Downregulation of nitric oxide formation by cytosolic phospholipase A2-released arachidonic acid

The oxidation of plasma LDLs (low-density lipoproteins) is a key event in the pathogenesis of atherosclerosis. LPC (lysophosphatidylcholine) and oxysterols are major lipid constitutents of oxidized LDLs. In particular, 7-oxocholesterol has been found in plasma from cardiac patients and atherosclerotic plaque. In the present study, we investigated the ability of 7-oxocholesterol and LPC to regulate the activation of eNOS (endothelial nitric oxide synthase) and cPLA2 (cytosolic phospholipase A2) that synthesize two essential factors for vascular wall integrity, NO (nitric oxide) and arachidonic acid. In endothelial cells from human umbilical vein cords, both 7-oxocholesterol (150 µM) and LPC (20 µM) decreased histamine-induced NO release, but not the release activated by thapsigargin. The two lipids decreased NO release through a PI3K (phosphoinositide 3-kinase)-dependent pathway, and decreased eNOS phosphorylation. Their mechanisms of action were, however, different. The NO release reduction was dependent on superoxide anions in LPC-treated cells and not in 7-oxocholesterol-treated ones. The Ca2+ signals induced by histamine were abolished by LPC, but not by 7-oxocholesterol. The oxysterol also inhibited (i) the histamine- and thapsigargin-induced arachidonic acid release, and (ii) the phosphorylation of both cPLA2 and ERK1/2 (extracellular-signal-regulated kinases 1/2). The results show that 7-oxocholesterol inhibits eNOS and cPLA2 activation by altering a Ca2+-independent upstream step of PI3K and ERK1/2 cascades, whereas LPC desensitizes eNOS by interfering with receptor-activated signalling pathways. This suggests that 7-oxocholesterol and LPC generate signals which cross-talk with heterologous receptors, effects which could appear at early stage of atherosclerosis.

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Amyloid beta enhances cytosolic phospholipase A2 level and arachidonic acid release via nitric oxide in APP-transfected PC12 cells.

Acta Biochimica Polonica ◽

10.18388/abp.2007_3235 ◽

2007 ◽

Vol 54 (3) ◽

pp. 611-623 ◽

Cited By ~ 21

Author(s):

Małgorzata Chalimoniuk ◽

Anna Stolecka ◽

Magdalena Cakała ◽

Susane Hauptmann ◽

Kris Schulz ◽

...

Keyword(s):

Nitric Oxide ◽

Arachidonic Acid ◽

Phospholipase A2 ◽

Pc12 Cells ◽

Cell Lines ◽

Amyloid Beta ◽

Protein Level ◽

Cytosolic Phospholipase A2 ◽

Double Mutation ◽

Cytosolic Phospholipase

Cytosolic phospholipase A2 (cPLA2) preferentially liberates arachidonic acid (AA), which is known to be elevated in Alzheimer's disease (AD). The aim of this study was to investigate the possible relationship between enhanced nitric oxide (NO) generation observed in AD and cPLA2 protein level, phosphorylation, and AA release in rat pheochromocytoma cell lines (PC12) differing in amyloid beta secretion. PC12 control cells, PC12 cells bearing the Swedish double mutation in amyloid beta precursor protein (APPsw), and PC12 cells transfected with human APP (APPwt) were used. The transfected APPwt and APPsw PC12 cells showed an about 2.8- and 4.8-fold increase of amyloid beta (Abeta) secretion comparing to control PC12 cells. An increase of NO synthase activity, cGMP and free radical levels in APPsw and APPwt PC12 cells was observed. cPLA2 protein level was higher in APPsw and APPwt PC12 cells comparing to PC12 cells. Moreover, phosphorylated cPLA2 protein level and [3H]AA release were also higher in APP-transfected PC12 cells than in the control PC12 cells. An NO donor, sodium nitroprusside, stimulated [3H]AA release from prelabeled cells. The highest NO-induced AA release was observed in control PC12 cells, the effect in the other cell lines being statistically insignificant. Inhibition of cPLA2 by AACOCF3 significantly decreased the AA release. Inhibitors of nNOS and gamma-secretase reduced AA release in APPsw and APPwt PC12 cells. The basal cytosolic [Ca2+](i) and mitochondrial Ca2+ concentration was not changed in all investigated cell lines. Stimulation with thapsigargin increased the cytosolic and mitochondrial Ca2+ level, activated NOS and stimulated AA release in APP-transfected PC12 cells. These results indicate that Abeta peptides enhance the protein level and phosphorylation of cPLA2 and AA release by the NO signaling pathway.

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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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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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