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R W Bonser

Publications and source records attributed to R W Bonser.

16 recordsLinked to original sources
PubMed1992

Tyrosine phosphorylation is involved in receptor coupling to phospholipase D but not phospholipase C in the human neutrophil.

The tyrosine kinase inhibitors ST271, ST638 and erbstatin inhibited phospholipase D (PLD) activity in human neutrophils stimulated by fMet-Leu-Phe, platelet-activating factor and leukotriene B4. These compounds did not inhibit phorbol ester-stimulated PLD, indicating that they do not inhibit PLD per se, but probably act at a site between the receptor and the phospholipase. In contrast, the protein kinase C inhibitor Ro-31-8220 inhibited phorbol 12,13-dibutyrate- but not fMet-Leu-Phe-stimulated PLD activity, arguing against the involvement of protein kinase C in the receptor-mediated activation of PLD. ST271 did not inhibit Ins(1,4,5)P3 generation, but did inhibit protein tyrosine phosphorylation stimulated by fMet-Leu-Phe. The phosphotyrosine phosphatase inhibitor pervanadate increased tyrosine phosphorylation and stimulated PLD. These results suggest that tyrosine kinase activity is involved in receptor coupling to PLD but not to PtdIns(4,5)P2-specific phospholipase C in the human neutrophil.

Enzyme Activation↗
PubMed1991

A quantitative investigation into the dependence of Ca2+ mobilisation on changes in inositol 1,4,5-trisphosphate levels in the stimulated neutrophil.

1. The coupling of N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) receptor stimulation to Ca2+ mobilisation has been investigated in the human neutrophil by measuring the concentration-effect curves for inositol 1,4,5-trisphosphate (IP3) formation and Ca2+ mobilisation. 2. fMet-Leu-Phe-dependent mobilisation of intracellular Ca2+ has been monitored in fluo-3-loaded human neutrophils by measuring increases in the cytoplasmic free Ca2+ concentration ([Ca2+]i) in the presence of extracellular EGTA. Fluo-3 was used in preference to fura-2 because it was found to be more sensitive to the high Ca2+ levels seen in stimulated neutrophils. 3. fMet-Leu-Phe induced a rapid mobilisation of intracellular Ca2+ (EC50 = 2.9 +/- 0.1 nM) and increased [Ca2+]i to a maximum of 1286 +/- 184 nM. 4. The amount of IP3 in fMet-Leu-Phe-stimulated neutrophils was determined by competition with [3H]-IP3 for a specific IP3 binding protein isolated from bovine adrenocortical microsomes. Basal IP3 levels of 13.3 +/- 2.0 pmol per 10(7) cells were increased nearly 4 fold by maximally effective concentrations of fMet-Leu-Phe. 5. The EC50 for the IP3 response (95 +/- 18 nM) was much higher than that for mobilisation of intracellular Ca2+, such that only a doubling in the concentration of IP3 was required to fully mobilise intracellular Ca2+. 6. As a result of this relationship IP3 production was more sensitive than Ca2+ mobilisation to inhibition by demethoxyviridin, an inhibitor of phospholipase activation.

Aniline Compounds↗
PubMed1991

Demethoxyviridin and wortmannin block phospholipase C and D activation in the human neutrophil.

1. The fungal metabolite, wortmannin, has recently been shown to inhibit fMet-Leu-Phe-stimulated superoxide production and phospholipase D (PLD) activation in the human neutrophil. 2. We have found that a close structural analogue of wortmannin, demethoxyviridin, has a similar inhibitory profile but in addition blocks phosphatidylinositol 4,5-bisphosphate-specific phospholipase C and hence inositol 1,4,5-trisphosphate (IP3) formation. 3. Inhibition of fMet-Leu-Phe-stimulated PLD by demethoxyviridin was characteristically non-competitive (IC50 = 31 +/- 10 nM). 4. Inhibition of fMet-Leu-Phe-stimulation IP3 formation required concentrations almost 10 times higher (IC50 = 250 +/- 130 nM). 5. Surprisingly, demethoxyviridin only inhibited fMet-Leu-Phe-induced intracellular calcium mobilization at concentrations 100 times greater than those needed to block IP3 formation. 6. Demethoxyviridin also inhibited PLD activation induced by sodium fluoride or phorbol myristate acetate (PMA) but the concentrations required were 100 times those needed to block fMet-Leu-Phe-stimulated PLD. 7. These observations support the contention that PLD plays an important role in signal transduction in the human neutrophil and indicate that wortmannin and demethoxyviridin inhibit PLD activation at a common step in the signalling pathway. 8. Furthermore, these results suggest that demethoxyviridin may block the interaction between the chemotactic peptide receptor and a GTP-binding protein that is intimately involved in PLD activation.

Androstadienes↗
PubMed1990

The temporal relationship between phospholipase activation, diradylglycerol formation and superoxide production in the human neutrophil.

Fluctuations in the amounts of choline, inositol 1,4,5-trisphosphate (IP3) and diradylglycerol have been used to monitor phospholipase activation in the human neutrophil. Stimulation of human neutrophils by formylmethionyl-leucylphenylalanine (fMet-Leu-Phe) resulted in a rapid activation of both phosphatidylinositol 4,5-bisphosphate breakdown by phospholipase C and phosphatidylcholine breakdown by phospholipase D. Diradylglycerol accumulation occurred more slowly than that of either choline or IP3 and was inhibited by 30 mM-butanol, suggesting that the bulk was derived from the phospholipase D pathway via phosphatidate phosphohydrolase. Consistent with this is the observation that choline and diradylglycerol are produced in similar amounts. 1,2-Diacylglycerol (DAG) and 1-O-alkyl-2-acyl-sn-glycerol species accumulated with different time courses, indicating that one or more steps in the phospholipase D pathway was selective for the diacyl species. Superoxide production by fMet-Leu-Phe-stimulated neutrophils paralleled DAG accumulation over the first 5 min, but thereafter this production stopped, despite the fact that DAG remained elevated. We conclude that DAG derived from the phospholipase D pathway is only one of the second messengers important in controlling this functional response.

Choline↗
PubMed1990

A novel and sensitive assay for phospholipase D in intact cells.

A novel and sensitive assay for phospholipase D (PLD) that measures the incorporation of high specific activity [3H]butan-1-ol into [3H]phosphatidylbutanol has been developed. The assay has been used to measure PLD activation in human neutrophils and platelets. Both the chemotactic peptide fMet-Leu-Phe and opsonised-zymosan stimulated PLD in the human neutrophil. In the platelet, PLD was stimulated by thrombin and collagen but responses were small and only occurred at high agonist concentrations. This assay has a number of advantages over existing techniques and should be valuable for investigating PLD activation in a variety of isolated cells and possibly intact tissues.

1-Butanol↗
PubMed1990

Selectivity of protein kinase inhibitors in human intact platelets.

The specificity of commonly used protein kinase inhibitors has been evaluated in the intact human platelet. Protein kinase C (PKC) and cyclic AMP-dependent protein kinase (PKA) were activated selectively by treating platelets with phorbol dibutyrate (PDBu) or prostacyclin (PGl2). PKC activity was quantitated by measuring PDBu-specific phosphorylation of a 47,000 molecular weight protein, and PKA activity monitored by measuring prostacyclin-dependent phosphorylation of a 22,000 molecular weight protein. Staurosporine and 1-(5-isoquinolinylsulphonyl)-2-methyl-piperazine (H-7) were found to be non-specific inhibitors in the intact platelet, consistent with their effects on the isolated enzymes. Tamoxifen inhibited PKC activity (IC50 = 80 microM) but increased PKA-dependent protein phosphorylation. These results support the use of human platelets for measuring the specificity of protein kinase inhibitors and indicate that tamoxifen might have value for experimental purposes as a relatively selective PKC inhibitor.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1989

Phospholipase D activation is functionally linked to superoxide generation in the human neutrophil.

Neutrophils stimulated with formylmethionyl-leucylphenylalanine (fMet-Leu-Phe) in the presence of butanol and ethanol formed phosphatidyl alcohols through a phospholipase D mechanism. The alcohols inhibited phosphatidic acid and diradylglycerol (DRG) formation, but did not block inositol 1, 4, 5-trisphosphate release. fMet-Leu-Phe-stimulated superoxide production was inhibited by alcohol concentrations which blocked DRG formation, whereas opsonized-zymosan-stimulated superoxide production was only partially decreased. These results suggest that phospholipase D activation is functionally linked to superoxide production in the human neutrophil.

1-Butanol↗
PubMed1988

Ca2+-independent binding of [3H]phorbol dibutyrate to protein kinase C is supported by protamine and other polycations.

The activity of the Ca2+- and phospholipid-dependent protein kinase, protein kinase C (PKC), can be modulated by diacylglycerols and phorbol esters. The association of these agents with PKC is, in turn, generally understood to be dependent on Ca2+ and phospholipids. Certain substrates, e.g. protamine sulphate, are known to undergo cofactor-independent phosphorylation by PKC. We report here that, in the presence of such substrates, PKC bound 1,2-dihexanoylglycerol and phorbol dibutyrate in a Ca2+-independent manner. Histone IIIs, which is phosphorylated by PKC only in the presence of Ca2+ and phospholipid, also supported Ca2+-independent binding of 1,2-dihexanoylglycerol and phorbol dibutyrate to PKC, but to a lesser extent than did protamine. Support for Ca2+-independent binding was also exhibited by non-peptide polycations (e.g. DEAE-cellulose DE52), indicating that recognition of the catalytic site is not a prerequisite for this effect. The natural polyamines spermine and putrescine did not have this property, however. The affinity of PKC for phorbol dibutyrate and 1,2-dihexanoylglycerol was found to be unchanged by the presence of substrates or DE52. It is proposed that, in the absence of Ca2+, certain polycations favour expression of the diacylglycerol/phorbol ester binding site by stabilizing the active conformation of PKC.

Animals↗
PubMed1988

Evidence that a second stereochemical centre in diacylglycerols defines interaction at the recognition site on protein kinase C.

The interaction of novel diacylglycerol analogues at the recognition site on protein kinase C has been evaluated using a modified [3H]phorbol dibutyrate binding assay and an established kinase activation assay. Studies with the 3-methyl analogues of 1,2-dihexanoyl-sn-glycerol have revealed a preferred stereochemical configuration at the C-3 position. Other chemical modifications have extended existing structure/activity relationships by showing that carbamates and sulphonyl esters cannot substitute for carboxylate esters and that cyclic acyl groups are active. Thus, most, if not all of the functionalities in the diacylglycerol molecule are required for interaction at the receptor on protein kinase C. Stereochemical specificity is required at C2 and C3.

Binding Sites↗
PubMed1987

Decrease of cellular ATP by dihexanoylglycerol may limit responses to protein kinase C activation.

1,2-sn-Dihexanoylglycerol (HHG) reduced the ATP content of HL-60 cells. This concentration-related (10-100 microM) effect reached a maximum of over 90%, was enantiomerically specific and not accompanied by release of lactate dehydrogenase. Oleoylacetylglycerols (3-100 microM) had no effect on ATP levels while phorbol dibutyrate (PDBu, 0.01-1 microM) decreased ATP content of HL-60 cells by up to 40%. Responses stimulated by HHG became limited as the concentration was increased above 10 microM, this being manifest as either a low maximum response compared to PDBu (superoxide release) or a bell-shaped concentration-effect curve (degranulation). HHG (30-100 microM) inhibited PDBu-stimulated superoxide release, this inhibition being enantiomerically specific. It is probable that the effect of HHG on ATP content impairs cellular responses mediated through protein kinase C activation.

Adenosine Triphosphate↗
PubMed1986

Inhibition of phorbol ester stimulated superoxide production by 1-oleoyl-2-acetyl-sn-glycerol (OAG); fact or artefact?

OAG-stimulated superoxide (O2) production by HL-60 granulocytes showed enantiomeric specificity but reached a maximum of only 5% of that produced by either phorbol myristate acetate (PMA) or phorbol dibutyrate (PDBu). At 10-100 microM, OAG displaced specifically-bound [3H]PDBu from intact HL-60 cells by only 25%, suggesting limited cell penetration. OAG (10-100 microM) also inhibited PDBu-stimulated O2 production by 25%; this inhibition was enantiomerically specific. However, at a lower concentration (3 microM), both enantiomers of OAG fully blocked O2 production stimulated by PMA (0.5 microM). This inhibition is probably artefactual, due to the hydrophobic PMA physically associating with OAG in the extracellular fluid.

Animals↗
PubMed1985

Stimulation and inhibition of secretion by phorbol myristate acetate in different cell types.

In washed human platelets and in HL60 granulocytes phorbol myristate acetate (PMA, 1-2000nM) synergised with threshold concentrations of secretogogues to induce a sustained maximum secretory response. Likewise, superoxide production from HL60 cells maintained a maximal response at PMA concentrations between 30-300nM. At concentrations up to 10nM PMA also augmented calcium ionophore, A23187, stimulated histamine release from rat peritoneal mast cells. However, in the mast cell PMA concentrations above 10nM reduced maximum histamine release in a dose-dependent manner.

Animals↗