Arachidonate metabolite(s) increase the permeability of the plasma membrane of the neutrophils to calcium

The relationship between arachidonate metabolism and stimulated calcium fluxes in rabbit peritoneal neutrophils has been investigated. The addition of arachidonate to the neutrophils was found to cause a rapid and significant increase in the permeability of the plasma membrane to calcium. This effect is specific to calcium, concentration dependent and sensitive to inhibitors of the lipoxygenase mediated metabolic pathway(s). These results strongly suggest that arachidonate metabolites are directly involved in the mechanisms underlying calcium gating in the neutrophils.     

Decreased prostaglandin production by cholesterol-rich macrophages

The regulation of prostaglandin production by macrophages enriched in cholesterol was examined. Mouse peritoneal macrophages were incubated for 18 h with 25 micrograms/ml of human acetyl-LDL (low density lipoprotein) and trace amounts of labeled arachidonic acid. After cholesterol enrichment, the cells were incubated with phorbol 12-myristate 13-acetate (PMA), calcium ionophore, or zymosan to stimulate endogenous arachidonic acid metabolism. A high performance liquid chromatography profile of the eicosanoids released revealed no qualitative differences between unmodified and modified macrophages. Cholesterol-rich cells, however, released less prostacyclin (PGI2) and prostaglandin E2 (PGE2) compared to unmodified cells, and products from the lipoxygenase pathway became the predominant metabolites. A decrease in the synthesis of PGI2 and PGE2 by cholesterol-rich macrophages was confirmed by radioimmunoassay and radiolabeled experiments. The activity of prostaglandin synthetase was modestly increased in the cholesterol-modified macrophages compared to controls. As an estimation of phospholipase activity, the release of labeled arachidonic acid from membrane phospholipids, however, was significantly decreased in cholesterol-rich macrophages. The phosphatidylinositol fraction was particularly resistant to arachidonate release in response to calcium ionophore and PMA in the modified cells. The measurement of membrane phospholipid fatty acid composition before and after calcium ionophore supported the observation that less arachidonate was released by cholesterol-enriched cells in response to the ionophore. Based on these observations, we propose that prostaglandin synthesis from endogenous arachidonate stores is decreased in the cholesterol-rich macrophage. A decrease in agonist-induced activation of the phospholipase activity is proposed as a mechanism for this effect.     

Differential effects of anti-inflammatory drugs on lipoxygenase and cyclo-oxygenase activities of neutrophils from a reverse passive Arthus reaction

Rat neutrophils isolated from four-hour reverse passive Arthus reaction pleural exudates actively metabolize arachidonic acid. Production of 11-hydroxy- and 15-hydroxy-icosatetraenoic acid and 12-hydroxy-heptadecatrienoic acid is inhibited by indomethacin, benoxaprofen, BW 755C, piroxicam, ibuprofen, timegadine, and naproxen, suggesting that production of these arachidonic acid metabolites occurs at similar enzymic active sites. In addition, in the presence of the calcium inophore A23187 or the non-ionic detergent, BRIJ 56, rat neutrophils also produce the lipoxygenase products 5-hydroxy-icosatetraenoic acid and leukotriene B. The production of these metabolites is calcium dependent. Moreover, the calcium ionophore A23187 and BRIJ 56 synergistically act to augment the metabolism of exogenously added arachidonic acid via lipoxygenase. The formation of these metabolites is inhibited by BW 755C, benoxaprofen and timegadine but not by other non-steroidal anti-inflammatory drugs tested. In fact, at doses which inhibit cyclo-oxygenase activity, indomethacin, naproxen, and ibuprofen stimulate arachidonic acid metabolism via lipoxygenase.     

Synthesis of eicosanoids by gamma-interferon-differentiated U937 cells

Interferons induce morphological, biochemical and functional alterations in monocyte macrophage and myeloid cell lines. We studied the effect of 3 days incubation with gamma-interferon from human buffy coats on the global synthesis of arachidonic acid metabolites by U937 cells. Interferon-induced morphologic changes including cytoplasmic and nuclear changes and the appearance of multiple lysosomal-like granules consistent with cellular differentiation were observed by electron microscopy. The labeling of phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine was increased and that of phosphatidylinositol, free fatty acids as 3H-arachidonic acid and neutral lipids reduced, when interferon-treated cells were incubated with 3H-arachidonic acid. Interferon caused qualitative and quantitative changes in the synthesis of cyclooxygenase and lipoxygenase products. A23187, a calcium ionophore, and the tumor promotor, phorbol myristate acetate, greatly increased the synthesis by interferon-differentiated cells of 2 cyclooxygenase products; synthesis of lipoxygenase products was reduced. In the presence of indomethacin, 'shunting' into putative lipoxygenase products occurred. The relationship between interferon-induced morphologic and functional changes, the development of altered phospholipid and eicosanoid metabolism and the identity of these metabolites are yet to be established.     

Detection of a novel cyclooxygenase metabolite produced by human promyelocytic leukemia (HL-60) cells

Arachidonic acid metabolism via the lipoxygenase pathway was examined in HL-60 cells before and after N,N-dimethylformamide induced differentiation along granulocytic lines. Untreated HL-60 cells produced small amounts of the 5-lipoxygenase products, 5-hydroxy-eicosatetraenoic acid and leukotriene B4 upon stimulation with calcium ionophore A23187. N,N-dimethylformamide treatment, caused a 10 to 20 fold increase in the amount of ionophore A23187-induced 5-lipoxygenase metabolites. An additional, and as yet unidentified arachidonic acid metabolite was routinely observed during reverse-phase high pressure liquid chromatography analyses of lipoxygenase products. Sensitivity to inhibition by less than 10(-7)M indomethacin coupled with other characteristics of its production, strongly suggest the compound is a cyclooxygenase product. The unusual UV absorbance and chromatographic elution pattern, however, suggest that it is not a typical prostaglandin, thromboxane or prostacyclin product.     

Modulation of tumor necrosis factor-alpha cytotoxicity in L929 cells by bacterial toxins, hydrocortisone and inhibitors of arachidonic acid metabolism

L929 cells were incubated with tumor necrosis factor-alpha (TNF-alpha) in the presence or absence of various inhibitors of arachidonic acid metabolism. The addition of either hydrocortisone or nordihydroguaiaretic acid (NDGA) decreased the cytotoxic effect of TNF-alpha but exogenously added arachidonate or linoleate, indomethacin and eicosatetraynoic acid (ETYA) were without effect. While it was found that TNF-alpha stimulated arachidonic acid release, no metabolites of this fatty acid could be evidenced. Cytotoxicity of TNF-alpha could also be decreased by the addition of either cholera or pertussis toxin. These results suggest that a GTP-binding protein is involved in the cytotoxic action of TNF-alpha. Arachidonic acid, released possibly by a phospholipase A2, might also play a role, but probably not via its conversion to known metabolites.     

Signal transduction by bFGF, but not TGF beta 1, involves arachidonic acid metabolism in endothelial cells.

We investigated the stimulation of early cellular events resulting from the interaction of the growth factor basic FGF (bFGF) and of the growth inhibitor transforming growth factor beta-type 1 (TGFβ1), with their specific receptors on bovine endothelial cells. At mitogenic concentrations, bFGF stimulated the rapid release of arachidonic acid and its metabolites from (³H)-arachidonic acid labeled cells. When arachidonic acid metabolism was stimulated by addition of the calcium ionophore A23187, the effect of bFGF was amplified. Nordihydroguaïaretic acid, an inhibitor of the lipoxygenase pathway of arachidonic acid metabolism, decreased the mitogenic effect of bFGF, whereas indomethacin, an inhibitor of the cyclooxygenase pathway, was ineffective. These findings suggest that metabolism of arachidonic acid to lipoxygenase products may be necessary for the mitogenic effect of bFGF. Basic FGF did not stimulate the production of inositol phosphates from cells labelled with myo-(2-³H)-inositol nor did it induce calcium mobilization, as measured by fura-2 fluorescence, indicating that bFGF does not activate phosphoinositide specific phospholipase C in endothelial cells, but rather, that bFGF-induced arachidonic acid metabolism is mediated by another phospholipase. TGFβ1, which inhibits basal and bFGF-induced endothelial cell growth, had no effect on arachidonic acid matabolism and inositol phosphate formation and did not prevent bFGF-induced arachidonic acid metabolism. These results suggest that the inhibitory action of TGFβ1 on endothelial cell growth occurs through different mechanisms.     

Effect of arachidonic acid and the chemotactic factor F-Met-Leu-Phe on cation transport in rabbit neutrophils

A detailed examination of the effects of exogenous arachidonate on cation metabolism in rabbit neutrophils was undertaken. Arachidonic acid stimulates the movement of ⁴⁵Ca into and out of the neutrophils with a net result, in the presence of extracellular calcium, of increasing the steady-state level of ⁴⁵Ca. Arachidonate also increases the uptake of ²²Na. These effects of arachidonate are specific to these cations, concentration-dependent, and sensitive to lipoxygenase inhibitors. At the concentrations used in this study arachidonate does not influence the permeability of human erythrocytes to ⁴⁵Ca. Furthermore, both arachidonic acid and F-Met-Leu-Phe release calcium from a previously unexchangeable intracellular pool and the effect of the two stimuli are not additive. Arachidonic acid-dependent, but not F-Met-Leu-Phe-dependent, calcium release is sensitive to lipoxygenase inhibitors. These two stimuli thus appear to release is sensitive to lipoxygenase inhibitors. These two stimuli thus appear to release calcium from the same pool(s) by separate mechanisms. The results summarized above are consistent with the hypothesis that one or more arachidonate metabolites are involved in the mechanism underlying the chemotactic factor induced permeability changes in rabbit neutrophils.     

Human peritoneal macrophages synthesize leukotrienes B4 and C4

Macrophages were isolated from the dialysis fluid of patients undergoing continuous ambulatory peritoneal dialysis and separated by gradient centrifugation and purification on 50% Percoll. The cells were prelabeled with [14C]arachidonic acid for 1.5 h. The labeled cells were then incubated with calcium ionophore A23187 (1 microM), serum-treated zymosan (200 micrograms/ml), and a lipoxygenase inhibitor, nordihydroguairetic acid (1 X 10(-5) M). The arachidonate metabolites in the medium were separated on Sep-Pak columns, and finally purified by reverse-phase high-pressure liquid chromatography (HPLC). The labeled products co-chromatographed with authentic leukotriene B4 and leukotriene C4 standards. Serum-treated zymosan and A23187 significantly stimulated and nordihydroguairetic acid significantly inhibited leukotriene synthesis. Leukotriene D4 was not detected, which suggests that these cells contain low gamma-glutamyltranspeptidase or high dipeptidase activity. These results establish, for the first time, that human peritoneal macrophages synthesize the lipoxygenase products, leukotriene B4 and leukotriene C4.     

Leukotriene B formation by neutrophils from essential fatty acid-deficient rats

Analysis of neutrophil phospholipids from rats fed an essential fatty acid-deficient diet revealed a 33% reduction in arachidonate and a 90% reduction in linoleate compared to neutrophil phospholipids of rats fed a normal diet. The neutrophil phospholipids from rats fed the essential fatty acid-deficient diet also contained significant amounts of 5,8,11-eicosatrienoate, a fatty acid not found in the neutrophils of rats fed a normal diet. Analysis of the production of leukotrienes of the B series by ionophore-stimulated neutrophils from rats fed an essential fatty acid-deficient diet revealed a 87% reduction in leukotriene B4 compared to neutrophils from rats fed a normal diet even though the arachidonate content was reduced by only 34%. Essential fatty acid-deficient neutrophils converted endogenous 5,8,11-eicosatrienoic acid to leukotriene A3 and its nonenzymatic degradation products, but little or no leukotriene B3 was formed. Neutrophils from rats fed a normal diet incubated with ionophore and exogenous 5,8,11-eicosatrienoate also produced leukotriene A3 and its nonenzymatic degradation products but little or no leukotriene B3. Exogenous 5,8,11-eicosatrienoate incubated with ionophore-stimulated normal neutrophils caused a dose-dependent inhibition of leukotriene A hydrolase resulting in diminished production of leukotriene B4 from endogenous arachidonate. Assays of leukotriene A hydrolase in the 10,000 X g supernatant fraction of a homogenate of RBL-1 cells revealed that a lipoxygenase metabolite of 5,8,11-eicosatrienoate rather than 5,8,11-eicosatrienoate itself is the inhibitor of leukotriene A hydrolase. Thus the finding that leukotriene B4 production by neutrophils from essential fatty acid-deficient rats is diminished out of proportion to the decrease in arachidonate content appears to be due to inhibition of leukotriene A hydrolase by a lipoxygenase metabolite.     

Role for protein kinase C in A23187 induced glomerular arachidonate release and PGE2 production

A high concentration of the calcium ionophore A23187 (10 uM) stimulated release of labeled arachidonate and immunoreactive PGE2 from isolated superfused glomeruli. A lower concentration of A23187 (1 uM) or 12-0-tetradecanoyl phorbol-13-acetate (TPA, 0.1 uM), a direct activator of protein kinase C, when added alone was without effect on these parameters. Combined addition of A23187 (1 uM) and TPA (0.1 uM) synergistically stimulated arachidonate release and PGE2 production. 1-(5-isoquinolinyl)-2-methylpiperazine (H-7) a known inhibitor of protein kinase C in other tissues, suppressed increases in arachidonate release and PGE2 production mediated by A23187 (10 uM) or TPA plus A23187 (1 uM). H-7 inhibited while TPA stimulated protein kinase C activity that had been partially purified from soluble fractions of glomerular homogenates. These results support a role for protein kinase C in A23187 mediated arachidonate release.     

Effects of calmodulin and lipoxygenase inhibitors on LH (lutropin)- and LHRH (luliberin)-agonist-stimulated steroidogenesis in rat Leydig cells.

The results of this study, carried out with purified rat Leydig cells, indicate that there are no major differences in the stimulating effects of lutropin (LH) and luliberin (LHRH) agonists on steroidogenesis via mechanisms that are dependent on Ca2+. This was demonstrated by using inhibitors of calmodulin and the lipoxygenase pathways of arachidonic acid metabolism. All three calmodulin inhibitors used (calmidazolium, trifluoperazine and chlorpromazine) were shown to block LH- and LHRH-agonist-stimulated steroidogenesis. This probably occurred at the step of cholesterol transport to the mitochondria. Similarly, three lipoxygenase inhibitors (nordihydroguaiaretic acid, BW755c and benoxaprofen), inhibited both LH- and LHRH-agonist-stimulated steroidogenesis. The amounts of the inhibitors required were similar for LH- and LHRH-agonist-stimulated steroidogenesis. Steroidogenesis stimulated by the Ca2+ ionophore A23187 was also inhibited, but higher concentrations of the inhibitors were required. Indomethacin (a cyclo-oxygenase inhibitor) increased LHRH-agonist-stimulated steroidogenesis;this is consistent with the role of the products of arachidonic acid metabolism via the alternative, lipoxygenase, pathway. The potentiation of LH-stimulated testosterone production by LHRH agonist was unaffected by indomethacin or by lipoxygenase inhibitors at concentrations that inhibited LH-stimulated testosterone production by 75-100%. It was not possible to eliminate a role of calmodulin in modulating the potentiation, although higher concentrations of the inhibitors were generally required to negate the potentiation than to inhibit LH- or LHRH-agonist-stimulated testosterone production.     

Arachidonic acid metabolism in rat pancreatic acinar cells: calcium-mediated stimulation of the lipoxygenase system

Isolated rat pancreatic acini were employed to demonstrate that the exocrine pancreas can metabolize [14C]-arachidonic acid by way of the lipoxygenase pathway as well as the cyclooxygenase pathway. Analysis by high performance liquid chromatography delineated a monohydroxy acid, presumably 12-L-hydroxy-5,8-10,14-eicosatetraenoic acid (12-HETE) as the major lipoxygenase product. The formation of this hydroxy arachidonate derivative was stimulated by the calcium ionophore ionomycin. Stimulation of the lipoxygenase pathway by ionomycin was confirmed by thin layer chromatography. In addition, 6-keto-PGF1 alpha, PGF2 alpha, and PGE2 were identified; and ionomycin, carbamylcholine, and caerulein enhanced the formation of these metabolites of the cyclooxygenase pathway. Ionomycin induced stimulation of HETE formation was inhibited by ETYA and nordihydroguaiaretic acid, but spontaneous and evoked enzyme secretion was unaffected. Thus, although ionomycin, a pancreatic secretagogue, stimulates the lipoxygenase pathway, the precise role of these arachidonate metabolites in the physiology of the exocrine pancreas is still obscure.     

The role of arachidonate lipoxygenase in the release of SRS-A from guinea-pig chopped lung

The immunological release of SRS-A was investigated in guinea-pig chopped lung. A number of unsaturated fatty acids, all of which are substrates for arachidonate lipoxygenase were found to potentiate the release of SRS-A. This potentiation was enhanced by indomethacin, a cyclo-oxygenase inhibitor, and completely reversed by nordihydroguaiaretic acid (NDGA) and eicosatetraynoic acid (ETA) which inhibit lipoxygenase. This suggests that some aspect of arachidonate lipoxygenase action stimulates release of SRS-A and that release of SRS-A is increased by redirection of arachidonic acid (AA) metabolism via the lipoxygenase pathway (Hamberg, 1976). However, although exogenous ¹⁴C-AA increased SRS-A output it was not incorporated into SRS-A.     

The effect of eicosapentaenoic acid on leukotriene B production by human neutrophils

Eicosapentaenoic acid, which is a major fatty acid in fish oil, previously has been shown to competitively inhibit the cyclooxygenase-catalyzed metabolism of arachidonic acid in platelets. In the present study the effect of eicosapentaenoic acid on the production of leukotriene B via the lipoxygenase pathway in human neutrophils was examined. Eicosapentaenoate was incorporated into complex lipids of neutrophils at the same rate as arachidonate; release of the two homologous fatty acids in response to calcium ionophore A23187 was equivalent and both fatty acids were metabolized to a leukotriene B. The products derived from eicosapentaenoic acid were identified as leukotriene B5 and its stereoisomers. Eicosapentaenoate was a less favorable substrate for leukotriene B5 synthesis (94 ng/10(7) cells/5 min at 20 microM exogenous fatty acid) than arachidonate was for leukotriene B4 (401 ng under the same conditions). However, eicosapentaenoate or an oxygenated product inhibited arachidonate metabolism since at equimolar concentrations of eicosapentaenoate and arachidonate leukotriene B4 production was decreased by 68%. The inhibitory effect occurred at the level of leukotriene A hydrolase. The biological activity of eicosapentaenoate -derived products was tested; leukotriene B5 was found to have only approximately 10% of the potency of leukotriene B4 in inducing the aggregation of neutrophils, and the stereoisomers of leukotriene B5 were inactive. These data suggest that diets enriched in eicosapentaenoic acid affect neutrophils by decreasing the quantity of leukotriene B and by the production of a less potent leukotriene.     

Reactive oxygen production associated with arachidonic acid metabolism by peritoneal macrophages

The nature of the calcium-dependent chemiluminescence observed in peritoneal macrophages after exposure to the calcium ionophore A23187 or during the phagocytosis of zymosan has been investigated. Eicosatetraynoic acid, an inhibitor of the lipoxygenase and cyclooxygenase pathways of arachidonic acid metabolism, inhibited the calcium-dependent chemiluminescence whereas indomethacin, a selective inhibitor of the cyclooxygenase pathway, did not. Arachidonic acid induced chemiluminescence only in phagocytosing cells, whilst 15-HPETE, an intermediate of the lipoxygenase pathway, generated a similar, transient chemiluminescent response in either unstimulated or phagocytosing cells. The results suggest that the lipoxygenase pathway may be a significant source of the reactive species of oxygen that give rise to chemiluminescence. Prostaglandin E₁ inhibited the chemiluminescence induced by zymosan and A23187, but did not affect that generated in response to 15-HPETE or arachidonic acid, suggesting that the inhibition is directed at a step either connected with or occurring prior to the release of free arachidonic acid by the cells.     

Suppression of cholesteryl ester accumulation in cultured human monocyte-derived macrophages by lipoxygenase inhibitors

Atherosclerotic lesions and xanthomas are characterized by the occurrence of cholesteryl ester (CE)-laden foam cells, which partly originate from macrophages. Little is known about the role of cyclo-oxygenase or lipoxygenase metabolites of arachidonic acid in the development of foam cells. In this study we investigated the influence of prostaglandins and inhibitors of the cyclo-oxygenase or the lipoxygenase pathway on CE accumulation in cultured human monocyte-derived macrophages. Accumulation of CE was achieved by incubation of the cells with acetylated low density lipoprotein (AcLDL). The stable prostacyclin analogue ZK 36 374 and prostaglandin E2 showed no effect on cellular CE storage. Similarly, the cyclo-oxygenase inhibitor indomethacin failed to influence AcLDL-induced CE accumulation. By contrast, however, the inhibitors of lipoxygenase activity nordihydroguaiaretic acid (NDGA) and BW 755 C markedly suppressed the accumulation of CE in monocyte-derived macrophages. The inhibitory effect of NDGA was dose-dependent. Incubation of the cells with the anti-oxidant vitamin E gave no significant reduction of CE accumulation. Our results indicate that inhibition of the lipoxygenase pathway of arachidonic acid metabolism in cultured monocyte-derived macrophages effectively decreases the rate of experimentally-induced CE accumulation.     

Inhibitors of arachidonic acid lipoxygenase impair the stimulation of inositol phospholipid hydrolysis by the T lymphocyte mitogen phytohaemagglutinin

Piriprost and nordihydroguiaretic acid (NDGA), specific inhibitors of arachidonate lipoxygenase, inhibited phytohaemagglutinin (PHA)-stimulated breakdown of inositol lipids in human T lymphocytes. The dual inhibitors eicosatetraynoic acid (ETYA) and BW 755C, which inhibit both lipoxygenase and cyclooxygenase, also had similar actions, whereas indomethacin and acetylsalicyclic acid, which inhibit cyclooxygenase alone, did not. The effects of lipoxygenase inhibitors and dual inhibitors were reversible. These agents did not inhibit phosphatidylinositol-4,5-bisphosphate-specific phospholipase C (PIP2-PLC) in vitro. Bromophenacyl bromide, and irreversible inhibitor of phospholipase A2, also abolished PHA-stimulated inositol lipid breakdown without affecting PIP2-PLC in vitro. The results are consistent with a role for the PHA-stimulated generation of arachidonic acid and its conversion to lipoxygenase metabolites (e.g. leukotrienes and/or hydroxyeicosatetraenoic acids) as intermediate steps in the signal transduction pathway between cell-surface mitogen receptors and the stimulation of PIP2-PLC in lymphocytes.