Ammer H, Schulz R
Institute of Pharmacology, Toxicology and Pharmacy, University of Munich, Germany.
J Pharmacol Exp Ther. 1997 Jan;280(1):512-20.
Chronic opioid regulation of stimulatory beta-2 adrenoceptor (beta-2 AR) signaling was investigated in human mammary epidermoid carcinoma A431 cells stably expressing the cloned rat mu opioid receptor. In the cell clone used (A431/mu 13; Bmax = 302.9 +/- 46 fmol/mg membrane protein), the addition of morphine acutely attenuated basal as well as (-)-isoproterenol-stimulated cAMP accumulation. Prolonged exposure of the cells to morphine (10 microM; 2 d) resulted in homologous desensitization of MOR function as well as heterologous sensitization of adenylate cyclase (AC). Up-regulation of AC in A431/mu 13 cells is characterized by an increased capacity rather than an increased sensitivity of beta-2 AR-stimulated AC. Moreover, opioid withdrawal falls to precipitate a cAMP overshoot in this cell system. Sensitization of stimulatory AC signaling by chronic morphine develops in a time- and dose-dependent manner and is blocked by both naloxone and pertussis toxin. Investigation into the mechanism leading to up-regulation of AC revealed a 40% increase in the number of beta-2 ARs as assessed by [125I]-cyanopindolol binding experiments. No additional quantitative changes were found for stimulatory G proteins and the effector enzyme itself. Sensitization of AC appears to be mediated solely by the increase in beta-2 AR numbers, because (+/-)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3- [(1-methylethyl)amino]-2-butanol hydrochloride, which acts as an "inverse agonist" at the beta-2 AR, completely reversed elevated basal AC activities, and because the ratio between functional active beta-2 ARs and stimulatory G proteins remained unchanged. In conclusion, chronic exposure of clonal A431/ mu13 cells to morphine increases the capacity of stimulatory AC signaling by up-regulating beta-2 AR number. These results demonstrate participation of stimulatory receptor systems in the cellular mechanisms underlying opioid dependence.
在稳定表达克隆大鼠μ阿片受体的人乳腺表皮样癌A431细胞中，研究了慢性阿片类药物对刺激性β-2肾上腺素能受体（β-2 AR）信号传导的调节作用。在所使用的细胞克隆（A431/mu 13；Bmax = 302.9 +/- 46 fmol/mg膜蛋白）中，吗啡的加入急性减弱了基础以及（-）-异丙肾上腺素刺激的cAMP积累。细胞长期暴露于吗啡（10 microM；2天）导致MOR功能的同源脱敏以及腺苷酸环化酶（AC）的异源敏化。A431/mu 13细胞中AC的上调表现为β-2 AR刺激的AC的能力增加而非敏感性增加。此外，在该细胞系统中，阿片类药物戒断并未引发cAMP过冲。慢性吗啡对刺激性AC信号的敏化以时间和剂量依赖性方式发展，并被纳洛酮和百日咳毒素阻断。对导致AC上调的机制的研究表明，通过[125I]-氰胍洛尔结合实验评估，β-2 AR的数量增加了40%。对于刺激性G蛋白和效应酶本身，未发现其他定量变化。AC的敏化似乎仅由β-2 AR数量的增加介导，因为作为β-2 AR的“反向激动剂”的（+/-）-1-[2,3-（二氢-7-甲基-茚-4-基）氧基]-3-[(1-甲基乙基)氨基]-2-丁醇盐酸盐完全逆转了基础AC活性的升高，并且因为功能性活性β-2 AR与刺激性G蛋白之间的比例保持不变。总之，克隆的A431/mu13细胞长期暴露于吗啡通过上调β-2 AR数量增加了刺激性AC信号传导的能力。这些结果证明了刺激性受体系统参与了阿片类药物依赖的细胞机制。
