Abstract
We showed previously that OX1 orexin receptor stimulation produced a strong 3H overflow response from [3H]arachidonic acid (AA)-labeled cells. Here we addressed this issue with a novel set of tools and methods, to distinguish the enzyme pathways responsible for this response. CHO-K1 cells heterologously expressing human OX1 receptors were used as a model system. By using selective pharmacological inhibitors, we showed that, in orexin-A-stimulated cells, the AA-derived radioactivity was released as two distinct components, i.e., free AA and the endocannabinoid 2-arachidonoyl glycerol (2-AG). Two orexin-activated enzymatic cascades are responsible for this response: cytosolic phospholipase A2 (cPLA2) and diacylglycerol lipase; the former cascade is responsible for part of the AA release, whereas the latter is responsible for all of the 2-AG release and part of the AA release. Essentially only diacylglycerol released by phospholipase C but not by phospholipase D was implicated as a substrate for 2-AG production, although both phospholipases were strongly activated. The 2-AG released acted as a potent paracrine messenger through cannabinoid CB1 receptors in an artificial cell-cell communication assay that was developed. The cPLA2 cascade, in contrast, was involved in the activation of orexin receptor-operated Ca2+ influx. 2-AG was also released upon OX1 receptor stimulation in recombinant HEK-293 and neuro-2a cells. The results directly show, for the first time, that orexin receptors are able to generate potent endocannabinoid signals in addition to arachidonic acid signals, which may explain the proposed orexin-cannabinoid interactions (e.g., in neurons).
Footnotes
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The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.This study was supported by the Academy of Finland, the Magnus Ehrnrooth Foundation, the University of Helsinki Research Funds, the Biomedicum Helsinki Foundation, and the Research Foundation of the University of Helsinki.
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
ABBREVIATIONS:
- AA
- arachidonic acid
- 2-AG
- 2-arachidonoyl glycerol
- AM-251
- 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide
- CAY10499
- [4-[5-methoxy-2-oxo-1,3,4-oxadiazol-3(2H)-yl]-2-methylphenyl]carbamic acid phenylmethyl ester
- CAY10593
- N-[2-[4-(5-chloro-2,3-dihydro-2-oxo-1H-benzimidazol-1-yl)-1-piperidinyl]-1-methylethyl]-2-naphthalenecarboxamide
- CCPA
- N-cyclohexanecarbonylpentadecylamine
- CHO
- Chinese hamster ovary
- cPLA2
- cytosolic (Ca2+-independent) phospholipase A2
- DAG
- diacylglycerol
- DAGL
- diacylglycerol lipase
- FAAH
- fatty acid amide hydrolase
- FKGK11
- 1,1,1,2,2-pentafluoro-7-phenyl-heptan-3-one
- HBM
- HEPES-buffered medium
- HU-210
- 3-(1,1′-dimethylheptyl)-6aR,7,10,10aR-tetrahydro-1-hydroxy-6,6-dimethyl-6H-dibenzo[b,d]pyran-9-methanol
- IBMX
- 3-isobutyl-1-methylxanthine
- iPLA2
- intracellular (Ca2+-independent) phospholipase A2
- JZL184
- 4-nitrophenyl-4-[dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl]piperidine-1-carboxylate
- MAFP
- methyl arachidonyl fluorophosphonate
- MAGL
- monoacylglycerol lipase
- PA
- phosphatidic acid
- PLA2
- phospholipase A2
- PLC
- phospholipase C
- PLD
- phospholipase D
- RHC-80267
- 1,6-bis(cyclohexyloximinocarbonylamino)hexane
- SB-334867
- 1-[2-methylbenzoxazol-6-yl]-3-[1,5]naphthyridin-4-yl-urea HCl
- S-BSA
- stripped bovine serum albumin
- THL
- tetrahydrolipstatin
- TLC
- thin-layer chromatography
- U-73122
- 1-[6-([(17b)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino)hexyl]-1H-pyrrole-2,5-dione
- URB597
- [3-(3-carbamoylphenyl)phenyl] N-cyclohexylcarbamate.
- Received February 1, 2012.
- Accepted May 1, 2012.
- Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics
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