scholarly journals Na+/H+ exchange modulates the production of leukotriene B4 by human neutrophils

1989 ◽  
Vol 257 (3) ◽  
pp. 751-758 ◽  
Author(s):  
M Osaki ◽  
H Sumimoto ◽  
K Takeshige ◽  
E J Cragoe ◽  
Y Hori ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Leukotriene B4 ◽  
Human Neutrophils ◽  
Ionophore A23187 ◽  
Cytoplasmic Ph ◽  
Chemotactic Peptide ◽  
Trans Isomers ◽  
Lipoxygenase Pathway ◽  
Exogenous Arachidonic Acid ◽  
Specific Inhibitors

Human neutrophils produce various compounds of the 5-lipoxygenase pathway, including (5S)-hydroxyeicosatetraenoic acid, leukotriene B4, its 6-trans isomers and omega-oxidation metabolites of LTB4, when the cells are stimulated with the Ca2+ ionophore A23187. The elevation in the extracellular pH (pHo) facilitated the cytoplasmic alkalinization induced by the ionophore as determined fluorometrically using 2',7'-bis(carboxyethyl)carboxyfluorescein and enhanced the production of all the 5-lipoxygenase metabolites. The production decreased when the alkalinization was blocked by the decrease in the pHo, the removal of the extracellular Na+ or the addition of specific inhibitors of the Na+/H+ exchange, such as 5-(NN-hexamethylene)amiloride, 5-(N-methyl-N-isobutyl)amiloride and 5-(N-ethyl-N-isopropyl)amiloride. The alkalinization of the cytoplasm with methylamine completely restored the production suppressed by the removal of Na+ from the medium. These findings suggest that the change in the cytoplasmic pH (pHi) mediated by the Na+/H+ exchange regulates the production of the lipoxygenase metabolites. The site of the metabolism controlled by the pHi change seemed to be the 5-lipoxygenase, because the production of all the metabolites decreased in parallel and the release of [3H]arachidonic acid from the neutrophils in response to the ionophore was not affected by the pHi change. Furthermore, the production of the 5-lipoxygenase metabolites stimulated by A23187 with or without exogenous arachidonic acid showed a similar pHo-dependence and the production induced by N-formylmethionyl-leucylphenylalanine (chemotactic peptide) with exogenous arachidonic acid also decreased when the cytoplasmic alkalinization was inhibited.

Human alveolar macrophage arachidonic acid metabolism

1988 ◽  
Vol 254 (6) ◽  
pp. C809-C815 ◽  
Author(s):  
G. P. Brown ◽  
M. M. Monick ◽  
G. W. Hunninghake
Keyword(s):  
Arachidonic Acid ◽  
Alveolar Macrophages ◽  
Biological Effects ◽  
Leukotriene B4 ◽  
Arachidonic Acid Metabolism ◽  
Ionophore A23187 ◽  
Lipoxygenase Pathway ◽  
Cyclooxygenase Inhibition ◽  
Human Alveolar Macrophage ◽  
Lipoxygenase Products

Metabolites of arachidonic acid are potent modulators of many biological events, and their release from macrophages appears to play an important role in immune and inflammatory processes. In addition, metabolites of the cyclooxygenase or lipoxygenase pathway exhibit distinct biological effects. We used a method to determine if human alveolar macrophages (HAM) could be selectively activated to release products of cyclooxygenase or lipoxygenase pathway of arachidonic acid. HAM obtained by bronchoalveolar lavage from individuals were [3H]arachidonic acid labeled and then stimulated with lipopolysaccharide (LPS) or Ca ionophore A23187. Essentially no arachidonate metabolites were released by unstimulated cells. LPS caused dose- and time-dependent release of arachidonate and only cyclooxygenase products; no lipoxygenase products were detected, even in presence of cyclooxygenase inhibition. Metabolites released in response to LPS included thromboxane B2, prostaglandins D2, F2a, E2, and hydroxyheptadecatrienoic acid. A23187 caused a rapid release of arachidonate and 5-lipoxygenase products, leukotriene B4 and 5-hydroxyeicosatetraenoic acid; no cyclooxygenase inhibition. This demonstrates that HAM are specifically activated to release metabolites derived from cyclooxygenase or lipoxygenase pathway of arachidonic acid. Additionally, shunting down an alternate pathway is not induced by use of inhibitors of either pathway. This suggests alveolar macrophages may enhance or suppress various inflammatory or immune processes in lung, in part, by selective release of various derivatives of arachidonic acid.

scholarly journals Leukotriene generation by eosinophils.

1982 ◽  
Vol 155 (2) ◽  
pp. 390-402 ◽  
Author(s):  
A Jörg ◽  
W R Henderson ◽  
R C Murphy ◽  
S J Klebanoff
Keyword(s):  
Arachidonic Acid ◽  
Calcium Ionophore ◽  
Leukotriene B4 ◽  
Arachidonic Acid Metabolism ◽  
Calcium Ionophore A23187 ◽  
Eicosatetraenoic Acid ◽  
Gas Chromatography Mass Spectrometry ◽  
Ionophore A23187 ◽  
Lipoxygenase Pathway ◽  
Leukotriene D4

Horse eosinophils purified to greater than 98% generated slow reacting substance (SRS) when incubated with the calcium ionophore A23187. On a per cell basis, eosinophils generated four to five times the SRS produced by similarly treated horse neutrophils. Eosinophil SRS production was inhibited by 5,8,11,14-eicosatetraynoic acid and augmented by indomethacin and arachidonic acid, suggesting that it was a product(s) of the lipoxygenase pathway of arachidonic acid metabolism. Compounds with SRS activity were purified by high-pressure liquid chromatography (HPLC) and identified by ultraviolet spectra, spectral shift on treatment with lipoxygenase, incorporation of [14C]arachidonic acid, gas chromatography-mass spectrometry, and comparison of retention times on HPLC to authentic standards. The eosinophil products characterized were 5-(S), 12-(R)-dihydroxy-6-cis-8, 10-trans-14-cis-eicosatetraenoic acid (leukotriene B4) and its 5-(S), 12-(R)-6-trans and 5-(S), 12-(S)-6-trans isomers, 5-(S)-hydroxy-6-(R)-S-glutathionyl-7,9-trans-11, 14-cis-eicosatetraenoic acid (leukotriene C4) and its 11-trans isomer, and 5-(S)-hydroxy-6-(R)-S-cysteinylglycine-7,9-trans-11,14-cis-eicosatetraenoic acid (leukotriene D4).

scholarly journals Leukotriene B4 synthesis and metabolism by neutrophils and granule-free cytoplasts

1986 ◽  
Vol 233 (2) ◽  
pp. 583-588 ◽  
Author(s):  
K A Haines ◽  
K N Giedd ◽  
G Weissmann
Keyword(s):  
Arachidonic Acid ◽  
Plasma Membrane ◽  
Leukotriene B4 ◽  
Ionophore A23187 ◽  
Tetraenoic Acid ◽  
Exogenous Arachidonic Acid ◽  
Neutrophil Degranulation ◽  
Arachidonate Metabolites

Leukotriene B4 [LTB4, (5S,12R)-hydroxyeicosa-6,14-cis-8,10-trans-tetraenoic acid], a potent mediator of inflammation, is released from neutrophils by agonists that provoke degranulation of the cell. To examine whether degranulation is a necessary requirement for synthesis and metabolism of LTB4 (or of other arachidonate metabolites), we prepared neutrophil-derived cytoplasts (neutroplasts), organelle-depleted vesicles of cytoplasm surrounded by the plasma membrane. In the presence of extracellular Ca2+ with or without exogenous arachidonic acid (150 microM), neutroplasts were exposed to the Ca2+ ionophore A23187 (10 microM) and the resultant lipoxygenation products of arachidonate were determined. Neutrophils metabolize arachidonic acid to 5-HETE greater than 15-HETE greater than LTB4 greater than all-trans-LTB4 isomers. Neutroplast products of arachidonate lipoxygenation were 15-HETE greater than 5-HETE greater than LTB4 greater than all-trans-LTB4 isomers. Neutroplasts, like neutrophils, were capable of converting LTB4 into its 20-hydroxy and 20-carboxy metabolites. Finally, neutroplasts could utilize intrinsic arachidonate, since the neutroplasts synthesized LTB4 (30 pmol/mg of protein) in the absence of added arachidonic acid. The data demonstrate that neutrophil degranulation is not required for synthesis or metabolism of LTB4 by neutrophils.

scholarly journals Effect of dietary supplementation with n-9 eicosatrienoic acid on leukotriene B4 synthesis in rats: a novel approach to inhibition of eicosanoid synthesis.

1993 ◽  
Vol 178 (6) ◽  
pp. 2261-2265 ◽  
Author(s):  
M J James ◽  
R A Gibson ◽  
M A Neumann ◽  
L G Cleland
Keyword(s):  
Arachidonic Acid ◽  
Fatty Acid ◽  
Cell Membranes ◽  
Essential Fatty Acids ◽  
Dietary Supplementation ◽  
Eicosatrienoic Acid ◽  
Leukotriene B4 ◽  
Trans Isomers ◽  
Practical Advantage ◽  
Acid Analogue

Studies were undertaken to assess the biochemical effects of dietary supplementation with n-9 eicosatrienoic acid (ETrA), an arachidonic acid analogue that is normally present in cell membranes at very low levels but is raised in the presence of essential fatty acid deficiency (EFAD). The incorporation of dietary ETrA into rat neutrophils and its effect on A23187-stimulated 5-lipoxygenase metabolism in these cells was examined; in addition, the effect of ETrA was compared with that of another arachidonic acid analogue, eicosapentaenoic acid (EPA), which is known to accumulate in cell membranes and inhibit synthesis of leukotriene B4 (LTB4) a product of the 5-lipoxygenase metabolic pathway. Rats were fed a defined diet that was sufficient in essential fatty acids and that contained EPA or ETrA (0.014% of energy) or no added fatty acid, for 3 wk. In the cells from ETrA-fed rats, LTB4 synthesis was inhibited relative to control values, but synthesis of the other products of 5-lipoxygenase metabolism, 5-hydroxyeicosatetraenoic acid (5-HETE) and the all-trans isomers of LTB4, were not inhibited. This pattern indicates inhibition of LTA hydrolase in ETrA-fed rats. In EPA-fed rats, there was inhibition of LTB4 and the all-trans isomers of LTB4, but there was no inhibition of 5-HETE. This pattern indicates inhibition of LTA synthase in EPA-fed rats. The results establish that dietary ETrA effectively inhibits synthesis of the inflammatory mediator, LTB4, and suggest that ETrA may confer antiinflammatory benefits similar to those observed with EFAD or dietary fish oil (which contains EPA). Because ETrA is substantially less unsaturated than EPA, it can be expected to have greater chemical stability, which could be an important practical advantage when used as a dietary constituent or supplement.

Studies of the inhibitory activity of MK-0591 (3-[1-(4-chlorobenzyl)-3-(t-butylthio)-5-(quinolin-2-yl-methoxy)-indol-2-yl]-2,2-dimethyl propanoic acid) on arachidonic acid metabolism in human phagocytes

1992 ◽  
Vol 70 (6) ◽  
pp. 808-813 ◽  
Author(s):  
Luc Ménard ◽  
Michel Laviolette ◽  
Pierre Borgeat
Keyword(s):  
Arachidonic Acid ◽  
Inhibitory Activity ◽  
Platelet Activating Factor ◽  
Specific Inhibitor ◽  
Oxidation Products ◽  
Propanoic Acid ◽  
Ionophore A23187 ◽  
Colony Stimulating Factor ◽  
Trans Isomers ◽  
Stimulating Factor

We have investigated the inhibitory activity of compound MK-0591 (3-[1-(4-chlorobenzyl)-3-(t-butylthio)-5-(quinolin-2-yl-methoxy)-indol-2-yl]-2,2-dimethyl propanoic acid) on 5-lipoxygenase (5-LO) product synthesis in various human phagocytes stimulated with either the ionophore A23187, opsonized zymosan (OPZ), platelet-activating factor (PAF), or forrnyl-methionyl-leucyl-phenylalanine (fMLP). The lipoxygenase products were analyzed by reversed-phase HPLC. MK-0591 inhibited the formation of 5-hydroxyeicosatetraenoic acid, leukotriene (LT) B4, its Ω-oxidation products, and 6-trans-isomers with IC50 values of 2.8–4.8 nM in A23187-stimulated neutrophils. In these conditions, arachidonic acid at a concentration of 10 μM had no effect on MK-0591 inhibitory activity. In neutrophils stimulated with OPZ, the synthesis of LTB4, its Ω-oxidation products, and 6-trans-isomers was inhibited with IC50 values of 9.5–11.0 nM. MK-0591 inhibited 5-LO product synthesis in A23187-stimulated blood monocytes, eosinophils, and alveolar macrophages with IC50 values of 0.3–0.9, 3.7–5.3, and 8.5–17.3 nM, respectively. In neutrophils primed with granulocyte – macrophage colony-stimulating factor and stimulated with PAF, lipoxygenase product synthesis was inhibited with IC50 values of 7.7–8.7 nM. At the concentration of 1 μM, MK-0591 had no inhibitory effect on 15-lipoxygenase activity in human polymorphonuclear leukocytes, nor on human platelet 12-lipoxygenase and cyclooxygenase. In conclusion, MK-0591 is a very potent and specific inhibitor of 5-LO product synthesis in various types of human phagocytes.Key words: 5-lipoxygenase inhibition, colony-stimulating factor, leukotriene, neutrophil, eosinophil, monocyte, macrophage, platelet-activating factor.

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

1985 ◽  
Vol 232 (1) ◽  
pp. 55-59 ◽  
Author(s):  
M H Sullivan ◽  
B A Cooke
Keyword(s):  
Arachidonic Acid ◽  
Leydig Cells ◽  
Acid Metabolism ◽  
Arachidonic Acid Metabolism ◽  
Ionophore A23187 ◽  
Lhrh Agonist ◽  
Lipoxygenase Pathway ◽  
Lipoxygenase Inhibitors ◽  
Testosterone Production

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.

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