Gβγ亚基在去甲肾上腺素α2A受体抑制慢性炎性疼痛中的作用

发布时间：2018-07-10 10:44

本文选题：去甲肾上腺素α2A受体 + C-末端Src激酶　；参考：《兰州大学》2015年硕士论文

【摘要】：目的：去甲肾上腺素α2受体和抑制性Gi蛋白相偶联,通过Gi蛋白α亚基(Giα)和βγ亚基(Gβγ)触发多种细胞内信号转导通路。α2受体包含多种亚型；其中,A型去甲肾上腺素α2受体(α2A受体)表达于脊髓背角、并在痛觉信息的调控中发挥着至关重要的作用。目前为止,绝大多数实验探讨了Giα亚基诱发的cAMP信号通路与痛觉调制的关系；但Gβγ亚基的作用尚不清楚。本研究的目的,在于探讨Gβγ通路在α2A受体抑制慢性炎性疼痛中的作用及其分子机制。方法：本研究给小鼠后足底皮下注射完全弗氏佐剂(Complete Freund's Adjuvant, CFA)、建立慢性炎性疼痛模型；构建腺病毒表达载体,使脊髓背角表达Gβγ亚基的特异性阻断剂PARKct(即：195个氨基酸组成的β-肾上腺素受体激酶-1的胞浆C-末端序列)；鞘内注射a2A受体的选择性激动剂Guanfacine,通过痛行为学测试、免疫印迹、膜片钳电生理记录、免疫沉淀等方法,探讨了PARKct对 Guanfacine效应的影响。结果：(1)鞘内注射α2A受体激动剂Guanfacine (0.28μg),能够有效缓解CFA诱发的热痛觉过敏和机械性痛觉超敏,但这一作用可被PARKct拮抗；(2)α2A受体可能通过Gβγ亚基下调N-甲基-D-天冬氨酸(N-Methyl-D-Aspartate; NMD A)型谷氨酸受体的突触表达,因为βARKct会阻断Guanfacine对突触小体膜结构中NMDA受体GluNl/GluN2B亚基的抑制效应；(3)免疫印迹实验显示：α2A受体通过Gβγ亚基降低GluN2B亚基第1472位酪氨酸残基(GluN2B-Tyrl472)的磷酸化水平,从而诱发包含GluN2B亚基的NMDA受体的内陷；(4)免疫沉淀实验显示：GluN2B亚基的催化激酶---Src,可能是α2A受体/Gβγ亚基的重要调控靶点,因为：Guanfacine能够降低Src的第418位酪氨酸残基(Tyr418)的磷酸化、同时升高其529位酪氨酸残基(Tyr529)的磷酸化,而预先表达βARKct则拮抗Guanfacine对Src活性的影响；(5)α2A受体/Gβγ亚基对Src的调节作用,可能与C-末端-Src激酶(CSK)有关；我们发现：βARKct能够取消Guanfacine诱导的炎性疼痛小鼠脊髓背角CSK的突触再分布；(6)为直接探讨CSK的痛觉调控作用,我们构建重组腺病毒载体,使脊髓表达无活性的CSK突变体CSK(K222R),结果发现：在正常动物,CSK(K222R)能够升高GluN2B-Tyr1472的磷酸化水平；而在炎性疼痛动物,CSK(K222R)会阻断Guanfacine对突触NMDA受体的调节效应；(7)尤为重要的是：通过重组腺病毒载体、使炎性疼痛动物的脊髓背角表达野生型CSK[CSK(WT)],能够产生和Guanfacine相似的效应,即：降低GluNl/GluN2B亚基的突触表达水平,逆转GluN2B-Tyr1472的磷酸化,阻断NMDA受体介导的痛觉突触传递,有效缓解慢性炎性疼痛症状。结论：去甲肾上腺素α2A受体,能够通过Gβγ/CSK信号通路,抑制Src对GluN2B的磷酸化、降低GluN2B的突触表达,这可能是α2A受体干预慢性炎性疼痛的一条重要途径。
[Abstract]:Objective: to couple norepinephrine 伪 2 receptor with inhibitory GI protein, and trigger many intracellular signal transduction pathways via GI 伪 subunit (GI 伪) and 尾纬 subunit (G 尾纬). Type A norepinephrine 伪 2 receptor (伪 2A receptor) is expressed in the dorsal horn of spinal cord and plays an important role in the regulation of pain information. Up to now, most experiments have explored the relationship between camp signaling pathway induced by GI 伪 subunit and pain modulation, but the role of G 尾纬 subunit is not clear. The purpose of this study was to investigate the role of G 尾纬 pathway in 伪 2A receptor inhibiting chronic inflammatory pain and its molecular mechanism. Methods: in this study, a chronic inflammatory pain model was established by subcutaneous injection of complete FreundsAdjuvant (CFA) into the posterior plantar of mice, and adenovirus expression vector was constructed. PARKct, a specific inhibitor of G 尾纬 subunit in spinal dorsal horn, was injected intrathecally with Guanfacine, a selective agonist of a2A receptor. The effects of PARKct on Guanfacine effect were investigated by patch clamp electrophysiological recording and immunoprecipitation. Results: (1) Intrathecal injection of 伪 2A receptor agonist Guanfacine (0. 28 渭 g),) could effectively relieve thermal hyperalgesia and mechanical hyperalgesia induced by CFA, but this effect could be antagonized by PARKct. (2) 伪 2A receptor may down-regulate the synaptic expression of N-Methyl-D-Aspartate (NMD A) glutamate receptor through G 尾纬 subunit, because 尾 ARKct can block the inhibitory effect of Guanfacine on the NMDA receptor GluNl / GluN2B subunit in the membrane structure of synaptosome. (3) Western blot showed that 伪 2A receptor reduced the phosphorylation level of the 1472 tyrosine residue (GluN2B-Tyrl472) of GluN2B subunit through G 尾纬 subunit, which induced the invagination of NMDA receptor containing GluN2B subunit. (4) Immunoprecipitation assay showed that the catalytic kinase -Src of the 1: GluN2B subunit may be an important regulatory target of 伪 2A receptor / G 尾纬 subunit, because the tyrosine residue (Tyr418) phosphorylation of the 418th position of Src can be reduced by the weight Guanfacine. At the same time, the phosphorylation of Tyr529 tyrosine residue (Tyr529) was increased, and the effect of Guanfacine on SRC activity was antagonized by 尾 ARKct. (5) the regulation of SRC by 伪 2A receptor / G 尾纬 subunit may be related to C-terminal -Src kinase (CSK). We found that 尾 ARKct could cancel the synaptic redistribution of CSK in the spinal dorsal horn of Guanfacine induced inflammatory pain mice. (6) in order to investigate the pain regulation of CSK directly, we constructed recombinant adenovirus vector. CSK mutant CSK (K222R), which made spinal cord expression inactive, was found that CSK (K222R) could increase the phosphorylation level of GluN2B-Tyr1472 in normal animals, while in inflammatory pain animals, CSK (K222R) blocked the regulatory effect of Guanfacine on synaptic NMDA receptor. (7) it is particularly important that the expression of wild type CSK [CSK (WT)] in the spinal dorsal horn of inflammatory pain animals can be induced by recombinant adenovirus vector, which can produce a similar effect to Guanfacine, that is, to reduce the synaptic expression level of GluNl / GluN2B subunit and reverse the phosphorylation of GluN2B-Tyr1472. Blocking NMDA receptor mediated nociceptive synaptic transmission effectively alleviates chronic inflammatory pain symptoms. Conclusion: norepinephrine 伪 2A receptor can inhibit Src phosphorylation of GluN2B and decrease the synaptic expression of GluN2B through G 尾纬 -CSK signaling pathway, which may be an important pathway for 伪 2A receptor to interfere with chronic inflammatory pain.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R402

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