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.     

Phospholipase C-diglyceride lipase is a major pathway for arachidonic acid release in macrophages

Macrophages are a rich source of arachidonic acid oxygenated metabolites and play a remarkable role in a number of physiopathological situations. The synthesis and secretion of arachidonic acid metabolites are triggered at the cytoplasmic membrane level. The present study was outlined to further investigate the cellular mechanisms controlling arachidonic acid release in macrophages. The results presented here strongly suggest that the amount of arachidonic acid released in macrophages in response to phagocytic challenge could be accounted for by a phospholipase C-diglyceride lipase system being unnecessary the presence of phospholipase A2 whose activity, on the other hand, was found vanishingly small in macrophage homogenates.     

Dual effects of staurosporine on arachidonic acid metabolism in rat peritoneal macrophages

Staurosporine is a microbial anti-fungal alkaloid having a most potent inhibitory activity on protein kinase C and is recently found as a non-12-O-tetradecanoylphorbol-13-acetate (non-TPA)-type tumor promoter of mouse skin, although tumor promotion induced by a TPA-type tumor promoter teleocidin is suppressed by staurosporine. When rat peritoneal macrophages were incubated in the medium containing various concentrations of staurosporine, prostaglandin E2 production and release of radioactivity from [3H]arachidonic acid-labeled macrophages were stimulated at concentrations of 1 and 10 ng/ml. But higher concentrations of staurosporine such as 100 and 1000 ng/ml showed no stimulative effect on prostaglandin E2 production although cytoplasmic free calcium levels were increased in a dose-dependent manner. Staurosporine-induced stimulation of prostaglandin E2 production was inhibited by treatment with cycloheximide, suggesting that a certain protein synthesis is prerequisite for the stimulation of arahcidonic acid metabolism. At higher concentrations (100 and 1000 ng/ml), staurosporine inhibited TPA-type tumor promoter (TPA, teleocidin and aplysiatoxin)-induced stimulation of arachidonic acid metabolism probably due to the inhibition of protein kinases. Tumor promotion activity and anti-tumor promotion activity of staurosporine might be explained by the fact that the lower concentrations of staurosporine stimulate arachidonic acid metabolism and the higher concentrations of staurosporine inhibit the tumor promoter-induced arachidonic acid metabolism, respectively.     

Ultrastructural localization of arachidonic acid in stimulated macrophages

The distribution of 3[H] arachidonic acid incorporated into cultured mouse peritoneal macrophages was assessed upon stimulation of the cells with either the calcium ionophore A23187 or zymosan. After a labeling time of 24 h, cells were stimulated and processed for light and electron microscopic autoradiography. Grains were primarily localized over the plasma membrane and lipid-containing vesicles of both control and stimulated cells. In macrophages stimulated with ionophore, a decreased labeling density was evident in both of these cell compartments. Similar alterations in labeling pattern were observed in zymosan treated cells, although a larger decline in grain density occurred from the plasma membrane compartment. Immunocytochemical localization of PGE2, a major eicosanoid product released upon ionophore stimulation, revealed the presence of the prostaglandin in clear vesicular structures, many of which appear to be continuous with the plasma membrane. These results provide morphological evidence that different cellular pools of arachidonic acid may be differentially mobilized for eicosanoid production as a function of the mode of stimulation.     

Platelet-activating factor-stimulated hepatic glycogenolysis is not mediated through cyclooxygenase-derived metabolites of arachidonic acid

Platelet-activating factor (1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC)) is a potent lipid mediator which stimulates hepatic glycogenolysis, causes hepatic vasoconstriction, and stimulates the production of cyclooxygenase-derived metabolites of arachidonic acid, primarily prostaglandin (PG) D2 in the perfused liver. Following infusion of platelet-activating factor (1 nM) in the perfused rat liver the production of PGD2, measured in the effluent perfusate, increased 4-fold after only 2 min. Infusion of the cyclooxygenase inhibitor, ibuprofen (50 microM), abolished the stimulated production of PGD2 and thromboxane B2 in response to AGEPC without significantly affecting the hepatic glycogenolytic or vasoconstrictive responses to AGEPC. Contrary to previous reports, these observations do not support the suggestion that cyclooxygenase-derived metabolites mediate directly either the glycogenolytic or the vasoactive effects of AGEPC in the perfused rat liver.     

Inhibitors of the 5-lipoxygenase arachidonic acid pathway induce ATP release and ATP-dependent organic cation transport in macrophages

We have previously described that arachidonic acid (AA)-5-lipoxygenase (5-LO) metabolism inhibitors such as NDGA and MK886, inhibit cell death by apoptosis, but not by necrosis, induced by extracellular ATP (ATPe) binding to P2X7 receptors in macrophages. ATPe binding to P2X7 also induces large cationic and anionic organic molecules uptake in these cells, a process that involves at least two distinct transport mechanisms: one for cations and another for anions. Here we show that inhibitors of the AA-5-LO pathway do not inhibit P2X7 receptors, as judged by the maintenance of the ATPe-induced uptake of fluorescent anionic dyes. In addition, we describe two new transport phenomena induced by these inhibitors in macrophages: a cation-selective uptake of fluorescent dyes and the release of ATP. The cation uptake requires secreted ATPe, but, differently from the P2X7/ATPe-induced phenomena, it is also present in macrophages derived from mice deficient in the P2X7 gene. Inhibitors of phospholipase A2 and of the AA-cyclooxygenase pathway did not induce the cation uptake. The uptake of non-organic cations was investigated by measuring the free intracellular Ca^2 + concentration ([Ca^2 +]_i) by Fura-2 fluorescence. NDGA, but not MK886, induced an increase in [Ca^2 +]_i. Chelating Ca^2 + ions in the extracellular medium suppressed the intracellular Ca^2 + signal without interfering in the uptake of cationic dyes. We conclude that inhibitors of the AA-5-LO pathway do not block P2X7 receptors, trigger the release of ATP, and induce an ATP-dependent uptake of organic cations by a Ca^2 +- and P2X7-independent transport mechanism in macrophages.     

Hypotensive effects in cats caused by horseradish peroxidase mediated by metabolites of arachidonic acid

Horseradish peroxidase (HRP) given intravenously was shown to possess a marked hypotensive effect in cats. The effect was not noticeably influenced by antihistamines. Indomethacin and acetylsalicylic acid completely blocked the hypotensive effect of HRP. It is concluded that the effect of HRP on blood pressure is due to stimulation of the synthesis of metabolites of arachidonic acid.     

Cardioprotective effects of high-intensity interval training are mediated through microRNA regulation of mitochondrial and oxidative stress pathways

Human studies have shown high-intensity interval training (HIIT) has beneficial cardiovascular effects and is typically more time-efficient compared with traditional endurance exercise. The main goal of this study is to show the potential molecular and functional cardiovascular benefits of HIIT compared with endurance training (ET). Three groups of mice were used including sedentary-control, ET mice, and HIIT mice groups. Results indicated ejection fraction was increased in HIIT compared with ET while fractional shortening was increased in the HIIT group compared with both groups. Blood flow of the abdominal aorta was increased in both exercise groups compared with control. Increases in cross-sectional area and mitochondrial and antioxidative markers in HIIT compared with control were observed, along with several microRNAs. These findings indicate HIIT has specific cardiac-protective effects and may be a viable alternative to traditional ET as a cardiovascular preventative medicine intervention.     

Antiplatelet effect of green tea catechins: a possible mechanism through arachidonic acid pathway

We have previously reported that green tea catechins (GTC) showed an antithrombotic activity, which might be due to antiplatelet effect rather than anticoagulation. The present study was performed to investigate the effect of GTC on the arachidonic acid (AA) metabolism in order to elucidate a possible antiplatelet mechanism. GTC inhibited the collagen-, AA- and U46619-induced rabbit platelet aggregation in vitro in a concentration-dependent manner, with IC50 values of 61.0+/-2.5, 105.0+/-4.9 and 67.0+/-3.2 microg/ml, respectively. Moreover, GTC administered orally into rats inhibited the AA-induced platelet aggregation ex vivo by 46.9+/-6.1% and 95.4+/-2.2% at the doses of 25 and 50 mg/kg, respectively. [3H]AA liberation induced by collagen in [3H]AA incorporated rabbit platelets was significantly suppressed by GTC compared to the control. GTC also significantly inhibited the thromboxane A2 (TXA2) and prostaglandin D2 (PGD2) generations induced by addition of AA in intact rabbit platelets. GTC significantly inhibited TXA2 synthase activity in a concentration-dependent manner. Moreover, adenosine triphosphate (ATP) release from dense granule was inhibited by GTC in washed platelets. These results suggest that the antiplatelet activity of GTC may be due to the inhibition of TXA2 formation through the inhibition of AA liberation and TXA2 synthase.     

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

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

Metabolism of exogenous arachidonic acid by mouse peritoneal macrophages

On incubation of resident mouse peritoneal macrophages with arachidonic acid several hydroxyacyl derivatives detectable in cellular supernatants are formed. As main products monohydroxyarachidonic acids (monoHETE's) were identified. In addition, smaller amounts of dihydroxyarachidonic acids (diHETE's) supernatants by reversed phase HPLC, normal phase HPLC in combination with UV-spectroscopy and combined gas-chromatography / masspectrometry revealed the presence of 5-, 8-, 12- and 15 - mono-HETE's, two distinct 5, 12-diHETE's, several 8, 15-diHETE's and 14, 15-diHETE. Among the 5, 12-diHETE's, only small amounts of a compound with the characteristics of LTB, were detected. Under the conditions employed, the cycloxygenase products PGE₂ and PGI₂ (as 6-keto-PGF_1g) were only minor metabolites. In contrast, when macrophage cultures were stimulated with the phagocytic stimulus zymosan, PGI₂, PGE₂ and LTC₄ were found as the major conversion products of arachidonic acid, whereas mono- and diHETE's were not formed in detectable amounts.     

Metabolism of exogenous arachidonic acid by mouse peritoneal macrophages

On incubation of resident mouse peritoneal macrophages with arachidonic acid several hydroxyacyl derivatives detectable in cellular supernatants are formed. As main products monohydroxyarachidonic acids (monoHETE's) were identified. In addition, smaller amounts of dihydroxyarachidonic acids (diHETE's) were formed. A detailed analysis of cell culture supernatants by reversed phase HPLC, normal phase HPLC in combination with UV-spectroscopy and combined gas-chromatography/masspectrometry revealed the presence of 5-, 8-, 12- and 15- monoHETE's, two distinct 5,12-diHETE's, several 8,15-diHETE's and 14,15-diHETE. Among the 5,12-diHETE's, only small amounts of a compound with the characteristics of LTB4 were detected. Under the conditions employed, the cyclooxygenase products PGE2 and PGI2 (as 6-keto-PGF1 alpha) were only minor metabolites. In contrast, when macrophage cultures were stimulated with the phagocytic stimulus zymosan, PGI2, PGE2 and LTC4 were found as the major conversion products of arachidonic acid, whereas mono- and diHETE's were not formed in detectable amounts.     

Effect of ethanol on the arachidonic acid metabolism in mouse peritoneal macrophages

Macrophages play an important role in the development of chronic inflammatory states. Ethanol has been shown to impair a number of membrane-linked phenomena. The synthesis and secretion of oxygenated metabolites of arachidonic acid is triggered at the cytoplasma membrane level. The present study was carried out in order to investigate the effect of ethanol on the arachidonic acid metabolism in mouse peritoneal macrophages. Two types of experiments were performed: with endogenous radiolabeled arachidonic acid and with exogenously added radiolabeled arachidonic acid. Our data show that ethanol in vitro activates the release of arachidonic acid from intracellular pools, while the proportion of endogenous substrate metabolized in the presence of ethanol is similar to that in controls. From the exogenous it seems clear that ethanol induces different effects depending whether the arachidonic acid is endogenous or added exogenously.     

Mast cell mediators stimulate synthesis of arachidonic acid metabolites in macrophages

Mast cells and macrophages were isolated from human lung tissues by using density gradient centrifugation, cell sorter, and adherence techniques. Passively sensitized mast cells in the absence of exogenous arachidonic acid (AA) released leukotriene (LT)C4, LTD4, PGD2, and thromboxane-B2 when challenged with Ag, and in the presence of AA, released 5-hydroxyeicosatetraenoic acid (HETE) and 15-HETE in addition to the above metabolites. Passively sensitized macrophages did not release significant amounts of AA metabolites when challenged with Ag. However, these cells released LTB4, LTC4, LTD4, LTE4, 5-HETE, PGE2 and 6-keto-PGF1 alpha when co-incubated with activated mast cells. During co-incubation, mast cells also generated greater amount of AA metabolites than when they were activated alone. The stimulatory action of mast cells on macrophages was shown to be due to the extracellular factor(s) present in the supernatant of the activated mast cells. Both heat and trypsin inhibited the biologic activity of mast cell-derived stimulatory factor. In addition, extraction of mast cells' materials with chloroform or ether showed no activity associated with the organic phase, suggesting it possibly possesses a protein nature, such as peptides, protease, or peptidase. These results suggest that mast cell-macrophage interaction might be important in the generation of multiple mediators in the airways during immediate hypersensitivity reactions.