Pardaxin Stimulation of Phospholipases A2 and Their Involvement in Exocytosis in PC-12 Cells
- Eugenia Bloch-Shilderman1,
- Saleh Abu-Raya1,1,
- Victoria Trembovler1,
- Hassia Boschwitz2,
- Arie Gruzman1,
- Michal Linial2 and
- Philip Lazarovici1
- 1Department of Pharmacology and Experimental Therapeutics, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel (E.B.-S., S.A.-R., V.T., A.G., P.L.); and 2Department of Biological Chemistry, Life Sciences Institute, The Hebrew University of Jerusalem, Israel (H.B., M.L.)
- Prof. Philip Lazarovici, Department of Pharmacology and Experimental Therapeutics, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, P.O. Box 12065, Jerusalem 91120, Israel. E-mail: lazph{at}md2.huji.ac.il
Abstract
Pardaxin (PX) is a voltage-dependent ionophore that stimulates catecholamine exocytosis from PC-12 pheochromocytoma cells both in the presence and absence of extracellular calcium. Using a battery of phospholipase A2 inhibitors we show that PX stimulation of phospholipase A2 (PLA2) enzymes is coupled with induction of exocytosis. We investigated the relationship between PX-induced PLA2 activity and neurotransmitter release by measuring the levels of arachidonic acid (AA), prostaglandin E2 (PGE2), and dopamine release. In the presence of extracellular calcium, the cytosolic PLA2inhibitor arachidonyl trifluoromethyl ketone (AACOCF3) inhibited by 100, 70, and 73%, respectively, the release of AA, PGE2, and dopamine induced by PX. The mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor 2′-amino-3′-methoxyflavone (PD98059) reduced by 100 and 82%, respectively, the release of AA and PGE2 induced by PX. In the absence of extracellular calcium, the calcium-independent PLA2 (iPLA2) inhibitors methyl arachidonyl fluorophosphonate, AACOCF3, and bromoenol lactone (BEL) inhibited by 80 to 90% PX stimulation of AA release, by 65 to 85% PX stimulation of PGE2 release, and by 80 to 90% PX-induced dopamine release. Using vesicle fusion-based enzyme-linked immunosorbent assay we found similar levels of inhibition of PX-induced exocytosis by these inhibitors. Also, PX induced the formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor complexes, an effect that was augmented byN-methylmaleimide. This complex formation was completely inhibited by BEL. Botulinum toxins type C1 and F significantly inhibited the release of AA, PGE2, and dopamine induced by PX. Our data suggest that PX stimulates exocytosis by activating cystolic PLA2 and iPLA2, leading to the generation of AA and eicosanoids, which, in turn, stimulate vesicle competence for fusion and neurotransmitter release.
Footnotes
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↵1 Current address: Hadassah Academic College, Haneviim Street 37, P.O. Box 1114, Jerusalem 91010, Israel.
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E.B.-S. and S.A.-R. contributed equally to this work.
- Abbreviations:
- SNARE
- solubleN-ethylmaleimide-sensitive factor attachment protein receptor (SNAP receptor)
- SNAP-25
- synaptosomal associated protein of 25 kDa
- VAMP
- vesicle-associated membrane protein (synaptobrevin)
- PX
- pardaxin
- AA
- arachidonic acid
- PLA2
- phospholipase A2
- sPLA2
- secretory phospholipase A2
- cPLA2
- cytosolic phospholipase A2
- iPLA2
- calcium-independent phospholipase A2
- MAPK
- mitogen-activated protein kinase
- AACOCF3
- arachidonyl trifluoromethyl ketone
- MAFP
- methyl arachidonyl fluorophosphonate
- BEL
- bromoenol lactone
- BSA
- bovine serum albumin
- NEM
- N-ethylmaleimide
- 5-HETE
- 5-hydroxyeicosatetraenoic acid
- HPLC
- high-pressure liquid chromatography
- DMEM
- Dulbecco's modified Eagle's medium
- ELISA
- enzyme-linked immunosorbent assay
- PBS
- phosphate-buffered saline
- RIA
- radioimmunoassay
- NGF
- nerve growth factor
- ERK
- extracellular regulated kinase
- PGE2
- prostaglandin E2
- NSF
- N-ethylmaleimide-sensitive factor
- BoNT/C1
- botulinum toxin type C1
- BoNT/F
- botulinum toxin type F
- O.D.
- optical density
- UO126
- 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio) butadiene
- PD98059
- 2′-amino-3′-methoxyflavone
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- Received December 10, 2001.
- Accepted February 27, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
