Department of Biological Sciences, Simon Fraser University, 8888 University Drive, Burnaby, British Columbia, Canada.
Neurochem Int. 2011 Oct;59(5):706-13. doi: 10.1016/j.neuint.2011.06.019. Epub 2011 Jul 7.
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.
本研究考察了邻苯二甲酸二酯和单酯与 G 蛋白偶联大麻素 1（CB（1））受体的体外相互作用，该受体是参与调节哺乳动物大脑中突触活动的突触前复合物。二酯，正丁基苄基邻苯二甲酸酯（nBBP）、二正己基邻苯二甲酸酯（DnHP）、二正丁基邻苯二甲酸酯（DnBP）、二-2-乙基己基邻苯二甲酸酯（DEHP）、二异辛基邻苯二甲酸酯（DiOP）和二正辛基邻苯二甲酸酯（DnOP）以微摩尔浓度抑制 CB（1）受体激动剂 [（3）H]CP-55940 与小鼠全脑膜的特异性结合（IC（50）：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 和 nBBP 实现了 [（3）H]CP-55940 结合的完全（或接近完全）阻断，而 DEHP、DiOP 和 DnOP 产生了部分（55-70%）抑制。用苯甲磺酰氟（PMSF）进行的结合实验表明，nBBP 和 DnBP 的酯键在测定过程中保持完整。单酯单-2-乙基己基邻苯二甲酸酯（M2EHP）和单异己基邻苯二甲酸酯（MiHP）在 150 μM 时未达到 IC（50），而单正丁基邻苯二甲酸酯（MnBP）则没有活性。[（3）H]CP-55940 结合测定中的抑制效力与 CB（1）受体激动剂刺激的 [（35）S]GTPγS 与 G 蛋白结合的抑制呈正相关，表明邻苯二甲酸酯会导致该复合物的功能障碍。DnBP、nBBP 和 DEHP 还抑制 [（3）H]SR141716A 的结合，而 MiHP 的抑制作用相对较弱，MnBP 没有作用。用 [（3）H]SR141716A 进行的平衡结合实验表明，邻苯二甲酸酯降低了放射性配体的 B（max），而不改变其 K（d）。DnBP 和 nBBP 还迅速增强了 [（3）H]SR141716A 的解离。我们的数据与抑制的变构机制一致，邻苯二甲酸酯作为 CB（1）受体的相对低亲和力拮抗剂和大麻素激动剂依赖性 G 蛋白激活。由于一些邻苯二甲酸酯可能有潜力改变整个动物中 CB（1）受体依赖性行为和生理结果，因此需要进一步研究。
