Rinaldi-Carmona M, Le Duigou A, Oustric D, Barth F, Bouaboula M, Carayon P, Casellas P, Le Fur G
Sanofi Recherche, 371 rue du Professeur J. Blayac, 34184 Montpellier CEDEX 04, France.
J Pharmacol Exp Ther. 1998 Dec;287(3):1038-47.
We have investigated the adaptive changes of the human central cannabinoid receptor (CB1) stably expressed in Chinese hamster ovary cells (CHO-CB1), after agonist (CP 55,940) or selective CB1 inverse agonist (SR 141716) treatment. CB1 receptor density and affinity constant as measured by binding assays with both tritiated ligands remained essentially unchanged after varying period exposure of CHO-CB1 cells (from 30 min to 72 hr) to saturating concentrations of CP 55,940 or SR 141716. However, using a C-myc-tagged version of the CB1 receptor, FACS analysis and confocal microscopy studies on CB1 expression indicated that the agonist promoted a disappearance of cell surface receptor although inverse agonist increased its cell surface density. Taken together these results suggest that 1) agonist induces internalization of the receptor into a cellular compartment that would be still accessible to both the hydrophobic ligands CP 55,940 or SR 141716; 2) inverse-agonist promotes externalization of the receptor from an intracellular preexisting pool to the cell surface. In parallel, we also investigated the associated effects of CP 55,940 and SR 141716 on CB1 receptor-coupled second messengers. We showed that preexposure of cells to CP 55,940 induced a rapid desensitization of the CB1 to the agonist response. The ability of CP 55,940 to inhibit the forskolin-stimulated adenylyl cyclase and to activate the mitogen-activated protein kinase activity was dramatically reduced. By striking contrast, SR 141716 pretreatment of CHO-CB1 cells not only had no significant effect on the potency of CP 55,940 to inhibit the forskolin-stimulated adenylyl cyclase but also induced a significant enhancement of the CP 55,940 ability to stimulate the mitogen-activated protein kinase activity. These results suggest that the modulation of the number of cell surface receptor could lead to functional desensitization or sensitization of the CB1 receptors.
我们研究了在中国仓鼠卵巢细胞（CHO-CB1）中稳定表达的人中枢大麻素受体（CB1）在激动剂（CP 55,940）或选择性CB1反向激动剂（SR 141716）处理后的适应性变化。在用氚标记配体的结合试验测量中，CHO-CB1细胞在不同时间段（从30分钟到72小时）暴露于饱和浓度的CP 55,940或SR 141716后，CB1受体密度和亲和常数基本保持不变。然而，使用带有C-myc标签的CB1受体版本，对CB1表达进行的荧光激活细胞分选（FACS）分析和共聚焦显微镜研究表明，激动剂促使细胞表面受体消失，而反向激动剂则增加其细胞表面密度。综合这些结果表明：1）激动剂诱导受体内化进入一个细胞区室，疏水性配体CP 55,940或SR 141716仍可进入该区域；2）反向激动剂促使受体从细胞内预先存在的库中转运到细胞表面。同时，我们还研究了CP 55,940和SR 141716对CB1受体偶联的第二信使的相关影响。我们发现，细胞预先暴露于CP 55,940会导致CB1对激动剂反应的快速脱敏。CP 55,940抑制福斯高林刺激的腺苷酸环化酶和激活丝裂原活化蛋白激酶活性的能力显著降低。与之形成鲜明对比的是，对CHO-CB1细胞进行SR 141716预处理不仅对CP 55,940抑制福斯高林刺激的腺苷酸环化酶的效力没有显著影响，而且还显著增强了CP 55,940刺激丝裂原活化蛋白激酶活性的能力。这些结果表明，细胞表面受体数量的调节可能导致CB1受体的功能性脱敏或致敏。
