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E L Becker

Publications and source records attributed to E L Becker.

At least 55 records · Page 3Linked to original sources
PubMed1986

Biochemical events associated with the stimulation of rabbit neutrophils by platelet-activating factor.

The functional and biochemical responses evoked by the addition of platelet-activating factor (PAF) to a suspension of rabbit neutrophils have been characterized in an effort to define the mode of action of this lipid mediator. PAF was found to elicit a secretory response and to stimulate a rapid breakdown of the polyphosphoinositides, an increase in the cytoplasmic level of free calcium (as monitored by quin2), a decrease in the fluorescence of cell-associated chlortetracycline, an enhanced activity of the sodium/hydrogen antiport, a transient depolarization, and an increase in the level of cytoskeletal actin. The quin2 response to PAF was found to be detectable at concentrations as low as 0.01 nM, to be very dependent on the presence of extracellular calcium, and to be sensitive to inhibition by phorbol esters. On the other hand, the increase in free calcium induced by PAF in the presence of extracellular calcium was essentially unaffected by pertussis toxin. PAF-induced neutrophil degranulation was similarly extracellular calcium dependent and phorbol ester sensitive. The secretory activity of PAF was evident only at concentrations in excess of 1 nM. All of the other effects of PAF were found to be independent of the presence of external calcium and to be demonstrable only at concentrations larger than 1 nM. In addition, all neutrophil responses to PAF (with the above noted exception of quin2) were potently inhibited by pertussis toxin. These results are interpreted in terms of the possible existence of two functionally distinct populations of receptors. The occupation of one set (of apparent high affinity) induces an increase in permeability to calcium in a phorbol-ester-, but not pertussis-toxin-, sensitive manner. The activation of the other set of receptors at higher concentrations of PAF stimulates the polyphosphoinositide-specific phospholipase C and induces the attendant biochemical responses. These latter responses appear to be mediated by a guanine-nucleotide-binding regulatory protein.

Actins↗
PubMed1986

Is a rise in intracellular concentration of free calcium necessary or sufficient for stimulated cytoskeletal-associated actin?

The addition of the calcium ionophore A23187 to rabbit neutrophils increases the amount of actin associated with the cytoskeleton regardless of the presence or absence of calcium in the incubation medium. In the presence of extracellular calcium, the effect of A23187 is biphasic with respect to concentration. The action of the ionophore is rapid, transient, and is inhibited by pertussis toxin, hyperosmolarity, and quinacrine. On the other hand, the addition of pertussis toxin or hyperosmolarity has small if any, effect on the rise in intracellular calcium produced by A23187. While quinacrine does not affect the fMet-Leu-Phe-induced increase in cytoskeletal actin and the polyphosphoinositide turnover, its addition inhibits completely the stimulated increase in Ca-influx produced by the same stimulus. The results presented here suggest that a rise in the intracellular concentration of free calcium is neither necessary nor sufficient for the stimulated increase in cytoskeletal-associated actin. A possible relationship between the lipid remodeling stimulated by chemoattractants and the increased cytoskeletal actin is discussed.

Actins↗
PubMed1986

Heterogeneity of binding of chemotactic formyl peptides to their receptors on rabbit neutrophils: methodological and analytical considerations in the study of complex receptor-binding patterns.

The pattern of binding of chemotactic formyl peptides to their receptors on intact rabbit neutrophils was investigated using three different radiolabeled formyl peptides. The purpose of the study was both to establish suitable procedures for the analysis of complex receptor-binding data and to evaluate the heterogeneity of binding of the chemotactic formyl peptides. Radiolabeled formylmethionyl-leucyl-phenylalanine was found to require repurification by thin-layer chromatography before it was of sufficient purity for use in these studies. All three radioligands were essentially stable during the binding assay. The 'LIGAND' computer program proved suitable for a detailed and rigorous analysis of the binding data. Each of the three formyl peptides showed significant heterogeneity in its binding pattern and all appeared to interact with the same receptor sites. The degree of heterogeneity, however, was greater with formylnorleucyl-leucyl-phenylalanine than with the other two formyl peptides. The heterogeneity was close to the theoretical limit of discernibility; thus individual binding studies with one formyl peptide did not always show statistical evidence of the heterogeneity. The study also highlighted the severe uncertainty in estimates of the proportions of each type of binding site when such data are analyzed on the basis of the 'two binding site' model.

Animals↗
PubMed1986

Pertussis toxin as a probe of neutrophil activation.

In reviewing our own and other work, it is clear that pertussis toxin treatment of neutrophils causes a time- and concentration-dependent inhibition of granule enzyme secretion induced by formylmethionylleucylphenylalanine (fMet-Leu-Phe), C5a, leukotriene (LT) B4 and platelet-activating factor (PAF). Chemotaxis, O2- generation, aggregation, and arachidonic acid production induced by fMet-Leu-Phe are also inhibited by pertussis toxin. Granule enzyme release caused by A23187 or phorbol 12-myristate 13-acetate is not inhibited. The inhibition of neutrophil function correlates closely with the NAD-ribosylation of a 41,000-dalton protein in the neutrophil plasma membrane, presumably the GTP-binding regulatory protein Ni. Pertussis toxin treatment prevents or obtunds the increased influx of Ca2+ induced by fMet-Leu-phe and LTB4, but not that caused by stimulation of neutrophils with PAF. Pertussis toxin prevents the receptor-induced breakdown of polyphosphoinositides in intact neutrophils and isolated membrane and prevents or decreases the production of inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol. The hypothesis advanced by us and others is that pertussis toxin interacts with a GTP-binding regulatory protein identical or similar to Ni, which couples receptor-chemotactic factor interaction to phospholipase C activation. Inhibition of the activation prevents the production of IP3 and the resulting release of Ca2+ from intracellular stores and of 1,2-diacylglycerol and thus, the activation of protein kinase C. The lack of these two mediators is the immediate cause of the depression of neutrophil activation resulting from pertussis toxin. Some of the limitations and uncertainties of our present knowledge with respect to this hypothesis are discussed.

Animals↗
PubMed1985

Unique inhibitory profile of platelet activating factor induced calcium mobilization, polyphosphoinositide turnover and granule enzyme secretion in rabbit neutrophils towards pertussis toxin and phorbol ester.

Platelet activating factor has been found to increase the intracellular level of free calcium (as monitored by the fluorescent calcium indicator quin-2) and to stimulate the turnover of the polyphosphoinositides in rabbit neutrophils. Calcium mobilization induced by platelet activating factor, in contrast to previous reports with chemotactic factors, is unaffected by pertussis toxin; on the other hand, stimulated polyphosphoinositol hydrolysis and granule enzyme secretion are potently antagonized under the same conditions. The calcium, as well as the secretory responses to the lipid mediator are largely dependent on the presence of extracellular calcium. Internal contributions to the quin-2 signal are only detectable at relatively high concentrations of platelet activating factor. Calcium mobilization and secretion stimulated by platelet activating factor are inhibited following a short incubation with phorbol 12-myristate 13-acetate. These results are discussed in terms of the possibility that platelet activating factor activates neutrophils via dual pathways, the first involving direct interaction with phorbol ester inhibitable calcium channels and the other the stimulation in a manner dependent on a guanine nucleotide binding protein of the phospholipase C specific for polyphosphoinositides.

Aminoquinolines↗
PubMed1985

A highly active chemotactic peptide analog incorporating the unusual residue 1-aminocyclohexanecarboxylic acid at position 2.

Analogs of chemotactic peptides (Formyl-Met-X-Phe-OMe) containing the stereochemically constrained residues alpha-aminoisobutyric acid (Aib), 1-aminocyclopentanecarboxylic acid (Acc5) and 1-aminocyclohexanecarboxylic acid (Acc6) at position 2 are compared with the parent sequence (X = Leu) for their ability to induce lysozyme release in rabbit neutrophils. The Acc6 analog is about 78 times more active than the parent peptide, For-Met-Leu-Phe-OH, whereas Aib and Acc5 analogs are approximately 3 and 2 times, respectively, less active than the parent peptide. NMR and model building studies clearly favour a Met-Acc6 beta-turn solution conformation in the Acc6 analog, suggesting that the neutrophil receptor is capable of recognizing a folded peptide structure. The significant differences in the activities of the Acc5 and Acc6 analogs suggest an important role for the residue 2 sidechain in receptor interactions.

Amino Acids↗
PubMed1985

Pertussis but not cholera toxin inhibits the stimulated increase in actin association with the cytoskeleton in rabbit neutrophils: role of the "G proteins" in stimulus-response coupling.

Treatment of rabbit neutrophils with pertussis toxin, but not cholera toxin, inhibits the increases produced by formylmethionyl-leucyl-phenylalanine, leukotriene B4 and the calcium ionophore A23187 in the amounts of actin associated with the cytoskeletons. The increase in the cytoskeletal actin produced by phorbol 12-myristate, 13-acetate on the other hand is not affected by pertussis toxin. Incubation of the neutrophils with cholera toxin, unlike pertussis toxin, did not inhibit the fMet-Leu-Phe induced rise in the intracellular concentration of free calcium, and caused only a shift to the right of the dose-response curve of N-acetyl-beta-glucosaminidase release. This shift was more marked in the presence of 1-methyl-3-isobutylxanthine. In addition, the stimulated breakdown of phosphatidylinositol 4,5 bis-phosphate was inhibited by pertussis toxin. These results suggest that pertussis toxin acts at an early step in the signal transduction and does not affect the sequence of reactions initiated by the activation of the protein kinase C. Furthermore, the guanine nucleotide regulatory protein Gi, but not Gs, is closely involved in signal transduction in these cells.

Actins↗
PubMed1985

Pertussis toxin inhibits fMet-Leu-Phe- but not phorbol ester-stimulated changes in rabbit neutrophils: role of G proteins in excitation response coupling.

Addition of pertussis toxin to rabbit neutrophils inhibits the fMet-Leu-Phe- induced increases in Na+ influx and in intracellular pH. In addition, pretreatment of the cells with the toxin inhibits the decrease in the levels of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate and the enhanced production of phosphatidic acid produced by the chemotactic factor fMet-Leu-Phe. Furthermore, the fMet-Leu-Phe-induced changes in the phosphorylation of a 46-kDa protein and of several other proteins are also inhibited by the toxin. On the other hand, the phorbol 12-myristate 13-acetate (PMA)-induced increases in the phosphorylation of several proteins are not inhibited by the toxin. PMA, but not its inactive analogue 4 alpha-phorbol 12,13-didecanoate, was also found to stimulate Na+ influx and to increase the intracellular pH in rabbit neutrophils. These ionic effects, like those produced by fMet-Leu-Phe, are inhibited by amiloride. The stimulated Na+ influx and H+ efflux produced by the phorbol ester, on the other hand, are not inhibited by pertussis toxin. The results reported here suggest that the activity of the Na+/H+ antiport in neutrophils is regulated by protein kinase C; that the G-protein system, either directly or indirectly, is involved in the stimulus-response coupling sequence in these cells; and that the toxin acts at, or prior to, the steps responsible for the activation of phospholipase C, and it does not affect the sequence of reactions initiated by the activation of the protein kinase C.

Animals↗
PubMed1985

The inhibition of neutrophil granule enzyme secretion and chemotaxis by pertussis toxin.

Pertussis toxin treatment of rabbit peritoneal neutrophils causes a concentration-dependent inhibition of granule enzyme secretion induced by formylmethionyl-leucyl-phenylalanine, C5a, and leukotriene B4. It also inhibits chemotaxis induced by formylmethionyl-leucyl-phenylalanine. The same toxin treatment, however, has no effect on granule enzyme secretion induced by the calcium ionophore A23187 or phorbol 12-myristate 13-acetate. Moreover, pertussis toxin treatment does not affect either the number or affinity of the formylpeptide receptors on the neutrophil nor does it have any effect on the unstimulated levels of cyclic AMP (cAMP) or the transient rise in cAMP induced by chemotactic factor stimulation in these cells. We hypothesize that pertussis toxin, as in other cells, interacts with a GTP binding regulatory protein identical with or analogous to either Ni or transducin which mediates the receptor-induced inhibition or activation of a target protein or proteins required in neutrophil activation. The nature of the target protein is unknown, but it is not the catalytic unit of adenylate cyclase. The target protein acts after binding of chemotactic factor to its receptor in the sequence that leads to the receptor-induced rise in intracellular Ca2+. It does not affect the responses elicited by the direct introduction of calcium into the cells or the activity of protein kinase C.

Adenylate Cyclase Toxin↗
PubMed1985

Synthetic homo-oligomethionine chemoattractants. Conformation-activity study.

Using solution methods, we have synthesized several series of L-methionine homo-oligopeptides from the dipeptide to the hexapeptide with either a free alpha-carboxyl function or different C-blocking groups (methoxy, benzyloxy, benzylamino) and with N-blocking groups of either the amide type (formyl, pivaloyl) or the urethane type (tert.-butyloxycarbonyl). Compounds were compared to determine the combined effect of main-chain length and presence and nature of N- and C-blocking groups on conformation-activity relationship. Each peptide was tested for its ability to induce rabbit peritoneal polymorphonuclear leukocytes in the presence of cytochalasin B to secrete granule enzymes. In parallel, a conformational analysis was carried out in the solid state and in solution, using infrared absorption and circular dichroism. The biological and conformational data are discussed in relation to a recently proposed model of the chemotactic peptide receptor of rabbit neutrophils.

Animals↗
PubMed1985

Inhibition of neutrophil phospholipase A2 by p-bromophenylacyl bromide, nordihydroguaiaretic acid, 5,8,11,14-eicosatetraynoic acid and quercetin.

The lipoxygenase and/or cyclooxygenase inhibitors nordihydroguaiaretic acid (NDGA), 4,8,11,14-eicosatetraynoic acid (ETYA) and the bioflavonoid, quercetin, also inhibit phospholipase A2 (phosphatidase 2-acyl hydrolase; EC 3.1.1.4) activity of neutrophil acid extracts and sonicates. The IC50 are 13 microM for NDGA, 22 microM for ETYA, and 100 microM for quercetin when measured on the neutrophil acid extracts; the IC50 obtained with the sonicates are 11 microM, 12 microM and 57 microM, respectively. p-Bromophenylacyl bromide (BPB) inhibits the phospholipase A2 activity of neutrophil acid extracts with an IC50 of 10 microM. In contrast, intact neutrophils incubated for up to 1 h with BPB, washed to remove the drug, and sonicated to expose the phospholipase A2, lose less than 20% of their activity. This strongly suggests that BPB does not inhibit neutrophil function by preventing phospholipase action.

5,8,11,14-Eicosatetraynoic Acid↗
PubMed1985

Biochemical analysis of desensitization of mouse mast cells.

Biochemical mechanisms of desensitization were explored by using peritoneal mouse mast cells saturated with monoclonal mouse IgE anti-DNP antibody. It was found that a 1-min incubation of the sensitized cells with 0.01 micrograms/ml DNP-HSA in the absence of Ca2+ was sufficient to desensitize the cells completely. The treated cells failed to release a detectable amount of histamine upon incubation with an optimal concentration (0.1 to 1.0 micrograms/ml) of DNP-HSA and Ca2+. Determination of the number of antigen molecules bound to mast cells revealed that only a small (less than 10%) fraction of cell-bound IgE antibody molecules reacted with desensitizing antigen, and that desensitized cells and untreated (sensitized) cells could bind comparable amounts of antigen upon incubation with rechallenging antigen. However, the binding of antigen molecules to desensitized cells failed to induce any of the early biochemical events, i.e., phospholipid methylation, cAMP rise, and 45Ca uptake, as well as histamine release. It was also found that intracellular cAMP levels in desensitized cells were comparable to those in sensitized cells. Desensitization by a suboptimal concentration of DNP-HSA was prevented by inhibitors of methyltransferases, such as 3-deaza adenosine plus L-homocysteine thiolactone. Sensitized cells pretreated with 0.01 micrograms/ml DNP-HSA in the absence of Ca2+ and in the presence of the methyltransferase inhibitors responded to an optimal concentration of antigen for histamine release when they were rechallenged in the presence of Ca2+. Inhibition of desensitization by methyltransferase inhibitors was reversed by the addition of S-adenosyl-L-methionine to the system. The results indicated that the activation of methyltransferases, induced by receptor bridging, is involved in the process of desensitization. Desensitization was inhibited by reversible inhibitors of serine proteases, such as p-aminobenzamidine, indole, and synthesized substrates of rat mast cell proteases. It was also found that diisopropylfluorophosphate (DFP), an irreversible inhibitor of serine proteases, completely blocked desensitization at the concentration of 10 to 40 nM. This concentration of DFP did not affect the antigen-induced histamine release, whereas 100- to 1000-fold higher concentrations of DFP did inhibit histamine release. The results suggest that serine proteases are involved in both the induction of histamine release and desensitization, and that the protease involved in desensitization is distinct from that involved in triggering histamine release.

Animals↗
PubMed1984

Pertussis toxin inhibits the rise in the intracellular concentration of free calcium that is induced by chemotactic factors in rabbit neutrophils: possible role of the "G proteins" in calcium mobilization.

The addition of pertussis toxin to rabbit neutrophils inhibits the rise in the intracellular concentration of free calcium induced by the chemotactic factors fMet-Leu-Phe and leukotriene B4. At high concentrations of fMet-Leu-Phe, the inhibitory effect of the toxin is more on the stimulus-induced increase in membrane permeability to calcium than on calcium mobilization from internal stores. These results suggest that the "G protein" system either directly or indirectly is involved in the regulation of the stimulus-induced changes in the calcium mobilization and/or gating systems.

Animals↗
PubMed1984

Effect of phorbol 12-myristate 13-acetate and its analogue 4 alpha-phorbol 12,13-didecanoate on protein phosphorylation and lysosomal enzyme release in rabbit neutrophils.

The co-carcinogenic compound phorbol 12-myristate 13-acetate but not its inactive analogue 4 alpha-phorbol 12,13-didecanoate causes the phosphorylation of several rabbit neutrophil polypeptides whose molecular weights and isoelectric points (pI) are as follows: Mr = 40,000, pI = 6.4; Mr = 50,000, pI = 4.9; Mr = 55,000, pI = 6.3; Mr = 64,000, pI = 6.0; Mr = 70,000, pI = 5.6; Mr = 90,000, pI = 6.0. Most of these phosphorylated proteins are located exclusively in the cytosol; the 64,000 molecular weight protein is found both in the cytosol and the cytoskeleton, and the 40,000 molecular weight protein is found in the nuclear pellet. The 50,000 molecular weight protein is also phosphorylated in whole cells by the chemotactic peptide fMet-Leu-Phe and in cell-free systems by protein kinase C. Using limited proteolysis, one phosphopeptide fragment was phosphorylated by the three stimuli. In addition, phorbol 12-myristate 13-acetate but not 4 alpha-phorbol 12,13-didecanoate causes cell aggregation and the exocytotic release of the specific granules of rabbit neutrophils. In contrast, both compounds increase the amount of actin associated with the cytoskeleton. The divalent cation ionophore A23187 at low concentration and the compound phorbol 12-myristate 13-acetate act synergistically in causing neutrophil degranulation. Lysosomal enzyme release and the phosphorylation of the 50,000 molecular weight polypeptide produced by phorbl 12-myristate 13-acetate are inhibited by trifluoperazine, and these two responses seem to be causally related. These results are discussed in terms of the role of 1,2-diacylglycerol and activation of protein kinase C in specific granule release from rabbit neutrophils.

Animals↗
PubMed1984

Purification and identification of formyl-methionyl-leucyl-phenylalanine as the major peptide neutrophil chemotactic factor produced by Escherichia coli.

Chemotactic factor-enriched butanol extracts from Escherichia coli culture filtrates were fractionated and purified by high pressure liquid chromatography. The yield from individual fractions of biological activity (lysosomal enzyme secretion) and antigenic activity (competition with [3H]fMet-Leu-Phe for binding to rabbit anti-fMet-Leu-Phe) revealed an average 50% recovery of original material. Five peaks of biological activity were separated as demonstrated by enzyme-releasing activity. Three of these peaks coincided exactly with peaks of antigenic activity, suggesting that at least 3 and as many as 5 distinct formyl-methionyl peptides had been separated. The majority of recovered activity appeared in peak 3 and represented 70% of the total biological and antigenic activities recovered. The five peak fractions were subsequently analyzed by dipeptidyl carboxypeptidase gas chromatography-mass spectrometry (DCP/GC-MS) to determine amino acid sequences. After digestion, the formyl-Met peptide was demonstrated in only one of the five peak fractions (peak 3). Furthermore, both the GC retention times and mass spectra indicated that peak 3 contained formyl-methionyl-leucyl-phenylalanine. The DCP/GC and MS data were confirmed with tests made on authentic fMet-Leu-Phe. Butanol extracts from E. coli filtrates to which were added synthetic fMet-Leu-Phe resulted in increased biological and antigenic activity in the precise high pressure liquid chromatography fractions of peak 3 where the fMet-Leu-Phe produced by E. coli was found. Finally, the analysis of recovered biological and antigenic activities indicated that the formyl peptides were found in nanomolar concentrations in culture filtrates. These results demonstrate that the NH2-terminal formyl peptides produced by E. coli, of which formyl-methionyl-leucyl-phenylalanine appears to be the major component, are the peptide mediators responsible for leukocyte chemotactic activity in the bacterial culture extracts.

Animals↗
PubMed1984

Structural requirements for formyl homooligopeptide chemoattractants.

Using solution peptide synthesis, we have made three series of N alpha-formylated homooligopeptides, from the dipeptide to the heptapeptide, derived from L-methionine, L-norleucine, and S-methyl-L-cysteine and related to the chemotactic peptide N alpha-formylmethionylleucylphenylalanine. Compounds were prepared to determine the combined effects of the main-chain length and the presence of a sulfur atom in side-chain gamma- and delta-positions. Each peptide was tested for its ability to induce rabbit peritoneal polymorphonuclear leukocytes in the presence of cytochalasin B to secrete granule enzymes. In parallel, a conformational analysis was carried out in the solid state and in solution, using infrared absorption and circular dichroism. We examined these peptides in solvents of widely different polarities, i.e., chloroform, 2,2,2-trifluoroethanol, 1,1,1,3,3,3-hexafluoropropan-2-ol, and mixed organic-aqueous media. The tendencies to form antiparallel-chain beta-associated and folded structures were determined. The biological and conformational data are described in terms of a model of the chemotactic peptide receptor of rabbit neutrophils recently proposed by Freer et al. (1982) [Freer, R.J., Day, A.R., Muthukumarswamy, N., Pinon, D., Wu, A., Showell, H.J., & Becker, E.L. (1982) Biochemistry 21, 257-263]. In the three N alpha-formylated C-methoxy homooligopeptide series tested, the highest level of activity attained is at the tetrapeptide or pentapeptide stage, confirming the suggestion that the formylpeptide receptor is large enough to accommodate a peptide with at least four amino acid residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Chemotactic factors induced vimentin phosphorylation in rabbit peritoneal neutrophil.

Rabbit peritoneal neutrophils were reacted for 5-10 s with the chemotactic factors, fMet-Leu-Phe or (5S),(12R)-dihydroxy-6,8,11,14-(cis,trans,trans,cis)-eicosatetraenoic acid and then lysed with a solution of Triton X-100 and [gamma-32P]ATP. They showed an enhanced incorporation of 32P in Mr = 60,000- and 67,000-dalton polypeptides compared to control cells treated similarly. Another chemotactic factor, C5a, produced a similar but much lesser effect. The enhancement was not affected by the addition of the purified catalytic subunit of cAMP-dependent protein kinase, the inhibitor of cAMP-dependent protein kinase, or CaCl2, suggesting that the effect was not mediated by a cAMP-dependent or a Ca2+-dependent protein kinase. When analyzed by two-dimensional gel electrophoresis, the Mr = 60,000 phosphoprotein contained several phosphoproteins with different isoelectric points. The isoelectric point and molecular weight of one of them was similar to those of the intermediate filament protein, vimentin, purified from Chinese hamster ovary cells. Addition of the purified Chinese hamster ovary vimentin and [gamma-32P]ATP to the Triton X-100 lysate of fMet-Leu-Phe-treated neutrophils resulted as an enhanced incorporation of 32P into the vimentin. Addition of fMet-Leu-Phe to 32P-labeled intact neutrophils also enhanced incorporation of 32P into the vimentin of neutrophils. The results suggest that chemotactic factors stimulate vimentin phosphorylation in rabbit neutrophils.

Animals↗