Nakagawa T, Ozawa T, Watanabe T, Minami M, Satoh M
Department of Molecular Pharmacology, Faculty of Pharmaceutical Sciences, Kyoto University, Japan.
Nihon Yakurigaku Zasshi. 1998 Oct;112 Suppl 1:78P-82P. doi: 10.1254/fpj.112.supplement_78.
Chronic opioid treatment has been shown to develop supersensitization of adenylyl cyclase (AC) system or cAMP overshoot. In this study, we investigated the molecular mechanism of supersensitization of AC system using CHO cells expressing one of the cloned mu-, delta- and kappa-opioid receptors. In naive cells, acute treatment with an opioid agonist, but not antagonist, suppressed forskolin-stimulated cAMP accumulation. In the cells sustainedly (4 hr) treated with the agonist, the challenge by antagonist induced the cAMP overshoot over the naive level (supersensitization of AC system), but had no effect on GTPase activity. This supersensitization of AC system was not affected by pretreatment with cycloheximide, a protein synthesis inhibitor, or various protein kinase inhibitors (H7, H8, H89 and staurosporine). On the other hand, pretreatment with pertussis toxin blocked both inhibition of AC activity by acute agonist treatment and development of supersensitization of AC system. To examine an involvement of the interaction between G protein and AC in the supersensitization of AC system, we used CHO cells coexpressing the opioid receptor and some chimeric G alpha proteins between G alpha i2 and G alpha q. The results revealed that a specific region of G alpha i2, which is responsible for the interaction with AC, is closely related to the supersensitization. In addition, the supersensitization of AC system was induced by sustained muscarinic agonist treatment in CHO cells expressing the cloned m2 or m4 muscarinic receptor, suggesting this phenomenon is common to the members of the Gi-coupled receptor superfamily. In conclusion, these findings suggest that the development of supersensitization of AC system is attributed to a continuous inhibition of AC by G alpha i, but not to continuous activations of the Gi-coupled receptor and G protein themselves.
慢性阿片类药物治疗已被证明会导致腺苷酸环化酶（AC）系统超敏或环磷酸腺苷（cAMP）过冲。在本研究中，我们使用表达克隆的μ-、δ-和κ-阿片受体之一的CHO细胞，研究了AC系统超敏的分子机制。在未处理的细胞中，阿片类激动剂急性处理可抑制福斯高林刺激的cAMP积累，但拮抗剂则无此作用。在用激动剂持续处理（4小时）的细胞中，拮抗剂刺激会诱导cAMP超过未处理时的水平（AC系统超敏），但对GTP酶活性无影响。AC系统的这种超敏不受蛋白质合成抑制剂环己酰亚胺或各种蛋白激酶抑制剂（H7、H8、H89和星形孢菌素）预处理的影响。另一方面，百日咳毒素预处理可阻断激动剂急性处理对AC活性的抑制以及AC系统超敏的发生。为了研究G蛋白与AC之间的相互作用在AC系统超敏中的作用，我们使用了共表达阿片受体和一些Gαi2与Gαq之间的嵌合Gα蛋白的CHO细胞。结果显示，Gαi2中负责与AC相互作用的特定区域与超敏密切相关。此外，在表达克隆的M2或M4毒蕈碱受体的CHO细胞中，毒蕈碱激动剂持续处理可诱导AC系统超敏，这表明这种现象在Gi偶联受体超家族成员中很常见。总之，这些发现表明，AC系统超敏的发生归因于Gαi对AC的持续抑制，而非Gi偶联受体和G蛋白自身的持续激活。
