scholarly journals The receptors for ATP and fMetLeuPhe are independently coupled to phospholipases C and A2 via G-protein(s). Relationship between phospholipase C and A2 activation and exocytosis in HL60 cells and human neutrophils

1989 ◽  
Vol 263 (3) ◽  
pp. 715-723 ◽  
Author(s):  
S Cockcroft ◽  
J Stutchfield
Keyword(s):  
Arachidonic Acid ◽  
Phospholipase A2 ◽  
Phospholipase C ◽  
G Protein ◽  
Pertussis Toxin ◽  
Human Neutrophils ◽  
Ionophore A23187 ◽  
Arachidonic Acid Release ◽  
Hl60 Cells ◽  
Acid Release

The relationship between phospholipase A2 and C activation and secretion was investigated in intact human neutrophils and differentiated HL60 cells. Activation by either ATP or fMetLeuPhe leads to [3H]arachidonic acid release into the external medium from prelabelled cells. This response was inhibited when the cells were pretreated with pertussis toxin. When the [3H]arachidonic acid-labelled cells were stimulated with fMetLeuPhe, ATP or Ca2+ ionophore A23187, and the lipids analysed by t.l.c., the increase in free fatty acid was accompanied by decreases in label from phosphatidylinositol and phosphatidylcholine. Moreover, incorporation of label into triacylglycerol and to a lesser extent phosphatidylethanolamine was evident. Activation of secretion was evident with ATP and fMetLeuPhe but not with A23187. The pharmacological specificity of the ATP receptor in HL60 cells was investigated by measuring secretion of beta-glucuronidase, formation of inositol phosphatases and release of [3H]arachidonic acid. External addition of ATP, UTP, ITP, adenosine 5′-[gamma-thio]triphosphate (ATP[S]), adenosine 5′-[beta gamma-imido]triphosphate (App[NH]p), XTP, CTP, GTP, 8-bromo-ATP and guanosine 5′-[gamma-thio]triphosphate (GTP[S]) to intact HL60 cells stimulated inositol phosphate production, but only the first five nucleotides were effective at stimulating secretion or [3H]arachidonic acid release. In human neutrophils, addition of ATP, ITP, UTP and ATP[S] also stimulated secretion from specific and azurophilic granules, and this was accompanied by increases in cytosolic Ca2+ and in [3H]arachidonic acid release. The addition of phorbol 12-myristate 13-acetate (PMA; 1 nM) prior to the addition of either fMetLeuPhe or ATP led to inhibition of phospholipase C activity. In contrast, this had no effect on phospholipase A2 activation, whilst secretion was potentiated. Phospholipase A2 activation by either agonist was dependent on an intact cell metabolism, as was secretion. It is concluded that (1) activation of phospholipase C does not always lead to activation of phospholipase A2, (2) phospholipase A2 is coupled to the receptor independently of phospholipase C via a pertussis-toxin-sensitive G-protein and (3) for secretion to take place, the receptor has to activate both phospholipases C and A2.

Evidence for a Role for Phospholipase C, but not Phospholipase A2, in Platelet Activation in Response to Low Concentrations of Collagen

2001 ◽  
Vol 85 (05) ◽  
pp. 882-889 ◽  
Author(s):  
Leslie Lockhart ◽  
Caroline Pampolina ◽  
Brent Nickolaychuk ◽  
Archibald McNicol
Keyword(s):  
Arachidonic Acid ◽  
Phospholipase A2 ◽  
Phospholipase C ◽  
Platelet Activation ◽  
Cytosolic Phospholipase A2 ◽  
Arachidonic Acid Release ◽  
Cytosolic Phospholipase ◽  
Low Concentrations ◽  
Acid Release ◽  
Induced Aggregation

SummaryThe release of arachidonic acid is a key component in platelet activation in response to low concentrations (1-20 g/ml) of collagen. The precise mechanism remains elusive although a variety of pathways have been implicated. In the present study the effects of inhibitors of several potentially key enzymes in these pathways have been examined. Collagen (1-10 g/ml) caused maximal platelet aggregation which was accompanied by the release of arachidonic acid, the synthesis of thromboxane A2, and p38MAPK phosphorylation. Preincubation with the dual cyclooxygenase/lipoxygenase inhibitor BW755C inhibited aggregation and thromboxane production, and reduced p38MAPK phosphorylation. A phospholipase C inhibitor, U73122, blocked collagen-induced aggregation and reduced arachidonic acid release, thromboxane synthesis and p38MAPK phosphorylation. Pretreatment with a cytosolic phospholipase A2 inhibitor, AACOCF3, blocked collagen-induced aggregation, reduced the levels of thromboxane formation and p38MAPK phosphorylation but had no significant effect on arachidonic acid release. In contrast inhibition of PKC by Rö31-8220 inhibited collagen-induced aggregation, did not affect p38MAPK phosphorylation but significantly potentiated arachidonic acid release and thromboxane formation. Collagen caused the tyrosine phosphorylation of phospholipase C 2 which was inhibited by pretreatment with U73122, unaffected by AACOCF3 and enhanced by Rö31-8220. These results suggest that cytosolic phospholipase A2 plays no role in the arachidonic acid release in response to collagen. In contrast, the data are consistent with phospholipase C 2 playing a role in an intricately controlled pathway, or multiple pathways, mediating the release of arachidonic acid in collagen-stimulated platelets.

scholarly journals Permissive stimulation of Ca(2+)-induced phospholipase A2 by an adenosine receptor agonist in a pertussis toxin-sensitive manner in FRTL-5 thyroid cells: a new ‘cross-talk’ mechanism in Ca2+ signalling.

1994 ◽  
Vol 299 (3) ◽  
pp. 845-851 ◽  
Author(s):  
S Shimegi ◽  
F Okajima ◽  
Y Kondo
Keyword(s):  
Arachidonic Acid ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase A2 ◽  
Phospholipase C ◽  
Arachidonic Acid Release ◽  
Thyroid Cells ◽  
Kinase C ◽  
Adenosine Receptor Agonist ◽  
Acid Release

We have described the pertussis toxin (PTX)-sensitive potentiation of P2-purinergic agonist-induced phospholipase C activation, Ca2+ mobilization and arachidonic acid release by an adenosine receptor agonist, N6-(L-2-phenylisopropyl)adenosine (PIA), which alone cannot influence any of these cellular activities [Okajima, Sato, Nazarea, Sho and Kondo (1989) J. Biol. Chem. 264, 13029-13037]. In the present study we have found that arachidonic acid release was associated with lysophosphatidylcholine production, and conclude that arachidonic acid is produced by phospholipase A2 in FRTL-5 thyroid cells. This led us to assume that PIA augments P2-purinergic arachidonic acid release by increasing [Ca2+]i which, in turn, activates Ca(2+)-sensitive phospholipase A2. The arachidonic acid-releasing response to PIA was, however, always considerably higher (3.1-fold increase) than the Ca2+ response (1.3-fold increase) to the adenosine derivative. In addition, arachidonic acid release induced by the [Ca2+]i increase caused by thapsigargin, an endoplasmic-reticulum Ca(2+)-ATPase inhibitor, or calcium ionophores was also potentiated by PIA without any effect on [Ca2+]i and phospholipase C activity. This action of PIA was also PTX-sensitive, but not affected by the forskolin- or cholera toxin-induced increase in the cellular cyclic AMP (cAMP), suggesting that a PTX-sensitive G-protein(s) and not cAMP mediates the PIA-induced potentiation of Ca(2+)-generated phospholipase A2 activation. Although acute phorbol ester activation of protein kinase C induced arachidonic acid release, P2-purinergic and alpha 1-adrenergic stimulation of arachidonic acid release was markedly increased by the protein kinase C down-regulation caused by the phorbol ester. This suggests a suppressive role for protein kinase C in the agonist-induced activation of arachidonic acid release. We conclude that PIA (and perhaps any of the G1-activating agonists) augments an agonist (maybe any of the Ca(2+)-mobilizing agents)-induced arachidonic acid release by activation of Ca(2+)-dependent phospholipase A2 in addition to enhancement of agonist-induced phospholipase C followed by an increase in [Ca2+]i.

scholarly journals Stress stimuli increase calcium-induced arachidonic acid release through phosphorylation of cytosolic phospholipase A2

1999 ◽  
Vol 344 (2) ◽  
pp. 359-366 ◽  
Author(s):  
Marcus BUSCHBECK ◽  
Farideh GHOMASHCHI ◽  
Michael H. GELB ◽  
Steve P. WATSON ◽  
Angelika G. BÖRSCH-HAUBOLD
Keyword(s):  
Arachidonic Acid ◽  
Protein Kinase ◽  
Phospholipase A2 ◽  
Cytosolic Phospholipase A2 ◽  
Ionophore A23187 ◽  
Arachidonic Acid Release ◽  
High Osmolarity ◽  
Cytosolic Phospholipase ◽  
Acid Release ◽  
Sb 203580

Stress stimuli such as free radicals, high osmolarity or arsenite activate stress-activated protein kinases (SAPKs) in a wide variety of cells. In the present study, we have investigated the ability of several stress stimuli to activate SAPKs in platelets and to induce phosphorylation of their substrates. Treatment of human platelets with H2O2 stimulated SAPK2a and its downstream target mitogen-activated protein kinase-activated protein kinase-2 (MAPKAP-K2). Kinase activity reached a maximum after 2-5 min and declined towards basal levels after 15 min. Arsenite caused a steady increase of MAPKAP-K2 activity up to 15 min. The level of maximal kinase activation by H2O2 and arsenite was comparable with the effect caused by the physiological platelet stimulus thrombin. A high osmolarity solution of sorbitol induced comparatively small activation of SAPK2a and MAPKAP-K2. The 42-kDa extracellular signal-regulated kinase (ERK) 2 was not activated by H2O2, sorbitol or arsenite. None of these stimuli triggered significant arachidonic acid release on their own. However, H2O2 and sorbitol enhanced the release of arachidonic acid induced by the calcium ionophore A23187. This effect was reversed by the inhibitor of SAPK2a, 4-(4-fluorophenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl) imidazole (SB 203580), but not by the inhibitor of the ERK2-activating pathway, 2-(2-amino-3-methoxyphenyl)-oxanaphthalen-4-one (PD 98059). Both H2O2 and sorbitol increased phosphorylation of cytosolic phospholipase A2 (cPLA2) and its intrinsic activity; both responses were blocked by SB 203580. Phosphorylation of cPLA2 by H2O2 occurred on Ser-505, a reaction that is known to increase the intrinsic lipase activity of the enzyme. Our results demonstrate that activation of SAPKs by stress stimuli primes cPLA2 activation through phosphorylation. In vivo, this mechanism would lead to the sensitization of platelet activation and may be an important risk factor in thrombotic disease.

scholarly journals A major role for phospholipase A2 in antigen-induced arachidonic acid release in rat mast cells

1987 ◽  
Vol 247 (1) ◽  
pp. 95-99 ◽  
Author(s):  
K Yamada ◽  
Y Okano ◽  
K Miura ◽  
Y Nozawa
Keyword(s):  
Arachidonic Acid ◽  
Mast Cells ◽  
Phospholipase A2 ◽  
Histamine Release ◽  
Phospholipase C ◽  
Minor Role ◽  
Arachidonic Acid Release ◽  
Ige Receptors ◽  
Acid Release ◽  
A Minor

Cross-linking of IgE receptors by antigen stimulation leads to histamine release and arachidonic acid release in rat peritoneal mast cells. Investigators have reported a diverse distribution of [3H]arachidonate that is dependent on labelling conditions. Mast cells from rat peritoneal cavity were labelled with [3H]arachidonic acid for different periods of time at either 30 or 37 degrees C. Optimum labelling was found to be after 4 h incubation with [3H]arachidonate at 30 degrees C, as judged by cell viability (Trypan Blue uptake), responsiveness (histamine release) and distribution of radioactivity. Alterations in 3H-radioactivity distribution in mast cells labelled to equilibrium were examined on stimulation with antigen (2,4-dinitrophenyl-conjugated Ascaris suum extract). The results indicated that [3H]arachidonic acid was lost mainly from phosphatidylcholine and, to a lesser extent, from phosphatidylinositol. A transient appearance of radiolabelled phosphatidic acid and diacylglycerol indicated phosphatidylinositol hydrolysis by phospholipase C. Pretreatment with a phospholipase A2 inhibitor, mepacrine, substantially prevented the antigen-induced liberation of [3H]arachidonic acid from phosphatidylcholine. It can be thus concluded that, in the release of arachidonic acid by antigen-stimulated mast cells, the phospholipase A2 pathway, in which phosphatidylcholine is hydrolysed, serves as the major one, the phospholipase C/diacylglycerol lipase pathway playing only a minor role.

EVIDENCE FOR INVOLVEMENT OF GTP-BINDING PROTEIN IN ARACKIDONIC ACID RELEASE BY PHOSPHOLIPASE A2 IN PERMEABILIZED HUMAN PLATELETS

1987 ◽  
Author(s):  
S Nakashima ◽  
T Tohmatsu ◽  
H Hattori ◽  
A Suganuma ◽  
Y Nozawa
Keyword(s):  
Arachidonic Acid ◽  
Phospholipase A2 ◽  
Phospholipase C ◽  
Binding Protein ◽  
Human Platelets ◽  
Arachidonic Acid Release ◽  
Gtp Binding Protein ◽  
Diacylglycerol Lipase ◽  
Gtp Binding ◽  
Acid Release

Platelet activation is accompanied by the active metabolism of membrane phospholipids. Phosphoinositide breakdown by phospholipase C generates second messengers; inositol trisphosphate and diacylglycerol. Recently, it is suggested that GTP-binding protein is linked to the activation of phospholipase C as is true for adenylate cyclase. Although it is known that the receptor stimulation by agonists leads to generation of arachidonic acid, its molecular mechanism has not yet been clear. However, several studies in neutrophils and mast cells using pertussis toxin, have shown the possibility that a GTP-binding protein may act as an intermediary unit component between the receptor and phospholipase A2. The present study was therefore designed to examine the effect of GTP and its analogue GTPγS on the arachidonic acid release in saponin-permeabilized human platelets. GTP or GTPγS alone caused a small but significant liberation of arachidonic acid in permeabilized cells but not in intact cells. GTP or GTPγS was found to enhance thrombin-induced [3H]arachidonic acid release in saponi n-permeabi li zed human platelets. The release of arachidonic acid has been ascribed to activity of phospholipase A2 and/or to sequential action of phospholipase C and diacylglycerol lipase. Inhibitors of phospholipase C (neomycin)/ diacylglycerol lipase (RHC 80267) pathway of arachidonate liberation did not reduce the level of the [3H]arachidonic acid release. The loss of [3H]arachidonate radioactivity from phosphatidylcholine was almost complementary to the increment of released [3H]arachidonic acid, suggesting thrombin-induced hydrolysis of phosphatidylcholine by phospholipase A2. Although phospholipase A2 usually are described as having a requirement for calcium, the effect of GTPγS was more evident at lower calcium concentrations (buffer>0.1 mM>1.0 mM). These data thus indicate that release of arachidonic acid by phospholipase A2 in saponin-treated platelets is closely linked to GTP-binding protein which may decrease the calcium requirement for phospholipase A2 activation.

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