Abstract
The effect of Pardaxin, a neurotoxin that induces neurotransmitter release from neurons, on the arachidonic acid (AA) cascade was studied in PC12 cells. Both native and the synthetic Pardaxin selectively stimulated phospholipase A2 (PLA2) activity (measured by [3H]AA release) in the presence as well as in the absence of extracellular calcium. Pardaxin-stimulated PLA2 activity was also evident in the increased formation of lysophosphatidylcholine. Pardaxin analogs, lacking the α-helical structure that is essential for insertion into the plasma membrane, were ineffective in stimulating the AA cascade in PC12 cells. Pardaxin stimulation of PLA2 was markedly inhibited by the nonselective PLA2 inhibitors bromophenacyl bromide and mepacrine, by methyl arachidonyl fluorophosphonate, a dual inhibitor of calcium-dependent cytosolic PLA2 and the calcium-independent PLA2 and by bromoenol lactone[(E)-6-(bromoethylene)tetrahydro-3-(1-naphthalenyl-2H-pyran-2-one], a highly specific inhibitor of calcium-independent PLA2. After Pardaxin treatment, there was increased release of AA metabolites produced by the cyclooxygenase pathway as expressed in an 8-fold increase of PGE2 release. The release of other eicosanoids, such as 6-keto-PGF1α and thromboxane B2, was also augmented. Pardaxin-induced PGE2 release was observed in calcium-free medium and in the absence of any increase in cytosolic calcium. Dexamethasone partially inhibited Pardaxin-induced PGE2 release. This effect was reversed by the type II corticosteroid receptor antagonist RU-38486. Our results indicate that Pardaxin stimulates release of AA and eicosanoids, independently of calcium, and suggest that calcium-independent PLA2 plays an important role in Pardaxin stimulation of the AA cascade.
Footnotes
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Send reprint requests to: Lazarovici Philip, Department of Pharmacology and Experimental Therapeutics, School of Pharmacy, Faculty of Medicine, Hebrew University, P.O.B. 12065, Jerusalem, 91120, Israel.
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↵1 This study was supported in part by the David R. Bloom Center for Pharmacy at the Hebrew University.
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↵2 Present address: Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot, 76100, Israel.
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↵3 Present address: Department of Neurology, Hadassah University Hospital, P.O.B. 12000, Jerusalem, 91120, Israel.
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↵4 Present address: Department of Biochemistry, Faculty of Medicine, The Hebrew University, P.O.B. 12065, Jerusalem, 91120, Israel.
- Abbreviations:
- AA
- arachidonic acid
- PLA2
- phospholipase A2
- iPLA2
- calcium-independent phospholipase A2
- PLC
- phospholipase C
- PLD
- phospholipase D
- PA
- phosphatidic acid
- PC
- phosphatidylcholine
- LPC
- lysophosphatidylcholine
- PI
- phosphatidylinositol
- PS
- phosphatidylserine
- Fura 2-AM
- acetoxymethyl ester of fura 2
- PGE2-prostaglandin E2
- TXB2, thromboxane B2
- DMEM
- Dulbecco’s modified Eagle’s medium
- Dex
- dexamethasone
- PBS
- phosphate-buffered saline
- EGTA
- ethyleneglycol-bis-(β-amino-ethyl ether) N,N′-tetra acetic acid
- PMSF
- phenylmethanesulfonyl
- RIA
- radioimmunoassay
- [Ca]i
- cytosolic calcium
- pBPB-4
- bromophenacyl bromide
- [Ca]o
- extracellular calcium
- BSA
- bovine serum albumin
- DAG
- diacylglycerol
- RHC-80267
- 1,6-di[o-(carbamoyl)cyclohexaneoxim]hexane
- BEL
- bromoenol lactone[(E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one]
- MAFP
- methyl arachidonyl fluorophosphonate
- TLC
- thin-layer chromatography
- Received September 30, 1997.
- Accepted July 7, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
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