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Biomedical subjects

L G Garland

Publications and source records attributed to L G Garland.

At least 19 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↗
PubMed1992

New pathways of phagocyte activation: the coupling of receptor-linked phospholipase D and the role of tyrosine kinase in primed neutrophils.

Protein kinase C (PKC) appears to have a central role in the O2- response of neutrophils following stimulation of membrane receptors. The second messenger, diacylglycerol (DG), that activates PKC is derived from membrane phospholipids via activation of phosphatidylinositol 4,5-bisphosphate (PIP2)-phospholipase C (PLC) and phospholipase D (PLD), with the latter pathway being more prominent in primed cells. In resting cells receptor coupling to PLD is through a G-protein. Priming brings a cytoplasmic tyrosine kinase into the transducer sequence which, through protein phosphorylation, increases the efficiency of coupling between membrane receptors and PLD. Phosphatidic acid (PA), the initial product of the PLD pathway, also appears to act as a second messenger by directly activating the NADPH oxidase responsible for generating O2-. Interconversion of PA and DG by phosphatidate phosphohydrolase and DG kinase determines which of these second messengers has the dominant role.

GTP-Binding Proteins↗
PubMed1991

Selective 5-lipoxygenase inhibitor BW A4C does not influence progression of tissue injury in a canine model of regional myocardial ischaemia and reperfusion.

The effects of BW A4C, a selective arachidonate 5-lipoxygenase (5-LO) inhibitor, on the progression of myocardial tissue injury were examined in anaesthetised, open-chest beagle dogs subjected to 90-min occlusion of the left anterior descending coronary artery (LAD) followed by 120-min reperfusion. Regional myocardial blood flow (RMBF, microspheres), segment shortening (sonomicrometry), and infarct size (tetrazolium stain) as an index of tissue injury were measured. Control animals (group 1, n = 11) received an infusion of vehicle [50% vol/vol glycofurol and distilled water, 47 ml at 12 ml h-1, intravenously (i.v.)] beginning 15 min before ischaemia and continuing until the end of reperfusion. Treated animals received either 10 (group 2, n = 11) or 50 micrograms kg-1 min-1 (group 3, n = 5) BW A4C i.v. in the same period. The infarct/risk zone ratio (I/R) in group 1 (24.1 +/- 6.0%) was not significantly different from that of group 2 (28.0 +/- 8.4%) or group 3 (46.1 +/- 6.7%). The close inverse relationship observed in controls between I/R ratio and collateral flow was not altered by either dose of BW A4C. Segment shortening during ischaemia (-0.2 +/- 2.7, -2.4 +/- 1.7, and -1.5 +/- 1.7%) and reperfusion (4.9 +/- 2.8, 1.0 +/- 1.8, and -1.0 +/- 1.9%) and during an isoprenaline infusion to unmask stunned myocardium (14.7 +/- 3.0, 14.7 +/- 2.6, and 7.4 +/- 1.7%) were not significantly different between groups 1, 2, and 3.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
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↗
PubMed1991

Hydroxamic acids and hydroxyureas as novel, selective 5-lipoxygenase inhibitors for possible use in asthma.

Inhibition of 5-lipoxygenase (5-LO) is a potential target for therapeutic intervention in asthma. Acetohydroxamic acids such as BW A4C are potent and selective 5-LO inhibitors in vitro and also inhibit 5-LO activity in vivo following oral administration. In man, BW A4C is metabolised relatively rapidly (t1/2 = approx. 2h) but nevertheless inhibits 5-LO with reasonable persistence. Chemical modification of BW A4C has resulted in compounds, including the alpha-methyl analogues BW B218C and BW A360C and the hydroxyurea BW B70C, that retain high in vitro potency as selective 5-LO inhibitors and, compared to BW A4C, have a higher potency and longer duration of action in vivo. Members of both the hydroxamic acid and hydroxyurea series of 5-LO inhibitors are presently being considered as potential anti-asthma drugs.

Anaphylaxis↗
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↗
PubMed1990

The generation and metabolism of leukotrienes in the ionophore-stimulated blood of normal and asthmatic subjects.

The generation and metabolism of leukotrienes (LTs) B4, C4, D4, and E4 were studied in vitro in the A23187-stimulated whole blood of normal (N) and atopic asthmatic (AA) human subjects. Using a combination of reversed-phase high performance liquid chromatography and radioimmunoassay, we have demonstrated that the blood cells of atopic asthmatic patients have an enhanced ability to release LTB4 and LTC4 when compared to those of normal subjects. The release of LTB4 and LTC4 in response to ionophore is dose- and time-dependent. Half-maximal doses of ionophore caused the generation of high, sustained levels of LTB4, which are significantly higher in the AA blood than in N blood. Incubations of 3H-LTB4 in ionophore-stimulated N and AA blood revealed a slow metabolism to 20-OH-LTB4 and 20-COOH-LTB4. LTC4 is generated in smaller amounts than LTB4, with an early peak after 10 min which is significantly higher (p less than 0.01) in the AA blood compared to the N blood. Subsequent metabolism of LTC4 elicits significantly greater amounts of LTD4, and consistently higher levels of LTE4, in the AA blood. Parallel incubations of 3H-LTC4 in ionophore-stimulated N and AA blood demonstrated rapid metabolism of LTC4 by the glutathione detoxification pathway. The elevated production of LTB4 and LTC4 in AA blood was not accounted for by differences in leukocyte sub-type counts in the two groups, nor by differences in their rates of catabolism. The novel, selective 5-lipoxygenase inhibitor BW A4C [N-(3-phenoxycinnamyl) acetohydroxamic acid] caused dose-dependent inhibition of LTB4 and LTC4 generation and was equipotent in N and AA blood.

Asthma↗
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↗
PubMed1989

A method for the comparative assessment of antioxidants as peroxyl radical scavengers.

Antioxidants which are peroxyl radical scavengers have been compared in a model of lipid peroxidation based on the oxidation of a suspension of linoleic acid initiated by a thermolabile azo compound. By analysing the effect of antioxidant concentration on linoleic acid peroxidation we have defined the constant kAH which characterises the rate of the reaction of the antioxidant with the peroxyl radical. This allows quantitative comparison of the efficiency of different antioxidants as peroxyl radical scavengers. By using an initiation system which is not iron dependent we were able to show that the iron chelators desferrioxamine, BW A4C and U74500A are also peroxyl radical scavengers.

Antioxidants↗
PubMed1989

A phorbol ester and a daphnane ester stimulate a calcium-independent kinase activity from human mononuclear cells.

TPA and a non-promoting, pro-inflammatory ester RX were used to stimulate the forms of PKC isolated from human mononuclear cells. Three peaks of kinase activity corresponding to gamma, beta and alpha PKC were stimulated by TPA in the presence and absence of calcium and/or phosphatidylserine (PS) but were not activated by RX. A fourth peak eluted at high phosphate concentration was activated by TPA and RX in the presence of PS and the absence of calcium. Activity in this fraction was labile to freezing and thawing and was inhibited by staurosporine.

Alkaloids↗