Biosynthesis of paf-acether. X. Phorbol myristate acetate-induced paf-acether biosynthesis and acetyltransferase activation in human neutrophils

We tested the hypothesis that protein kinase C might play a role in the biosynthesis of platelet-activating factor (paf-acether) in human neutrophils. PMA but not its inactive analog 4-alpha-phorbol-12,13-didecanoate induced lyso paf-acether production, followed by acetyltransferase activation, leading to paf-acether synthesis and release. Moreover, PMA was twice as powerful compared to opsonized zymosan (OPZ). 1-Oleoyl-2-acetyl-glycerol also induced acetyltransferase activation and paf and lyso paf production. The paf-acether formed by PMA or OPZ stimulation was composed of alkyl chains C16:0 (84.3 +/- 5% and 80.7 +/- 3.5%, respectively, and C18:0 (15.7 +/- 5% and 19.3 +/- 3.5%, respectively, means +/- SEM) as assessed by gas chromatography-electron capture detection. The inhibitor of protein kinase C, D-sphingosine, markedly decreased paf and lyso paf production and acetyltransferase activation in PMA- as well as OPZ-stimulated neutrophils. These results strongly suggest the involvement of protein kinase C in signal transduction during cell stimulation, leading to the paf biosynthesis.     

Biosynthesis of paf-acether. Activators of protein kinase C stimulate cultured mast cell acetyltransferase without stimulating paf-acether synthesis.

A property common to many growth factors is that they must be present for several hours before the commitment to DNA synthesis and cell division occurs. The intracellular signals that are relevant during this period are poorly defined. We examined the formation of 1,2-diacylglycerol in IIC9 fibroblasts after stimulation with epidermal growth factor (EGF), and found that the mass of this lipid remained elevated for at least four hours. The concentration-dependence of EGF-stimulated 1,2-diacylglycerol production and [3H]thymidine incorporation were similar. Studies of phospholipid metabolism strongly suggested that phosphatidylcholine was the source of the 1,2-diacylglycerol generated in response to EGF. EGF did not stimulate the hydrolysis of other phospholipids, including the phosphoinositides, nor did it increase synthesis de novo of 1,2-diacylglycerol. This pattern of sustained 1,2-diacylglycerol formation from phosphatidylcholine may be important in the mitogenic signalling of EGF and potentially other growth factors.     

Biosynthesis of paf-acether. XI. Regulation of acetyltransferase by enzyme-substrate imbalance

Acetyl-CoA:1-O-alkyl-sn-glycero-3-phosphocholine acetyltransferase is the key enzyme in paf-acether (paf) biosynthesis, since it yields the active mediator from its nonacetylated precursor, lyso-paf. In microsomal fractions obtained from the ionophore A23187-stimulated human polymorphonuclear neutrophils, the optimal conditions allowing the full acetylation of lyso-paf were: 2-2.5 mg.ml-1 bovine serum albumin, 40 microM lyso-paf, 200 microM acetyl-CoA and acetyltransferase of high specific activity, at least 18 nmol.min-1.mg protein- -1. The reaction frequently stopped before the substrate was consumed due to spontaneous decay of the enzyme activity at 37 degrees C and inhibition of the enzyme by the paf formed in the reaction. However, low concentrations of acetyltransferase substrates (lyso-paf or lysophosphatidylcholine) and the antioxidant dithiothreitol, but not the inhibitors of proteinases or phosphatases, protected the enzyme against decay. In contrast, high concentrations of those lyso substrates inhibited the enzyme activity in the assay. This inhibition as well as that due to paf was overcome by raising the concentration of the enzyme contained in the microsomal fraction or the bovine serum albumin in the assay. These results suggest that the biosynthesis of paf in cell-free assay and most probably in intact cells might be controlled to a larger extent by the acetyltransferase concentration rather than by that of its substrates.     

Biosynthesis of paf-acether in cultured-mouse mast cells: the role of calcium and G proteins.

We examined the potential role of a guanine nucleotide-binding protein in the biosynthesis of paf-acether (paf) and the release of beta-hexosaminidase during antigenic stimulation of cultured mouse bone marrow-derived mast cells. Unlike pertussis toxin, cholera toxin treatment enhanced the antigen-stimulated production of paf and calcium mobilisation without affecting acetyltransferase activation and cell degranulation. The level of intracellular cAMP doubled in cholera toxin-treated cells. Our data suggest that a cholera toxin-sensitive guanine nucleotide-binding protein is involved in the IgE receptor-mediated signal transduction leading to paf production most probably at the level of Ca2+ influx.     

Heterogeneity of arachidonate and paf-acether precursor pools in mast cells.

In mammalian cells, arachidonate release and paf-acether formation are frequently associated. The alkyl-acyl-GPC has been proposed as an important source for released arachidonic acid and arachidonate-containing alkylacyl-GPC species as unique precursor for paf-acether. However, the specificity of precursor pools either concerning arachidonic acid or paf-acether is still a matter of controversy. We studied the relationship between the precursor pools for both autacoids in antigenically-stimulated cultured mast cells. We took advantage of the particular arachidonate turnover rate in each phospholipid to investigate the role of alkyl-arachidonyl-GPC in the supply of arachidonic acid by using newly and previously [14C]arachidonate-labeled cells. The specific activity of the released arachidonate was reduced 2-fold following overnight cell incubation, whereas labeling in alkyl-arachidonoyl-GPC was only slightly modified and never corresponded to that of released arachidonate when newly or previously labeled cells were triggered with the antigen. These results are not in favor of a major role for alkyl-arachidonoyl-GPC in supplying arachidonate. In contrast, by using previously labeled cells, we demonstrated that all arachidonate-containing phospholipids were involved in the release of arachidonic acid. The pattern of alkyl chains in alkyl-arachidonoyl-GPC, as well as in total alkylacyl-GPC, is unique since it consists mainly of 18:1 (more than 55%), whereas the 16:0 represents only about 30% of total alkyl chains. Therefore, we analyzed paf-acether molecular composition in order to compare it to the alkyl composition of the precursor pools. The content in 18:1 species of paf-acether, as measured by bioassay (aggregation of rabbit platelets), was always lower than that of 16:0 species and then did not correspond to the alkyl composition of the precursor. These data suggest that the enzymes involved in paf synthesis might be specific for 16:0 alkyl chains of precursor pool.     

Signalling mechanism in the lysophosphatidylserine-induced activation of mouse mast cells.

Lysophosphatidylserine (0.1-1 microM) elicits histamine release in isolated mouse peritoneal mast cells. The effect becomes manifest after a lag of 30 s and reaches completion in 5 min. Maximal activity is observed when serine is in L-configuration. As shown by the activity of a lysophosphatidylserine analogue lacking the OH group in C2 position of glycerol, conversion into phosphatidylserine is not required. When 32PO4-labeled mast cells are challenged 2-5 min with lysophosphatidylserine, the labeling of phosphatidate, phosphatidylinositol and phosphatidylcholine is increased. When [3H]arachidonate-labeled mast cells are used, lysophosphatidylserine increases the appearance of isotopic diacylglycerol and phosphatidate. Like the secretory response, these effects are independent of the presence of extracellular Ca2+. Incubations in the presence of [3H]glycerol show that lysophosphatidylserine does not activate the de novo synthesis of phospholipids. In agreement with a participation of phosphoinositidase C in the action of lysophosphatidylserine, we observe accumulation of inositol phosphates in [3H]inositol labeled mast cells incubated in the presence of Li+. The results suggest that lysophosphatidylserine delivers its stimulus to mast cells, by the activation of phosphoinositide-dependent signalling mechanism.     

Insulin-regulated aminopeptidase marks an antigen-stimulated recycling compartment in mast cells

Insulin-regulated aminopeptidase (IRAP) is a marker for insulin-sensitive recycling compartments of fat and muscle cells that contain the glucose transporter isoform GLUT4. Unlike GLUT4, IRAP is expressed in many other cell types. Thus, it is a potential marker for regulated recycling compartments that are analogous to GLUT4 vesicles. In bone marrow-derived mast cells, IRAP is highly expressed and localizes to an intracellular compartment different from secretory granules. Using cell-surface biotinylation, we determined that IRAP underwent rapid redistribution to the plasma membrane on antigen/immunoglobulin E (IgE) stimulation and was re-internalized within 30 min. When granule exocytosis was inhibited, by removing extracellular calcium, adding the protein kinase C inhibitor bisindolylmaleimide or the phosphatidylinositol 3-kinase inhibitor wortmannin, IRAP redistribution was still detected in stimulated cells. However, the redistribution of IRAP required intracellular calcium. By immunofluorescence, IRAP significantly co-localized with the transferrin receptor (TfR), a marker for constitutively recycling endosomes. However, antigen/IgE stimulation did not increase TfR on the cell surface, indicating that IRAP and TfR may follow different pathways to the plasma membrane. In rat peritoneal mast cells, the distributions of IRAP and TfR overlapped to only a limited extent, indicating that overlap may decrease with cell differentiation. We propose that IRAP vesicles represent a second IgE-sensitive exocytotic compartment in mast cells, which is regulated differently from secretory granules, and that these vesicles may be similar to GLUT4 vesicles.     

Interaction of PLD1b with actin in antigen-stimulated mast cells

Phosphatidic acid, the product of phospholipase D catalysed phosphatidylcholine hydrolysis is an important signalling molecule that has been implicated in regulation of actin cytoskeleton remodelling and secretion from mast cells. We show that human PLD1b (hPLD1b) is an actin-binding protein and the N-terminus is predominantly involved in this interaction. Protein kinase C (PKC) is a major upstream regulator of PLD activity and PKC phosphorylation sites have been identified within the N-terminus of PLD1b at serine 2 and threonine 147. Over-expression of wild type hPLD1b in mast cells showed that antigen stimulation significantly enhanced co-localisation of PLD1b with actin structures. Mutation of serine 2 to alanine abolished antigen-induced co-localisation whereas mutation of threonine 147 had less dramatic effects on co-localisation. The absence of co-localisation of PLD1b (S2A) with actin coincides with a significant decrease in PLD activity in cells expressing the PLD1b (S2A) mutant. In resting RBL-2H3 cells, mutation of serine 2 to aspartate resulted in constitutive co-localisation of PLD with the actin cytoskeleton, coincident with restored PLD activity. These results reveal that serine 2 is an important regulatory site involved in controlling PLD enzyme activity and the interaction between PLD and actin.     

Biosynthesis of paf-acether. XVI. Acetyltransferase specificity determines composition of paf-acether molecular species in human polymorphonuclear neutrophils.

In human neutrophils, the velocity of the lyso paf-acether:acetyl-CoA acetyltransferase reaction was almost 2-fold higher in the presence of lyso paf-acether bearing a 16:0 alkyl chain at the sn-1 position of glycerol than in that of its 18:0 analog. The paf-acether produced from an equimolar mixture of the two substrates was a 5:1 mixture, respectively, of the 16:0 and 18:0 species. The ratio of 16:0/18:0 lyso paf-acether in microsomal fractions, as analyzed by gas chromatography, was close to 1, whereas the paf-acether formed in these fractions from endogenous phospholipids was nearly exclusively of the 16:0 form. We conclude that acetyltransferase possesses a higher affinity for 16:0 than for 18:0 lyso-PAF and thus might control the molecular composition of paf-acether synthesized by stimulated human polymorphonuclear neutrophils.     

Elevated levels of cyclooxygenase-2 in antigen-stimulated mast cells is associated with minimal activation of p38 mitogen-activated protein kinase

We have investigated possible factors that underlie changes in the production of eicosanoids after prolonged exposure of mast cells to Ag. Ag stimulation of cultured RBL-2H3 mast cells resulted in increased expression of cyclooxygenase (COX-2) protein and message. Other eicosanoid-related enzymes, namely COX-1, 5-lipoxygenase, and cytosolic phospholipase A(2) were not induced. Activation of extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 mitogen-activated protein (MAP) kinase preceded the induction of COX-2, whereas phosphatidylinositol 3' kinase and its substrate, Akt, were constitutively activated in RBL-2H3 cells. Studies with pharmacologic inhibitors indicated that of these kinases, only p38 MAP kinase regulated expression of COX-2. The induction of COX-2 was blocked by the p38 MAP kinase inhibitor SB202190, even when added 12-16 h after stimulation with Ag when p38 MAP kinase activity had returned to near basal, but still minimally elevated, levels. Interestingly, expression of COX-2 as well as cytosolic phospholipase A(2) and 5-lipoxygenase were markedly reduced by SB202190 in unstimulated cells. Collectively, the results imply that p38 MAP kinase regulates expression of eicosanoid-related enzymes, passively or actively, at very low levels of activity in RBL-2H3 cells. Also, comparison with published data suggest that different MAP kinases regulate induction of COX-2 in inflammatory cells of different and even similar phenotype and suggest caution in extrapolating results from one type of cell to another.     

Biosynthesis of paf-acether. XVII. Regulation by the CoA-independent transacylase in human neutrophils

Treatment of intact human polymorphonuclear neutrophils (PMN) with low concentrations of phorbol myristate acetate (PMA, 1-10 ng/ml) induced paf-acether (paf) and lyso paf formation, arachidonate release, and simultaneous inhibition of CoA-independent lyso paf: transacylase as assayed in a cell-free system. Inhibition of [3H]lyso paf reacylation was also observed when it was exogenously added to the PMA-treated intact PMN. When higher concentrations of PMA (40-100 ng/ml) were used, paf biosynthesis was severely impaired and the level of the CoA-independent transacylase activity returned to basal level. Since lyso paf appears to be the substrate for PMA-activated paf formation (remodeling pathway), we showed that [14C]acetate was incorporated into the paf molecule. By contrast, labeling with [3H]choline was not appropriate in this model. The presented results are against the involvement of a de novo route in paf synthesis initiated by PMA and open a new possibility of an important role for the CoA-independent transacylase in controlling the level of lyso paf availability for paf formation.     

Biosynthesis of heparin. Effects of n-butyrate on cultured mast cells

Murine mastocytoma cells were incubated in vitro with inorganic [35S]sulfate, in the absence or presence of 2.5 mM n-butyrate, and labeled heparin was isolated. The polysaccharide produced in the presence of butyrate showed a lower charge density on anion exchange chromatography than did the control material and a 3-fold increased proportion (54 versus 17% for the control) of components with high affinity for antithrombin. Structural analysis of heparin labeled with [3H] glucosamine in the presence of butyrate showed that approximately 35% of the glucosamine units were N-acetylated, as compared to approximately 10% in the control material; the nonacetylated glucosamine residues were N-sulfated. The presence of butyrate thus leads to an inhibition of the N-deacetylation/N-sulfation process in heparin biosynthesis, along with an augmented formation of molecules with high affinity for antithrombin. Preincubation of the mastocytoma cells with butyrate was required for manifestation of either effect; when the preincubation period was reduced from 24 to 10 h the effects of butyrate were no longer observed. Assays for microsomal N-acetylheparosan deacetylase activity failed to show any significant inhibition of the enzyme at butyrate concentrations well above those found to affect heparin biosynthesis in intact mastocytoma cells. Moreover, a polysaccharide formed on incubating mastocytoma microsomal fraction with UDP-[3H]glucuronic acid, UDP-N-acetylglucosamine, and 3'-phosphoadenylylsulfate in the presence of 5 mM butyrate showed the same N-acetyl/N-sulfate ratio as did the corresponding control polysaccharide, produced in the absence of butyrate. These findings suggest that the effect of butyrate on heparin biosynthesis depends on the integrity of the cell.     

Mechanisms of mouse mast cell activation and inactivation for IgE-mediated histamine release.

The IgE-mediated histamine release from mouse mast cells requires Ca++, is optimal at 37 degrees C, and is enhanced by phosphatidylserine. The rate of release is relatively slow. The mast cells can be activated to release histamine by either anti-IgE or anti-Fab antibodies and, in the case of cells from sensitized mice, by the immunizing antigen. The incubation of mast cells with antigen in the absence of Ca++ or phosphatidylserine fails to release histamine. Such cells are desensitized to the further addition under optimal conditions of the same antigen. Desensitization is antigen specific, requires optimal levels of antigen, and occurs at both 30 degrees and 37 degrees C. In contrast, anti-IgE desensitizes all IgE-mediated histamine release reactions.     

Decrease in IgE Fc receptor expression on mouse bone marrow-derived mast cells and inhibition of paf-acether formation and of beta-hexosaminidase release by dexamethasone

The effect of dexamethasone (DM) on the immunologic and nonimmunologic release of paf-acether and of the granule marker beta-hexosaminidase (BHEX) from mouse bone marrow-derived mast cells (BMMC) was studied. BMMC (1 X 10(6] in a modified Tyrode's solution containing 0.25% bovine serum albumin (BSA) were sensitized with an optimal dose of dinitrophenyl (DNP)-specific monoclonal IgE, and were washed before challenge with 40 ng/ml of DNP coupled to BSA. Preincubation of BMMC for 24 hr with 1 nM to 1 microM DM inhibited in a dose-dependent fashion the immunologic release of paf-acether and of BHEX as compared with control cells, with a half-maximal effect at 20 nM and 4 nM respectively. By contrast, the ionophore A23187 (1 microM)-induced release of paf-acether and of BHEX was unaffected by DM pretreatment. Finally, the antigen-induced increase in acetyltransferase activity, used as an index of cellular activation, was inhibited by 37 +/- 16% in 1 microM DM-treated BMMC as compared with untreated cells. Preincubation of BMMC with DM for 24 hr caused a dose-dependent inhibition of 125I-IgE binding to the cells, with a half-maximal effect at 14 nM. As determined by Scatchard analysis, the number of IgE Fc receptors was decreased by 55% in 1 microM DM-treated BMMC as compared with untreated cells, although the dissociation constants were comparable (control: 12.6 +/- 4.1 nM; DM-treated cells: 14.1 +/- 6.7 nM; mean +/- 1 SD; n = 3). Cytofluorometer analysis of BMMC sensitized with a saturating amount of purified monoclonal IgE, followed by addition of a fluoresceinated anti-mouse IgG (heavy and light chains), revealed a single cellular population for both DM-treated and untreated BMMC. This demonstrates that the DM-induced decrease in IgE Fc receptor expression was exhibited by every BMMC. The possible link between the decreased sensitization of the cells consequent to the reduction in IgE Fc receptor expression and the alteration of the secretory response and acetyltransferase activity was investigated. BMMC were incubated with IgE under experimental conditions giving half-sensitization of the cells. Upon antigen challenge, a 10.5 +/- 3.7% decrease in acetyltransferase activity and a 29.2 +/- 3.5% decrease in paf-acether release were observed with half-sensitized cells as compared with cells sensitized with a saturating amount of IgE.(ABSTRACT TRUNCATED AT 400 WORDS)     

Biosynthesis of paf-acether factor-acether by human skin fibroblasts in vitro

The synthesis and release of paf-acether by fibroblasts from normal human skin was investigated in vitro. When fibroblasts in suspension (1 X 10(6) cells) were stimulated with 2 microM Ca1+ ionophore A23187 (Io), they synthesized a material that aggregated aspirin-treated washed rabbit platelets and was identified as paf because 1) the platelet aggregation it induced was inhibited by BN 52021, an antagonist of paf putative receptors; 2) the factor was inactivated by phospholipase A2 but was insensitive to lipase from Rhizopus arrhizus; 3) it exhibited the same retention time as synthetic paf during standard and reverse phase HPLC elution. Paf production by fibroblasts occurred as soon as the first min of Io stimulation (287 +/- 92 pg/1 X 10(6) cells), reached a maximum at 5 min (369 +/- 85 pg/1 X 10(6) cells) and decreased thereafter. Half of the fibroblast-produced paf was recovered in supernatants. Addition of exogenous 1-O-alkyl-sn-glycero-3-phosphocholine (lyso-paf) at 0.1 microM and/or acetyl-coenzyme A at 0.1 mM to fibroblasts during Io stimulation enhanced paf production by two- and three-fold, respectively. The paf precursors, i.e., 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (1-alkyl-2-acyl-GPC) and lyso-paf, were detected in fibroblasts either stimulated with Io or not. These precursors exhibited 80% hexadecyl and 20% octadecyl chains at the sn-1 position of the molecules, as determined by reverse phase HPLC and gas chromatography analysis. The present results are the first to demonstrate the synthesis and release of paf by fibroblasts from normal human skin. Such production within the dermis might account for the development of cutaneous inflammation and for the pathogenesis of many skin disorders.     

Biosynthesis of adrenocorticotropic hormone in mouse pituitary tumor cells.

A double antibody immunoprecipitation technique using affinity-purified adrenocorticotropic hormone (ACTH) antiserum was employed to investigate the biosynthesis of ACTH in a mouse pituitary tumor cell line. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of cell extracts resolved four forms of ACTH with apparent molecular weights of 4,500, 13,000, 23,000, and 31,000. These four forms of ACTH can be detected by radioimmunoassay of cell extracts or by immunoprecipitation of cell extracts following incubation of cultures in [3H] tryptophan, [3H] lysine, or [3H] tyrosine. The double antibody immunoprecipitation scheme developed is specific, quantitative, and reproducible. ACTH biosynthesis was examined in both steady and pulse-labeling experiments using [8H] tyrosine or [3H] lysine. The results of these experiments are consistent with the proposal that Mr=31,000 ACTH is the biosynthetic precursor for all three smaller forms of ACTH and that Mr=23,000 ACTH is a biosynthetic intermediate. Both Mr=13,000 ACTH and Mr=4,500 ACTH are derived from the intracellular processing of Mr=31,000 ACTH.     

Metabolism of macromolecular heparin in mouse neoplastic mast cells.

1. Polysaccharide in a heparin-producing mouse mastocytoma was pulse-labelled in vivo with [35S] sulphate, and after various periods of time was isolated from subcellular fractions. Such fractions were recovered from tissue homogenates by consecutive centrifugations at 1000g for 10min, 20000g for 20min and 100000g for 1h. Initially the 35S-labelled polysaccharide formed occurred principally in the second centrifugal fraction (20000g precipitate), with small amounts in the first (granular) and third (microsomal) fractions. Analysis for glycosyltransferase activity confirmed that glycosaminoglycans were formed chiefly in particles sedimenting at 20000g. Molecules of this newly synthesized polysaccharide were considerably larger than those of commercially available heparin, as judged from gel chromatography. 2. Within the first hour after injection of [35S]sulphate, most of the labelled polysaccharide was redistributed from the second to the first centrifugal fraction. During, and possibly also after, this shift, the macromolecular polysaccharide was degraded, ultimately to the size of commercial heparin. The degradation process appeared complete 6h after injection of [35S]sulphate. 3. Particulate subcellular fractions were incubated with macromolecular [35S]heparin and the products were analysed by gel chromatography. Significant degradation of the substrate occurred only with the second centrifugal fraction. Further characterization of this fraction, by density-gradient centrifugation in iso-osmotic colloidal silica, revealed a single visible band of particles, at approximately the same density at lysosomes. This band contained all the beta-glucuronidase, 35S-labelled endogenous polysacchride and heparin-degrading enzyme present in the second fraction.