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#beta#--淀粉肽(1--40)对海马神经元高电压激活钙电流的作用及银杏内酯B对该作用的影响

陈蕾, 刘长金, 唐明, 李爱, 胡新武, 周莹, JÜrgen Hescheler

华中科技大学同济医学院生理学系. 湖北, 武汉 430030；德国科隆大学神经生理研究所. 科隆D-50931

摘要

应用全细胞膜片钳技术探讨#beta#--淀粉肽(1--40)(#beta#--amyloid peptide_(1-40), A#beta#_(1-40))对新鲜分离的大鼠海马CA1区锥体神经元高电压依赖性钙通道电流(high voltage--activated calcium channel current, I_(HVA))的作用并观察银杏内酯B (ginkgolide B, GB)对该作用的影响。利用细胞外灌流或者电极内液给药的方法，比较加药前后电流幅度的变化以判断药物是否发挥作用。细胞外给予老化处理的A#beta#_(1-40)可以浓度依赖性地增强I_(HVA)的幅度，A#beta#_(1-40)的浓度为0.01---30 #mu#mol/L时可分别使IHVA幅度增加(5.43±3.01)% ({sl n}=8，{sl P}>0.05)、(10.49±4.13)% ({sl n}=11，{sl P}>0.05)、(40.69±8.01)% (n=16，{sl P}<0.01)、(58.32±4.85)% ({sl n}=12，{sl P}<0.01)和(75.45±5.81)% ({sl n}=6，{sl P}<0.01); 新鲜配制的A#beta#_(1-40)对I_(HVA)几乎没有影响({sl n}=5，{sl P}>0.05)。L--型钙通道阻断剂nifedipine可以抵消A#beta#_(1-40)对I_(HVA)的增强作用。A#beta#_(1-40)(1.0 #mu#mol/L)对I_(HVA)的增强作用可以被cAMP的类似物8--Br--cAMP和腺苷酸环化酶(adenylyl cyclase, AC)的激动剂forskolin增强[分别为(66.19±5.74)%，P<0.05和(73.21±6.90)%，{sl P}<0.05]，被蛋白激酶A (protein kinase A, PKA)的抑制剂H--89减弱[(20.08±2.18)%，{sl P}<0.05]。GB可有效地减弱A#beta#_(1-40)对I_(HVA)的增强作用。以上结果表明A#beta#_(1-40)可通过AC--cAMP--PKA增强I_(HVA)引起胞内钙超载，这可能是其产生神经毒性作用的机制之一。GB可通过抑制A#beta#_(1-40)引起的异常钙离子内流对神经元起一定保护作用。

关键词： 海马; #beta#-淀粉肽; 电压依赖性钙通道电流; 银杏内酯B; 膜片钳技术

Action of #beta#--amyloid peptide_(1-40) on I_(HVA) and its modulation by ginkgolide B

Chen Lei, Liu Changjin, Tang Ming, Li Ai, Hu Xinwu, Zhou Ying, JÜrgen Hescheler

Department of Physiology, Tongji Medical College, Huazhong University of Science and Technology. Wuhan 430030, China；Institute of Neurophysiology, University of Cologne, Cologne D-50931, Germany

Abstract

Whole-cell patch clamp recording was used to investigate the action of #beta#-amyloid peptide1-40 (A#beta#_(1-40)) on high voltage-activated calcium channel current (I_(HVA)) in acutely isolated hippocampal CA1 pyramidal neurons in rats and observe its modulation by ginkgolide B (GB). Drug was applied by extracellular bath or adding in the pipette solution, and its effect was determined by comparing the amplitude of (I_(HVA) before and after the drug application. Bath application of aggregated A#beta#_(1-40) at concentrations of 0.01--30 #mu#mol/L increased the amplitude of I_(HVA) in a dose-dependent manner by (5.43=3.01)% (n=8, P>0.05), (10.49±4.13)% (n=11, P>0.05), (40.69±8.01)% (n=16, P<0.01), (58.32±4.85)% (n=12, P<0.01), and (75.45±5.81)% (n=6, P<0.01), respectively, but had no effect on the I-V curve of I_(HVA); fresh A#beta#_(1-40) almost had no effect on I_(HVA) (n=5, P>0.05). L-type calcium channel antagonist nifedipine abolished the increase of IHVA by A#beta#_(1-40). The increase of I_(HVA) A#beta#_(1-40) (1.0 #mu#mol/L) was enhanced to (66.19±5.74)% (P<0.05) by 8-Br-cAMP (membrane permeable analogue of cAMP) and to (73.21±6.90)% (P<0.05) by forskolin, an adenylyl cyclase (AC) agonist, and reduced to (20.08±2.18)% (P<0.05) by H-89, cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) antagonist. GB effectively inhibited the increase of I_(HVA) by A#beta#_(1-40). The results indicate that A#beta#_(1-40) leads to an intracellular calcium overload by increasing I_(HVA) via AC-cAMP-PKA. This may be one of the mechanisms for its neurotoxicity. GB can prevent neurons from neurotoxicity by inhibiting abnormal calcium influx caused by A#beta#_(1-40).

Key words: Hippocampus;#beta#-amyloid peptide;Voltage-dependent calcium channel current;Ginkgolide B;Patch-clamp technique

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引用本文：

陈蕾, 刘长金, 唐明, 李爱, 胡新武, 周莹, JÜrgen Hescheler. #beta#--淀粉肽(1--40)对海马神经元高电压激活钙电流的作用及银杏内酯B对该作用的影响 [J]. 生理学报 2006; 58 (1): .

Chen Lei, Liu Changjin, Tang Ming, Li Ai, Hu Xinwu, Zhou Ying, JÜrgen Hescheler. Action of #beta#--amyloid peptide_(1-40) on I_(HVA) and its modulation by ginkgolide B . Acta Physiol Sin 2006; 58 (1): (in Chinese with English abstract).