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.     

Metabolism of arachidonic acid by human nasal and bronchial epithelial cells

Nasal and bronchial epithelium from normal human nasal turbinates was isolated from surgical specimens and used to study arachidonic acid metabolism. High-performance liquid chromatography analysis of cell incubations in the presence of calcium ionophore, A23187, showed the formation of 15-lipoxygenase products. The major arachidonic acid metabolite with bronchial and nasal tissue was 15-HETE identified by uv spectroscopy, coelution with the authentic standards by HPLC, and GC-mass spectrometry. The second major metabolite, formed from either arachidonic acid or 15-HPETE, was identified as 13-hydroxy-14,15-epoxy-5,8,11-eicosatetraenoic acid (15-alpha-HEPA) by uv spectroscopy, coelution with the authentic standard, and GC-mass spectrometry. In addition, two 8,15-diHETEs and two 8,15-LTs were identified by uv spectroscopy and coelution with the authentic standards by HPLC on both reverse-phase and normal-phase HPLC. Also isolated and identified were 14,15-diHETEs, and 12-HETE. Nasal epithelial cells appear to be more active than nasal bronchial cells in oxidizing arachidonic acid. However, the profile of metabolites from these normal tissue preparations was similar. The addition of 15-lipoxygenase products to nasal epithelium weakly stimulated Cl- ion secretion. These studies indicate that human pulmonary epithelial cells selectively oxidize arachidonic acid to 15-lipoxygenase metabolites.     

Colchicine inhibits ionophore-induced formation of leukotriene B4 by human neutrophils: the role of microtubules

Neutrophils which ingest particles (serum-treated zymosan, monosodium urate crystals) or are exposed to calcium ionophore A23187 generate leukotriene B4 (LTB4). Earlier work has shown that cells exposed to colchicine before exposure to monosodium urate crystals produce less LTB4; the formation of 5-HETE is unaffected. To determine whether inhibition by colchicine of LTB4 generation was stimulus-specific and was mediated by microtubule integrity, the effects of colchicine (10 microM, 60 min) on the release of lipoxygenase products from neutrophils exposed to ionophore A23187 (10 microM, 5 min) were examined. In the presence of exogenous arachidonic acid (100 microM, 15 min), colchicine decreased LTB4 to 48% +/- 11.7 of control and 5-HETE to 60.5% +/- 5.7 of control (mean +/- SEM); 15-HETE was also decreased to 61% +/- 10.3 of control. In the absence of exogenous arachidonate, LTB4 was decreased to 22.2% +/- 11.7 of control and 5-HETE to 13% +/- 4.8 of control. Lumicolchicine did not significantly affect formation of 5-HETE or LTB4. However, vinblastine sulfate (20 microM, 60 min), another microtubule-disruptive agent, decreased the formation of both 5-lipoxygenase products. The effects of colchicine and vinblastine were not due to impairment of cell viability because the release of cytoplasmic lactic dehydrogenase was unaffected. Ultrastructural analysis of centriolar microtubules showed that decrements in microtubule numbers of colchicine- and vinblastine-treated cells paralleled decrements in 5-lipoxygenase products. These pharmacologic manipulations suggested that functional microtubules might be required for optimal lipoxygenase activity. Consequently, we prepared neutrophil-derived cytoplasts, devoid of an intact microtubule system. No significant decreases in the 5- or 15-lipoxygenase products were found when cytoplasts were exposed to colchicine in the presence of exogenous arachidonate and A23187. The data show that colchicine inhibits the formation of lipoxygenase products from neutrophils stimulated with A23187, most likely via its effect on microtubules, the integrity of which appears necessary for full expression of 5- and 15-lipoxygenases.     

Development of a radioimmunoassay for 15-HETE and its application to 15-HETE production by reticulocytes

Mono-hydroxy-eicosatetraenoic acids (HETE's) are frequently the principal lipoxygenase-derived products in a number of cell types. This paper describes the development of a selective and sensitive radioimmunoassay procedure for 15-HETE, a metabolite which has previously been shown to be both an activator and inhibitor of leukotriene formation in various cells. Initially, rabbits were immunized with 15-HETE conjugated to bovine serum albumin. After seven months, the anti-plasma showed significant binding of tritiated 15-HETE (40-45% binding with a 1:600 dilution of the anti-plasma) which was displaceable by cold 15-HETE. The sensitivity of the assay was approximately 20 pg. of 15-HETE. The anti-plasma exhibited very little (less than 1%) cross-reactivity with arachidonic acid, 5-, 8-, 9-, 11- and 12-HETE's, HHT, TXB2, PGE2 and 6-Keto-PGF1 alpha. Significant cross-reactivity was observed with 5,15-diHETE (53%), 8, 15-diHETE (6.6%), and several other 15-hydroxy-eicosanoids. Rabbit reticulocytes have a very active 15S-lipoxygenase and converted arachidonic acid (final concentration 7 microM) principally to 15-HETE. Unstimulated reticulocytes were found to release negligible amounts of 15-HETE as determined by radioimmunoassay. Treatment of these cells with the calcium ionophore A23187 (0.16 to 4.0 micrograms/ml) elicited a level of 15-HETE release (8 - 14 ng/ml) that was twenty to forty times less than that obtained with exogenous arachidonic acid (2.5 micrograms/ml). The radioimmunoassay reported here may be useful for identifying factors which stimulate cellular release of 15-HETE and other 15-hydroxy-eicosanoids from endogenous arachidonic acid.     

Endogenous hydroxyeicosatetraenoic acids stimulate the human polymorphonuclear leukocyte 15-lipoxygenase pathway

Arachidonic acid metabolism in ionophore A23187-activated human polymorphonuclear leukocytes (PMNs) proceeds predominantly via the 5-lipoxygenase pathway in comparison to metabolism by the 15-lipoxygenase route. Products of both lipoxygenase pathways appear to be involved in the mediation of inflammatory reactions. Pretreatment of polymorphonuclear leukocytes with micromolar amounts of the platelet-derived 12-lipoxygenase product 12-hydroxy-5,8,10,14- eicosatetraenoic acid (12-HETE) prior to the addition of A23187 and [14C]arachidonic acid resulted in the unexpected dose-dependent stimulation of the 15-lipoxygenase pathway, as evidenced by the formation of [14C]15-HETE. A concomitant inhibition of the 5-lipoxygenase pathway was also observed. The structural identity of 15-HETE was confirmed by retention times on straight-phase and reverse-phase high pressure liquid chromatography in comparison with an authentic standard, radioimmunoassay, and chemical derivatization. When other isomeric HETEs were tested, the order of stimulatory potencies was 15-HETE greater than 12-HETE greater than 5-HETE. When arachidonic acid metabolism via the 5-lipoxygenase route was inhibited by nordihydroguaiaretic acid, previously ineffective concentrations of exogenous 12-HETE were now able to stimulate the polymorphonuclear leukocyte 15-lipoxygenase. Thus, blockade of the 5-lipoxygenase pathway appeared to be a prerequisite for the activation of the 15-lipoxygenase. The HETE-induced activation of the 15-lipoxygenase occurred within 1-2 min, was a reversible process, and was enhanced in the presence of A23187. In nine donors tested, up to 14-fold stimulation of [14C]15-HETE production was observed. Our results indicate that endogenous HETEs can have a dual role in the post-phospholipase regulation of arachidonic acid metabolism since they can act as physiological stimulators of the 15-lipoxygenase as well as inhibitors of the 5-lipoxygenase.     

1-[4-[3-[4-[bis(4-fluorophenyl)hydroxymethyl]-1- piperidinyl]propoxy]-3-methoxyphenyl]ethanone(AHR-5333): a selective human blood neutrophil 5-lipoxygenase inhibitor

In this study we report the in vitro inhibition of leukotriene synthesis in calcium ionophore (A23187)-stimulated, intact human blood neutrophils by AHR-5333. The results showed that AHR-5333 inhibits 5-HETE, LTB4 and LTC4 synthesis with IC50 values of 13.9, 13.7 and 6.9 microM, respectively. Further examination of the effect of AHR-5333 on individual reactions of the 5-lipoxygenase pathway (i.e. conversion of LTA4 to LTB4, LTA4 to LTC4, and arachidonic acid to 5-HETE) showed that this agent was not inhibitory to LTA4 epoxyhydrolase and glutathione-S-transferase activity in neutrophil homogenates. However, conversion of arachidonic acid (30 microM) to 5-HETE was half maximally inhibited by 20 microM AHR-5333 in the cell-free system. The inhibition of LTB4 and LTC4 formation in intact neutrophils by AHR-5333 appears to be entirely due to a selective inhibition of 5-lipoxygenase activity and an impaired formation of LTA4, which serves as substrate for LTA4 epoxyhydrolase and glutathione-S-transferase. AHR-5333 did not affect the transformation of exogenous arachidonic acid to thromboxane B2, HHT and 12-HETE in preparations of washed human platelets, indicating that this agent has no effect on platelet prostaglandin H synthase, thromboxane synthase and 12-lipoxygenase activity. The lack of inhibitory activity of AHR-5333 on prostaglandin H synthase activity was confirmed with microsomal preparations of sheep vesicular glands.     

Ionophore-stimulated lipoxygenase activity and histamine release in a cloned murine mast cell, MC9

A cloned murine mast cell line designated MC9 expresses a 5-lipoxygenase activity when stimulated with the ionophore A23187. Upon addition of 0.5 microM ionophore, MC9 cells produce 270 +/- 43 pmoles 5-HETE, 74 +/- 40 pmoles 5,12 diHETEs and 65 +/- 31 pmoles LTC4/10(6) cells from 37 microM exogenously added [1-14C]arachidonic acid in two minutes. 5-HETE and 5,12-diHETES, including LTB4 were identified by GC/MS whereas LTC4 was confirmed by HPLC mobility, bio-assay, RIA and enzymatic transformation. The principal cyclooxygenase products were PGD2 and TxB2 (8.5 +/- 2.4 and 5.4 +/- 1.2 pmoles/10(6) cells respectively). Prostanoids were identified by comigration with authentic standards on two-dimensional thin layer chromatograms. Production of arachidonic acid lipoxygenase metabolites stimulated with ionophore proved relatively insensitive to removal of extracellular Ca+2 and chelation by EGTA. In addition, MC9 5-lipoxygenase required only low micromolar amounts of exogenous arachidonic acid for maximal activity. Whereas production of arachidonic acid metabolites lasted only two to five minutes, histamine release stimulated with ionophore was not initiated until 5 minutes (12 +/- 3% cellular histamine) and continued for 30 minutes (37 +/- 7% cellular histamine). Although these cells metabolize arachidonic acid differently from the classic peritoneal-derived mast cell, they resemble subpopulations found in certain tissues (such as mucosa) and should be useful in understanding the biochemistry of mast cell mediator release.     

Leukotriene B4 level in neutrophils from allergic and healthy subjects stimulated by low concentration of calcium ionophore A23187. Effect of exogenous arachidonic acid and possible endogenous source.

Peripheral blood neutrophils from patients with allergic rhinitis and from normal subjects were incubated for 5 min at 37 degrees C with 0.15 microM calcium ionophore A23187 in the absence or presence of exogenous arachidonic acid (2.5 to 10 microM). In neutrophils from allergic patients, the leukotriene B4 (LTB4) level was significantly increased by exogenous arachidonic acid in a concentration-dependent manner (16.2 +/- 4.2 and 38.1 +/- 6.8 pmol/5 min per 2 X 10(6) cells in the absence and presence of 10 microM arachidonic acid, respectively; P less than 0.005; n = 8). The LTB4 level in neutrophils from healthy subjects was only 0.97 +/- 0.17 pmol/5 min per 2 x 10(6) cells (n = 5) and was not enhanced by exogenous arachidonate. When cells from allergic patients were challenged in the presence of exogenous [1-14C]arachidonic acid, released LTB4 was radiolabeled and the incorporated radioactivity increased with the labeled arachidonate concentration. Labeled LTB4 was never detectable after incubating neutrophils from normal donors with exogenous labeled arachidonate. When neutrophils were incubated with [1-14C]arachidonate for 1 h, the different lipid pools of the two cell populations were labeled but both types of neutrophils produced unlabeled LTB4 in response to ionophore stimulation. The hydrolysis of choline and ethanolamine phospholipids into diacyl-, alkenylacyl- and alkylacyl-species revealed that solely the alkylacyl-subclass of phosphatidylcholine was unlabeled. We conclude (i) that neutrophils from allergic patients stimulated by low ionophore concentration produce more LTB4 than neutrophils from healthy subjects and incorporate exogenous arachidonate, (ii) that endogenous arachidonate converted to LTB4 by the 5-lipoxygenase pathway may provide only from 1-O-alkyl-2-arachidonoyl-glycero-3-phosphocholine.     

15(S)-hydroxyeicosatetraenoic acid is the major arachidonic acid metabolite in human bronchi: association with airway epithelium

15(S)-Hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) was by far the most abundant metabolite of arachidonic acid in chopped human bronchi, as identified by reverse phase HPLC, uv spectrometry, and GC/MS. The quantitation of monohydroxyeicosatetraenoic acids (mono-HETEs) was performed by the use of 16(S)-hydroxy-9(Z),12(Z),14(E)-heneicosatrienoic acid as internal standard. Thus, significant amounts of 15-HETE were obtained in incubations of bronchi in buffer alone, but the addition of exogenous arachidonic acid (3-100 microM), dose-dependently increased the formation, with maximal levels reached at around 10 min. In contrast, challenge with ionophore A23187 or anti-human IgE did not stimulate the production of 15-HETE in the bronchi. Nordihydroguaiaretic acid inhibited the production of 15-HETE, whereas indomethacin did not. Small amounts of 8,15-diHETEs were detected in incubations with exogenous 15H(P)ETE. Lipoxins were however not detected under any of the incubation conditions used. Furthermore, removal of the airway epithelium substantially diminished the production of 15-HETE in the bronchi. Finally, bronchi were obtained from three patients with asthma, and the amounts of 15-HETE in these specimens were significantly higher than those found in tissues from nonasthmatics. Also, in peripheral lung parenchyma and pulmonary blood vessels 15-HETE was the major mono-HETE after stimulation with arachidonic acid but the levels were about 10 times lower than in the bronchi. As another difference, challenge of the parenchyma with the ionophore A23187 made 5-HETE the predominant mono-HETE. Taken together, airway epithelium appears to be the major source of 15-HETE in the human lung and the findings in specimens of asthmatics raise the possibility that 15-HETE somehow is involved in airway inflammation.     

Arachidonic acid metabolism by rabbit synovial cells in culture. Studies of non-cyclooxygenase pathways

We have investigated arachidonic acid (20:4) metabolism by rabbit synovial cells in culture. The lipoxygenase products 5-HETE, 12-HETE and 15-HETE were not detected, despite the presence of a cyclooxygenase inhibitor sodium meclofenamate (20 microM), nor after incubation with ionophore A23187 (1 microM), 20:4 (10 microM), prostaglandin E2, (1 microM), N-formylmethionylleucylphenylalanine (0.01 microM), or murine spleen cell-conditioned medium. [3H]20:4 (10 microM) was incorporated into phospholipids, triacylglycerols and diacylglycerols. A majority of the 3H content of phosphatidylinositol/phosphatidylserine and of diacylglycerols was already present at 1 min, in contrast to the slower accumulation of 3H in triacylglycerols, phosphatidylcholine and phosphatidylethanolamine. The diacylglycerol fraction contained sn-glycerol-1-acyl-2-20:4. These observations are consistent with phospholipase C activity in synovial cells under those culture conditions. The products generated by these enzymes may play important roles in the physiological processes of synovium.     

COX-1 dependent biosynthesis of 15-hydroxyeicosatetraenoic acid in human mast cells

15-hydroxyeicosatetraenoic acid (15-HETE) is an arachidonic acid derived lipid mediator which can originate both from 15-lipoxygenase (15-LOX) activity and cyclooxygenase (COX) activity. The enzymatic source determines the enantiomeric profile of the 15-HETE formed. 15-HETE is the most abundant arachidonic acid metabolite in the human lung and has been suggested to influence the pathophysiology of asthma. Mast cells are central effectors in asthma, but there are contradictory reports on whether 15-HETE originates from 15-LOX or COX in human mast cells. This prompted the current study where the pathway of 15-HETE biosynthesis was examined in three human mast cell models; the cell line LAD2, cord blood derived mast cells (CBMC) and tissue isolated human lung mast cells (HLMC). Levels and enantiomeric profiles of 15-HETE and levels of the downstream metabolite 15-KETE, were analyzed by UPLC-MS/MS after stimulation with anti-IgE or calcium ionophore A23187 in the presence and absence of inhibitors of COX isoenzymes. We found that 15-HETE was produced by COX-1 in human mast cells under these experimental conditions. Unexpectedly, chiral analysis showed that the 15(R) isomer was predominant and gradually accumulated, whereas the 15(S) isomer was metabolized by the 15-hydroxyprostaglandin dehydrogenase. We conclude that during physiological conditions, i.e., without addition of exogenous arachidonic acid, both enantiomers of 15-HETE are produced by COX-1 in human mast cells but that the 15(S) isomer is selectively depleted by undergoing further metabolism. The study highlights that 15-HETE cannot be used as an indicator of 15-LOX activity for cellular studies, unless chirality and sensitivity to pharmacologic inhibition is determined.     

Cell damage unmasks 15-lipoxygenase activity in human neutrophils

Metabolism of arachidonic acid (10 microM) into 15(S)-hydroxyl-5,8,11-cis-13-trans-eicosatetraenoic acid (15-HETE) was proportional to lactate dehydrogenase release from human neutrophils incubated with supratherapeutic concentrations of non-steroidal anti-inflammatory agents. In contrast to others (Vanderhoek, J., and Bailey, J. (1984) J. Biol. Chem. 259, 6752-6756), we report that increased 15-HETE formation was not uniquely attributable to 5 mM ibuprofen, and it did not originate from enzymatic activation. For instance, ibuprofen (1-5 mM) did not affect the isolated 15-lipoxygenase enzyme in the 100,000 X g supernatant from neutrophil lysates, and dose-dependent increases in 15-HETE biosynthesis, proportional to lactate dehydrogenase release, were evident with benoxaprofen, naproxen, flurbiprofen, or etodolac. At similar supratherapeutic concentrations (1-5 mM), aspirin and phenylbutazone did not influence lactate dehydrogenase release or 15-HETE production. In further contrast, neutrophils did not tolerate 1-5 mM ibuprofen. Biochemical, morphological, flow cytometric, and fluorochromatic analyses each indicated cytological damage. A correlation between lactate dehydrogenase release and increased 15-HETE formation was a dose-dependent property also exhibited by arachidonic acid alone (10-100 microM). We conclude that cytological damage, facilitating access of arachidonic acid to 15-lipoxygenase in a cytosolic compartment, accounts for this phenomenon.     

Lipoxygenase- and cyclooxygenase-reaction products and incorporation into glycerolipids or radiolabeled arachidonic acid in the bovine retina

The metabolism of radiolabeled arachidonic acid (AA) by the intact bovine retina in vitro has been studied. Synthesis of prostaglandins (PGs) and hydroxyeicosatetraenoic acids (HETEs), and incorporation of AA into glycerolipids has been measured by reverse-phase and straight-phase high performance liquid chromatography with flow scintillation detection, and by thin-layer chromatography. AA was actively acylated into glycerolipids, particularly triglycerides, phosphatidylcholine and phosphatidylinositol. AA was also converted to the major PGs, PGF2 alpha, PGE2, PGD2, 6-keto-PGF1 alpha and TXB2, and to the lipoxygenase reaction products, 12-HETE, 5-HETE, and other monohydroxy isomers. Approximately 6% of the radiolabeled AA was converted to eicosanoids. The synthesis of HETEs was inhibited in a concentration-dependent manner (IC50 = 8.3 nM) by nordihydroguaiaretic acid (NDGA). PG synthesis was inhibited by aspirin (10 microM), indomethacin (1 microM) and NDGA (IC50 = 380 nM). Metabolism of AA via lipoxygenase, cyclooxygenase and activation-acylation was inhibited by boiling retinal tissue prior to incubation. These studies demonstrate an active system for the uptake and utilization of AA in the bovine retina, and provide the first evidence of lipoxygenase-mediated metabolism of AA, resulting in the synthesis of mono-hydroxyeicosatetraenoic acids, in the retina.     

Human B lymphocytes possess 5-lipoxygenase activity and convert arachidonic acid to leukotriene B4.

Incubation of cell sonicates from monoclonal B cells with arachidonic acid led to the formation of leukotriene (LT) B4 and 5-hydroxy-eicosatetraenoic acid (5-HETE). In contrast, stimulation of intact B cells with the calcium ionophore A23187 +/- arachidonic acid did not, under similar conditions, lead to formation of LTB4. The identification of these products was based on reverse phase- and straight phase-HPLC analysis, UV-spectroscopy and gas chromatography-mass spectrometry. Cell sonicates of highly enriched human tonsillar B lymphocytes also converted arachidonic acid to LTB4 and 5-HETE. Activation of these cells with B cell mitogen and cytokines for three days led to an upregulation of 5-lipoxygenase activity. This study provides evidence for the biosynthesis of LTB4 from arachidonic acid in B cell lines and in normal human tonsillar B lymphocytes.     

Phospholipid metabolism in human neutrophil subfractions

We describe a procedure for isolating human neutrophil subfractions by sucrose density centrifugation following nitrogen cavitation. Using this procedure we were able to isolate and characterize a cytosolic fraction, two separate plasma membrane-enriched fractions, and specific and azurophilic granule fractions. We used this procedure to examine the subcellular localization of the enzymes and substrates involved in the release of arachidonic acid from cellular phospholipids in response to whole cell stimulation. Whole cells were prelabeled for 2 h with [3H]arachidonic acid and [14C] stearic acid. When prelabeled cells were challenged with calcium ionophore A23187 (2 microM) for 5 min at 37 degrees C, each membrane-associated fraction, including both plasma membrane fractions and specific and azurophilic granule fractions, exhibited deacylation of phosphatidylinositol (PI) and phosphatidylcholine (PC). The specific granule fraction exhibited the greatest proportion of deacylation from PI while the more dense plasma membrane fraction was deacylated to a much lower extent than the other fractions. In terms of mass, the azurophilic granules deacylated the greatest amount of radiolabeled arachidonic acid. Although all membrane fractions may be sources of arachidonic acid to some extent, the azurophilic granule fraction may contain the largest pool of radiolabeled arachidonic acid that is released upon cell stimulation.     

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.     

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.     

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.     

A calcium-independent 5-lipoxygenase system in mast/basophil PT-18 cells

Mammalian 5-lipoxygenase systems exist in inactive or cryptic states and have to be stimulated in order to metabolize exogenous [14C]arachidonic acid to 5-HETE and leukotrienes. In most cells, both the activation process and the 5-lipoxygenase activity are calcium-dependent. However, the cryptic 5-lipoxygenase system in the murine PT-18 mast/basophil cell line, which can be stimulated by 15-hydroxyeicosatetraenoic acid (15-HETE), is unusual. Studies with fura-2 loaded PT-18 cells indicate that increases in cytosolic calcium do not appear to correlate with enhanced 5-lipoxygenase product formation. Thus, both the calcium ionophore ionomycin and arachidonic acid increase cytosolic calcium levels but have very little effect on [14C]5-HETE formation, whereas 15-HETE induces large increases in [14C]5-HETE production but no concomitant enhancement in cytosolic calcium is observed. Chelation of extracellular calcium by 3 mM EGTA resulted in a 30-40% inhibition of [14C]5-HETE formation induced by 15 HETE, whereas 3 mM EGTA has no appreciable effect on a crude PT-18 5-lipoxygenase homogenate. These results indicate that in PT-18 cells, calcium does not appear to play an important role in either the 15-HETE-induced activation process, or the enzymatic activity of the cryptic 5-lipoxygenase system.