Biosynthesis of a linoleic acid allylic epoxide: mechanistic comparison with its chemical synthesis and leukotriene A biosynthesis

Biosynthesis of the leukotriene A (LTA) class of epoxide is a lipoxygenase-catalyzed transformation requiring a fatty acid hydroperoxide substrate containing at least three double bonds. Here, we report on biosynthesis of a dienoic analog of LTA epoxides via a different enzymatic mechanism. Beginning with homolytic cleavage of the hydroperoxide moiety, a catalase/peroxidase-related hemoprotein from Anabaena PCC 7120, which occurs in a fusion protein with a linoleic acid 9R-lipoxygenase, dehydrates 9R-hydroperoxylinoleate to a highly unstable epoxide. Using methods we developed for isolating extremely labile compounds, we prepared and purified the epoxide and characterized its structure as 9R,10R-epoxy-octadeca-11E,13E-dienoate. This epoxide hydrolyzes to stable 9,14-diols that were reported before in linoleate autoxidation (Hamberg, M. 1983. Autoxidation of linoleic acid: Isolation and structure of four dihydroxy octadecadienoic acids. Biochim. Biophys. Acta 752: 353–356) and in incubations with the Anabaena enzyme (Lang, I., C. Göbel, A. Porzel, I. Heilmann, and I. Feussner. 2008. A lipoxygenase with linoleate diol synthase activity from Nostoc sp. PCC 7120. Biochem. J. 410: 347–357). We also prepared an equivalent epoxide from 13S-hydroperoxylinoleate using a “biomimetic” chemical method originally described for LTA₄ synthesis and showed that like LTA₄, the C18.2 epoxide conjugates readily with glutathione, a potential metabolic fate in vivo. We compare and contrast the mechanisms of LTA-type allylic epoxide synthesis by lipoxygenase, catalase/peroxidase, and chemical transformations. These findings provide new insights into the reactions of linoleic acid hydroperoxides and extend the known range of catalytic activities of catalase-related hemoproteins.     

Synthesis of a metabolically stable modified long-chain fatty acid salt and its photolabile derivative

An analogue of the long-chain fatty acid salt, sodium stearate, was synthesized in which the hydrogen atoms at carbons 2, 3, and 18 were replaced by fluorine. The key step in the synthesis was the addition of 3-iodo-2,2,3,3-tetrafluoropropanoic acid amide to 15,15,15-trifluoro-1-pentadecene. Radioactivity was introduced by catalytic reduction of 2,2,3,3,18,18,18-heptafluoro-4-octadecenoic acid amide with carrier-free tritium gas yielding a product with the specific radioactivity of 2.63 TBq/mmol. The resulting 2,2,3,3,18,18,18-heptafluoro-4-octadecenoic acid has a pKa of about 0.5 and is completely dissociated under normal physiological conditions. The fluorinated fatty acid salt analogue is readily taken up into hepatocytes and proved to be metabolically inert. In an approach to the identification of proteins involved in long-chain fatty acid salt transport across membranes and intracellular compartments, the photolabile derivative 11,11-azo-2,2,3,3,18,18,18-heptafluoro[G-3H]octadecanoic acid sodium salt was synthesized with a specific radioactivity of 2.63 TBq/mmol. Photolysis of the photolabile derivative, using a light source with a maximum emission at 350 nm, occurred with a half-life of 1.5 min. The generated carbene reacted with 14C-labeled methanol and acetonitrile with covalent bond formation of 6-13%. Its efficacy for photoaffinity labeling was demonstrated by incorporation into serum albumin, the extracellular fatty acid salt-binding protein, as well as into the intracellular fatty acid salt-binding protein (FABP) of rat liver with the molecular weight of 14,000.     

The leukotriene B4 paradox: neutrophils can, but will not, respond to ligand-receptor interactions by forming leukotriene B4 or its omega-metabolites.

Leukotriene B4 (5S,12R-dihydroxy-6,14-cis,8,10-trans-eicosatetraenoic acid, LTB4) is released from neutrophils exposed to calcium ionophores. To determine whether LTB4 might be produced by ligand-receptor interactions at the plasmalemma, we treated human neutrophils with serum-treated zymosan (STZ), heat-aggregated IgG and fMet-Leu-Phe (fMLP), agonists at the C3b, Fc and fMLP receptors respectively. STZ (10 mg/ml) provoked the formation of barely detectable amounts of LTB4 (0.74 ng/10(7) cells); no omega-oxidized metabolites of LTB4 were found. Adding 10 microM-arachidonate did not significantly increase production of LTB4 or its metabolites. Addition of 50 microM-arachidonate (an amount which activates protein kinase C) before STZ caused a 40-fold increase in the quantity of LTB4 and its omega-oxidation products. Neither phorbol myristate acetate (PMA, 200 ng/ml) nor linoleic acid (50 microM), also activators of protein kinase C, augmented generation of LTB4 by cells stimulated with STZ. Neither fMLP (10(-6) M) nor aggregated IgG (0.3 mg/ml) induced LTB4 formation (less than 0.01 ng/10(7) cells). Moreover, cells exposed to STZ, fMLP, or IgG did not form all-trans-LTB4 or 5-hydroxyeicosatetraenoic acid; their failure to make LTB4 was therefore due to inactivity of neutrophil 5-lipoxygenase. However, adding 50 microM-arachidonate to neutrophil suspensions before fMLP or IgG triggered LTB4 production, the majority of which was metabolized to its omega-oxidized products (fMLP, 20.2 ng/10(7) cells; IgG, 17.1 ng/10(7) cells). The data show that neutrophils exposed to agonists at defined cell-surface receptors produce significant quantities of LTB4 only when treated with non-physiological concentrations of arachidonate.     

Selective inhibition of leukotriene C4 synthesis and natural killer activity by ethacrynic acid

We have investigated the role of arachidonic acid (AA) metabolism in natural killer (NK) cell activity. Human nonadherent (NA) peripheral blood lymphocytes were used as effector cells against 51Cr-labeled K562 target cells. Synthesis of leukotriene C4 (LTC4) is dependent on glutathione S-transferase (GST). We have chosen to study three putative GST inhibitors, namely, ethacrynic acid (ET), caffeic acid (CA), and ferulic acid (FA), with regard to NK activity and with regard to their effect on AA metabolism. The GST inhibitors inhibited NK lysis when added directly to the NK assay. The GST inhibitors inhibited LTC4 synthesis as induced by calcium ionophore A23187 in a dose-dependent manner similar to their inhibition of NK activity. However, only ET was selective, for it had little effect on LTB4, 5-hydroxyeicosatetraenoic acid, and prostaglandin E2 synthesis. LTC4 synthesis was associated with the NK-enriched fractions obtained from discontinuous Percoll gradients. NK-specific anti-Leu-11b antibody and C treatment could abrogate NK activity and LTC4 synthesis. ET was also inhibitory when NA cells were cultured at 37 degrees C for 18 hr. In this case, LTC4 could reverse the inhibitory effect of ET. Our data suggest that LTC4 plays an important role in NK activity.     

Binding of leukotriene C4 to rat lung fibroblasts and stimulation of collagen synthesis in vitro

Arachidonate metabolites are potent biological mediators affecting multiple cellular functions. Although prostaglandins of the E series, which are products of the cyclooxygenase pathway, have been known as inhibitors or down-regulators of fibroblast proliferation and collagen synthesis, the more recently discovered products of the 5-lipoxygenase pathway have not been as extensively investigated with regard to fibroblast function. In this study, a sulfidopeptide product of the lipoxygenase pathway, leukotriene C4 (LTC4), was examined for its ability to modulate rat lung fibroblast collagen synthesis and proliferation in vitro. The data revealed the ability of LTC4 and to a lesser extent leukotriene D4 (LTD4) to stimulate collagen synthesis in a dose-dependent (10(-11)-10(-8) M) manner without affecting cellular proliferation as determined by radiolabeled thymidine incorporation; 1 nM LTC4 caused an 85% (p less than 0.02) increase above untreated controls in [3H]proline incorporation into collagenous protein in the media, which was blocked by the putative leukotriene receptor antagonist FPL55712 (10 microM) and inhibited by cycloheximide and actinomycin D. This LTC4 stimulatory effect was slightly more specific for collagen synthesis vs noncollagenous protein synthesis but was not accompanied with any change in the collagen type composition. Binding of [3H]LTC4 to these cells was specific, reversible, and saturable, with a Kd of 1.8 +/- 0.95 nM. Under equilibrium conditions, there was an estimated 2.39 X 10(4) receptors per cell. This binding was also inhibited by 10 microM FPL55712. Competitive binding studies show specificity of this binding for LTC4 relative to LTD4 and FPL55712. Furthermore, no significant conversion of LTC4 to LTD4 or leukotriene E4 was noted during the binding studies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective inhibition of leukotriene C4 synthesis in human neutrophils by ethacrynic acid

Addition of glutathione S-transferase inhibitors, ethyacrynic acid (ET), caffeic acid (CA), and ferulic acid (FA) to human neutrophils led to inhibition of leukotriene C4 (LTC4) synthesis induced by calcium ionophore A23187. ET is the most specific of these inhibitors for it had little effect on LTB4, PGE2 and 5-HETE synthesis. The inhibition of LTC4 was irreversible and time dependent. ET also had little effect on 3H-AA release from A23187-stimulated neutrophils.     

Differences in arachidonic acid release, metabolism and leukotriene B4 synthesis in human polymorphonuclear leukocytes activated by different stimuli

Products of the 5-lipoxygenase pathway were analyzed after different stimuli in human polymorphonuclear leukocytes prelabeled with 3H-arachidonic acid. Upon stimulation with the Ca2+ ionophore, A23187, polymorphonuclear leukocytes generate 118.2 +/- 18 pg [3H]dihydroxyeicosatetraenoic acids (diHETEs, including 3H-leukotriene B4 and its 6-trans-stereoisomers), after exposure to serum coated zymosan (35.8 +/- 9 pg) and N-fMet-Leu-Phe (39.5 +/- 9 pg). Conversion of 3H-arachidonic acid paralleled its release after A23187 and fMet-Leu-Phe exposure leaving only 13.8 +/- 7% and 13.6 +/- 3% of the released 3H-arachidonic acid unmetabolized, respectively. In contrast, after stimulation with serum-coated zymosan only a small fraction of the released 3H-arachidonate was converted to 5-lipoxygenase products leaving 73.0 +/- 5% of the released 3H-arachidonic acid unmetabolized. In parallel, leukotriene B4 synthesis was studied in unlabeled polymorphonuclear leukocytes, resulting in 40 +/- 15 ng upon A23187 stimulation, 4 +/- 0.9 ng upon stimulation with fMet-Leu-Phe and 1.8 +/- 0.9 ng after serum-coated zymosan, showing a different ratio of leukotriene B4 to 3H-diHETE for A23187 in contrast to serum-coated zymosan and fMet-Leu-Phe. These results indicate that the coupling between the release of the precursor fatty acid and the metabolism via the 5-lipoxygenase pathway differs greatly between different stimuli.     

Binding of leukotriene B4 and its analogs to human polymorphonuclear leukocyte membrane receptors

LTB4-induced proinflammatory responses in PMN including chemotaxis, chemokinesis, aggregation and degranulation are thought to be initiated through the binding of LTB4 to membrane receptors. To explore further the nature of this binding, we have established a receptor binding assay to investigate the structural specificity requirements for agonist binding. Human PMN plasma membrane was enriched by homogenization and discontinuous sucrose density gradient purification. [3H]-LTB4 binding to the purified membrane was dependent on the concentration of membrane protein and the time of incubation. At 20 degrees C, binding of [3H]-LTB4 to the membrane receptor was rapid, required 8 to 10 min to reach a steady-state and remained stable for up to 50 min. Equilibrium saturation binding studies showed that [3H]-LTB4 bound to high affinity (dissociation constant, Kd = 1.5 nM), and low capacity (density, Bmax = 40 pmol/mg protein) receptor sites. Competition binding studies showed that LTB4, LTB4-epimers, 20-OH-LTB4, 2-nor-LTB4, 6-trans-epi-LTB4 and 6-trans-LTB4, in decreasing order of affinity, bound to the [3H]-LTB4 receptors. The mean binding affinities (Ki) of these analogs were 2, 34, 58, 80, 1075 and 1275 nM, respectively. Thus, optimal binding to the receptors requires stereospecific 5(S), 12(R) hydroxyl groups, a cis-double bond at C-6, and a full length eicosanoid backbone. The binding affinity and rank-order potency of these analogs correlated with their intrinsic agonistic activities in inducing PMN chemotaxis. These studies have demonstrated the existence of high affinity, stereoselective and specific receptors for LTB4 in human PMN plasma membrane.     

Stimulation of lipoxin synthesis from leukotriene A4 by endogenously formed 12-hydroperoxyeicosatetraenoic acid in activated human platelets

Human platelets are devoid of 5-lipoxygenase activity but convert exogenous leukotriene A₄ (LTA₄) either by a specific LTC₄ synthase to leukotriene C₄ or via a 12-lipoxygenase mediated reaction to lipoxins. Unstimulated platelets mainly produced LTC₄, whereas only minor amounts of lipoxins were formed. Platelet activation with thrombin, collagen or ionophore A23187 increased the conversion of LTA₄ to lipoxins and decreased the leukotriene production. Maximal effects were observed after incubation with ionophore A23187, which induced synthesis of comparable amounts of lipoxins and cysteinyl leukotrienes (LTC₄, LTD₄ and LTE₄). Chelation of intra- and extracellular calcium with quin-2 and EDTA reversed the ionophore A23187-induced stimulation of lipoxin synthesis from LTA₄ and inhibited the formation of 12-hydroxyeicosatetraenoic acid (12-HETE) from endogenous substrate. However, calcium did not affect the 12-lipoxygenase activity in the 100 000 × g supernatant of sonicated platelet suspensions. Furthermore, the stimulatory effect on lipoxin formation induced by platelet agonists could be mimicked in intact platelets by the addition of low concentrations of arachidonic acid, 12-hydroperoxyeicosatetraenoic acid (12-HPETE) or 13-hydroperoxyoctadecadienoic acid (13-HPODE). The results indicate that the elevated lipoxin synthesis during platelet activation is due to stimulated 12-lipoxygenase activity induced by endogenously formed 12-HPETE.     

Analogs of leukotriene B4: effects of modification of the hydroxyl groups on leukocyte aggregation and binding to leukocyte leukotriene B4 receptors

The syntheses and agonist and binding activities of 5(S)-hydroxy- 6(Z), 8(E), 10(E), 14(Z)-eicosatetraenoic acid (12-deoxy LTB4), 5(S), 12(S)-dihydroxy-6(Z), 8(E), 10(E), 14(Z)-eicosatetraenoic acid (12-epi LTB4), 12(R)-hydroxy-6(Z), 8(E), 10(E), 14(Z)-eicosatetraenoic acid (5-deoxy LTB4), 5(R), 12(S)-dihydroxy-6(Z), 8(E), 10(E), 14(Z)-eicosatetraenoic acid (5-epi LTB4), 6(Z), 8(E), 10(E), 14(Z)-eicosatetraenoic acid (5, 12-deoxy LTB4) are described. These leukotriene B4 analogs were all able to aggregate rat leukocytes and compete with [3H]-leukotriene B4 for binding to rat and human leukocyte leukotriene B4 receptors with varying efficacy. The analog in which the 12-hydroxyl group was removed was severely reduced both in agonist action (aggregation) and binding. The epimeric 12-hydroxyl analog demonstrated better agonist and binding properties than the analog without a hydroxyl at this position. In contrast, in the case of the 5-hydroxyl the epimeric hydroxyl analog had greatly reduced agonist and binding activities while the 5-deoxy analog demonstrated potency only several fold less than leukotriene B4 itself. The dideoxy leukotriene B4 analog was more than a thousand fold less active than leukotriene B4 as an agonist and in binding to the leukotriene B4 receptor. These results show that binding to the leukocyte leukotriene B4 receptor requires a hydroxyl group at the 12 position in either stereochemical orientation but that the presence of a hydroxyl at the 5 position is less important. However, the epimeric C5 leukotriene B4 analog clearly interacts unfavourably with the binding site of the leukotriene B4 receptor.     

Metabolism of leukotriene B4 to dihydro and dihydro-oxo products by porcine leukocytes

Porcine leukocytes contain a novel pathway for the metabolism of leukotriene B4 (LTB4) which results in reduction of the conjugated triene chromophore to a conjugated diene. These cells converted LTB4 to two major metabolites, both of which exhibited maximal absorbance at 230 nm in their UV spectra. These products were purified by high pressure liquid chromatography and identified as 10, 11-dihydro-LTB4 and 10,11-dihydro-12-oxo-LTB4 on the basis of the mass spectra of various derivatives. The position of the double bond of LTB4 which had been reduced was established by cleaving the remaining double bonds of 10, 11-dihydro-LTB4 with ozone followed by oxidation or reduction of the resulting ozonide and analysis of the products by mass spectrometry. Experiments with deuterium-labeled substrate indicated that LTB4 could be directly converted to 10, 11-dihydro-LTB4 without the prior oxidation of either of its hydroxyl groups, as is required for the formation of dihydro metabolites of prostaglandins. Incubation of porcine leukocytes with 10, 11-dihydro-LTB4 and 10, 11-dihydro-12-oxo-LTB4 indicated that these two products can be interconverted and are in equilibrium with one another. The dihydro-oxo metabolite can therefore be formed from 10, 11-dihydro-LTB4, although we have not ruled out the possibility that it is also produced via 12-oxo-LTB4, which could be a transitory intermediate. These results indicate that porcine leukocytes contain a novel reductase/dehydrogenase pathway distinct from the pathway responsible for the metabolism of prostaglandins. This pathway is also different from the pathway in human polymorphonuclear leukocytes which converts 6-trans-isomers of LTB4 to dihydro products, since the latter pathway involves 5-oxo intermediates and results in a shift in the positions of the remaining double bonds.     

Heat shock stimulates the release of arachidonic acid and the synthesis of prostaglandins and leukotriene B4 in mammalian cells

Heat shock has a profound influence on the metabolism and behavior of eukaryotic cells. We have examined the effects of heat shock on the release from cells of arachidonic acid and its bioactive eicosanoid metabolites, the prostaglandins and leukotrienes. Heat shock (42-45 degrees) increased the rate of arachidonic acid release from human, rat, murine, and hamster cells. Arachidonate accumulation appeared to be due, at least partially, to stimulation of a phospholipase A2 activity by heat shock and was accompanied by the accumulation of lysophosphatidyl-inositol and lysophosphatidylcholine in membranes. Induction of arachidonate release by heat did not appear to be mediated by an increase in cell Ca++. Stimulation of arachidonate release by heat shock in hamster fibroblasts was quantitatively similar to the receptor-mediated effects of alpha thrombin and bradykinin. The effects of heat shock and alpha thrombin on arachidonate release were inhibited by glucocorticoids. Increased arachidonate release in heat-shocked cells was accompanied by the accelerated accumulation of cyclooxygenase products prostaglandin E2 and prostaglandin F2 alpha and by 5-lipoxygenase metabolite leukotriene B4. Elevated concentrations of arachidonic acid and metabolites may be involved in the cytotoxic effects of hyperthermia, in homeostatic responses to heat shock, and in vascular and inflammatory reactions to stress.     

Inhibition of leukotriene B4 synthesis in human polymorphonuclear leukocytes after exposure to meningococcal lipopolysaccharide

Polymorphonuclear leukocytes (PMNL) were preincubated in the presence and absence of lipopolysaccharide (LPS) prior to stimulation of arachidonic acid (20:4) metabolism by addition of the divalent cation ionophore, A23187. Analysis of the products by high pressure liquid chromatography showed that LPS inhibited the formation of leukotriene B₄, 5-hydroxy-6,8,11,14- icosatetraenoic acid and 12-hydroxy-5,8,10,14-icosatetraenoic acid by about 70%. In the absence of ionophore, LPS had little effect on the basal synthesis of 20:4 metabolites. Preincubation with LPS also inhibited the formation of the above 3 products in the presence of an excess of exogenous 20:4, suggesting that its action was mediated by the inhibition of lipoxygenases rather than phospholipase.     

Kinetics of leukotriene A4 synthesis by 5-lipoxygenase from rat polymorphonuclear leukocytes

When arachidonic acid is added to lysates of rat polymorphonuclear leukocytes, it is oxidized to (5S)-hydroperoxy-6(E),8(Z),11(Z),14(Z)-eicosatetraenoic acid (5-HPETE). The 5-HPETE then partitions between reduction to the 5-hydroxyeicosanoid and conversion to leukotriene A4 (LTA4). Both steps in the formation of LTA4 are catalyzed by the enzyme 5-lipoxygenase. When [3H]arachidonic acid and unlabeled 5-HPETE were incubated together with 5-lipoxygenase, approximately 20% of the arachidonic acid oxidized at low enzyme concentrations was converted to LTA4 without reduction of the specific radioactivity of the LTA4 by the unlabeled 5-HPETE. A significant fraction of the [3H]-5-HPETE intermediate that is formed from arachidonic acid must therefore be converted directly to LTA4 without dissociation of the intermediate from the enzyme. This result predicts that even in the presence of high levels of peroxidase activity, which will trap any free 5-HPETE by reduction, the minimum efficiency of conversion of 5-HPETE to LTA4 will be approximately 20%, and this prediction was confirmed. 5-HPETE was found to be a competitive substrate relative to arachidonic acid, so that it is likely that the two substrates share a common active site.     

Optimization of assay conditions for leukotriene B4 synthesis by neutrophils or platelets isolated from peripheral blood of monogastric animals

Neutrophils are involved in inflammation through leukotriene (LT) production. The predominant proinflammatory leukotriene released from neutrophils is LTB4, which serves as a biological marker of inflammation. The purpose of this study was to optimize the conditions ex vivo for LTB4 production by neutrophils from horses and dogs, and platelets from chickens. Optimal production of LTB4 was characterized by incubation time (2.5, 5, 10, 15 or 20 min), temperature (25 or 37 degrees C), and calcium ionophore A23187 concentration (0.1, 1, 10 or 20 microM). Incubation longer than 2.5 min did not increase production of LTB4 in chickens or horses; in dogs, incubation for 2.5 and 10 min resulted in the highest concentrations of LTB4 (P相似文献   

Recognition of human polymorphonuclear leukocyte receptors for leukotriene B4 by rabbit anti-idiotypic antibodies to a mouse monoclonal antileukotriene B4

Rabbit anti-idiotypic IgG antibodies to the combining site of a mouse monoclonal IgG2b antibody to leukotriene B4 (LTB4) cross-reacted with human polymorphonuclear (PMN) leukocyte receptors for LTB4. Anti-idiotypic IgG and Fab both inhibited the binding of [3H]LTB4, but not [3H]N-formylmethionyl-leucylphenylalanine (fMLP), to PMN leukocytes with similar concentration-effect relationships, whereas neither nonimmune rabbit IgG nor Fab had any inhibitory activity. At a concentration of anti-idiotypic IgG that inhibited by 50% the binding of [3H] LTB4 to PMN leukocytes, the antibodies preferentially recognized high affinity receptors. Anti-idiotypic IgG and Fab inhibited PMN leukocyte chemotactic responses to LTB4, but not fMLP, with concentration-effect relationships resembling those characteristic of the inhibition of binding of [3H] LTB4, without altering the LTB4-induced release of beta-glucuronidase. Chemotaxis and increases in the cytoplasmic concentration of calcium equal in magnitude to those elicited by optimal concentrations of LTB4 were attained at respective concentrations of anti-idiotypic IgG equal to and 1/25 the level required for inhibition of binding of [3H]LTB4 by approximately 50%. Thus, the anti-idiotypic antibodies bound to PMN leukocyte receptors for LTB4 with a specificity, preference for high affinity sites, and capacity to alter PMN leukocyte functions that were similar to LTB4.     

Inhibition of fructose bisphosphatase and stimulation of phosphofructokinase by a stable isosteric phosphonate analog of fructose 2,6-bisphosphate

We describe the synthesis of a mixture of D-manno- and D-gluco-2,5-anhydro-1-deoxy-1-phosphonohexitol 6-phosphate via a Horner-Emmons reaction of 2,3,5-tri-O-benzyl-beta-D-arabinofuranose followed by phosphorylation of the equivalent 6-position and subsequent deprotection. This mixture inhibits fructose-1,6-bisphosphatase; the concentration required for half-maximal effect in the presence of 25 microM AMP is approximately 6 microM. The mixture of analogs also stimulates 6-phosphofructo-1-kinase from rabbit liver; the concentration required to reach one-half Vmax was found to be ca. 25 microM at 0.25 mM fructose 6-phosphate and 50 microM AMP. These analogs have replaced the labile anomeric phosphate of fructose 2,6-bisphosphate with a stable methylenephosphonate, and could be of great interest due to their appropriate physiological effects and their chemical stability.     

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.     

Immunoglobulin M and immunoglobulin G secretion by human B cells exposed to RU 41.740, a glycoprotein extract from Klebsiella pneumoniae

RU 41.740, a glycoprotein extract from Klebsiella pneumoniae, was seen to activate human B cells to immunoglobulin secretion in vitro. The effects of RU 41.740 on human B cells were compared to those induced by pokeweed mitogen, a T-cell-dependent polyclonal B-cell activator, and Epstein-Barr virus, a T-cell-independent polyclonal B-cell activator. Exposure of human B cells to all of these agents resulted in increased immunoglobulin M (IgM) and immunoglobulin G (IgG) secretion. IgM and IgG secretion induced by RU 41.740 appeared to be T cell dependent when B cells were isolated from human peripheral blood. However, this activity may have been T cell independent when B cells were isolated from human spleen. RU 41.740-induced IgM secretion by peripheral blood B cells was seen to peak after 6 days in culture; IgG secretion peaked after 7 days in culture. The optimal concentration of RU 41.740 for the induction of IgM and IgG secretion by human B cells in vitro was seen to be 200 micrograms/ml.