Ammer H, Schulz R
Institute of Pharmacology, Toxicology and Pharmacy, University of Munich, Germany.
Mol Pharmacol. 1997 Dec;52(6):993-9. doi: 10.1124/mol.52.6.993.
Chronic opioid treatment of stably mu-opioid receptor transfected human mammary epidermoid A431 carcinoma cells (clone A431/mu 13) results in sensitization of adenylyl cyclase (AC), a cellular adaptation associated with drug dependence. Up-regulation of AC is characterized by significantly increased levels of both basal and post-receptor-stimulated effector activities, which develop without any apparent change in the quantity of stimulatory G proteins and the maximum catalytic activity of AC. Here, we report that detergent extracts from membranes of chronically morphine-treated (10 microM; 2 days) A431/mu 13 cells display higher stimulatory AC activities as assessed in the S49cyc- reconstitution assay. This finding is most likely due to an increased functional activity of Gs alpha because the addition of exogenous G beta gamma subunits, which per se stimulate AC in S49cyc- membranes, failed to affect the difference in reconstitutive AC activity. Moreover, both chemical depalmitoylation by hydroxylamine and inhibition of palmitoyl-CoA transferase in vivo by tunicamycin treatment incresed the reconstitutive activity of detergent extracts and eliminated the differences between native and opioid-dependent cells, indicating that the increase in stimulatory activity is due to depalmitoylation of Gs alpha. Indeed, metabolic labelling studies with [3H]palmitic acid revealed that chronic opioid treatment reduces considerably the fraction of palmitoylated Gs alpha in the plasma membrane. Furthermore, high affinity [3H]forskolin binding experiments demonstrated that depalmitoylated Gs alpha is able to associated directly with AC during the state of opioid dependence even without preceding receptor activation. These results suggest that post-translational palmitoylation of Gs alpha provides a potential regulator of transmembrane signaling. Moreover, accumulation of the depalmitoylated form of Gs alpha in the plasma membrane as reported herein may contribute to the increase in stimulatory AC signaling, as is characteristic for the state of opioid dependence.
对稳定转染μ-阿片受体的人乳腺表皮样A431癌细胞（克隆A431/μ 13）进行慢性阿片类药物处理，会导致腺苷酸环化酶（AC）敏化，这是一种与药物依赖性相关的细胞适应性变化。AC的上调表现为基础效应和受体刺激后效应活性水平显著增加，而刺激型G蛋白的数量和AC的最大催化活性并未发生明显变化。在此，我们报告，在S49cyc-重组试验中评估发现，经慢性吗啡处理（10 microM；2天）的A431/μ 13细胞膜的去污剂提取物显示出更高的刺激AC活性。这一发现很可能是由于Gsα功能活性增加，因为添加外源性Gβγ亚基（其本身可刺激S49cyc-膜中的AC）并未影响重组AC活性的差异。此外，用羟胺进行化学去棕榈酰化以及用衣霉素在体内抑制棕榈酰辅酶A转移酶，均增加了去污剂提取物的重组活性，并消除了天然细胞和阿片类药物依赖性细胞之间的差异，表明刺激活性的增加是由于Gsα的去棕榈酰化。实际上，用[3H]棕榈酸进行的代谢标记研究表明，慢性阿片类药物处理可显著降低质膜中棕榈酰化Gsα的比例。此外，高亲和力[3H]福司可林结合实验表明，去棕榈酰化的Gsα即使在没有预先受体激活的情况下，在阿片类药物依赖性状态下也能够直接与AC结合。这些结果表明，Gsα的翻译后棕榈酰化提供了一种跨膜信号传导的潜在调节机制。此外，本文报道的去棕榈酰化形式的Gsα在质膜中的积累可能有助于刺激型AC信号传导的增加，这是阿片类药物依赖性状态的特征。
