Functional coupling, desensitization and internalization of virally expressed μ opioid receptors in cultured dorsal root ganglion neurons from μ opioid receptor knockout mice
Functional coupling, desensitization and internalization of virally expressed μ opioid receptors in cultured dorsal root ganglion neurons from μ opioid receptor knockout mice
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DOI：
10.1016/j.neuroscience.2003.08.060
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发表时间：
2004-01-01
期刊：
影响因子：
3.3
通讯作者：
Maidment, NT
中科院分区：
文献类型：
--
作者：
Walwyn, WM;Keith, DE;Maidment, NT
尽管μ倍体受体在体外多种细胞系中脱敏，但这种信号传导功效的变化与体内耐受性发展的关系仍不确定。很明显。需要一种系统，其中在适当的神经元中获得功能性μ阿片受体表达，以便可以测量、操纵脱敏并突变在此环境中表达的受体。我们开发了一种重组系统，其中带标记的 mu 阿片受体的表达在体外从 mu 阿片受体敲除小鼠的背根神经节神经元中恢复。流式细胞术分析表明，腺病毒介导的神经元中氨基末端标记的μ阿片受体的表达导致大约1.3 x 10(6)个受体/细胞。许多μ阿片受体细胞系表达相似的受体密度，但这比匹配的野生型神经元表达的内源性受体数量大约高7倍。 mu 激动剂、D-Ala(2)、N-MePhe(4)、Gly(5)-ol-脑啡肽 (DAMGO) 对背根神经节神经元中高电压激活的钙电流的抑制作用，在使用的几种 DAMGO 浓度下，内源性受体和标记受体之间没有差异。在 DAMGO 预孵育的前 6 小时内，两种受体的脱敏程度相同，但 24 小时后，内源性受体对 DAMGO 的反应比标记的受体进一步脱敏（分别为 71+/-3% vs 29+/-7%；P
Although mu oploid receptors desensitize in various cell lines in vitro, the relationship of this change in signaling efficacy to the development of tolerance in vivo remains uncertain. It is clear that. a system is needed in which functional mu opioid receptor expression is obtained in appropriate neurons so that desensitization can be measured, manipulated, and mutated receptors expressed in this environment. We have developed a recombinant system in which expression of a flag-tagged mu opioid receptor is returned to dorsal root ganglia neurons from mu opioid receptor knockout mice in vitro. Flow cytometry analysis showed that adenoviral-mediated expression of the amino-terminal flag-tagged mu opioid receptor in neurons resulted in approximately 1.3 x 10(6) receptors/cell. Many mu opioid receptor cell lines express a similar density of receptors but this is approximately 7x greater than the number of endogenous receptors expressed by matched wild-type neurons. Inhibition of the high voltage-activated calcium currents in dorsal root ganglia neurons by the mu agonist, D-Ala(2), N-MePhe(4), Gly(5)-ol-enkephalin (DAMGO), was not different between the endogenous and flag-tagged receptor at several concentrations of DAMGO used. Both receptors desensitized equally over the first 6 h of DAMGO pre-incubation, but after 24 h the response of the endogenous receptor to DAMGO had desensitized further than the flag-tagged receptor (71+/-3 vs 29+/-7% respectively; P