Effect of various lipoxygenase metabolites of arachidonic acid on degranulation of polymorphonuclear leukocytes

Lipoxygenase metabolites of guinea pig peritoneal polymorphonuclear leukocytes stimulated with 10 microM A23187 plus arachidonic acid were isolated and identified. These metabolites were compared with each other and to chemically synthesized arachidonate metabolites for their ability to stimulate leukocyte degranulation. 5(S),12(R)-Dihydroxy-6,8,10-(cis/trans/trans)14-cis-eicosatetraenoic acid (leukotriene B4) produced a significant release of lysozyme, but not beta-glucuronidase or beta-N-acetylglucosaminidase at low concentrations (EC50 = 6.5 x 10(-9) M), while the leukocyte nonenzymatically generated 5,12-or 5,6-dihydroxyeicosatetraenoic acids had no effect at these concentrations. Higher concentrations (1--10 microM) of all the dihydroxyeicosatetraenoic acids, 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) and its hydroperoxy precursor stimulated significant lysozyme release which was greater than that produced by 15-hydroxy-5,8,11-13-eicosatetraenoic acid, arachidonic acid, or its acetylene analogue, 5,8,11,14-eicosatetraynoic acid. Micromolar concentrations of leukotriene B4 and 5-HETE also stimulated significant release of beta-N-acetylglucosaminidase above controls, but not beta-glucuronidase. These results suggest that leukotriene B4 may play a role in regulating the release of certain granule-bound enzymes from polymorphonuclear leukocytes.     

Evidence for a novel pathway of leukotriene formation in human platelets

The metabolism of arachidonic acid via lipoxygenase-catalyzed reactions in washed human platelets was investigated. In addition to the previously discovered lipoxygenase metabolites, 12-hydroxyeicosatetraenoic acid, 15-hydroxyeicosatetraenoic acid, 8,15-dihydroxyeicosatetraenoic acid and 14,15-dihydroxyeicosatetraenoic acid, several other products were formed. The compounds were all dihydroxylated metabolites of arachidonic acid, containing a conjugated triene structure, and identified as 11,12-dihydroxyeicosatetraenoic acid (two isomers) and 5,12-dihydroxyeicosatetraenoic acid (four isomers). The identification was based on ultraviolet spectroscopy and gas chromatography-mass spectrometry of native and hydrogenated compounds. Stereochemical analysis of the hydroxyl groups of the 5,12-dihydroxyeicosatetraenoic acids and experiments with 18O2 indicated that the compounds were formed by the 12-lipoxygenase pathway, probably via an unstable epoxide.     

Arachidonic acid metabolism by erythrocytes

Rabbit, chicken, rat, and dog erythrocytes (10(9) cells/ml) synthesized immunologically active 12-hydroxyeicosatetraenoic acid (12-HETE) when stimulated by the Ca2+ ionophore, A-23187. The levels of immunologically active hydroxyeicosatetraenoic acid were independent of the number of white blood cells and platelets in the erythrocyte suspensions. Two products were resolved by high performance liquid chromatography; one product was identified as 12-HETE, while a second product appeared to be a dihydroxyeicosatetraenoic acid. Radiolabeled arachidonic acid was incorporated into phospholipids. Phosphatidylcholine and phosphatidylethanolamine were primary sources of the 12-HETE and dihydroxyeicosatetraenoic acid, all of which were released from the cells.     

Enhanced tumor cell adhesion to the subendothelial matrix resulting from 12(S)-HETE-induced endothelial cell retraction

A 12-lipoxygenase metabolite of arachidonic acid, 12(S)-hydroxyeicosatetraenoic acid (12[S]-HETE), which is produced by platelets and tumor cells, was tested for its ability to induce retraction of endothelial cell monolayers. The induction of endothelial cell retraction is a critical step in tumor cell metastasis. Endothelial cells demonstrated reversible retraction in response to 12(S)-HETE, but did not respond to the stereoisomer 12(R)-HETE or to unrelated 5-lipoxygenase (i.e., 5[S]-HETE) or 15-lipoxygenase (i.e., 15[S]-HETE) metabolites. Endothelial cells did not demonstrate loss of viability in response to 12(S)-HETE. The induction of retraction was both dose and time dependent. Scanning electron microscopy confirmed that 12(S)-HETE induced endothelial cell retraction and revealed collapsed filopodia on their surface, the appearance of spaces between endothelial cells and the underlying subendothelial matrix, in addition to large gaps between adjacent endothelial cells. Tumor cell adhesion to endothelial cell monolayers was enhanced 1 h after pretreatment of monolayers with 12(S)-HETE but not after pretreatment with other lipoxygenase metabolites. Tumor cell adhesion to endothelial cell monolayers 36 h after pretreatment with 12(S)-HETE was not different from adhesion to untreated monolayers. Therefore we suggest that 12(S)-HETE generated during tumor cell-platelet-endothelial cell interactions may induce reversible endothelial cell retraction, allowing tumor cell access to the subendothelial matrix, which is a critical step in their eventual extravasation from the microvasculature during hematogenous metastasis.     

Stereospecificity of the hydroxyeicosatetraenoic and hydroxyoctadecadienoic acids produced by cultured bovine endothelial cells

Characterization of the stereospecificity of the derivatives of arachidonic acid and linoleic acid produced by endothelial cells is needed to define the enzymatic origin of these compounds and their role in vascular physiology. In studies utilizing two bovine endothelial cell lines (CPAE and AG04762), both free 15-hydroxyeicosatetraenoic acid (15-HETE) and 11-hydroxyeicosatetraenoic acid (11-HETE) were generated during incubations with exogenous arachidonic acid and both free 9-hydroxyoctadecadienoic acid (9-HODE) and 13-hydroxyoctadecadienoic acid (13-HODE) were generated during incubations with exogenous linoleic acid. Esterification of 15-HETE, 9-HODE and 13-HODE during these incubations was demonstrated. The analyses included reversed-phase high performance liquid chromatography of the free acid and its methyl ester and chiral separation of the methyl ester on straight phase chiral columns. The ratio of 9-HODE/13-HODE averaged 2.7 in the chromatographic analyses of the extracts of the incubations with linoleic acid. The combined production of 13-HODE and 9-HODE from linoleic acid was four times greater than that of 15-HETE and 11-HETE from arachidonic acid. With regard to the products of the CPAE endothelial cell line, the S/R ratio of the stereoisomers averaged 1.5 for free 15-HETE, 5.7 for free 13-HODE and 0.2 for free 9-HODE. The 11-HETE had strict (R) stereospecificity. The products from the AG04762 endothelial cell line had similar stereochemistry. All these stereochemical findings point to the activity of a cyclooxygenase rather than that of a lipoxygenase.     

Enhancement of platelet 12-HETE production in the presence of polymorphonuclear leukocytes during calcium ionophore stimulation.

This study investigates the effect of platelet/neutrophil interactions on eicosanoid production. Human platelets and polymorphonuclear leukocytes (PMNs) were stimulated alone and in combination, with calcium ionophore A23187 and the resulting eicosanoids 12S-hydroxy-(5Z,8Z,10E,14Z)-eicosatetraenoic acid (12-HETE), 12S-heptadecatrienoic acid (HHT), 5S,12R-dihydroxy-(6Z,8E,10E,14Z)-eicosatetraenoi c acid (LTB4) and 5S-hydroxy-(6E,8Z,11Z,14Z)-eicosatetraenoic acid (5-HETE) were measured by HPLC. The addition of PMNs to platelet suspensions caused a 104% increase in 12-HETE, a product of 12-lipoxygenase activity, but had only a modest effect on the cyclooxygenase product HHT (increase of 18%). By using PMNs labelled with [14C]arachidonic acid it was shown that the increases in these platelet eicosanoids could be accounted for by translocation of released arachidonic acid from PMNs to platelets and its subsequent metabolism. The observation that 12-lipoxygenase was about five times more efficient than cyclooxygenase at utilising exogenous arachidonic acid during the platelet/PMN interactions was confirmed in experiments in which platelets were stimulated with A23187 in the presence of [14C]arachidonic acid. Stimulations of platelets with thrombin in the presence of PMNs resulted in a decrease in 12-HETE and HHT levels of 40% and 26%, respectively. The presence of platelets caused a small increase in neutrophil LTB4 output but resulted in a decrease in 5-HETE production of 43% during stimulation with A23187. This study demonstrates complex biochemical interactions between platelets and PMNs during eicosanoid production and provides evidence of a mechanism to explain the large enhancement in 12-HETE production.     

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.     

Hydroxyeicosatetraenoic acids are increased in bronchoalveolar lavage fluid of endotoxemic pigs

We hypothesized that lipoxygenase metabolites of arachidonic acid might be produced during endotoxin-induced acute respiratory failure (ARF) observed in young pigs. We used radioimmunoassay (RIA) to determine the presence of 5-hydroxyeicosatetraenoic acid (5-HETE), 12-HETE, and 15-HETE in bronchoalveolar lavage fluid (BALF) of saline (n = 12)- and endotoxin (n = 18)-treated pigs. Endotoxin, infused at 5 micrograms/kg for 1 hr followed by 2 micrograms/kg/hr for an average of 3 hrs, caused pulmonary hypertension, a biphasic increase in pulmonary vascular resistance, hypoxemia, bronchoconstriction, leukopenia, and thrombocytopenia. Relative to saline controls, the levels of immunoreactive (i)-5-HETE (816 +/- 209 pg/ml), i-12-HETE (1589 +/- 517 pg/ml), and i-15-HETE (448 +/- 78 pg/ml) were significantly (P less than 0.05) increased in BALF recovered from endotoxemic pigs at postmortem. Relative to control BALF i-HETE concentrations, the endotoxin values were 3.5x, 5.1x, and 2.8x higher for i-5-HETE, i-12-HETE, and i-15-HETE, respectively. We conclude that during porcine endotoxemia, the 5-, 12-, and 15-lipoxygenase pathways are activated and that HETES might be involved in the pathophysiology of endotoxin-induced ARF.     

Stereospecificity of the products of the fatty acid oxygenases derived from psoriatic scales

The principal in vivo oxygenase products of arachidonic acid and linoleic acid in psoriatic skin scales are 12-hydroxyeicosatetraenoic acid (R/S ratio = 5.7), 13-hydroxyoctadecadienoic acid (S/R = 1.9), and 9-hydroxyoctadecadienoic acid (R/S = 2.4). Definition of the enzymatic origin of these fatty acid derivatives is an important step in assessing their possible role in the pathogenesis of psoriasis. Psoriatic skin scales were incubated with radiolabeled arachidonic acid and linoleic acid and the monohydroxylated derivatives produced in vitro were characterized. The products of incubation with [3H]arachidonic acid were an enantiopure 15(S)-[3H]hydroxyeicosatetraenoic acid and a nonracemic mixture of the 12-[3H]hydroxyeicosatetraenoic acid steroisomers (R/S ratio = 4.5). An enantiopure 13(S)-[14C]hydroxyoctadecadienoic acid was produced from [14C]linoleic acid. No radiolabeled products were derived from incubations with heat-denatured scales. These results provide evidence for two distinct oxygenase activities that are preserved in psoriatic skin scales. One is that of an omega-6 oxygenase with strict (S) stereospecificity, consistent with the activity of a lipoxygenase. This enzyme activity appears to be similar to that of the 15-lipoxygenase which has been described in cultured human keratinocytes. The second activity is that of an arachidonic acid 12(R)-oxygenase that has not been observed in normal human epidermis but which appears to be expressed in psoriatic epidermis.     

Structural requirements in hydroxyeicosanoids for the activation of the 5-lipoxygenase in PT-18 mast/basophil cells

We have previously reported that 15-hydroxyeicosatetraenoic acid (15-HETE) stimulated the 5-lipoxygenase in the murine PT-18 mast/basophil cell line to produce leukotriene B4 and 5-HETE from exogenously added arachidonic acid. In order to determine the structural requirements in the HETE molecule that are necessary for the activation of this 5-lipoxygenase, various isomeric HETEs, derivatives and analogs were prepared, purified and tested. The order of stimulatory potencies was: 15-HETE acetate greater than 15-HETE = 15-hydroperoxyeicosatetraenoic acid (15-HPETE) greater than 5-HPETE = 12-HPETE greater than 5-HETE. 15-HETE methyl ester, 12-HETE and prostaglandin E2 were ineffective over the concentration range tested. Several diHETEs were also tested. 5S,15S-DiHETE was somewhat less potent than 15-HETE, whereas both 8S,15S-diHETE and leukotriene B4 were inactive. The calcium ionophore A23187 was much less effective than 15-HETE. These structure-activity studies indicate the importance of the nature, position and location of the various functional groups in the HETE molecule and suggest that a specific recognition site is involved in the activation of the 5-lipoxygenase in PT-18 cells.     

Hydroxyeicosatetraenoic acids are increased in bronchoalveolar lavage fluid of endotoxemic pigs

We hypothesized that lipoxygenase metabolites of arachidonic acid might be produced during endotoxin-induced acute respiratory failure (ARF) observed in young pigs. We used radioimmunoassay (RIA) to determine the presence of 5-hydroxyeicosatetraenoic acid (5-HETE), 12-HETE, and 15-HETE in bronchoalveolar lavage fluid (BALF) of saline (n=12)- and endotoxin (n=18)- treated pigs. Endotoxin, infused at 5 μg/kg for 1 hr followed by 2 μg/kg/hr for an average of 3 hrs, caused pulmonary hypertension, a biphasic increase in pulmonary vascular resistance, hypoxemia, bronchoconstriction, leukopenia, and thrombocytopenia. Relative to saline controls, the levels of immunoreactive (i)-5-HETE (816 ± 209 pg/ml), i-12-HETE (1589 ± 517 pg/ml), and i-15-HETE (448 ± 78 pg/ml) were significantly ) increased in BALF recovered from endotoxemic pigs at postmortem. Relative to control BALF i-HETE concentrations, the endotoxin values were 3.5x, 5.1x, and 2.8x higher for i-5-HETE, i-12-HETE, and i-15-HETE, respectively. We conclude that during porcine endotoxemia, the 5-, 12-, and 15-lipoxygenase pathways are activated and that HETES might be involved in the pathophysiology of endotoxin-induced ARF.     

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.     

15-Hydroxyeicosatetraenoic acid stimulates migration of human retinal microvessel endothelium in vitro and neovascularization in vivo.

We evaluated 15-hydroxyeicosatetraenoic acid (15-HETE), a major arachidonic acid product of vascular endothelium and leukocytes, for its effect on neovascularization. In a modified Boyden chamber assay, 15-HETE (10-7 M) stimulated human retinal microvessel endothelial cell migration by 42 +/- 10% (mean +/- S.E.M., p less than 0.01). 12-HETE, a major arachidonic acid metabolite of platelets, had no such effect. Further studies in the rabbit corneal pocket assay revealed that 15-HETE stimulated neovascularization in vivo. Concentrations at which the in vivo effects were observed are within the range generated by several cell types and are achievable in human serum. 15-HETE stimulation of human endothelial cell migration in vitro and neovascularization in vivo suggests that it may play a role in vasoproliferative disorders.     

Metabolism of arachidonic acid in neutrophils from alloxan-diabetic rabbits

The production of 5-lipoxygenase products from arachidonic acid was investigated in polymorphonuclear leukocytes (PMNL) isolated from non-diabetic and alloxan-induced diabetic rabbits: (i) production of 5-hydroxyeicosatetraenoic acid, leukotriene B4, and the two 6-trans-leukotriene B4 isomers were significantly decreased in the PMNL of diabetic rabbits when compared to non-diabetic rabbits; (ii) production of LTB4 and 5-HETE from diabetic PMNL required the addition of Ca2+ and A23187 to a greater degree than control incubations; and (iii) the availability of substrate in the PMNL of diabetics was not a limiting factor for 5-lipoxygenase product formation. Alternative pathways of arachidonic acid metabolism were also evaluated: the recovery of exogenous leukotriene B4 and 5-hydroxyeicosatetraenoic acid were identical using PMNL from control and diabetic rabbits and peptido-leukotrienes were not detected by radioimmunoassay. The data suggest that the activity of 5-lipoxygenase and the production of 5-hydroperoxyeicosatetraenoic acid in the diabetic PMNL may be limiting factors since the formation of leukotriene B4, leukotriene B4 isomers, and 5-hydroxyeicosatetraenoic acid are depressed in PMNL of diabetic rabbits. Alternative pathways do not account for the conversion of arachidonic acid to other products nor are the elimination pathways for LTB4 and 5-HETE different. Decreased formation of 5-hydroxyeicosatetraenoic acid and leukotriene B4 could predispose diabetic subjects to infection due to a decrease in mediators leading to the local accumulation of PMNL in the inflammatory response.     

The epithelium, endothelium, and stroma of the rabbit cornea generate (12S)-hydroxyeicosatetraenoic acid as the main lipoxygenase metabolite in response to injury

Previous work has shown that, shortly after rabbit corneas are injured, arachidonic acid metabolism is activated, and 12-hydroxyeicosatetraenoic acid (12-HETE) is one of the main products formed (Bazan, H. E. P., Birkle, D. L., Beuerman, R., and Bazan, N. G. (1985) Invest. Ophthalmol. & Visual Sci. 26, 474-480; Bazan, H. E. P. (1987) Invest. Ophthalmol. Visual Sci. 28, 314-319). In order to determine whether this metabolite is a lipoxygenase product, anesthetized rabbit corneas injured in vivo, either cryogenically or by 1 M NaOH, were subsequently incubated in vitro with [14C] arachidonic acid in the presence of indomethacin. 12-HETE was the main metabolite produced, as established by gas chromatography-mass spectrometry. The (R)- and (S)-enantiomers of novel naphthoyl-pentafluorobenzoyl derivatives of 12-HETE were resolved by chiral-phase high performance liquid chromatography. The radiolabeled 12-HETE from whole cornea and from isolated epithelium, endothelium, or stroma eluted as a single peak co-chromatographing with the (S)-enantiomer and was detected both by UV absorbance at 234 nm and by radioactivity. In noninjured corneas a smaller peak of radiolabeled (12S)-HETE was also eluted from the chiral column. The stereochemistry was additionally confirmed by liquid chromatography-mass spectrometry. These studies suggest that (12S)-lipoxygenase is activated in the injured rabbit cornea.     

Identification of lipoxygenase products from arachidonic acid metabolism in stimulated murine eosinophils

The presence of arachidonic acid lipoxygenase pathways in murine eosinophils was demonstrated by the isolation and identification of several lipoxygenase products from incubations of these cells. The most abundant arachidonate metabolite from murine eosinophils stimulated with ionophore A23187 and exogenous arachidonic acid was 12-S-hydroxyeicosatetraenoic acid (12-S-HETE), and the next most abundant was 15-HETE. Two families of leukotrienes were also recovered from these incubations. One family comprised the hydrolysis products of leukotriene A4, and the other included products derived from the 14,15-oxido analog of leukotriene A4 (14,15-leukotriene A4). Two double oxygenation products of arachidonate were also identified. These compounds were a 5,15-dihydroxyeicosatetraenoic acid (5,15-diHETE) and a 5,12-dihydroxyeicosatetraenoic acid (5,12-diHETE). Eosinophil stimulation promoter is a murine lymphokine which enhances the migration of eosinophils. When murine eosinophils were incubated with eosinophil stimulation promoter in concentrations sufficient to produce a migration response, a 2-3-fold increase in the production of 12-HETE was observed compared to unstimulated cells. Coupled with the recent demonstration that arachidonic acid lipoxygenase inhibitors suppress the migration response to eosinophil stimulation promoter and that 12-HETE induces a migration response, this observation provides further evidence in support of the hypothesis that eosinophil stimulation promoter stimulation of eosinophils results in the generation of lipoxygenase products which modulate the migratory activity of the cells.     

The differences in 12-hydroxyeicosatetraenoic acid and thromboxane B2 release from guinea pig platelets.

Anti-12(S)-hydroxyeicosatetraenoic acid (12-HETE)-antibody and anti-thromboxane B2 (TXB2)-antibody were generated and applied to the radioimmunoassay. The detection limit for 12-HETE was 16 pg. The cross-reactivities of anti-12-HETE-antibody were 4.6% for 15-HETE, 0.18% for 5-HETE and below 0.15% for leukotrienes and prostaglandins (PGs). 12-HETE and TXB2 released from guinea pig platelets were measured by radioimmunoassay. Platelet activating factor (PAF) at 10(-9) M induced the aggregation of platelets, the releases of immunoreactive-12-HETE (1.8 +/- 1.2 ng/10(8) platelets, mean +/- S.D.) and immunoreactive-TXB2 (18.5 +/- 17.3 ng/10(8) platelets). Collagen at 1 microgram/ml also evoked platelet aggregation, the releases of immunoreactive-12-HETE (2.7 +/- 1.1 ng/10(8) platelets) and immunoreactive-TXB2 (11.8 +/- 4.6 ng/10(8) platelets). By the stimulation with these compounds, TXB2 was produced in a greater amount than 12-HETE from guinea pig platelets. Although 10(-7) M and 10(-6) M U46619, a TXA2 mimetic, caused platelet aggregation, arachidonic acid metabolites were not released. These data suggest the presence of different mechanisms of platelet activation depending on each stimulus.     

Ethanol-enhanced transmembrane penetration of arachidonic acid and activation of the 5-lipoxygenase pathway in human leukocytes

Enhanced penetration by ethanol of exogenous arachidonic acid into human leukocyte preparations results in the production of large amounts of eicosanoids including 5-, 12- and 15-hydroxyeicosatetraenoic acids as well as the leukotrienes C4 delta 6-trans-leukotriene B4, 12-epi-delta 6-trans-leukotriene B4, leukotriene B4 and 5(S), 12(S)-dihydroxyeicosatetraenoic acid. The production of these compounds is affected by the concentrations of both ethanol and arachidonic acid independently in a complex manner with stimulation at lower concentrations and later relative inhibition. It was shown that the resulting leukotriene B4 exhibited the same specific activity as exogenous arachidonic acid when labelled substrate was used.     

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

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

Feedback regulation of platelet function by 12S-hydroxyeicosatetraenoic acid: inhibition of arachidonic acid liberation from phospholipids

We have proposed a mechanism that platelet aggregation is regulated by its 12-lipoxygenase product, 12S-hydroxyeicosatetraenoic acid (12-HETE) (Sekiya, F., Takagi, J. and Saito, Y. (1989) Thrombos. Res. 56, 407-415). Inhibition of endogenous 12-HETE production by 15-HETE, a specific inhibitor of 12-lipoxygenase, accelerated aggregation of bovine platelets in response to collagen and arachidonic acid liberation from phospholipids was enhanced. Exogenously added 12-HETE suppressed collagen-induced liberation of arachidonic acid and the aggregation was also inhibited. On the other hand, 12-HETE did not interfere with thromboxane synthesis from free arachidonic acid in a cell-free system. These observations suggest that 12-HETE exerts a negative feedback to prevent excess aggregation through interference with arachidonic acid liberation from membrane phospholipids.