Abstract
1-(4-Chlorophenyl)-3-[3-(6-pyrrolidin-1-ylpyridin-2-yl)phenyl] urea (PSNCBAM-1) has recently been described as a cannabinoid CB1 receptor allosteric antagonist associated with hypophagic effects in vivo; however, PSNCBAM-1 effects on CB1 ligand-mediated modulation of neuronal excitability remain unknown. Here, we investigate PSNCBAM-1 actions on CB1 receptor-stimulated guanosine 5′-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding in cerebellar membranes and on CB1 ligand modulation of presynaptic CB1 receptors at inhibitory interneuron-Purkinje cell synapses in the cerebellum using whole-cell electrophysiology. PSNCBAM-1 caused noncompetitive antagonism in [35S]GTPγS binding studies, with higher potency against the CB receptor agonist (−)-cis-3-[2-hydroxy-4-(1,1-dimethyl heptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol (CP55940) than for R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]-pyrrolo[1,2,3,-de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone mesylate] [WIN55,212-2 (WIN55)]. In electrophysiological studies, WIN55 and CP55940 reduced miniature inhibitory postsynaptic currents (mIPSCs) frequency but not amplitude. PSNCBAM-1 application alone had no effect on mIPSCs; however, PSNCBAM-1 pretreatment revealed agonist-dependent functional antagonism, abolishing CP55940-induced reductions in mIPSC frequency but having no clear effect on WIN55 actions. The CB1 antagonist/inverse agonist N-(piperidin-1-yl)-1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-1H-multipyrazole-3-carboxamide (AM251) increased mIPSC frequency beyond control; this effect was reversed by PSNCBAM-1. PSNCBAM-1 pretreatment also attenuated AM251 effects. Thus, PSNCBAM-1 reduced CB1 receptor ligand functional efficacy in the cerebellum. The differential effect of PSNCBAM-1 on CP55940 versus WIN55 actions in [35S]GTPγS binding and electrophysiological studies and the attenuation of AM251 effects are consistent with the ligand-dependence associated with allosteric modulation. These data provide the first description of functional PSNCBAM-1 allosteric antagonist effects on neuronal excitability in the mammalian central nervous system (CNS). PSNCBAM-1 allosteric antagonism may provide viable therapeutic alternatives to orthosteric CB1 antagonists/inverse agonists in the treatment of CNS disease.
Footnotes
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The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material.
This work was funded by an Ataxia UK Studentship and by a University of Reading International Postgraduate Research Studentship.
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/mol.110.068197.
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ABBREVIATIONS:
- GPCR
- G protein-coupled receptor
- AM251
- N-(piperidin-1-yl)-1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-1H-multipyrazole-3-carboxamide
- CP55940
- (−)-cis-3-[2-hydroxy-4-(1,1-dimethyl heptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol; PSNCBAM-1,1-(4-chlorophenyl)-3-[3-(6-pyrrolidin-1-ylpyridin-2-yl)phenyl]urea
- WIN55,212-2 (WIN55)
- R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]-pyrrolo[1,2,3,-de]-1,4-benzoxazinyl]-(1-naphthalenyl) methanone mesylate
- IN
- interneuron
- PC
- Purkinje cell
- mIPSC
- miniature inhibitory postsynaptic current
- CB
- cannabinoid
- aCSF
- artificial cerebrospinal fluid
- TTX
- tetrodotoxin
- ANOVA
- analysis of variance
- HSD
- honestly significant difference
- Δ9-THCV
- Δ9-tetrahydrocannabivarin
- NBQX
- 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline
- SR141716A
- N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride
- [35S]GTPγS
- guanosine 5′-O-(3-[35S]thio)triphosphate
- O-1602
- 5-methyl-4-[(1R,6R)-3-methyl-6-(1-methylethenyl)-2-cyclohexen-1-yl]-1,3-benzenediol.
- Received August 21, 2010.
- Accepted December 15, 2010.
- Copyright © 2011 The American Society for Pharmacology and Experimental Therapeutics
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