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N 型胆碱能受体参与淀粉样β 蛋白片段引起的大鼠大脑皮层神经元胞内钙水平升高

武美娜1, 李新毅, 郭芬, 祁金顺

山西医科大学 生理学系；第一临床医学院神经内科，太原030001

摘要

淀粉样β 蛋白(amyloid β-protein, Aβ)诱导的细胞内钙稳态失调被认为是Aβ 所致神经元损伤的最后通路。然而，Aβ导致钙超载的机制，尤其是Aβ 所致的细胞内钙浓度([Ca2+]i)升高是否与烟碱受体相关尚需进一步研究。本实验用激光扫描共聚焦显微成像技术观察了Aβ25-35 和Aβ31-35 对原代培养大鼠皮层神经元[Ca2+]i 的作用，探讨了其N 型胆碱能受体机制。结果显示：(1) Aβ25-35 可剂量依赖性地引起皮层神经元[Ca2+]i 升高；(2) Aβ 的更小片段Aβ31-35 也能增加[Ca2+]i，其效应与Aβ25-35 相比没有显著性差异，但Aβ31-35 的反序列即Aβ35-31 对[Ca2+]i 无明显影响；(3)使用烟碱受体的非特异性拮抗剂美加明(mecamylamine,MCA)预处理后，Aβ 25-35 和Aβ31-35 诱导的[Ca2+]i 升高效应被明显抑制，并表现出一定程度的剂量依赖性；(4)使用α4β2 亚型烟碱受体拮抗剂二氢-β- 刺桐啶碱(dihydro-β-erythroidine, D-β-E)同样能够部分阻断Aβ25-35 和Aβ31-35 诱导的[Ca2+]i 升高，但其作用弱于相同浓度的MCA。这些结果表明，两种Aβ 片段均能引起培养大鼠皮层神经元[Ca2+]i 增高，Aβ31-35 序列是Aβ 分子中具有同样生物活性的更短片段；中枢烟碱受体的过度激活参与了Aβ25-35 和Aβ31-35 诱导的 [Ca2+]i 升高。由此提示，阿尔茨海默病(Alzheimer’s disease, AD)时发生的认知功能障碍可能与中枢烟碱受体激活引起的细胞内钙超载有关，N 型胆碱能受体可能 是AD 时Aβ 发挥神经毒作用的靶点之一。

关键词： N 型胆碱能受体; 淀粉样β 蛋白; 皮层神经元; 细胞内钙浓度

分类号：R329

Involvement of nicotinic acetylcholine receptors in amyloid β-fragmentinduced intracellular Ca2+ elevation in cultured rat cortical neurons

WU Mei-Na, LI Xin-Yi, GUO Fen, QI Jin-Shun

Department of Neurobiology； Department of Neurology, the First Hospital, Shanxi Medical University, Taiyuan 030001, China

Abstract

The amyloid β-protein (Aβ)-induced disturbance of intracellular calcium homeostasis has been regarded as the final route whereby Aβ insults neurons. However, the mechanism of Aβ-induced Ca2+ overloading is still unclear so far. Especially, it remains to be clarified whether nicotinic acetylcholine receptors (nAChRs) are involved in the Aβ-induced elevation of intracellular calcium concentration ([Ca2+]i). In the present study, we observed the effects of Aβ fragments 25-35 (Aβ25-35) and 31-35 (Aβ31-35) on [Ca2+]i in primary cultured rat cortical neurons using laser-scanning confocal calcium imaging technique, and investigated its probable cholinergic mechanism. The results showed that: (1) Both Aβ25-35 and Aβ31-35 induced similar and significant [Ca2+]i elevation in a concentrationdependent manner, and no statistical difference was found between the effects of both peptides; (2) The reverse peptide of Aβ31-35, i.e. Aβ35-31, had no effect on [Ca2+]i elevation; (3) Mecamylamine (MCA), a non-specific nAChRs antagonist, significantly and dosedependently blocked the [Ca2+]i elevation induced by Aβ25-35 or Aβ31-35; (4) Dihydro-β-erythroidine (D-β-E), a specific α4β2 subtype nAChRs antagonist, also significantly inhibited the [Ca2+]i elevation induced by Aβ25-35 and Aβ31-35, but the effect was weaker than the effect of MCA at the same concentration. These results indicate that Aβ31-35 may be a shorter active sequence in full length of Aβ molecule, and the overactivation of nAChRs, including α4β2 subtype, may be, at least partly, responsible for the Aβ-induced elevation of [Ca2+]i in cultured rat cortical neurons. Thus, the present study suggests a new potential target of Aβ in the brain, and provides a new insight into the mechanisms by which Aβ impairs the cognitive function in Alzheimer’s disease.

Key words: nicotinic acetylcholine receptors; amyloid β-protein; cortical neurons; intracellular calcium concentration

收稿日期：2009-09-29　　录用日期：2009-11-16

通讯作者：祁金顺　　E-mail: jinshunqi2006@yahoo.com

引用本文：

武美娜1, 李新毅, 郭芬, 祁金顺. N 型胆碱能受体参与淀粉样β 蛋白片段引起的大鼠大脑皮层神经元胞内钙水平升高[J]. 生理学报 2009; 61 (6): 517-525.

WU Mei-Na, LI Xin-Yi, GUO Fen, QI Jin-Shun. Involvement of nicotinic acetylcholine receptors in amyloid β-fragmentinduced intracellular Ca2+ elevation in cultured rat cortical neurons. Acta Physiol Sin 2009; 61 (6): 517-525 (in Chinese with English abstract).