Amplification Mechanisms of Inflammation: Paracrine Stimulation of Arachidonic Acid Mobilization by Secreted Phospholipase A2 Is Regulated by Cytosolic Phospholipase A2-Derived Hydroperoxyeicosatetraenoic Acid

In this study we have verified the existence of a cytosolic phospholipase A2 (cPLA2) in rat-liver macrophages. Stimulation of these cells with phorbol 12-myristate 13-acetate (PMA), zymosan and lipopolysaccharide (LPS), but not with the Ca(2+)-ionophore A23187, leads to phosphorylation of cPLA2 and activation of mitogen-activated protein (MAP) kinase, supporting the hypothesis that MAP kinase is involved in cPLA2 phosphorylation. We show furthermore, that the tyrosine kinase inhibitor genistein prevents the LPS- but not the PMA- or zymosan-induced phosphorylation of cPLA2 and activation of MAP kinase, indicating that tyrosine kinases participate in LPS- but not in PMA- and zymosan-induced cPLA2 phosphorylation and MAP kinase activation. Phosphorylation of cPLA2 does not strongly correlate with stimulation of the arachidonic acid (AA) cascade: (1) A23187, a potent stimulator of AA release, fails to induce cPLA2 phosphorylation; (2) withdrawal of extracellular Ca2+, which inhibits PMA-stimulated AA release (Dieter, Schulze-Specking and Decker (1988) Eur. J. Biochem. 177, 61-67), has no effect on PMA-induced phosphorylation of cPLA2; (3) LPS induces cPLA2 phosphorylation within minutes, whereas increased AA release upon treatment with LPS is detectable for the first time after 4 h; and (4) genistein, which prevents LPS-induced cPLA2 phosphorylation, does not inhibit AA release in response to LPS. From these data we suggest that a rise in intracellular Ca2+, but not phosphorylation of cPLA2, is essential for activation of the AA cascade in rat-liver macrophages.

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Stimulation of cytosolic phospholipase A2-catalyzed arachidonic acid liberation by low dose tert-butyl hydroperoxide without an influence on the enzyme activity in rabbit platelets

IUBMB Life ◽

10.1080/15216549700204611 ◽

1997 ◽

Vol 43 (4) ◽

pp. 799-806

Author(s):

Satoshi Akiba ◽

Ryo Nagatomo ◽

Misako Hayama ◽

Takashi Sato

Keyword(s):

Enzyme Activity ◽

Arachidonic Acid ◽

Phospholipase A2 ◽

Low Dose ◽

Cytosolic Phospholipase A2 ◽

Butyl Hydroperoxide ◽

Cytosolic Phospholipase ◽

Tert Butyl Hydroperoxide ◽

Rabbit Platelets ◽

Stimulation Of

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Regulation of Cytosolic Phospholipase A2 in Arachidonic Acid Release of Rat-Liver Macrophages

Advances in Experimental Medicine and Biology - Eicosanoids and other Bioactive Lipids in Cancer, Inflammation, and Radiation Injury 3 ◽

10.1007/978-1-4899-1813-0_71 ◽

1997 ◽

pp. 479-483 ◽

Cited By ~ 1

Author(s):

P. Ambs ◽

M. Baccarini ◽

H. Schwende ◽

E. Fitzke ◽

P. Dieter

Keyword(s):

Arachidonic Acid ◽

Phospholipase A2 ◽

Rat Liver ◽

Cytosolic Phospholipase A2 ◽

Arachidonic Acid Release ◽

Liver Macrophages ◽

Cytosolic Phospholipase ◽

Acid Release

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The immediate phase of c-kit ligand stimulation of mouse bone marrow-derived mast cells elicits rapid leukotriene C4 generation through posttranslational activation of cytosolic phospholipase A2 and 5-lipoxygenase.

Journal of Experimental Medicine ◽

10.1084/jem.182.1.197 ◽

1995 ◽

Vol 182 (1) ◽

pp. 197-206 ◽

Cited By ~ 86

Author(s):

M Murakami ◽

K F Austen ◽

J P Arm

Keyword(s):

Bone Marrow ◽

Arachidonic Acid ◽

Mast Cells ◽

Cell Membrane ◽

Phospholipase A2 ◽

Mouse Bone Marrow ◽

Kit Ligand ◽

Cytosolic Phospholipase ◽

Delayed Phase ◽

Stimulation Of

c-kit ligand (KL) activated mouse bone marrow-derived mast cells (BMMC) for the dose- and time-dependent release of arachidonic acid from cell membrane phospholipids, with generation of leukotriene (LT) C4 in preference to prostaglandin (PG)D2. KL at concentrations of 10 ng/ml elicited half-maximal eicosanoid generation and at concentrations of > 50 ng/ml elicited a maximal generation of approximately 15 ng LTC4 and 1 ng PGD2 per 10(6) cells, with 20% net beta-hexosaminidase release 10 min after stimulation. Of the other cytokines tested, none, either alone or in combination with KL, elicited or modulated the immediate phase of mediator release by BMMC, indicating strict specificity for KL. Activation of BMMC in response to KL was accompanied by transient phosphorylation of cytosolic phospholipase A2 and reversible translocation of 5-lipoxygenase to a cell membrane fraction 2-5 min after stimulation, when the rate of arachidonic acid release and LTC4 production were maximal. BMMC continuously exposed to KL in the presence of IL-10 and IL-1 beta generated LTC4 in marked preference to PGD2 over the first 10 min followed by delayed generation of PGD2 with no LTC4 over several hours. Pharmacologic studies revealed that PGD2 generation in the immediate phase depended on prostaglandin endoperoxide synthase (PGHS)-1 and in the delayed phase on PGHS-2. Thus, KL provided a nonallergic stimulus for biphasic eicosanoid generation by mast cells. The immediate phase is dominated by LTC4 generation with kinetics and postreceptor biosynthetic events similar to those observed after cell activation through the high affinity IgE receptor, whereas the delayed phase of slow and selective PGD2 production is mediated by induction of PGHS-2.

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Role of Phosphorylation Sites and the C2 Domain in Regulation of Cytosolic Phospholipase A2

The Journal of Cell Biology ◽

10.1083/jcb.145.6.1219 ◽

1999 ◽

Vol 145 (6) ◽

pp. 1219-1232 ◽

Cited By ~ 154

Author(s):

Miguel A. Gijón ◽

Diane M. Spencer ◽

Alan L. Kaiser ◽

Christina C. Leslie

Keyword(s):

Arachidonic Acid ◽

Nuclear Envelope ◽

Phospholipase A2 ◽

Okadaic Acid ◽

Cytosolic Phospholipase A2 ◽

Phosphorylation Sites ◽

C2 Domain ◽

Arachidonic Acid Release ◽

Cytosolic Phospholipase ◽

Acid Release

Cytosolic phospholipase A2 (cPLA2) mediates agonist-induced arachidonic acid release, the first step in eicosanoid production. cPLA2 is regulated by phosphorylation and by calcium, which binds to a C2 domain and induces its translocation to membrane. The functional roles of phosphorylation sites and the C2 domain of cPLA2 were investigated. In Sf9 insect cells expressing cPLA2, okadaic acid, and the calcium-mobilizing agonists A23187 and CryIC toxin induce arachidonic acid release and translocation of green fluorescent protein (GFP)-cPLA2 to the nuclear envelope. cPLA2 is phosphorylated on multiple sites in Sf9 cells; however, only S505 phosphorylation partially contributes to cPLA2 activation. Although okadaic acid does not increase calcium, mutating the calcium-binding residues D43 and D93 prevents arachidonic acid release and translocation of cPLA2, demonstrating the requirement for a functional C2 domain. However, the D93N mutant is fully functional with A23187, whereas the D43N mutant is nearly inactive. The C2 domain of cPLA2 linked to GFP translocates to the nuclear envelope with calcium-mobilizing agonists but not with okadaic acid. Consequently, the C2 domain is necessary and sufficient for translocation of cPLA2 to the nuclear envelope when calcium is increased; however, it is required but not sufficient with okadaic acid.

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

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615763 ◽

2001 ◽

Vol 85 (05) ◽

pp. 882-889 ◽

Cited By ~ 21

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.

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