title: The G protein-coupled cannabinoid-1 (CB1) receptor of mammalian brain: inhibition by phthalate esters in vitro.
abstract: This research examines the in vitro interaction of phthalate diesters and monoesters with the G protein-coupled cannabinoid 1 (CB(1)) receptor, a presynaptic complex involved in the regulation of synaptic activity in mammalian brain. The diesters, n-butylbenzylphthalate (nBBP), di-n-hexylphthalate (DnHP), di-n-butylphthalate (DnBP), di-2-ethylhexylphthalate (DEHP), di-isooctylphthalate (DiOP) and di-n-octylphthalate (DnOP) inhibited the specific binding of the CB(1) receptor agonist [(3)H]CP-55940 to mouse whole brain membranes at micromolar concentrations (IC(50)s: nBBP 27.4 μM; DnHP 33.9 μM; DnBP 45.9 μM; DEHP 47.4 μM; DiOP 55.4 μM; DnOP 75.2 μM). DnHP, DnBP and nBBP achieved full (or close to full) blockade of [(3)H]CP-55940 binding, whereas DEHP, DiOP and DnOP produced partial (55-70%) inhibition. Binding experiments with phenylmethane-sulfonylfluoride (PMSF) indicated that the ester linkages of nBBP and DnBP remain intact during assay. The monoesters mono-2-ethylhexylphthalate (M2EHP) and mono-isohexylphthalate (MiHP) failed to reach IC(50) at 150 μM and mono-n-butylphthalate (MnBP) was inactive. Inhibitory potencies in the [(3)H]CP-55940 binding assay were positively correlated with inhibition of CB(1) receptor agonist-stimulated binding of [(35)S]GTPγS to the G protein, demonstrating that phthalates cause functional impairment of this complex. DnBP, nBBP and DEHP also inhibited binding of [(3)H]SR141716A, whereas inhibition with MiHP was comparatively weak and MnBP had no effect. Equilibrium binding experiments with [(3)H]SR141716A showed that phthalates reduce the B(max) of radioligand without changing its K(d). DnBP and nBBP also rapidly enhanced the dissociation of [(3)H]SR141716A. Our data are consistent with an allosteric mechanism for inhibition, with phthalates acting as relatively low affinity antagonists of CB(1) receptors and cannabinoid agonist-dependent activation of the G-protein. Further studies are warranted, since some phthalate esters may have potential to modify CB(1) receptor-dependent behavioral and physiological outcomes in the whole animal.
authors.affiliation: Department of Biological Sciences, Simon Fraser University, 8888 University Drive, Burnaby, British Columbia, Canada.
authors.forename: Kathleen M
authors.initials: KM
authors.lastname: Bisset
authors.forename: Amey S
authors.initials: AS
authors.lastname: Dhopeshwarkar
authors.forename: Chengyong
authors.initials: C
authors.lastname: Liao
authors.forename: Russell A
authors.initials: RA
authors.lastname: Nicholson
mesh_terms: D000818:Animals; D048588:Benzoxazines / Q000378:metabolism; D003511:Cyclohexanols / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D051379:Mice; D009025:Morpholines / Q000378:metabolism; D009281:Naphthalenes / Q000378:metabolism; D010795:Phthalic Acids / Q000494:pharmacology*; D010880:Piperidines / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding; D011720:Pyrazoles / Q000378:metabolism / Q000494:pharmacology; D043884:Receptor, Cannabinoid, CB1 / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D000077285:Rimonabant
chemical_list: D048588:Benzoxazines; D003511:Cyclohexanols; D009025:Morpholines; D009281:Naphthalenes; D010795:Phthalic Acids; D010880:Piperidines; D011720:Pyrazoles; D043884:Receptor, Cannabinoid, CB1; D043562:Receptors, G-Protein-Coupled; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C103505:AM 251; C070417:(3R)-((2,3-dihydro-5-methyl-3-((4-morpholinyl)methyl)pyrrolo-(1,2,3-de)-1,4-benzoxazin-6-yl)(1-naphthalenyl))methanone; C054649:3-(2-hydroxy-4-(1,1-dimethylheptyl)phenyl)-4-(3-hydroxypropyl)cyclohexanol; D000077285:Rimonabant