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.