scholarly journals Induction of cytosolic phospholipase A2 activity by phosphatidic acid and diglycerides in permeabilized human neutrophils: interrelationship between phospholipases D and A2

1997 ◽  
Vol 322 (2) ◽  
pp. 353-363 ◽  
Author(s):  
Sue A. BAULDRY ◽  
Rhonda E. WOOTEN
Keyword(s):  
Phosphatidic Acid ◽  
Phospholipase A2 ◽  
Second Messengers ◽  
Platelet Activating Factor ◽  
Human Neutrophils ◽  
Partial Restoration ◽  
Permeabilized Cells ◽  
Cpla2 Activation ◽  
Factor Α ◽  
Cytosolic Pla2

Relationships between phospholipases are poorly understood, but phosphatidic acid (PA) and diglycerides (DGs), produced by phospholipase D (PLD) and phosphatidate phosphohydrolase actions, might function as second messengers coupling cell stimulation to cellular responses. This study investigates the role of PLD-mediated PA and DG formation in inducing phospholipase A2 (PLA2) activity in intact human neutrophils (PMNs) and in PMNs permeabilized with Staphylococcus aureusα-toxin. PMNs were labelled with [3H]arachidonic acid (AA) to assess AA release and metabolism and diacylglycerol formation, or with [3H]1-O-hexadecyl-2-lyso-glycerophosphatidylcholine for the determination of platelet-activating factor (PAF), PA and alkylacylglycerol production. In intact PMNs primed with tumour necrosis factor α before stimulation with N-formyl-Met-Leu-Phe, AA release and metabolism and PAF formation increased in parallel with enhanced PA and DG formation, and inhibition of PA and DG production led to a decrease in both AA release and PAF accumulation. In α-toxin-permeabilized PMNs, AA release and PAF production result from the specific activation of cytosolic PLA2 (cPLA2). In this system, PA and DG formation were always present when cPLA2 activation occurred; blocking PA and DG production inhibited AA release and PAF accumulation. Adding either PA or DG back to permeabilized cells (with endogenous PA and DG formation blocked) led to a partial restoration of AA release and PAF formation; a combination of PA and DGs reconstituted full cPLA2 activity. These results strongly suggest that products of PLD participate in activating cPLA2 in PMNs.

Phospholipase A2 activation in human neutrophils requires influx of extracellular Ca2+ and leukotriene B4

1995 ◽  
Vol 268 (1) ◽  
pp. C138-C146 ◽  
Author(s):  
S. Reddy ◽  
R. Bose ◽  
G. H. Rao ◽  
M. Murthy
Keyword(s):  
Ethylene Glycol ◽  
Phospholipase A2 ◽  
Platelet Activating Factor ◽  
Leukotriene B4 ◽  
Lipid Mediators ◽  
Oxidation Products ◽  
Human Neutrophils ◽  
Tetraacetic Acid ◽  
A 23187 ◽  
Transient Elevation

We have demonstrated that phospolipase A2 (PLA2) activation in human neutrophils requires both the influx of extracellular Ca2+ and leukotriene B4 (LTB4). Surprisingly, the eicosanoids (LTB4 and its omega-oxidation products) formed were quantitatively very similar in both thapsigargin (Thap)- and A-23187-stimulated neutrophils. In contrast, Thap had very little effect on the activation of PLA2 when 5-lipoxygenase (5-LO) was blocked by BW755C or MK-886, whereas A-23187 caused a substantial activation. The lack of PLA2 activation in Thap-stimulated neutrophils results from the inhibition of LTB4 formation in the presence of 5-LO inhibitors. It appears that A-23187 activates both LTB4-dependent and -independent PLA2, whereas Thap activates LTB4-dependent PLA2. Experiments with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid demonstrated that activation of Thap-sensitive PLA2 and 5-LO requires the influx of Ca2+. Neither the transient elevation of cytosolic Ca2+ from intracellular stores nor the sustained Ca2+ influx alone without LTB4 appears sufficient to cause the activation of LTB4-dependent PLA2. We suggest that the activation of LTB4-dependent PLA2 involves 1) a sustained elevation of cytosolic Ca2+ coupled to the influx of extracellular Ca2+ and 2) a coupling between LTB4 and its receptor. We conclude that LTB4-dependent PLA2 plays an important role in the poststimulatory formation of lipid mediators such as prostaglandins, leukotrienes, and platelet-activating factor.

scholarly journals Accumulation of phosphatidic acid mass and increased de novo synthesis of glycerolipids in platelet-activating-factor-activated human neutrophils

1991 ◽  
Vol 280 (3) ◽  
pp. 625-629 ◽  
Author(s):  
J S Tou ◽  
J R Jeter ◽  
C P Dola ◽  
S Venkatesh
Keyword(s):  
Phosphatidic Acid ◽  
Cytochalasin B ◽  
De Novo ◽  
Platelet Activating Factor ◽  
Human Neutrophils ◽  
Total Lipid Extract ◽  
De Novo Synthesis ◽  
Predominant Species ◽  
Rapid Accumulation ◽  
Mass Accumulation

Incubation of human neutrophils with 100 nM-platelet-activating factor (PAF) but without cytochalasin B resulted in a rapid (5 s) accumulation (1.6-fold) of phosphatidic acid (PtdOH) mass. The increased PtdOH mass reached a maximum (2.8-fold) at 1 min and remained elevated (1.7-fold) at 10 min. No methylamine-stable lyso-PtdOH was detectable in the total lipid extract from control or from PAF-activated cells, suggesting that diacyl-PtdOH was the predominant species. In PAF-activated cells, changes in 1,2-diacylglycerol (DG) mass were not detectable at 5 or 15 s. Increased DG mass (1.7-fold) was detected between 30 s and 2 min, but then it declined to basal levels by 10 min. PAF enhanced [3H]glycerol incorporation into PtdOH and DG by 2- and 3-fold respectively during 1-10 min incubations. PAF also increased the radioactivity but not the mass of phosphatidylinositol and of choline glycerophospholipid by 8-fold and 4-fold respectively at 10 min. In addition, PAF-activated cells showed increased (2-fold) glycerol incorporation into triacylglycerol. These results demonstrate that PAF enhances rapid accumulation of diacyl-PtdOH mass, and that increased de novo synthesis may contribute to PtdOH mass accumulation.

scholarly journals Activation of phospholipase A2 in human neutrophils by polyunsaturated fatty acids and its role in stimulation of superoxide production

1998 ◽  
Vol 336 (3) ◽  
pp. 611-617 ◽  
Author(s):  
Brenton S. ROBINSON ◽  
Charles S. T. HII ◽  
Antonio FERRANTE
Keyword(s):  
Fatty Acids ◽  
Polyunsaturated Fatty Acids ◽  
Phospholipase A2 ◽  
Saturated Fatty Acids ◽  
Superoxide Production ◽  
Eicosatetraenoic Acid ◽  
Human Neutrophils ◽  
Dependent Manner ◽  
Biological Responses ◽  
Cytosolic Pla2

Although polyunsaturated fatty acids (PUFA) have been shown to stimulate neutrophil responses such as the oxygen-dependent respiratory burst (superoxide production), the mechanisms involved still remain undefined. Here we investigate the effect of PUFA on the phospholipase A2 (PLA2)-signal transduction process in human neutrophils. Exogenous eicosatetraenoic acid [arachidonic acid; C20:4(n-6)] or docosahexaenoic acid [C22:6(n-3)] promoted the release of [3H]C20:4(n-6) from prelabelled neutrophils in a time- and dose-dependent manner, which is indicative of PLA2 activation. The release of [3H]C20:4(n-6) from the cells by C20:4(n-6) and C22:6(n-3) was suppressed by PLA2 inhibitors. Other PUFA {eicosapentaenoic [C20:5(n-3)], octadecatrienoic [γ-linolenic; C18:3(n-6)] and octadecadienoic [linoleic; C18:2(n-6)] acids} also had the ability to release [3H]C20:4(n-6); however, certain C20:4(n-6) derivatives [15-hydroperoxyeicosatetraenoic acid, 15-hydroxyeicosatetraenoic acid and C20:4(n-6) methyl ester] and saturated fatty acids [octadecanoic (stearic; C18:0) and eicosanoic (arachidic; C20:0) acids] had no significant effect. Treatment of the neutrophils with exogenous C22:6(n-3) caused the mass of endogenous unesterified C20:4(n-6) to increase. Incubation of the leucocytes with C20:4(n-6) or C22:6(n-3) evoked activation of the 85 kDa cytosolic PLA2 (cPLA2) and the 14 kDa secretory PLA2 (sPLA2), but not the cytosolic Ca2+-independent PLA2. In contrast, C20:0 did not activate any of the PLA2 isoforms. Activation of cPLA2 by PUFA was found to precede that of sPLA2. C22:6(n-3), C20:4(n-6) and other PUFA induced punctate localization of cPLA2 in the cells, which was not observed with saturated fatty acids. Pretreatment of the leucocytes with PLA2 inhibitors markedly decreased superoxide production induced by C20:4(n-6). These results show that PUFA activate PLA2 in neutrophils, which might have a mandatory role in biological responses.

scholarly journals Roles of Rac and p38 kinase in the activation of cytosolic phospholipase A2 in response to PMA

2005 ◽  
Vol 388 (2) ◽  
pp. 527-535 ◽  
Author(s):  
Hye Jin YOU ◽  
Chang-Hoon WOO ◽  
Eun-Young CHOI ◽  
Sung-Hoon CHO ◽  
Yung Joon YOO ◽  
...  
Keyword(s):  
Phospholipase A2 ◽  
Dependent Manner ◽  
Perinuclear Region ◽  
P38 Kinase ◽  
Cytosolic Phospholipase ◽  
Cpla2 Activation ◽  
Kinase Cascade ◽  
Cytosolic Pla2 ◽  
Dependent Pathway

The roles of Rac and p38 kinase in the activation of cPLA2 (cytosolic PLA2) in Rat-2 fibroblasts were investigated. In the present study, we found that PMA activates cPLA2 by a Rac-p38 kinase-dependent pathway. Consistent with this, Rac, if activated, was shown to stimulate cPLA2 in a p38 kinase-dependent manner. In another experiment to understand the signalling mechanism by which the Rac-p38 kinase cascade mediates cPLA2 activation in response to PMA, we observed that PMA-induced cPLA2 translocation to the perinuclear region is completely inhibited by the expression of Rac1N17 or treatment with SB203580 (inhibitor of p38 kinase), suggesting that Rac-p38 kinase cascade acts in this instance by mediating the translocation of cPLA2. The mediatory role of p38 kinase in cPLA2 activation was further demonstrated after a treatment with anisomycin, a very effective activator of p38 kinase. Consistent with the mediatory role of p38 kinase in stimulating cPLA2, anisomycin induced the translocation and activation of cPLA2 in a p38 kinase-dependent manner.

scholarly journals Activation of phospholipase D by interleukin-8 in human neutrophils

Blood ◽  
1994 ◽  
Vol 84 (11) ◽  
pp. 3895-3901 ◽  
Author(s):  
S Sozzani ◽  
DE Agwu ◽  
MD Ellenburg ◽  
M Locati ◽  
M Rieppi ◽  
...  
Keyword(s):  
Phosphatidic Acid ◽  
Phospholipase D ◽  
Pertussis Toxin ◽  
Oxidative Burst ◽  
Platelet Activating Factor ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Interleukin 8 ◽  
Human Neutrophils ◽  
Bordetella Pertussis Toxin ◽  
Human Polymorphonuclear Leukocytes

Interleukin 8 (IL-8), a member of the C-X-C branch of the chemokine superfamily, stimulated the breakdown of 1-O-[3H]alkyl-2-acyl-sn- glycero-3-phosphocholine ([3H]EAPC) and the formation of 1-O-[3H]alkyl- 2-acyl-phosphatidic acid ([3H]-EAPA) in human polymorphonuclear leukocytes (PMN) in the presence of cytochalasin B. In addition, the mass of diradyl-PA was increased with similar kinetics. In the presence of ethanol, 1-O-[3H]alkyl-2-acyl-phosphatidylethanol ([3H]EAPEt) was formed at the expense of [3H]EAPA formation, indicating the activation of phospholipase D by the cytokine. The effect was time- and concentration-dependent, reaching a plateau at 30 seconds with the maximally activating concentration of 120 nmol/L IL-8. Preincubation of cells with 1 microgram/mL Bordetella pertussis toxin inhibited the breakdown of [3H]EAPC and [3H]EAPA formation, indicating a role for a pertussis toxin-sensitive guanosine triphosphate-binding protein. Formation of phosphatidic acid (PA) correlated with activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, the oxidative burst enzyme, with both events occurring in the same concentration range. Inhibition of PA formation, by the presence of ethanol, also inhibited the oxidative burst stimulation by IL-8. Pretreatment of PMN with 10 nmol/L platelet-activating factor potentiated both [3H]EAPA accumulation and activation of NADPD oxidase by IL-8. Collectively, these data show that IL-8 stimulates the metabolism of choline-containing phosphoglycerides in human PMN and support a role for PA in the signaling mechanisms used by IL-8 to stimulate PMN function.

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