Abstract
This research examines the in vitro interaction of phthalate diesters and monoesters with the G protein-coupled cannabinoid 1 (CB1) 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 CB1 receptor agonist [3H]CP-55940 to mouse whole brain membranes at micromolar concentrations (IC50s: nBBP 27.4μM; DnHP 33.9μM; DnBP 45.9μM; DEHP 47.4μM; DiOP 55.4μM; DnOP 75.2μM). DnHP, DnBP and nBBP achieved full (or close to full) blockade of [3H]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 IC50 at 150μM and mono-n-butylphthalate (MnBP) was inactive. Inhibitory potencies in the [3H]CP-55940 binding assay were positively correlated with inhibition of CB1 receptor agonist-stimulated binding of [35S]GTPγS to the G protein, demonstrating that phthalates cause functional impairment of this complex. DnBP, nBBP and DEHP also inhibited binding of [3H]SR141716A, whereas inhibition with MiHP was comparatively weak and MnBP had no effect. Equilibrium binding experiments with [3H]SR141716A showed that phthalates reduce the Bmax of radioligand without changing its Kd. DnBP and nBBP also rapidly enhanced the dissociation of [3H]SR141716A. Our data are consistent with an allosteric mechanism for inhibition, with phthalates acting as relatively low affinity antagonists of CB1 receptors and cannabinoid agonist-dependent activation of the G-protein. Further studies are warranted, since some phthalate esters may have potential to modify CB1 receptor-dependent behavioral and physiological outcomes in the whole animal.
Published Version
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