha simultaneously to SMC resulted in a dose-dependent synergistic increase of HETEs. Macrophages dose-dependently transformed extremely low concentrations of exogenous LTA(4) into LTB(4). Incubation of vascular SMC with various numbers of AM in the presence of IL-1beta (5 units/ml) and TNFalpha (10 units/ml) induced a great increase of LTB(4) synthesis in comparison with the detectable levels of LTB(4) produced by macrophages alone. Pretreatment of SMC with NDGA, cycloheximide, and actinomycin not only inhibited IL-1 and TNT induced HETEs synthesis but also abolished LTB(4) production when co-incubated with macrophages. These results suggest that LTB(4) in a mixture of SMC and macrophages could originate from a transcellular metabolism, i.e. macrophages transforming SMC-derived LTA(4) into LTB(4).     

Characterization of leukotriene B4 synthesis in canine polymorphonuclear leukocytes.

The synthesis and release of leukotriene B4 (LTB4) from canine polymorphonuclear leukocytes (PMNs) was characterized in terms of incubation time, temperature and effects of calcium ionophore A23187 concentrations. Maximal LTB4 concentrations were determined when canine PMNs were incubated with 10 microM A23187. Increasing LTB4 concentrations were determined through 10 min incubation. The maximal LTB4 concentrations (310 +/- 30 pg LTB4/2.5 x 10(5) cells) determined at 10 min did not change through a 55 min incubation period. Greater LTB4 concentrations were synthesized by canine PMNs at 37 degrees C (268 +/- 12 pg LTB4/2.5 x 10(5) cells) than at 25 degrees C (206 +/- 11 pg LTB4/2.5 x 10(5) cells) or 5 degrees C (59 +/- 3 pg LTB4/2.5 x 10(5) cells). The synthesis of LTB4 in canine PMNs was inhibited by incubation of the cells with either of two known lipoxygenase inhibitors, BWA4C or BW755C. BWA4C inhibited LTB4 synthesis with an approximate IC50 = 0.1 microM, whereas BW755C inhibited LTB4 synthesis with an approximate IC50 = 10 microM. These results indicate canine PMNs have the capability to synthesize large quantities of LTB4 when stimulated with calcium ionophore A23187. Furthermore, the 5-lipoxygenase inhibitors BWA4C, an acetohydroxyamic acid, and BW755C, a phenyl pyrazoline, can readily inhibit LTB4 synthesis in canine PMNs.     

Release of eicosanoids from cultured rat aortic endothelial cells; studies with arachidonic acid and calcium ionophore A23187

The release of prostanoids from the three different vascular cell types derived from rat aortic explants has been studied in vitro. Under resting conditions and when incubated with exogenous arachidonic acid (AA, 10 microM), the endothelial cells (EC) produced the highest concentration of prostacyclin (PGI2 PGE2 PGF2 alpha TxA2). In contrast, PGE2 was the major prostanoid produced by the smooth muscle cells and fibroblasts. Pretreatment of EC with aspirin (10 microM) or indomethacin (10 microM) effectively inhibited the production of prostanoids by these cells. Incubation with the calcium ionophore A23187 (10 microM) did not stimulate production of PGI2 or leukotriene B4 (LTB4) by EC. However, treatment of EC with a combination of A23187 and AA led to production of amounts of both PGI2 and LTB4 which were greater than the summed values for the different drug treatments. These findings indicate that the concentration of substrate, AA, is a limiting factor in prostanoid formation by these cultured vascular cells but that rat EC are relatively poor in the enzymes required for leukotriene formation.