切口痛大鼠脊髓背角AMPA受体GluR1和GluR2亚基在突触膜表达的差异变化

doi:10.3969/j.issn.1006-7795.2013.05.001

摘要/Abstract

摘要：

目的研究切口痛大鼠术后不同时间点使君子酸(α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid, AMPA)受体亚基谷氨酸受体1(GluR1)和受体2(GluR2)在脊髓背角组织突触膜中的表达变化。方法将32只成年雄性SD大鼠,采用抽签法随机分成4组(n=8):Ⅰ组(正常对照组)、Ⅱ组(术后3 h组)、Ⅲ组(术后1 d组)和Ⅳ组(术后3 d组)。Ⅱ~Ⅳ组大鼠按照Brennan法制作右足底切口痛模型,并分别在术后3 h、1 d和3 d检测大鼠累计疼痛评分(cumulative pain scores, CPS)和机械痛阈(paw withdrawal thresholds,PWT)后处死大鼠,取切口同侧L3~L6段脊髓背角组织。每组各取3只大鼠的脊髓背角组织,Western blotting法检测GluR1和GluR2在突触膜以及全细胞的表达。另选取每组各3只大鼠的脊髓背角组织,Western blotting检测全细胞Stargazin及磷酸化Stargazin蛋白的表达变化。结果术后3 h组(Ⅲ组)累计CPS显著高于Ⅰ组,随后下降但术后3 d组(Ⅲ组)仍稍高于Ⅰ组(P<0.05);Ⅱ~Ⅳ组大鼠PWT均显著低于Ⅰ组(P<0.01);与Ⅰ组相比,术后3 h、1 d和3 d切口同侧脊髓背角组织突触膜中GluR1表达显著增加,其中术后3 h GluR1在突触膜表达达高峰(P<0.05);同时,I~Ⅳ组大鼠GluR1全细胞水平表达差异无统计学意义(P>0.05)。Ⅰ~Ⅳ组GluR2在突触膜及全细胞水平的表达,差异无统计学意义(P>0.05)。与Ⅰ组比,Ⅱ~Ⅳ,全细胞水平Stargazin蛋白(Ser240/241)的磷酸化水平变化,差异无统计学意义(P>0.05)。结论组织损伤可诱导脊髓背角含GluR1亚基的AMPA受体向突触后膜募集,不改变突触后膜含GluR2亚基AMPA受体的表达水平,最终导致突触后膜Ca²⁺通透性AMPA受体含量增加,从而导致脊髓背角突触后膜兴奋性增加,有利于疼痛信号在脊髓背角的传导。同时,本实验证实,辅助AMPA受体突触靶向的Stargazin蛋白(Ser 240/241)磷酸化过程不参与AMPA受体的突触靶向过程。

关键词: AMPA受体, 突触转运, 疼痛, 手术

Abstract:

Objective To investigate the synaptic targeting changes of AMPA receptor GluR1 and GluR2 subunits in the spinal cord dorsal horns in rats with incisional pain.Methods Thirty-two adult male SD rats were randomly divided into four groups (n=8 in each): Group Ⅰ (control group), Group Ⅱ (3 h after incision), Group Ⅲ (1 d after incision), and Group Ⅳ (3 d after incision). The rats in Group Ⅱ-Ⅳ received right plantar incision and the cumulative pain scores (CPS) and paw withdrawal thresholds (PWT) were tested at 3 h, 1 d, and 3 d after incision, respectively. All rats were euthanized for harvesting L3-L6 spinal cord dorsal horns tissue ipsilateral to incision after pain behaviors were measured. Western blotting was used to test the expression of GluR1 and GluR2 in the crude synaptosomal membrane and total soluble fraction of spinal cord dorsal horns in each group (n=3). Western blotting was also used to test the Stargazin phosphorylation level (Ser240/241) in the spinal cord dorsal horns of each group (n=3). Results The CPSs peaked at 3 h after incision, and then was gradually decreased, but were a bit higher than those of in Group Ⅰ at 3d after incision. The PWTs in incisional groups were significantly lower than those of in Group Ⅰ. The expression of GluR1 in synaptosomal membrane was significantly increased in incisional groups as compared with Group Ⅰ and peaked in Group Ⅱ. In contrast, the expression of GluR1 in total solution of Group Ⅱ~Ⅳ has not significant changes compared to Group Ⅰ. The expression of GluR2 in synaptosomal membrane and total solution did not change among the groups. Compared with Group Ⅰ, Stargazin phosphorylation level of total solution in spinal cord dorsal horns in Group Ⅱ~Ⅳ has no significant differences. Conclusion Surgical injuries may induce the synaptic targeting of GluR1-containing subunit AMPA receptor into the synaptosomal membrane in the spinal cord dorsal horns, but not change the expression of GluR2-containing subunit AMPA receptor, which eventually increase GluR1/GluR2 ratio and Ca²⁺ permeable AMPA receptors in the synaptosomal membrane,and thus enhance the excitation of AMPA receptors in synaptosomal membrane which lead to the transduction of pain signals in spinal cord dorsal horns. Meanwhile, this experiment confirms, synaptic targeting of AMPA receptors assisted by Stargazin is not associated with the phosphorylation of Stargazin(Ser240/241).

Key words: α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor, synaptic trafficking, pain, surgery

中图分类号:

R614

时蓉, 王云, 郭瑞娟, 吴安石, 岳云. 切口痛大鼠脊髓背角AMPA受体GluR1和GluR2亚基在突触膜表达的差异变化[J]. 首都医科大学学报, 2013, 34(5): 639-645.

SHI Rong, WANG Yun, GUO Ruijuan, WU Anshi, YUE Yun. Differential changes of AMPA receptor GluR1 and GluR2 subunits synaptic targeting in the spinal cord dorsal horns in rats with incisional pain[J]. Journal of Capital Medical University, 2013, 34(5): 639-645.

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