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N T Thompson

Publications and source records attributed to N T Thompson.

13 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↗
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↗
PubMed1975

The variable fine structure of elastin visualized with Verhoeff's iron hematoxylin.

Verhoeff's iron hematoxylin (VIH) followed by lead citrate (LC) applied to epoxy thin sections stained the dense component of elastic fibers heavily and the peripheral microfibrillar component lightly in guinea pig trachea and mouse testis fixed with a glutaraldehyde-osmium tetroxide sequence. This method stained large fimbriated fibers beneath tracheal epithelium, small fibers and stacked aggregates thereof in the deep lamina propria, cartilage and adventitia of the trachea and large stacked fibers in the fibroelastic band of the trachea. Fibers of the fetus differed from those of the adult, especially in the subepithelial elastic lamina of the trachea. Elastic fibers were intimately associated with fibroblasts and particularly slender fibroblast processes in tracheal stroma and with chondrocytes in tracheal cartilage. Fibroblasts associated with elastic fibers in the tracheal subepithelial lamina propria were often closely bordered by eosinophils, mast cells, or monocytes. Occasional mast cells extended slender processes around elastic fibers in the subepithelial lamina propria. In mouse testis and in many regions of the trachea, small elastic fibers were identified which were below the limits of resolution for the light microscope and were not apparent at the ultrastructural level in routinely stained thin sections.

Animals↗
PubMed1975

Variability of dermal elastin visualized ultrastructurally with iron hematoxylin.

The Verhoeff iron hematoxylin-lead citrate (VIH-LC) method demonstrated vertical elastic fibers that were often composed only of microfibrillar component extending into the epidermal basement membrane in human skin. These fibers connected with a network of trabeculae composed of microfibrils and elastin fibrils in varying proportions. The large elastic fibers in the deep two thirds of the dermis consited mainly of compact bundles of small elstin fibrils in infants and of solid elastin cores with a fimbriated periphery in adults; Dermis of a 6-month-old fetus contained very few small elastic fibrils except around blood vessels. Skin of an elderly subject revealed exteme proliferation of unusual reticulated elastic fibers in various areas and disclosed abnormal nodules of elastin or collagen fibrils in finely particulate matter. Small elastin fibrils, abundant microfibrils, and intermixed individual collagen fibrils comprised an adventitial collar between sweat glands and fibroblasts. Elastin fibrils were absent from this collar in the fetus and increased with the subject's age. A permanganate-high iron diamine sequence appeared to impart density to the microfibrillar component of elastic fibers.

Adolescent↗