Ammer H, Schulz R
Institute of Pharmacology, Toxicology and Pharmacy, University of Munich, Germany.
Brain Res. 1996 Jan 29;707(2):235-44. doi: 10.1016/0006-8993(95)01265-6.
Chronic exposure of all-trans-retinoic acid-differentiated SH-SY5Y cells to morphine (10 mu M; 2 days) results in sensitization of adenylate cyclase as characterized by a significant increase in both PGE1 receptor-mediated as well as receptor-independent (NaF, 10 mM; forskolin, 100 mu M) stimulation of effector activity. To investigate the underlying biochemical alterations, chronic opioid regulation of each of the components comprising the stimulatory PGE1 receptor system was examined. On receptor level, chronic morphine treatment was found to reduce PGE1 receptor number (Bmax) by approximately 40%, whereas their affinity slightly increased. Binding experiments performed in the presence of GTPgammaS (100 mu M) further indicate that the decrease in PGE1 receptor density is associated with a loss of functionally G protein-coupled receptors. On post-receptor level, chronic morphine treatment substantially increased the abundance and functional activity of stimulatory G proteins, as assessed by cholera toxin-catalyzed ADP-ribosylation of GSalpha and S49 cyc- reconstitution assays. No changes were found on the level of adenylate cyclase. Evaluation of the functional interaction between PGE1 receptors and GS in situ by application of a C-terminal anti-GSalpha antibody revealed a more intense coupling efficiency between these two entities, since a significant higher amount of antibody (2.3-fold) was required in morphine dependent cell membranes to half-maximally attenuate PGE1 receptor-stimulated adenylate cyclase activity. In addition, limitation of the amount of functionally available GSalpha within the PGE1 receptor/adenylate cyclase signal transduction cascade abolished the generation of a supersensitive adenylate cyclase response during the state of naloxone (100 mu M)-precipitated withdrawal. These data demonstrate that in human neuroblastoma SH-SY5Y cells chronic morphine-induced sensitization of adenylate cyclase is associated with distinct quantitative and qualitative adaptations within the stimulatory adenylate cyclase-coupled PGE1 receptor system. Thus, alterations in the functional activity of stimulatory receptor systems are suggested to contribute to the cellular mechanisms underlying opioid dependence.
将全反式维甲酸分化的SH-SY5Y细胞长期暴露于吗啡（10 μM；2天）会导致腺苷酸环化酶致敏，其特征是PGE1受体介导的以及受体非依赖性（NaF，10 mM；福司可林，100 μM）效应器活性刺激均显著增加。为了研究潜在的生化改变，检测了构成刺激性PGE1受体系统的各组分的慢性阿片类药物调节情况。在受体水平上，发现慢性吗啡处理可使PGE1受体数量（Bmax）减少约40%，而其亲和力略有增加。在存在GTPγS（100 μM）的情况下进行的结合实验进一步表明，PGE1受体密度的降低与功能性G蛋白偶联受体的丧失有关。在受体后水平上，慢性吗啡处理显著增加了刺激性G蛋白的丰度和功能活性，这通过霍乱毒素催化的GSα ADP核糖基化和S49 cyc重组试验进行评估。在腺苷酸环化酶水平上未发现变化。通过应用C末端抗GSα抗体原位评估PGE1受体与GS之间的功能相互作用，发现这两个实体之间的偶联效率更高，因为在吗啡依赖的细胞膜中，需要显著更多量的抗体（2.3倍）才能将PGE1受体刺激的腺苷酸环化酶活性半最大程度地减弱。此外，限制PGE1受体/腺苷酸环化酶信号转导级联中功能性可用GSα的量，消除了纳洛酮（100 μM）诱发的戒断状态期间超敏腺苷酸环化酶反应的产生。这些数据表明，在人神经母细胞瘤SH-SY5Y细胞中，慢性吗啡诱导的腺苷酸环化酶致敏与刺激性腺苷酸环化酶偶联的PGE1受体系统内明显的定量和定性适应性变化有关。因此，刺激性受体系统功能活性的改变被认为有助于阿片类药物依赖的细胞机制。
