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Cytosolic phospholipase A2 alpha inhibition prevents neuronal NMDA receptor-stimulated arachidonic acid mobilization and prostaglandin production but not subsequent cell death.

ABSTRACT: Phospholipase A(2) (PLA(2)) enzymes encompass a superfamily of at least 13 extracellular and intracellular esterases that hydrolyze the sn-2 fatty acyl bonds of phospholipids to yield fatty acids and lysophospholipids. The purpose of this study was to characterize which phospholipase paralog regulates NMDA receptor-mediated arachidonic acid (AA) release. Using mixed cortical cell cultures containing both neurons and astrocytes, we found that [(3)H]-AA released into the extracellular medium following NMDA receptor stimulation (100 microM) increased with time and was completely prevented by the addition of the NMDA receptor antagonist MK-801 (10 microM) or by removal of extracellular Ca(2+). Neither diacylglycerol lipase inhibition (RHC-80267; 10 microM) nor selective inhibition of Ca(2+)-independent PLA(2) [bromoenol lactone (BEL); 10 microM] alone had an effect on NMDA receptor-stimulated release of [(3)H]-AA. Release was prevented by methyl arachidonyl fluorophosphonate (MAFP) (5 microM) and AACOCF(3) (1 microM), inhibitors of both cytosolic PLA(2) (cPLA(2)) and Ca(2+)-independent PLA(2) isozymes. This inhibition effectively translated to block of NMDA-induced prostaglandin (PG) production. An inhibitor of p38MAPK, SB 203580 (7.5 microM), also significantly reduced NMDA-induced PG production providing suggestive evidence for the role of cPLA(2)alpha. Its involvement in release was confirmed using cultures derived from mice deficient in cPLA(2)alpha, which failed to produce PGs in response to NMDA receptor stimulation. Interestingly, neither MAFP, AACOCF(3) nor cultures derived from cPLA(2)alpha null mutant animals showed any protection against NMDA-mediated neurotoxicity, indicating that inhibition of this enzyme may not be a viable protective strategy in disorders of the cortex involving over-activation of the NMDA receptor.

SUBMITTER: Taylor AL

PROVIDER: S-EPMC2582587 | biostudies-literature | 2008 Aug

REPOSITORIES: biostudies-literature

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Cytosolic phospholipase A2 alpha inhibition prevents neuronal NMDA receptor-stimulated arachidonic acid mobilization and prostaglandin production but not subsequent cell death.

Taylor Ava L AL Bonventre Joseph V JV Uliasz Tracy F TF Hewett James A JA Hewett Sandra J SJ

Journal of neurochemistry 20080628 4

Phospholipase A(2) (PLA(2)) enzymes encompass a superfamily of at least 13 extracellular and intracellular esterases that hydrolyze the sn-2 fatty acyl bonds of phospholipids to yield fatty acids and lysophospholipids. The purpose of this study was to characterize which phospholipase paralog regulates NMDA receptor-mediated arachidonic acid (AA) release. Using mixed cortical cell cultures containing both neurons and astrocytes, we found that [(3)H]-AA released into the extracellular medium follo ...[more]

PMID: 18564366

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Project description:In interphase HeLa cells, incubation with histamine or thapsigargin led to the rapid release of arachidonic acid. The release was absolutely dependent on Ca2+, consistent with the activation of an 85 kDa cytosolic phospholipase A2 (cPLA2). In metaphase-arrested HeLa cells, by contrast, the stimulation of arachidonate release by these agents was inhibited by more than 90%. The lack of arachidonic acid release by mitotic cells was at least partly expected, since histamine- or thapsigargin-induced Ca2+ influx and elevations of cytosolic free Ca2+ are known to be strongly inhibited during mitosis [Preston, Sha'afi and Berlin (1991) Cell Regul. 2, 915-925]. Indeed, incubation of interphase cells with the Ca2+ ionophore A23187 alone induced a high level of arachidonate release. However, even A23187 failed to elicit release from mitotic cells. Since the Ca(2+)-dependent release of arachidonate by many cell types is promoted by preincubation with ligands that activate receptors of the tyrosine kinase class, and tumour promoters that lead to the phosphorylation of cPLA2, we determined if the responses of mitotic HeLa cells could be modified by this 'priming' process. We first established that epidermal growth factor and phorbol 12-myristate 13-acetate were effective priming agents in interphase cells: cells preincubated with the hormone or tumour promoter showed a 2-fold stimulation of thapsigargin- or A23187-induced arachidonic acid release. However, none of the priming agents reversed the lack of mitotic cell response. This refractoriness was not caused by destruction of cPLA2 during mitosis: by Western blotting, cPLA2 of interphase and mitotic cells was shown to be present in comparable amounts. Moreover, cPLA2 activities measured in extracts of interphase and mitotic cells were also comparable. Surprisingly, mitotic cPLA2 appeared to be constitutively phosphorylated in non-hormone-treated (control) cells. The results indicate a novel mechanism of regulation by cPLA2 activity in mitotic cells.

Project description: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 H(2)O(2) 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 H(2)O(2) 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 H(2)O(2), sorbitol or arsenite. None of these stimuli triggered significant arachidonic acid release on their own. However, H(2)O(2) 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 H(2)O(2) and sorbitol increased phosphorylation of cytosolic phospholipase A(2) (cPLA(2)) and its intrinsic activity; both responses were blocked by SB 203580. Phosphorylation of cPLA(2) by H(2)O(2) 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 cPLA(2) 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.

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Cytosolic phospholipase A2 alpha inhibition prevents neuronal NMDA receptor-stimulated arachidonic acid mobilization and prostaglandin production but not subsequent cell death.

Publications

Cytosolic phospholipase A2 alpha inhibition prevents neuronal NMDA receptor-stimulated arachidonic acid mobilization and prostaglandin production but not subsequent cell death.

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