【作者】 李享元；

【导师】 杨宗凯； 吴鸿修；

【作者基本信息】 华中科技大学 ， 信息与通信工程， 2005， 博士

【副题名】Study on the Modulation by Bis(7)-tacrine of Potassium Channel and Calcium Channel

【摘要】 阿尔茨海默病(Alzheimer’s Disease,AD)是老年性痴呆中的一个主要类型,发达国家对此疾病的发病机制及预后研究较深入。许多研究表明胆碱能神经的功能缺失在AD 发病机制中具有重要的作用。目前,AD 已经和心、脑血管疾病、恶性肿瘤并列为导致老年人死亡的四大杀手。乙酰胆碱酯酶抑制药AChEI 获许用于AD 治疗,且今后仍是主要治疗用药。华中科技大学、香港科技大学、美国、台湾和上海药物研究所的不同学科和专业背景的学者,探索神经退行性疾病的发病机制和药物开发,取得了阶段性成果,并推出了第二代新型的乙酰胆碱酯酶抑制剂药物Bis(7)-tacrine。Bis(7)-tacrine [bis(7)-tetrahydroaminacrine] 是一种新型的二聚体乙酰胆碱酯酶抑制剂,两个tacrine 分子通过庚烯键连接而成。实验证明它的药理作用比tacrine 强150倍,对抑制乙酰胆碱酯酶的选择性是tacrine 的250 倍。Tacrine 是首先用于缓解治疗Alzheimer’s disease 的乙酰胆碱酯酶抑制剂药物。Bis(7)-tacrine 可以有效逆转老鼠AF64A 诱导方向性记忆缺陷。Bis(7)-tacrine 是一种多功能/多靶点乙酰胆碱酯酶抑制剂,可以作用于酶、神经递质、受体和离子通道等靶点。除了乙酰胆碱酯酶抑制作用,Bis(7)-tacrine 对几种配体门控离子通道也有调制作用,如大鼠海马神经元的GABAAR 受体,大鼠三叉神经元的5-HT3R 受体,和大鼠海马神经元的NMDAR 受体等等。本文的研究旨在探索bis(7)-tacrine 是否象FDA(美国食品和药物管理局)批准的乙酰胆碱酯酶抑制剂tacrine 一样对多种神经元的通道和受体具有调制作用及其对离子通道的动力学参数的影响。为此,综合利用膜片钳、电压钳和基因表达技术,进行了相应实验工作,验证了bis(7)-tacrine 神经药理作用,并且比tacrine 强两个数量级左右。本文创新性的研究工作如下: (1) 充分利用电子技术和计算机技术的成就和进步,研制了国内首台基于USB(Universal Serial Bus)的卵母细胞双电极电压钳和数据采集分析系统。在广泛听取电生理科研人员的意见和建议的基础上,研制的仪器易于操作,软件界面友好,并在试验中验证其性能指标,具有高输入阻抗、低噪声、低漂移、精度高和跟随响应速度快且性价比高。由于采用USB 接口,使仪器便于安装和调试。卵母细胞双电极电压钳系统,国内一直依赖进口,如NPI 的一个电压钳放大器价格达到八千到一万美更多还原

【Abstract】 In the new century, one of the serious social problems is the growing percentage of the aged population in the world, and most of the elder have been suffered by the age diseases such as Alzheimer’s disease (AD), which is a progressive degenerative disease of the brain from which there is no recovery. AD is the most common form of dementia, which primarily affects older people. Dementia is a loss of intellectual function (cognition and memory) so severe that it interferes with an individual’s daily activities and eventually results in death. AD is the fourth leading cause of death in adults, after heart disease, cancer and stroke. The causation of Alzheimer’s disease are unknown. However, scientific research has begun to point in several directions. Neurotransmitter deficits have been implicated, with a deficiency of the neurotransmitter acetylcholine being a prominent and consistently identified deficit. The cerebral cholinergic neurotransmission has been abundantly targeted for drug development in AD in view of the well-characterized degeneration of the acetylcholine (ACh) synthesizing neurones. Available treatments of AD produce an increase in the ACh levels by inhibiting the enzymes responsible for ACh breakdown (cholinesterases). Acetylcholines-terase inhibitors (AChEI) was approved in clinical AD treatment, some are being studied in clinical trials to find out whether they can slow the progression of the disease or improve memory for a period of time. Tacrine(THA) was approved by FDA in September 1993 for the treatment of mild to moderate Alzheimer’s disease. With the collaboration of researchers from China, America, Hong Kong and Taiwan, a novel acetylcholinesterase inhibitor, bis(7)-tacrine was synthesized and applied to clinical trial to treat AD. Bis(7)-tacrine [bis(7)-tetrahydroaminacrine] is a dimeric acetylcho-lineesterase inhibitor (AChEI), in which two tacrine molecules are linked by a heptylene chain. It has been identified that bis(7)-tacrine is more potent (up to 150 fold) and more selective (around 250 fold) in inhibiting AChE than tacrine, the first anticholinesterasic drugs approved by FDA for the palliative treatment of Alzheimer’s disease. Bis(7)-tacrine was also proved to effectively reverse AF64A-induced deficits in navigational memory in rats. Bis(7)-tacrine has been considered to be a multi-functional/multi-target AChE inhibitor, which can act on many targets: enzymes, neurotransmitter receptors and also ion channels. Besides its inhibitory effect on AChE, bis(7)-tacrine is capable to exert modulatory actions on several kinds of ligand-gated ion channels, including GABAAR in rat hippocampal neurons, nAChR in Torpedo electric organ, 5-HT3R in rat trigeminal ganglion neurons and NMDAR on rat hipocampal neurons. The present study was aimed to explore whether bis(7)-tacrine is able to modulate voltage-gated potassium and calcium channel as tacrine to do in rat primary sensory neurons, and it does so on the Kv4.2 potassium channel expressed on Xenopus laevis oocyte, too. In the whole-cell patch experiments, it has been identified that on the preparation of dorsal root ganglion (DRG) neurons, the suppression by bis(7)-tacrine of voltage-gated potassium channels including both transient (IA) and sustained (IKD) potassium channels and L-type calcium channel was much more potent than that by tacrine. The two-electrode voltage clamp experiments were carried out with the drugs of bis(7)-tacrine and tacrine, and USB-based oocyte voltage clamp amplifier was designed for the experiment on the expressed Kv4.2 potassium channel on oocyte. The data and results obtained in the present study were not reported elsewhere. (1) In our present investigation, the first domestic oocyte two-electrode voltage clamp amplifier was designed and developed based USB (Universal Serial Bus) interface, which has features of low noise, low drift, and fast response time for recording from high impedance electrolyte-filled glass electrodes. An USB-based fast high-resolution data acquisition and analyzed system was implemented suitable for the amplifier data recording studies in the research lab. It offers 12-bit 8 analog input channels and 8-bit digital to analog output channels, interfaced to PC via USB, so setup is plug-and-play easy. (2) Using the two-electrode oocyte voltage clamp amplifier system, Kv4.2 mRNA was microinjected into defolliculated Xenopus laevis oocytes to express transient potassium channel. Bis(7)-tacrine and tacrine were applied to expressed transient potassium channels, It was observed that both drugs exert inhibitory action on have inhibitive effect on transient potassium channel current IA. Bis(7)-tacrine was found more potent in inhibiting of IA than tacrine, and the inhibition of IA (IC50=1.12 ±0.04 μM(n=7)) by bis(7)-tacrine was almost two orders more potent than that by tacrine (IC50=121.5 ±4.4 μM(n = 7)). (3) The next experiment was performed on neurons acutely isolated from rat dorsalroot ganglion (DRG) to explore the modulation of both drugs on potassium channel. Potassium currents including transient (IA) and sustained (IKD) components were recorded using whole-cell patch clamp technique. Both IA and IKD were suppressed by bis(7)-tacrine, a novel dimeric AChE inhibitor, which are much more potent (IC50 = 21.1 ±2.3 μM for IKD, IC50 = 0.625 ±0.04 μM for IA) than that by tacrine (IC50 = 2.11 ±0.25 mM for IKD, IC50 = 73.5 ±4.4 uM for IA). It was identified that bis(7)-tacrine (1uM) shifts steady state activation curve and inactivation curve of IA and IKD to the hyperpolarizing direction. In the presence of bis(7)-tacrine no change is in time constant of rising phase (τon) but that in decay phase (τdec) of IA, which decays down more slowly evidently, and the recovery curve of IA shifts downwards significantly. These results suggest that the inhibition by bis(7)-tacrine of IA and IKD may be attributed to reduction of potential ranges of steady state activation and inactivation and delay of decay phase as well as recovery from inactivation of IA. (4) The effects of drugs on calcium channel were identified on neurons acutely isolated from rat dorsal root ganglion (DRG). Calcium channel currents including L-type, N-type and P/Q-type components were recorded using whole-cell patch clamp technique. The drug inhibitory effect on L-type is the most potent, while that on other types is weak. L-type was suppressed by bis(7)-tacrine, a novel dimeric AChE inhibitor, which are much more potent (IC50 =1.077 X10-7M) than that by tacrine (IC50 =2.92 X10-5M). These results suggest that the inhibition by bis(7)-tacrine of may be attributed to reduction of potential ranges of steady state activation and inactivation of calcium channel, therefore block associated increases in Ca2+ influx, calcium overload, hyperexcitability of the neurons. From these experimental data and results, it raise the intriguing possibility by bis(7)-tacrine that reduction of outward K+ current and Ca2+ influx may play a pivotal role on alleviation of AD owing to its neuro-protection.更多还原