Requirement of free arachidonic acid for leukotriene B4 biosynthesis by 12-hydroperoxyeicosatetraenoic acid-stimulated neutrophils

Stimulation of human neutrophils with 12-hydroperoxyeicosatetraenoic acid (12-HPETE) led to formation of 5S, 12S-dihydroxyeicosatetraenoic acid (DiHETE), but leukotriene B4 (LTB4) or 5-hydroxyeicosatetraenoic acid (5-HETE) was not detectable by reversed-phase high-performance liquid chromatography analysis. N-formylmethionylleucylphenylalanine (FMLP) induced the additional synthesis of small amounts of LTB4 in 12-HPETE-stimulated neutrophils. The addition of arachidonic acid greatly increased the synthesis of LTB4 and 5-HETE by neutrophils incubated with 12-HPETE. In experiments using [1-14C]arachidonate-labeled neutrophils, little radioactivity was released by 12-HPETE alone or by 12-HPETE plus FMLP, while several radiolabeled compounds, including LTB4 and 5-HETE, were released by A23187. These findings demonstrate that LTB4 biosynthesis by 12-HPETE-stimulated neutrophils requires free arachidonic acid which may be endogenous or exogenous.     

Evidence that hydrolysis of ethanolamine plasmalogens triggers synthesis of platelet-activating factor via a transacylation reaction

Addition of 1-O-alk-1'-enyl-2-lyso-sn-glycero-3-phosphoethanolamine (alkenyl-lyso-GPE) to human neutrophil membrane preparations containing 1-O-[3H]hexadecyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (1-O-[3H]alkyl-2-arachidonoyl-GPC) resulted in rapid deacylation of the 1-O-[3H]alkyl-2-arachidonoyl-GPC to 1-O-[3H]alkyl-2-lyso-GPC (lyso-platelet-activating factor, lyso-PAF). When acetyl-CoA was included in the incubation mixture, the [3H]lyso-PAF was converted to [3H]PAF. Studies of [3H]arachidonate-labeled neutrophils permeabilized with Staphlococcus aureus alpha-toxin revealed a major shift of labeled [3H]arachidonate from the choline to the ethanolamine-containing phosphoglycerides upon addition of alkenyl-lyso-GPE. The studies indicated that lyso-PAF is formed in the system by the transfer of arachidonate from 1-O-alkyl-2-arachidonoyl-GPC to the alkenyl-lyso-GPE by a CoA-independent transacylase reaction. Mass measurements revealed a rapid loss of arachidonate from 1-radyl-2-acyl-GPE and a concomitant increase in alkenyl-lyso-GPE upon stimulation of the neutrophils by ionophore A23187. Based on these and other findings, a pathway is proposed that may play a significant, if not obligatory, role in the synthesis of PAF in intact stimulated neutrophils. It has been widely accepted that phospholipase A2 acts directly on 1-O-alkyl-2-arachidonoyl-GPC as the first step in the synthesis of PAF via formation of lyso-PAF. In the proposed scheme, phospholipase A2, upon stimulation, acts rapidly on ethanolamine plasmalogen selectively releasing arachidonic acid and generating alkenyl-lyso-GPE. The CoA-independent transacylase then selectively transfers arachidonate from 1-radyl-2-arachidonoyl-GPC to the alkenyl-lyso-GPE generating lyso-PAF, which is then acetylated to form PAF. The interactions outlined can account for the synthesis of 1-acyl-2-acetyl-GPC, 1-O-alk-1'-enyl-2-acetyl-GPE, and eicosanoids, in parallel with PAF.     

Characterization and separation of the arachidonic acid 5-lipoxygenase and linoleic acid omega-6 lipoxygenase (arachidonic acid 15-lipoxygenase) of human polymorphonuclear leukocytes

The cytosolic fraction of human polymorphonuclear leukocytes precipitated with 60% ammonium sulfate produced 5-lipoxygenase products from [14C]arachidonic acid and omega-6 lipoxygenase products from both [14C]linoleic acid and, to a lesser extent, [14C]- and [3H]arachidonic acid. The arachidonyl 5-lipoxygenase products 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE) and 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) derived from [14C]arachidonic acid, and the omega-6 lipoxygenase products 13-hydroperoxy-9,11-octadecadienoic acid (13-OOH linoleic acid) and 13-hydroxy-9,11-octadecadienoic acid (13-OH linoleic acid) derived from [14C]linoleic acid and 15-hydroxyperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE), and 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) derived from [14C]- and [3H]arachidonic acid were identified by TLC-autoradiography and by reverse-phase high-performance liquid chromatography (RP-HPLC). Products were quantitated by counting samples that had been scraped from replicate TLC plates and by determination of the integrated optical density during RP-HPLC. The arachidonyl 5-lipoxygenase had a pH optimum of 7.5 and was 50% maximally active at a Ca2+ concentration of 0.05 mM; the Km for production of 5-HPETE/5-HETE from arachidonic acid was 12.2 +/- 4.5 microM (mean +/- S.D., n = 3), and the Vmax was 2.8 +/- 0.9 nmol/min X mg protein (mean +/- S.D., n = 3). The omega-6 linoleic lipoxygenase had a pH optimum of 6.5 and was 50% maximally active at a Ca2+ concentration of 0.1 mM in the presence of 5 mM EGTA. When the arachidonyl 5-lipoxygenase and the omega-6 lipoxygenase were separated by DEAE-Sephadex ion exchange chromatography, the omega-6 lipoxygenase exhibited a Km of 77.2 microM and a Vmax of 9.5 nmol/min X mg protein (mean, n = 2) for conversion of linoleic acid to 13-OOH/13-OH linoleic acid and a Km of 63.1 microM and a Vmax of 5.3 nmol/min X mg protein (mean, n = 2) for formation of 15-HPETE/15-HETE from arachidonic acid.     

Formation of leukotrienes and other hydroxy acids during platelet-neutrophil interactions in vitro

Interactions of human platelets with neutrophils were studied in suspensions of [³H]arachidonate-labeled platelets and unlabeled neutrophils stimulated with ionophore A23187. Several radioactive arachidonate metabolites, not produced by platelets alone, were detected, including [³H]-labeled leukotriene B₄ (LTB₄), dihydroxyeicosatetraenoic acid (DHETE) and 5-hydroxyeicosatetraenoic acid (5-HETE). When [³H]12-HETE, a platelet product, was added to stimulated neutrophils, DHETE was formed. Similarly, when [³H]5-HETE, a neutrophil product, was added to stimulated platelets, DHETE was the major product. These results suggest that upon stimulation: 1) platelet-derived arachidonate may serve as precursor for the neutrophil-derived eicosanoids LTB₄ and 5-HETE, and 2) that platelet-derived 12-HETE can be converted to DHETE by human neutrophils. The present investigation documents cell-cell interactions via the lipoxygenase pathway, which may be important in hemostasis, thrombosis and inflammation.     

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.     

Phorbol myristate acetate and the calcium ionophore A23187 synergistically induce release of LTB4 by human neutrophils: involvement of protein kinase C activation in regulation of the 5-lipoxygenase pathway

Phorbol myristate acetate (PMA), a tumor-promoting phorbol ester, and the calcium ionophore A23187 synergistically induced the noncytotoxic release of leukotriene B4 (LTB4) and other 5-lipoxygenase products of arachidonic acid metabolism from human neutrophils. Whereas neutrophils incubated with either A23187 (0.4 microM) or PMA (1.6 microM) alone failed to release any 5-lipoxygenase arachidonate products, neutrophils incubated with both stimuli together for 5 min at 37 degrees C released LTB4 as well as 20-COOH-LTB4, 20-OH-LTB4, 5-(S),12-(R)-6-trans-LTB4, 5-(S),12-(S)-6-trans-LTB4, and 5-hydroxyeicosatetraenoic acid, as determined by high pressure liquid chromatography. This synergistic response exhibited concentration dependence on both PMA and A23187. PMA induced 5-lipoxygenase product release at a concentration causing a half-maximal effect of approximately 5 nM in the presence of A23187 (0.4 microM). Competition binding experiments showed that PMA inhibited the specific binding of [3H]phorbol dibutyrate ([3H]PDBu) to intact neutrophils with a 50% inhibitory concentration (IC50) of approximately 8 nM. 1-oleoyl-2-acetyl-glycerol (OAG) also acted synergistically with A23187 to induce the release of 5-lipoxygenase products. 4 alpha-phorbol didecanoate (PDD), an inactive phorbol ester, did not affect the amount of lipoxygenase products released in response to A23187 or compete for specific [3H]PDBu binding. PMA and A23187 acted synergistically to increase arachidonate release from neutrophils prelabeled with [3H]arachidonic acid but did not affect the release of the cyclooxygenase product prostaglandin E2. Both PMA and OAG, but not PDD, induced the redistribution of protein kinase C activity from the cytosol to the membrane fraction of neutrophils, a characteristic of protein kinase C activation. Thus, activation of protein kinase C may play a physiologic role in releasing free arachidonate substrate from membrane phospholipids and/or in modulating 5-lipoxygenase activity in stimulated human neutrophils.     

Biosynthesis and metabolism of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine in rat glomerular mesangial cells

The ability of rat mesangial cells to synthesize 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (1-O-alkyl-2-acetyl-GPC), also known as platelet activating factor (PAF), was studied in mesangial cell cultures originating from isolated rat glomeruli. In response to the phospholipase A2 agonist A23187 mesangial cells synthesized PAF primarily via an acetyltransferase utilizing either [3H]lyso-PAF or [3H]acetate/[3H]acetyl-CoA substrates. The major PAF species synthesized was 1-O-hexadecyl-2-acetyl-GPC. PAF was also synthesized from 1-O-[3H]alkyl-2-acetyl-sn-3-glycerol, indicating the presence of a CDP-cholinephosphotransferase. Mesangial cells incorporated [3H]lyso-PAF to 1-O-[3H]alkyl-2-acyl-GPC. Subsequent stimulation with A23187 (2 microM) resulted in formation and release of [3H]PAF following 3 h, and this was associated with concomitant decrements in intracellular 1-O-[3H]alkyl-2-acyl-GPC and [3H]lyso-PAF levels, indicating a precursor-product relationship among these alkyl ether lipids. Mesangial cells rapidly converted exogenous [3H]PAF to [3H]lyso-PAF and 1-O-[3H]alkyl-2-acyl-GPC, and this process was inhibited by diisopropyl fluorophosphate (10 microM). The demonstration of PAF activation-inactivation pathways in mesangial cells may be of importance in regulating their function and in glomerular injury.     

Modulation of insulin secretion by lipoxygenase products of arachidonic acid. Relation to lipoxygenase activity of pancreatic islets

Glucose (16.7 mM)-induced insulin secretion from isolated pancreatic islets of rats was inhibited by nordihydroguaiaretic acid (NDGA), 1-phenyl-3-pyrazolidinone (phenidone), 3-amino-1-(3-trifluoromethylphenyl)-2-pyrazoline (BW755C), 2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone (AA861), and 2,6-di-tert-butyl-4-methylphenol (BHT). Indomethacin and aspirin, however, failed to inhibit the glucose-induced insulin secretion but rather tended to enhance it. The glucose-induced insulin secretion was inhibited by 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) (50 microM), 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE) (100 microM), and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) (100 microM), but not by 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) (100 microM). Exogenous 5-HETE (10 microM) induced significant insulin secretion in a low glucose (3.3 mM) medium. Racemic 5-HETE also showed insulinotropic effect in a concentration-dependent manner with the concentrations 20 microM or above, whereas 12-HETE, 15-HETE, 15-HPETE, 5,12-dihydroxy-6,8,10,14-eicosatetraenoic acid, 5-hydroxy-6-glutathionyl-7,9,11,14-eicosatetraenoic acid, 5-hydroxy-6-cysteinylglycinyl-7,9,11,14-eicosatetraenoic acid, prostaglandin E2, and prostaglandin F2 alpha failed to induce insulin secretion. Although significant insulin release was observed with arachidonic acid (greater than or equal to 100 microM), reduce cell viability was evident at 200 microM. When the 10,000 X g supernatant of isolated pancreatic islet homogenate was incubated with [3H]arachidonic acid at 37 degrees C in the presence of GSH and Ca2+, and the labeled metabolites then extracted with ethyl acetate and subjected to reverse phase high pressure liquid chromatography, several radioactive peaks, coeluted with authentic 15-, 12-, and 5-HETE, were observed. The radioactive peaks were completely suppressed by the addition of either NDGA, BW755C, or phenidone into the medium. The results support our contention i.e. the involvement of lipoxygenase product(s) in the secretory mechanism of insulin, and further suggest that 5-lipoxygenase system may play a role.     

Transformation of arachidonic acid by 5- and 15-lipoxygenase pathways in bovine adrenal fasciculata cells

[1-14C]Arachidonic acid was incubated with isolated bovine adrenal fasciculata cells for 15 min at 37gC. The metabolites were separated and purified by reverse- and straight-phase high performance liquid chromatography, and identified by gas chromatography-mass spectrometry or radioimmunoassay. Identified metabolites were 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE), 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE), leukotriene B4 and 11,14,15-trihydroxy-5,8,12-eicosatrienoic acid (11,14,15-THET). Addition of 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE), an intermediate metabolite of 15-lipoxygenase pathway to microsomes of bovine adrenal fasciculata cells resulted in the formation of 11,14,15-THET. The formation of 11,14,15-THET by microsomes was not dependent on the presence of NADPH, while it was dose-dependently suppressed by ketoconazole, a potent inhibitor of cytochrome P-450 dependent enzymes. These results indicate that 5- and 15-lipoxygenase pathways of arachidonic acid may exist in bovine adrenal fasciculata cells and that 15-HPETE is further metabolized to 11,14,15-THET by adrenal microsomal cytochrome P-450.     

Possible influence of lysophospholipase on the production of 1-acyl-2-acetylglycerophosphocholine in macrophages.

The rate of production of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF) and 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (acylPAF) was measured in macrophages following the incorporation of [3H]acetate. Upon activation by A23187, guinea pig alveolar macrophages incorporated [3H]acetate into PAF, but a little radioactivity was found in acylPAF. However, labeling of acylPAF and PAF with [3H]acetate was greatly enhanced in A23187-stimulated alveolar macrophages that had been pretreated with phenylmethanesulphonyl fluoride (PMSF). [3H]PAF was predominantly converted to 1-[3H]alkyl-2-acyl glycerophosphocholine, but [14C]acylPAF rapidly hydrolyzed to 14C-labeled free fatty acid by the incubation with lysates prepared from macrophages. The deacetylation of [14C]acylPAF and [3H]PAF by acetylhydrolase and also the hydrolysis of [14C]lysoPC by lysophospholipase were strongly inhibited in macrophages that had been pretreated with PMSF, while PMSF failed to inhibit the activities of acetyltransferase and acyltransferase. The relative proportions of PAF and acylPAF were quite different in different types of cells. In contrast to alveolar macrophages, peritoneal macrophages, neutrophils and spleen cells from guinea pigs incorporated 2-4 times more [3H]acetate into acylPAF than into PAF. The presence of high levels of acylPAF in peritoneal macrophages was confirmed by GLC-MS analysis. The activities of lysophospholipase, acetylhydrolase and acetyltransferase were measured in alveolar and peritoneal macrophages to determine whether the preferential formation of acylPAF as compared to PAF in peritoneal macrophages was due to differences in these activities between alveolar and peritoneal macrophages. The activity of acetylhydrolase of peritoneal macrophages was almost the same as that in alveolar macrophages. The activity of acetyltransferase in peritoneal macrophages was about half of that in alveolar macrophages. However, the activity of lysophospholipase in peritoneal macrophages was one-sixth of that in alveolar macrophages. These results suggest that lysophospholipase is one of the primary factors involved in the control of the production of acylPAF in activated cells, and that it acts by modulating the availability of lysoPC for the synthesis of acylPAF. Furthermore, high levels of activity of lysophospholipase allow the preferential formation of PAF, via the rapid hydrolysis of lysoPC which would act as a competitive inhibitor of the incorporation of acetate into lysoPAF.     

Stimulation of platelet-activating factor synthesis by a nonmetabolizable bioactive analog of platelet-activating factor and influence of arachidonic acid metabolites

Platelet-activating factor (PAF) is a potent neutrophil agonist operating through specific receptors located on the cell surface. Binding of PAF to its receptor may also stimulate further PAF synthesis, thus providing a means of amplifying the PAF signal for the cell of origin and/or other responsive cells. In this report we demonstrate that 1-O-alkyl-2-N-methylcarbamyl-sn-glycero-3-phosphocholine (C-PAF), a nonmetabolizable bioactive analog of PAF, stimulates human neutrophils to synthesize PAF, as detected by [3H]acetate incorporation into PAF. This approach allowed us to conclude that [3H]acetate-labeled PAF was formed from endogenous precursor rather than mere turnover of the stimulatory dose of PAF. PAF's ability to initiate further PAF synthesis was confirmed by measuring the PAF-stimulated conversion of 1-O-[3H]alkyl-2-acylglycerophosphocholine to 1-O-[3H]alkyl-2-acetylglycerophosphocholine by prelabeled human neutrophils and by determining the molecular species of 1-O-alkyl-2-[3H]acetylglycerophosphocholine produced by cells stimulated with a single molecular species of PAF (C15:0). Degradation of exogenously added [3H]PAF was not inhibited by C-PAF/5-hydroxyeicosatetraenoic acid treatment. Thus, inhibition of PAF degradation was ruled out as the mechanism accounting for the appearance of labeled PAF in the stimulated cells. Synthesis of PAF in response to C-PAF was not dependent on cytochalasin B pretreatment but was dramatically potentiated by 5-hydroxyeicosatetraenoic acid, which alone was without effect. Additionally, we have demonstrated that another major arachidonate metabolite of neutrophils, leukotriene B4, stimulates PAF production. Thus, at least three products of activated neutrophils, including PAF itself, can promote PAF synthesis by these cells. This positive feedback effect may amplify autacoid production and the final cellular response.     

Inhibition of platelet-activating factor biosynthesis via the acetyltransferase by arachidonic and oleic acids in ionophore A23187-stimulated bovine neutrophils

Platelet-activating factor (PAF) is a phospholipid mediator of inflammation and allergy that is synthesized by several inflammatory cells including neutrophils. Addition of exogenous arachidonic acid to ionophore A23187-stimulated bovine neutrophils led to the inhibition of PAF biosynthesis assayed by incorporation of [3H]acetate into PAF and by bioassay; under the same conditions, leukotriene B4 (LTB4) formation was not decreased. The activities of the PAF metabolism enzymes indicated that the PAF synthesis inhibition by arachidonic acid is mediated via the acetyltransferase inhibition which is the last enzyme of the PAF formation. Another unsaturated fatty acid, oleic acid, exhibited the same inhibitory effect on [3H]acetate-PAF formation; however, the saturated stearic acid did not lead to any inhibition. These findings suggest that liberation of unsaturated fatty acids from membrane phospholipids, as a consequence of phospholipase A2 activation, would modulate PAF formation via inhibition of the acetyltransferase. In addition, the utilization of arachidonic acid oleic acids in activated neutrophils furnishes an easy means of blocking PAF synthesis in order to understand the role of this mediator in cellular processes.     

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.     

C20 polyunsaturated fatty acids and phorbol myristate acetate enhance agonist-stimulated synthesis of 1-radyl-2-acetyl-sn-glycero-3-phosphocholine in vascular endothelial cells

This study has investigated the effect of supplementation of vascular endothelial cells with arachidonate and other polyunsaturated fatty acids on the agonist-stimulated synthesis of platelet activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; 1-alkyl-2-acetyl-GPC). Incubation of calf pulmonary artery endothelial cells for 48 h in medium containing 40 microM arachidonate resulted in a 2-3-fold enhancement of [3H]acetate incorporation into 1-radyl-2[3H]acetyl-GPC in response to either bradykinin or calcium ionophore A23187. The effects of arachidonate supplementation were both dose- and time-dependent, requiring a minimum exogenous arachidonate concentration of 2.5 microM and an incubation time of 4-6 h. Eicosapentaenoate and docosahexaenoate also enhanced the synthesis of 1-radyl-2-[3H]acetyl-GPC, but were less potent than arachidonate; alpha-linolenate, linoleate and oleate were without effect. Although not effective as an agonist, phorbol myristate acetate potentiated A23187- and bradykinin-stimulated synthesis of 1-radyl-2-[3H]acetyl-GPC. The effects of arachidonate supplementation were synergistic with potentiation by phorbol myristate acetate. Sphingosine inhibited agonist-stimulated incorporation of [3H]acetate into 1-radyl-2-[3H]acetyl-GPC both in the presence and absence of PMA. Characterization of the radiolabeled material indicated that the primary product was the acyl analogue of PAF (1-acyl-2-acetyl-GPC) rather than PAF. The results from this study suggest that agonist-stimulated synthesis of 1-radyl-2-acetyl-GPC in vascular endothelial cells is modulated both by cellular fatty acyl composition and activation of protein kinase C. Enrichment of vascular endothelial cells with fatty acids, which are mobilized by agonist-stimulated phospholipase A2, may enhance subsequent deacylation of choline phospholipids and, thus, increase synthesis of both 1-acyl-2-acetyl-GPC and PAF.     

An arachidonoyl (polyenoic)-specific phospholipase A2 activity regulates the synthesis of platelet-activating factor in granulocytic HL-60 cells

Human promyelocytic leukemia cells (HL-60) were used as a cell model to determine how arachidonic acid stimulates the synthesis of platelet-activating factor (PAF) synthesized via the remodeling pathway. In these studies HL-60 cells were cultured over 30 passages in fatty acid-free medium to deplete them of arachidonic acid. Even though the phospholipid classes from these cells contained no arachidonate, they could still be differentiated into granulocytes by dimethyl sulfoxide (1.25%). When the differentiated HL-60 cells, depleted of arachidonic acid, were stimulated with calcium ionophore A23187 in the presence of Ca2+ and [3H]acetate, only minimal amounts of [3H]PAF were produced. In contrast, if the differentiated HL-60 cells were supplemented with 10 microM arachidonic acid for 24 h and then stimulated with the ionophore, there was a large amount of [3H]PAF formed. The increase in PAF synthesis depended on the length of time the cells were supplemented with arachidonic acid; only a small increase in PAF synthesis occurred during the early hours of supplementation whereas stimulation of PAF synthesis was maximal (3-5-fold) after a 24-h period of the 20:4 supplementation. Other polyenoic fatty acid supplements (20:5, 22:4, and 22:6 for 24 h) also stimulated PAF production in the ionophore-treated HL-60 cells depleted of 20:4, but the amount of PAF was significantly less than found for the supplements of 20:4 under identical experimental conditions. Also noteworthy is that undifferentiated cells supplemented with 20:4 or their unsupplemented controls could not be stimulated by the calcium ionophore to produce PAF. Addition of indomethacin (cyclooxygenase inhibitor), A63162 (5'-lipoxygenase inhibitor), or eicosatetraynoic acid (cyclooxygenase/lipoxygenase inhibitor) to the incubations caused little change in the production of [3H]PAF in the differentiated cells supplemented with 20:4 for 24 h. On the other hand, the addition of mepacrine, bromophenacyl bromide, or U26384 (phospholipase A2 inhibitors) resulted in very large decreases (80-90% lower than controls) in the amount of [3H]PAF produced under the same conditions. Analysis of the molecular species of [3H]alkylacyl-GroPCho (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine, the precursor of PAF in the remodeling pathway) in 20:4-supplemented cells prelabeled with [3H]alkyl-lyso-GroPCho revealed that only the alkylarachidonoyl-GroPCho species were preferentially decreased after stimulation with the A23187 ionophore.These results demonstrate that arachidonate must be at the sn-2 position of alkylacyl-GroPCho in order for it to serve as a precursor of PAF.(ABSTRACT TRUNCATED AT 400 WORDS)     

Specific high affinity binding of lipoxygenase metabolites of arachidonic acid by liver fatty acid binding protein

Liver fatty acid binding protein (L-FABP) binds avidly the arachidonic acid metabolites, hydroperoxyeicosatetraenoic acids (HPETEs) and hydroxyeicosatetraenoic acids (HETEs). Binding of 15-[3H]HPETE was specific, saturable, reversible, and rapid. Protein specificity was indicated by the following order: L-FABP greater than bovine serum albumin greater than ovalbumin = beta-lactoglobulin greater than ribonuclease. Ligand specificity was evidenced by the following order of apparent competition: 15-HPETE greater than or equal to 5-HETE greater than or equal to 5-HPETE = oleic acid greater than 12-HETE greater than 12-HPETE greater than or equal to 15-HETE greater than prostaglandin E1 much greater than leukotriene C4 greater than prostaglandin E2 much greater than thromboxane B2 = leukotriene B4. Once bound, 15-HPETE was reversibly displaced. Ligand was recovered from the protein complex and confirmed to be 15-[3H]HPETE by TLC. L-FABP bound HPETE with a dissociation constant of 76 nM,5-HETE at 175 nM, and 15-HETE at 1.8 microM, and the reference fatty acids oleic acid at 1.2 microM and arachidonic acid at 1.7 microM. Thus, the affinity was approximately 16-fold greater for 15-HPETE, and 7-fold higher for 5-HETE, than for oleic acid. The need exists for studies of complexes of L-FABP with the HPETEs and HETEs in hepatocytes, especially since L-FABP has previously been associated with mitosis in normal hepatocytes, and shown to be the target protein of two liver carcinogens, and these arachidonic acid metabolites have been found to be able to modulate activities related to cell growth.     

The effects of the phospholipase A2 inhibitors aristolochic acid and PGBx on A23187-stimulated mobilization of arachidonate in human neutrophils are overcome by diacylglycerol or phorbol ester.

Aristolochic acid and PGBx, two structurally unrelated, protein-targeted inhibitors of isolated phospholipases A2, are effective antagonists of calcium ionophore A23187-stimulated mobilization of [3H]arachidonate from human neutrophils. We now report that preincubation of neutrophils with oleoylacetylglycerol (OAG, 15 microM) substantially reverses the inhibitory effect of 200 microM aristolochic acid (from 70 to 24% inhibition). Similarly, OAG increases the IC50 for PGBx from 2.5 to greater than 20 microM. The effects of OAG on inhibition by either aristolochic acid or PGBx are dose-dependent, with an ED50 of 2.5 microM. Protection against inhibition by either aristolochic acid or PGBx is also observed with phorbol myristate acetate (PMA, ED50 3 nM), but not 4-alpha-phorbol didecanoate. Aristolochic acid and PGBx do not inhibit PMA-stimulated superoxide generation, and are thus not protein kinase C inhibitors. Furthermore, neither aristolochic acid nor PGBx inhibit diglyceride generation through the phospholipase D/phosphatidate phosphohydrolase pathway. A23187-stimulated [3H]arachidonate mobilization is increased by 20-50% when neutrophils are preincubated with OAG or PMA. The present results indicate that OAG and PMA also modulate the A23187-stimulated [3H]arachidonate mobilization so as to render it less sensitive to inhibitors of phospholipase A2.     

Comparison of the production of eicosanoids by human and rat peritoneal macrophages and rat Kupffer cells

Human and rat peritoneal macrophages and rat Kupffer cells were labelled with [1-14C] arachidonic acid and stimulated with the calcium ionophore A23187. The metabolites formed were separated by high pressure liquid chromatography (HPLC). Human peritoneal macrophages formed especially leukotriene B4, 5-hydroxy-6,8,11,14 eicosatetraenoic acid and small amounts of leukotriene C4 and thromboxane B2, 12-hydroxy-5,8,10 heptadecatrienoic acid and 6-keto-prostaglandin F1 alpha, whereas rat peritoneal macrophages mainly produced cyclooxygenase products and in particular thromboxane B2 and 12-hydroxy-5,8,10 heptadecatrienoic acid. Rat Kupffer cells synthesized mainly cyclooxygenase products such as prostaglandin F2 alpha, prostaglandin D2 and prostaglandin E2. These results indicate that the profile of eicosanoids production by macrophages is dependent both on the species and on the tissue from which the macrophage is derived.     

Activation of the abl oncogene in murine and human leukemias

Deuterium-labelled standards of four regionally isomeric epoxyeicosatrienoic acids (EETs) and their hydrolysis products, the dihydroxyeicosatrienoic acids (DHETs), have been prepared and analyzed by capillary column gas chromatography (GC)-negative ion (NI)-methane chemical ionization (MCI)-mass spectrometry (MS) as the pentafluorobenzyl esters. As little as 40 pg of these compounds were readily visualized by these methods, and the deuterium-labelled standards were used in a stable isotope dilution mass spectrometric assay which was linear from near the detection limit over several orders of magnitude. NADPH-dependent synthesis of both EETs and DHETs from arachidonate by hepatic microsomal cytochrome P-450-mono-oxygenase activity was demonstrable with these methods and was significantly suppressed by the compound BW755C (500 microM), but not by eicosa-5,8,11,14-tetraynoic acid (ETYA, 20 microM) or by nordihydroguaiaretic acid (NDGA, 50 microM). All three compounds suppress glucose-induced insulin secretion and 12-hydroxyeicosatetraenoic acid (12-HETE) synthesis by isolated pancreatic islets with similar concentration dependence. Microsomes derived from isolated pancreatic islets synthesized less than 3% of the EET and DHET compounds as a comparable amount of hepatic microsomes. Intact islets synthesized less than 3% by mass of the EET and DHET compounds compared to the mass of 12-HETE produced by the islets. Islets also failed to convert 3H-labelled arachidonate to 3H-labelled EETs or DHETs under conditions where conversion to [3H]12-HETE and to [3H]prostaglandin E2 (but not to [3H]leukotriene C4, D4, or E4) was clearly demonstrable. Neither exogenous EETs nor leukotriene C4 stimulated insulin secretion from the isolated islets or reversed the suppression of glucose-induced secretion by the lipoxygenase inhibitor BW755C. The cytochrome P-450-monooxygenase inhibitor, metyrapone (50 microM), did not influence insulin secretion from the isolated islets under conditions where the lipoxygenase inhibitor, NDGA, suppressed glucose-induced secretion. These observations argue against the recently suggested hypothesis that EETs derived from arachidonate by monooxygenase action participate in glucose-induced insulin secretion by isolated pancreatic islets.     

Protein kinase C activity appears to be required for the synthesis of platelet-activating factor and leukotriene B4 by human neutrophils

Human neutrophils synthesize platelet-activating factor (PAF) and leukotriene B4 (LTB4) when stimulated with the Ca2+ ionophore A23187. These processes are enhanced to a variable extent by phorbol 12-myristate 13-acetate (PMA), a direct activator of protein kinase C. The long chain amines sphingosine, stearylamine (Hannun, Y.A., Loomis, C.R., Merrill, A.H., Jr., and Bell, R.M. (1986) J. Biol. Chem. 261, 12604-12609), and palmitoylcarnitine competitively inhibit activation of purified protein kinase C in vitro and inhibit protein kinase C-mediated activation of the respiratory burst in human neutrophils (Wilson, E., Olcott, M.C., Bell, R.M., Merrill, A.H., Jr., and Lambeth, J.D. (1986) J. Biol. Chem. 261, 12616-12623). These amines were found to inhibit A23187-induced PAF and LTB4 synthesis. Inhibition of PAF and LTB4 synthesis occurred in parallel; half-maximal inhibition by sphingosine occurred at 7 microM, with complete inhibition at 15 microM. PMA by itself did not induce the synthesis of PAF or LTB4, although it did enhance PAF and LTB4 synthesis at suboptimal concentrations of A23187. PMA reversed long chain amine inhibition of PAF and LTB4 accumulation. Reversal of the inhibition of PAF and LTB4 accumulation occurred in parallel, was concentration-dependent, and was complete by approximately 3 x 10(-8) M PMA. The inactive 4 alpha-phorbol didecanoate ester did not reverse inhibition at these concentrations. Sphingosine completely prevented the A23187-induced release of [3H]arachidonate and its various metabolites from [3H]arachidonate-labeled cells. PMA, but not 4 alpha-phorbol didecanoate, restored arachidonate release and its metabolism. Therefore, while activation of protein kinase C is not sufficient to induce PAF and LTB4 synthesis, its action appears to be required to couple a rise in intracellular Ca2+ to their synthesis. This coupling occurs at the level of the initial reaction in the production of lipid mediators, a phospholipase A2-like activity that mobilizes the two substrates 1-O-alkyl-sn-glycero-3-phosphocholine and arachidonic acid from complex lipids.