马桑内酯对大鼠海马锥体神经元钙激活钾通道的影响

背景:神经细胞的电活动以细胞膜离子通道的活动为基础.神经元异常放电是癫痫的基本特征,其基础是细胞膜离子通道的激活与离子的跨膜运动,然而马桑内酯致痫机制中是否存在钙激活钾通道的激活还不清楚.目的:以大鼠海马锥体神经元为靶细胞,了解马桑内酯在其致痫机制中对神经元钙激活钾通道的作用.设计:非随机对照实验.单位:四川大学华西医院神经内科和泸州医学院心肌电教研室.材料:实验于2000-05/12在四川省泸州医学院完成.选择出生24h之内Wistar乳鼠100只.方法:Wistar乳鼠在麻醉状态下和无菌条件下分离出海马组织,以培养第7～10天,生长良好、形态典型的锥体神经元进行膜片钳试验.将培养皿随机分成9组:①正常对照组,19皿,加入DMEM培养基,给以不同的钳制电压,以后加入四乙基胺.②10-8,10-7,10-6mol/L钙浓度组;加入含不同浓度钙离子的DMEM培养基,以后加入四乙基胺,每一浓度8皿,共24皿;马桑内酯0,0.25,0.5,1.0,2.0 mL/L致痫组,加入不同浓度含马桑内酯的DMEM培养基,以后加入四乙基胺.每一浓度26皿,共130皿.运用膜片钳制技术贴附式和内面向外式方法记录神经元单通道电活动,并分析通道活动的开放概率、平均开放时间、平均关闭时间、电流幅值等.主要观察指标:①观察并记录正常,不同钳制电压、不同钙离子浓度对锥体神经元钙激活钾通道的激活作用和四乙基胺的影响.②观察并记录致痫剂马桑内酯对锥体神经元细胞膜钙激活钾通道的激活作用及四乙基胺的影响.结果:①在钳制电压为0 mV时,锥体神经元钙激活钾通道有少量的随机开放,具有明显的电压依赖性,通道电导值为(122.79±21.68)pS,可被钾通道阻断剂四乙基胺所阻断.②在内面向外式膜片下,钙激活钾通道表现出钙离子的浓度依赖性.当钙浓度为10-8,10-7,10-6 mol/L时,平均开放概率分别为0.022±0.006,0.040±0.007,0.142±0.049(P＜0.01).③在细胞贴附式膜片下,浴液中游离钙离子浓度10-8 mol/L,膜电位在20 mV时,发现马桑内酯能明显增加钙激活钾通道的开放概率.④与马桑内酯0 mL/L比较,马桑内酯1.0 mL/L能增加钙激活钾通道平均开放时间(1.867±0.210,6.900±0.120,P＜0.01),减少平均关闭时间(78.505±7.192,6.233±0.854,P＜0.01).结论:在马桑内酯诱导的癫痫发病中,钙激活钾通道活化可能起重要的负反馈调节作用.

Influence of Coriaria Lactone on Ca2+-activated K+ channels in rat hippocampal pyramidal neurons

BACKGROUND: Electrical activity of nerve cells is based on the ion channel activity on cell membrane. Epilepsy is basically characterized by abnormal neuronal discharge. The foundation is ion channel activation on cell membrane and ion transmembrane movement, however, whether Ca2+-activated K+ channel involves in epilepsy induced by Coriaria Lactone is still unclear.OBJECTIVE: Considering rat hippocampal pyramidal neurons as target,we investigate the effect of Coriaria Lactone on neuronal Ca2+-activated K+ channels in epilepsy induced by Coriaria Lactone.DESIGN: Randomized controlled experimental trials.SETTING: Department of Neurology, West China Hospital, Sichuan University and Institute. of Myocardium Electrophysiology of Luzhou Medical College.MATERIALS: This experiment was carried out in Luzhou Medical College, Sichuan Province, from May to December 2000. Totally 100 Wistar infant rats within 24-hour ages were selected.METHODS: Wistar infant rats were anaesthetized and its hippocampus was obtained under disinfected state, pyramidal neurons were cultured for 7-10 days, neurons growing well with typical shape model were colleted normal control group, 19 dishes were added with DMEM culture medium,given different membrabe voltage and then followed by adding in te3 subgroups with 8 dishes each one. Added seperately DMEM culture medium containing f0-8, 10-7, 10~ mol/L concentration of calcium ion, and 2.0 mL/L Coriaria Lactone induced epilepsy group: added with DMEM culture medium with different dosages of Coriaria Lactone and finally tetraethylamine in each concentration of 26 dishes for totally 130 dishes.Cell-attache method and inside-out method of patch-clamp technique were used to record the neuronal single channel electricity. The open probability, average opening hour and closing hour, electric current amplitude of channel were analyzed.activated K+ channels of pyramidal neurons at normal, various membrane To observe and record the influence of Coriaria Lactone on the activation of pyramidal neuronal cell membrane, as well as the role of tetraethylamine.were only small amount of pyramidal neurons randomly opening its Ca2+-activated K+ channels and it displayed obvious voltage-dependent property.The channel electric conductance was (122.79±21.68) pS. The channels the inside-out condition, Ca2+-activated K+ channel displayed calcium iondependent property. The average opening rate was 0.022±0.006, 0.040±0.007, 0.142±0.049 when the calcium concentration was 10-8, 10-7,aria Lactone could increase the opening rate of Ca2+-activated K+ channels when the free calcium ion in bath solution was 10-8 mol/L and memLactone, 1.0 mL/L Coriaria Lactone could increase the average opening time of Ca2+-activated K+ channels (1.867±0.210, 6.900±0.120, P ＜ 0.01), and reducing the average closing time (78.505±7.192,6.233±0.854, P ＜ 0.01).CONCLUSION: In epilepsy induced by Coriaria Lactone, the activation of Ca2+-activated K+ channels might play an important role of negative modulation.