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P Borgeat

Publications and source records attributed to P Borgeat.

At least 73 records · Page 4Linked to original sources
PubMed1991

Synthesis of eicosanoids by isolated guinea pig tracheocytes.

Epithelial cells from the guinea pig trachea (tracheocytes) have been separated from the lamina propria by incubation with EDTA. Contaminating eosinophils were removed by plating on guinea pig immunoglobulin G (IgG)-coated plates. Purity of the cell suspension was assessed by electron microscopy. Enzyme immunoassay analyses of supernatants of guinea pig tracheocytes incubated with 10 microns arachidonic acid (AA) in the presence or absence of 2 microns ionophore A23187 showed that the cells released up to eight times more thromboxane, prostaglandin E2, and 6-ketoprostaglandin F1 alpha than control cells. Ionophore A23187 by itself stimulated to a smaller extent the release of these eicosanoids and did not potentiate the effect of AA. The release of cyclooxygenase products by these cells was highly modified by cell densities in the incubation media. The release of leukotrienes (LT) by stimulated epithelial cells was also investigated using reverse-phase high-performance liquid chromatography. Our results showed that the cells could not release LT on incubation with ionophore A23187 and/or AA. However, the cells released LTB4 in the medium on incubation with 2 microns LTA4. Peptido-leukotrienes were not detected. These results indicated that guinea pig tracheocytes have the cyclooxygenase and the LTA4 hydrolase but not the 5-lipoxygenase. It is postulated that tracheocytes may participate in transcellular metabolism of LTA4 generated by other cell types.

Animals↗
PubMed1991

Leukotriene B4 receptors on guinea pig alveolar eosinophils.

The existence of receptors for LTB4 on highly purified guinea pig alveolar eosinophils was investigated. Massive infiltration of eosinophils in alveolar spaces was induced in guinea pigs by i.v. injections of Sephadex beads G50 (16 mg/kg). Alveolar eosinophils (50 x 10(6) cells) were purified to approximately 98% by Percoll continuous density gradient centrifugation. The binding studies indicated that alveolar eosinophils bind LTB4 in a saturable, reversible and specific manner. Scatchard analysis indicated the existence of high-affinity binding sites (Kd1 = 1.00 +/- 0.22 nM; Bmax1 = 966 +/- 266 sites/cell) and low-affinity binding sites (Kd2 = 62.5 +/- 8.9 nM; Bmax2 = 5557 +/- 757 sites/cell). The metabolism of LTB4 by alveolar eosinophils in binding conditions was assessed by RP-HPLC and no significant degradation of [3H]LTB4 was observed. LTB4 dose-dependently stimulated eosinophil migration in both chemokinesis and chemotaxis assays with an EC50 value of 1.30 +/- 0.14 and 18.14 +/- 1.57 nM, respectively. LTB4 caused a dose-dependent increase in the production of superoxide anion with an apparent EC50 value of 50 X 10(-9) M in our experimental conditions. LTB4 also induced a dose-dependent increase in the generation of TxA2 with an EC50 value of 46.2 X 10(-9) M. Taken together, our results demonstrated that guinea pig alveolar eosinophils express two classes of specific receptors for LTB4. The high-affinity binding sites seem associated to chemokinesis and chemotaxis whereas the low-affinity binding sites seem associated to superoxide anion production and generation of TxA2. The existence of LTB4 receptors in eosinophils could explain the presence of these cells in hypersensitivity reactions.

Animals↗
PubMed1991

Specific binding sites for leukotriene B4 in guinea pig brain membranes.

The presence of specific leukotriene B4 (LTB4) binding sites was investigated in membranes prepared from mouse, rat, guinea pig and rabbit brain. Specific binding sites have been found in guinea pig brain membranes (GPBM) but not in the other species studied. Specific binding of LTB4 to GPBM was proportional to membrane concentration, saturable and reversible, reaching a steady-state suggesting equilibrium after 30 min of incubation. Analysis of the binding data showed a single binding site with a Kd = 2.27 +/- 0.55 nM and a Bmax = 576 +/- 89.6 fmol/mg protein. The relative potencies of LTB4 analogs to displace the tritiated ligand were LTB4 greater than 20-hydroxy-LTB4 much greater than 20-carboxy-LTB4 greater than 12-(S)hydroxy-5,8,10,14(Z,Z,E,Z)-eicosatetraenoic acid; LTD4 and LTC4 did not displace the ligand in concentrations up to 50 microM. The binding was inhibited by monovalent cations and GTP[gamma S] and increased by divalent cations. Specific binding was associated to the cerebral cortex. The present results suggest that these specific binding sites could represent functional LTB4 receptors in the guinea pig cerebral cortex with unknown biological effects.

Animals↗
PubMed1990

Involvement of a phospholipase D in the mechanism of action of granulocyte-macrophage colony-stimulating factor (GM-CSF): priming of human neutrophils in vitro with GM-CSF is associated with accumulation of phosphatidic acid and diradylglycerol.

The generation of diradylglycerol (DRG) and phosphatidic acid (PdtOH) was investigated in neutrophils primed with granulocyte-macrophage colony-stimulating factor (GM-CSF). Mass accumulation of DRG and PdtOH was measured using reversed-phase high performance liquid chromatography and thin layer chromatography, respectively. GM-CSF had no direct effect on the levels of PdtOH and DRG, but it increased PdtOH generation and the late phase of DRG accumulation in human neutrophils stimulated with FMLP. The elevation of the mass of PdtOH peaked approximately 100 s and clearly preceded that of DRG, which peaked at 150 s. The diacylglycerol kinase inhibitor R59022 enhanced the sustained increase in DRG but did not produce a parallel inhibition in PdtOH production. GM-CSF was without effect on the level of inositol 1,4,5-triphosphate [Ins(1,4,5)P3] and did not affect the liberation of Ins(1,4,5)P3 induced by FMLP. These findings exclude the involvement of the PtdIns(4,5)P2-specific phospholipase C/diacylglycerol pathway in the sustained phase of DRG accumulation. The early (30-s) appearance of PdtOH clearly suggests that GM-CSF enhanced FMLP receptor-linked phospholipase D (PLD) generation of PdtOH. PLD was assessed more directly by formation of labeled phosphatidylethanol (PEt) through PLD capacity of catalyzing a trans-phosphatidylation in presence of ethanol. The formation of PEt associated with a concomitant decrease in PdtOH directly demonstrated that the mechanism by which GM-CSF enhances PdtOH production is activation of a PLD active on phosphatidylcholine. This study provides evidence that the mechanism of action of GM-CSF involves upregulation of PLD activity leading to enhanced generation of PdtOH and DRG in FMLP-stimulated neutrophils. These findings may provide the basis for several of the priming effects of GM-CSF.

Chromatography, High Pressure Liquid↗
PubMed1990

Guinea pig alveolar eosinophils and macrophages produce leukotriene B4 but no peptido-leukotriene.

The metabolism of arachidonic acid (AA) was investigated in purified guinea pig alveolar eosinophils and macrophages. Alveolar eosinophils produced 12S-hydroxy-5,8,10-heptadecatraenoic acid (HHT) and small amounts only of 5-lipoxygenase products when stimulated by AA (10 microM) or ionophore A23187 (2 microM). However, when the cell suspensions were stimulated with both AA and A23187, the cells produced HHT, leukotriene (LT) B4, and 5S-hydroxy-6,8,11,14-eicosatetraenoic acid, whereas LTC4, D4, and E4 were undetectable. Similarly, alveolar macrophages stimulated with A23187 produced HHT, 5-hydroxy-6,8,11,14-eicosatetraenoic acid, and LTB4 but no peptido-leukotrienes. When LTA4 was added to suspensions of eosinophils and macrophages, only LTB4 was formed, whereas in parallel experiments, intact human platelets incubated with LTA4 produced LTC4. These data suggest that guinea pig alveolar eosinophils and macrophages contain both cyclooxygenase and 5-lipoxygenase, but do not produce peptido-leukotrienes, probably lacking LTA4 glutathione transferase activity. These studies demonstrate that guinea pig eosinophils differ from eosinophils of other animal species which have been shown to be major sources of leukotriene C4. The present data imply that eosinophils and macrophages are not the source of peptido-leukotrienes in anaphylactic guinea pig lungs.

Animals↗
PubMed1990

Biosynthesis and biological activity of leukotriene B4.

The leukotrienes are a family of biologically active molecules derived from arachidonic acid. While prostaglandins and thromboxanes are products of the cyclooxygenase pathway of arachidonic acid metabolism, the leukotrienes are formed by arachidonate 5-lipoxygenase, an enzyme present in phagocytes, mast cells, and basophils. Inflammatory stimuli, such as chemotactic peptides, platelet-activating factor, phagocytic particles, and immunological stimuli, which activate phagocytes and mast cells, stimulate leukotriene synthesis. Leukotriene B4, a dihydroxy derivative of arachidonic acid, has a unique stimulatory activity on important functional responses of phagocytes; leukotriene B4 exerts chemotactic and chemokinetic activity towards phagocytes in vitro and in vivo, and it is a putative mediator of inflammation.

Animals↗
PubMed1990

Nomenclature.

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Arachidonic Acids↗
PubMed1990

Inhibitory effects of MK-886 on arachidonic acid metabolism in human phagocytes.

1. We have investigated the inhibitory activity of compound MK-886 (formerly L-663,536), an indole derivative, on 5-lipoxygenase product synthesis in various human phagocytes stimulated with either the ionophore A23187, in the presence and absence of exogenous arachidonic acid, or platelet-activating factor (PAF). The lipoxygenase products were analysed by reversed-phase h.p.l.c. 2. MK-886 inhibited the formation of 5-hydroxy-eicosatetraenoic acid (5-HETE), leukotriene B4 (LTB4), its omega-oxidation products and 6-trans-isomers with an IC50 value of 10-14 nM in A23187-stimulated neutrophils. In the same system, nordihydroguaiaretic acid (NDGA), AA-861 and L-655,240 showed IC50 values of 250-510, 110-420 nM and 1.7-3.9 microM, respectively. 3. MK-886 inhibited 5-lipoxygenase product synthesis in A23187-stimulated blood eosinophils and monocytes, and in neutrophils primed with granulocyte-macrophage colony-stimulating factor and stimulated with PAF with IC50 values of 1-13 nM. 4. The inhibitory activity of MK-886 was not reversed by addition of 10 microM arachidonic acid to A23187-stimulated neutrophils. 5. Compound MK-886 had no effect on 15-lipoxygenase product synthesis in blood eosinophils and neutrophils up to a concentration of 1 microM. 6. At 100 nM compound MK-886 had no significant effects on calcium ion mobilization, superoxide anion production and actin polymerization in neutrophils. 7. In conclusion, MK-886 is a very potent and specific inhibitor of both LTB4 and LTC4 synthesis in various types of human phagocytes.

Actins↗
PubMed1990

Inhibition of human alveolar macrophage production of leukotriene B4 by acute in vitro and in vivo exposure to tobacco smoke.

The number of neutrophils in the lungs of cigarette smokers is increased. This could be a consequence of the chemotactic mediator synthesis by alveolar macrophages (AM). In order to evaluate the possible role of leukotriene B4 (LTB4) in this condition, we studied the formation of LTB4 by nonsmokers' AM exposed in vitro and in vivo to cigarette smoke and by smokers' AM. In the absence of stimulus or upon stimulation, nonsmokers' AM exposed in vitro to tobacco smoke formed less LTB4 than did nonexposed AM, e.g., the cells incubated with arachidonic acid and ionophore produced, respectively, 241 +/- 132 and 425 +/- 106 pmol LTB4/10(6) cells (mean +/- SEM) (P less than 0.01). In other experiments, smokers' AM were incubated in absence of stimulus and produced less LTB4 than did nonsmokers' AM; during a 3-h incubation, smokers' and nonsmokers' adherent AM released, respectively, 3 +/- 2 and 40 +/- 28 pmol LTB4/10(6) cells (P less than 0.05). Similarly stimulated smokers' AM produced less LTB4 than did nonsmokers' AM, e.g., the cells incubated with arachidonic acid and ionophore formed, respectively, 225 +/- 41 and 573 +/- 150 pmol LTB4/10(6) cells (P less than 0.05). In a group including mild smokers and nonsmokers, in vivo exposure to the smoke of 4 cigarettes produced a decrease in the release of LTB4 by AM, e.g., in the presence of arachidonic acid and ionophore, nonexposed and exposed AM produced, respectively, 198 +/- 38 and 143 +/- 38 pmol LTB4/10(6) cells (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1990

Enhancement of platelet-activating factor-induced leukotriene synthesis in neutrophils by granulocyte-macrophage colony-stimulating factor (GM-CSF): studies on the mechanism of action of GM-CSF.

Preincubation with granulocyte-macrophage colony-stimulating factor (GM-CSF) increased the synthesis of leukotriene B4 and its omega-oxidation products by neutrophils in response to 10(-7) M platelet-activating factor (PAF) by more than 10-fold compared to untreated cells. Pretreatment with GM-CSF also enabled the detection of these products in response to lower concentrations of PAF (greater than or equal to 10(-9) M), under which conditions synthesis of 5-lipoxygenase products was not observed in non-GM-CSF-treated cells. While PAF induced the release of arachidonic acid from neutrophils, GM-CSF alone did not stimulate the release of detectable amounts of the fatty acid. However, the levels of free arachidonic acid in response to PAF were enhanced in neutrophils pretreated with GM-CSF. Similarly, GM-CSF alone did not directly activate the 5-lipoxygenase as determined by the 5-lipoxygenase-mediated transformation of exogenous 15-hydroperoxyeicosatetraenoic acid into 5,15-dihydroxyeicosatetraenoic acid (5,15-diHETE). However, stimulation of neutrophils with PAF resulted in the transformation of the 15-hydroperoxy acid into the 5,15-diHETE, and furthermore, preincubation of neutrophils with GM-CSF resulted in enhanced formation of 5,15-diHETE in response to PAF. Taken together, these data indicate that GM-CSF does not enhance leukotriene synthesis in neutrophils in response to PAF by either directly activating the 5-lipoxygenase or stimulating the liberation of endogenous arachidonic acid, but rather by augmenting the effect of PAF on these two responses.

Arachidonate 5-Lipoxygenase↗
PubMed1990

Dietary linoleate supplementation modulates formyl-peptide receptor expression and functional responses of rat neutrophils.

We studied effects of dietary supplementation with the essential fatty acid (EFA) linoleic acid (LA) to see if neutrophil responses would be modulated. Neutrophils from rats maintained on a diet supplemented with EFA to 10% of the energy content--the high EFA (HEFA) group--showed a significantly higher LA concentration (but similar arachidonic acid content) compared with neutrophils from control rats maintained on a standard diet with 3% of the energy content as EFA. The HEFA group showed a significantly higher neutrophil oxidative metabolism compared with controls in response to N-formyl-methionyl-leucyl-phenylalanine (FMLP), but this response was equal to control values when stimulated by ionophore A23187, sodium fluoride, or phorbol myristate acetate. Similarly, FMLP conferred a more pronounced increase of intracellular Ca2+ in HEFA neutrophils, whereas this response to ionomycin was equal to that in controls. In contrast, HEFA rat neutrophil migration was decreased to 71% of the value in controls in response to FMLP. Similar results were observed for aggregation responses. On A23187 stimulation, HEFA and control neutrophils generated equal amounts of leukotriene B4 and other 5-lipoxygenase products as well as thromboxane B2 and 6-keto-prostaglandin F1 alpha. However, assessment of binding of FMLP labeled with tritium revealed an increase of the low affinity state of the FMLP receptor population. Thus an increased intake of one unsaturated fatty acid, LA, leading to its accumulation in neutrophils, conferred alterations in formyl-peptide-elicited responses, not associated with the formation of the assessed arachidonate-derived mediators, but most likely through the observed modulation of FMLP receptor subpopulations.

6-Ketoprostaglandin F1 alpha↗
PubMed1989

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and opsonization synergistically enhance leukotriene B4 (LTB4) synthesis induced by phagocytosis in human neutrophils.

Normal human blood neutrophils were studied for their capacity to synthesize leukotriene B4 (LTB4) and its omega-oxidized metabolites after phagocytosis of zymosan. Phagocytosis of serum-opsonized particles led to a higher release of LTs than did unopsonized zymosan. The most striking effect of phagocytosis was observed when neutrophils were primed with granulocyte-macrophage colony-stimulating factor (GM-CSF): opsonization and GM-CSF synergistically increased LTB4 synthesis by neutrophils.

Arachidonate 5-Lipoxygenase↗
PubMed1989

Syntheses and comparison of three reagents for allyldimethylsilylation of prostaglandins and steroids: a gas chromatographic method.

Three reagents used for allyldimethylsilylation of prostaglandins and steroids have been prepared: N,O-bis(allyldimethylsilyl)trifluoroacetamide, N-methylallyl-dimethylsilyltrifluoroacetamide and allyldimethylsilyl-N,N-diphenylamine. The reaction kinetics of the reagents have been studied at room temperature in several solvents, and the optimal conditions for the derivatization of prostaglandins and steroids are discussed.

Acetamides↗
PubMed1989

Arachidonic acid-induced mobilization of calcium in human neutrophils: evidence for a multicomponent mechanism of action.

1. The mechanism(s) involved in the mobilization of calcium induced by arachidonic acid in human neutrophils was investigated. 2. The addition of arachidonic acid to a suspension of human neutrophils led to a time- and concentration-dependent mobilization of calcium which was the result of two separate and experimentally differentiable processes. The latter consisted of a rapid and transient phase followed by a slower and more sustained response. 3. The initial phase of calcium mobilization elicited by arachidonic acid was decreased in the presence of EGTA, inhibited by pertussis toxin as well as by nordihydroguaiaretic acid (NDGA), and diminished following a pre-incubation with leukotriene B4, but not platelet-activating factor. 4. The characteristics of the first phase of the mobilization of calcium were consistent with an interaction of the fatty acid with the leukotriene B4 receptors, either directly or indirectly following the synthesis of leukotriene B4, as well as with a release of internal calcium. 5. The second, slower and more sustained phase of calcium mobilization was more apparent at high concentrations (greater than or equal to 8-16 microM) of arachidonic acid, and was relatively insensitive to pertussis toxin, EGTA or NDGA. 6. The characteristics of the 'slow' phase of calcium mobilization by arachidonic acid are consistent with its being associated primarily with a release of calcium from internal storage pools. 7. The data presented indicate that the mechanism of mobilization of calcium by arachidonic acid in human neutrophils is complex and involves specific activation pathways employed, in part at least, by other neutrophil agonists. These findings may have relevance to various inflammatory situations in which the elevated levels of extracellular arachidonic acid known to be present could modulate the functional responsiveness of the neutrophils to other stimuli.

Arachidonic Acid↗