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，然而马桑内酯致病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^-6 mol／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0，0．25．0．5,1．0，2．0mL／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0mV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&;#177;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&;#177;0．006，0．040&;#177;0．007，0．142&;#177;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^-8mol／L，膜电位在20mV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0ml／L比较，马桑内酯1．0ml／L能增加钙激活钾通道平均开放时间（1．867&;#177;0．210,6．900&;#177;0．120，P〈0．01），减少平均关闭时间（78．505&;#177;7．192，6．233&;#177;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。     

马桑内酯对大鼠海马锥体神经元钙激活钾通道的影响(英文)

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0,0.25,0.5,1.0,2.0mL/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0mV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10-8,10-7,10-6mol/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10-8mol/L,膜电位在20mV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0mL/L比较,马桑内酯1.0mL/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。     

吡拉西坦对缺氧大鼠海马神经元钙激活钾通道的作用

目的：研究吡拉西坦对缺氧大鼠海马神经元钙激活钾通道的作用，以揭示吡拉西坦抗缺血性脑损伤的电生理途径。 方法：实验于2004—03／2005—06在泸州医学院心肌电生理研究室完成。应用膜片钳制技术分别记录吡拉西坦和缺氧对海马神经元钙激活钾通道的单通道电流活动的影响，并经P-clamp软件进行微机采样、储存数据和数据的分析处理。 结果：（1）吡拉西坦对钙激活钾通道具有明显的激活作用，随着药物浓度（2．5—7．5mmol／L）的增加，通道开放概率明娃增加，由用药前的0．032&;#177;0．010增加到0．272&;#177;0．038（P〈0.01，n=6），伴随有通道平均关闭时间明显缩短，而电流幅值及平均开放时间无明显变化。（2）应用氰化钠20μmol／L造成细胞急性缺氧。在缺氧早期（5-15min）通道开放概率增加，由缺氧前的0．024&;#177;0．009增加至0．090&;#177;0．026（P〈0．01，n=6），而缺氧后期通道开放概率明显降低，表明缺氧时间对通道的开放概率有明显影响。（3）吡拉西坦能明显增加缺氧神经元钙激活钾通道的开放概率，而以缺氧20min时为最明显，缺氧40min时为最低，即由对照组的0．038&;#177;0.008逐渐增加至最高时的0．148&;#177;0．060，然后又逐渐降低至最低时的0．033&;#177;0．005（P〈0．01，n=6）。 结论：吡拉西坦对海马锥体神经元大电导钙激活钾通道具有明显的激活作用。吡拉西坦可能通过激活钙激活钾通道等机制发挥对缺氧神经元的保护作用。     

马桑内酯对锥体神经元三磷酸腺苷敏感钾通道的作用

背景：神经元异常放电的基础是细胞膜离子通道的激活与离子的跨膜运动。三磷酸腺苷敏感钾通道是将细胞电活动与代谢联系在一起的重要通道。三磷酸腺苷敏感钾通道是否参与癫痫的发病过程，马桑内酯是否具有调节三磷酸腺苷敏感钾通道的作用尚不清楚。 目的：了解致痫剂马桑内酯对大鼠海马锥体神经细胞膜三磷酸腺苷敏感钾通道的影响及三磷酸腺苷敏感钾通道在癫痫发病中的作用。 设计：随机对照实验。 单位：四川大学华西医院神经内科和四川大学华西基础医学与法医学院生理学教研室。 材料：实验于2000-05／12在泸州医学院完成。将Wistar乳鼠的培养的海马锥体神经元，随机分为正常对照组，四乙基胺组，二磷酸核苷组，马桑内酯组，电导与动力学组。 方法：Wistar乳鼠在麻醉和无菌条件下分离出海马组织，接种、培养24h后加入10μmol／L的阿糖胞苷，选择培养7-10d、生长良好、形态典型的锥体神经元进行膜片钳试验。正常对照组加入生理盐水；四乙基胺组加入5mmol／L氯化四乙基胺；二磷酸核苷组先加入30μmol/L的二磷酸核苷，后加入0．5mol／L的三磷酸腺苷；致痫组先加入1．0mL／L的马桑内酯，后加入1μmol／L的优降糖；对电导与动力学组，先调节钳制电压的大小，了解通道开放及通道形态，后加入马桑内酯。 主要观察指标：①观察神经元三磷酸腺苷敏感钾通道的活动及形态。（参观察不同钳制电压对通道活动的影响；了解二磷酸核苷、三磷酸腺苷和氯化四乙铵对通道的影响。②观察致痫剂马桑内酯对神经元细胞膜三磷酸腺苷敏感钾通道的激活作用及优降糖的影响。 结果：①对称性高钾溶液条件下，通道的翻转电位接近0mV。三磷酸腺苷敏感钾通道开放随着钳制电压绝对值的增大而增多，具有电压依赖性，该通道可被氯化四乙铵阻断。②其电流-电压（I-V）曲线可被直线拟合，电导值为（78．23&;#177;12．04）pS。③30μmol／L的二磷酸核苷可使通道开放增多，0．5mol／L的三磷酸腺苷可抑制通道活动。④1．0mL／L的马桑内酯诱导通道开放数量明显增多，1μmol／L的优降糖可抑制通道活动。⑤通道开放时间，致痫神经元T01为（1．754&;#177;0．060）ms，正常神经元为（1．733&;#177;0．046）ms，无显著性差异（n=25，t=0．147，P〉0．05）；而T02正常组为（2．441&;#177;0．265）ms，致痫组延长，为（10．446&;#177;0．579）ms（n=25,t=0．000，P〈0．01）。 结论：在马桑内酯诱导的癫痫发作中，三磷酸腺苷敏感钾通道开放的作用是降低动作电位频率、保护神经元，可能起一种负反馈调节作用。     

吡拉西坦对缺氧大鼠海马神经元钙激活钾通道的作用

目的:研究吡拉西坦对缺氧大鼠海马神经元钙激活钾通道的作用,以揭示吡拉西坦抗缺血性脑损伤的电生理途径。方法:实验于2004-03/2005-06在泸州医学院心肌电生理研究室完成。应用膜片钳制技术分别记录吡拉西坦和缺氧对海马神经元钙激活钾通道的单通道电流活动的影响,并经P-clamp软件进行微机采样、储存数据和数据的分析处理。结果:①吡拉西坦对钙激活钾通道具有明显的激活作用,随着药物浓度(2.5～7.5mmol/L)的增加,通道开放概率明显增加,由用药前的0.032±0.010增加到0.272±0.038(P<0.01,n=6),伴随有通道平均关闭时间明显缩短,而电流幅值及平均开放时间无明显变化。②应用氰化钠20μmol/L造成细胞急性缺氧,在缺氧早期(5～15min)通道开放概率增加,由缺氧前的0.024±0.009增加至0.090±0.026(P<0.01,n=6),而缺氧后期通道开放概率明显降低,表明缺氧时间对通道的开放概率有明显影响。③吡拉西坦能明显增加缺氧神经元钙激活钾通道的开放概率,而以缺氧20min时为最明显,缺氧40min时为最低,即由对照组的0.038±0.008逐渐增加至最高时的0.148±0.060,然后又逐渐降低至最低时的0.033±0.005(P<0.01,n=6)。结论:吡拉西坦对海马锥体神经元大电导钙激活钾通道具有明显的激活作用。吡拉西坦可能通过激活钙激活钾通道等机制发挥对缺氧神经元的保护作用。     

马桑内酯对锥体神经元三磷酸腺苷敏感钾通道的作用

背景神经元异常放电的基础是细胞膜离子通道的激活与离子的跨膜运动.三磷酸腺苷敏感钾通道是将细胞电活动与代谢联系在一起的重要通道.三磷酸腺苷敏感钾通道是否参与癫痫的发病过程,马桑内酯是否具有调节三磷酸腺苷敏感钾通道的作用尚不清楚.目的了解致痫剂马桑内酯对大鼠海马锥体神经细胞膜三磷酸腺苷敏感钾通道的影响及三磷酸腺苷敏感钾通道在癫痫发病中的作用.设计随机对照实验.单位四川大学华西医院神经内科和四川大学华西基础医学与法医学院生理学教研室.材料实验于2000-05/12在泸州医学院完成.将Wistar乳鼠的培养的海马锥体神经元,随机分为正常对照组,四乙基胺组,二磷酸核苷组,马桑内酯组,电导与动力学组.方法Wistar乳鼠在麻醉和无菌条件下分离出海马组织,接种、培养24 h后加入10 μmol/L的阿糖胞苷,选择培养7～10 d、生长良好、形态典型的锥体神经元进行膜片钳试验.正常对照组加入生理盐水;四乙基胺组加入5 mmol/L氯化四乙基胺;二磷酸核苷组先加入30 μmoL/L的二磷酸核苷,后加入0.5 mol/L的三磷酸腺苷;致痫组先加入1.0 mL/L的马桑内酯,后加入1 μmol/L的优降糖;对电导与动力学组,先调节钳制电压的大小,了解通道开放及通道形态,后加入马桑内酯.主要观察指标①观察神经元三磷酸腺苷敏感钾通道的活动及形态.②观察不同钳制电压对通道活动的影响;了解二磷酸核苷、三磷酸腺苷和氯化四乙铵对通道的影响.③观察致痫剂马桑内酯对神经元细胞膜三磷酸腺苷敏感钾通道的激活作用及优降糖的影响.结果①对称性高钾溶液条件下,通道的翻转电位接近0 mV.三磷酸腺苷敏感钾通道开放随着钳制电压绝对值的增大而增多,具有电压依赖性,该通道可被氯化四乙铵阻断.②其电流-电压(Ⅰ-Ⅴ)曲线可被直线拟合,电导值为(78.23±12.04)pS.③30μmol/L的二磷酸核苷可使通道开放增多,0.5 mol/L的三磷酸腺苷可抑制通道活动.④1.0mL/L的马桑内酯诱导通道开放数量明显增多,1μmol/L的优降糖可抑制通道活动.⑤通道开放时间,致痫神经元τ01为(1.754±0.060)ms,正常神经元为(1.733±0.046)ms,无显著性差异(n=25,t=0.147,P＞0.05);而τ02正常组为(2.441±0.265)ms,致痫组延长,为(10.446±0.579)ms(n=25,t=0.000,P＜0.01).结论在马桑内酯诱导的癫痫发作中,三磷酸腺苷敏感钾通道开放的作用是降低动作电位频率、保护神经元,可能起一种负反馈调节作用.     

羟丁酸钠对海马神经细胞钙激活钾通道的作用

应用膜片钳技术研究了羟相酸钠对海马神经元中电导钙激活通道的作用，实验选用培养３～７天的锥体神经元，在对称性高钾溶液中，应用细胞贴附式膜片和内面向外式膜片记录通道电流，结果发现：（１）该通道电导为６５ｐＳ左右，对胞内（Ｃａ＾２＋）１敏感，特异钾通道阻断剂（ＴＥＡ），能抑制或阻断该通道活动；（２）在细胞贴附式膜片上，羟丁酸钠明显增加该通道的开放概率。因经，羟丁酸钠对海马神经细胞中电导钙激活钾通道具有激     

新生大鼠海马神经元的急性分离与膜片钳技术

目的：获得适用于膜片钳技术的单个海马神经元。 方法：实验于2005-09／11在新乡医学院机能学研究室完成。应用酶消化及机械分离法，急性分离出生3～6d的SD大鼠海马神经元，在倒置显微镜下，选取已贴壁直径约10～15μm的锥体形神经元观察其急性分离效果和细胞活性状态。电生理学的鉴定采用全细胞膜片钳的模式记录电流反应，首先在大鼠海马CA1区锥体神经元诱发出内向离子流。在大鼠海马CA1锥体神经元诱发出内向离子流后，通过程控灌流系统在细胞外液中加1μmol／L的钠通道阻断剂河豚毒素灌流同一神经元，记录内向离子流的变化。 结果：①该法可获得形态良好的单个海马神经元，海马锥体细胞具有锥形胞体（10～20μm）和厚墩的顶树突特征。活性较好的海马神经元，胞体呈三角形或椭圆形，表面光亮，细胞膜完整、清晰的，立体感较强。一般有1个较长的顶树突和2个以上较短的基树突。②利用膜片钳技术内向电流被诱发，用1μmol／L河豚毒素完全阻断该电流，说明该内向电流为钠电流。 结论：该法可获得形态和生理特性良好的单个海马神经元，证实该方法适用于海马神经元膜片钳研究，对深入探讨药物和毒物对海马离子通道及信号转导机制的作用有重要价值。     

次声对视网膜微血管内皮细胞钙激活钾通道的影响

背景：次声暴露导致大鼠血-视网膜屏障通透性增加。但由于视网膜微血管内皮细胞来源困难。关于其屏障损伤的离子机制报道较少。目的：探讨次声对视网膜微血管内皮细胞钙激活钾通道的影响。设计：完全随机实验对照的开放性研究。地点和材料：实验在第四军医大学航空临床教研室膜片钳实验室进行，实验对象为培养牛视网膜微血管内皮细胞。干预：取传代的牛视网膜微血管内皮细胞8Hz，130dB次声暴露0．5h。主要观察指标：视网膜微血管内皮细胞钙激活钾通道的活动情况。结果：8Hz，130dB次声暴露0.5h后，视网膜微血管内皮细胞KCa通道活性增加，暴露后置于孵箱内0．5h再行膜片钳离子电流的俭测，则离子通道的活性也有所下降。结论：次声通过增加视网膜微血管内皮细胞钙激活钾通道的活性，导致膜去极化，引起钙离子进入细胞，内皮细胞收缩，造成一定程度的血-视网膜屏障通透性的损害。     

新生大鼠海马神经元的急性分离与膜片钳技术

目的:获得适用于膜片钳技术的单个海马神经元。方法:实验于2005-09/11在新乡医学院机能学研究室完成。应用酶消化及机械分离法,急性分离出生3～6d的SD大鼠海马神经元,在倒置显微镜下,选取已贴壁直径约10～15μm的锥体形神经元观察其急性分离效果和细胞活性状态。电生理学的鉴定采用全细胞膜片钳的模式记录电流反应,首先在大鼠海马CA1区锥体神经元诱发出内向离子流。在大鼠海马CA1锥体神经元诱发出内向离子流后,通过程控灌流系统在细胞外液中加1μmol/L的钠通道阻断剂河豚毒素灌流同一神经元,记录内向离子流的变化。结果:①该法可获得形态良好的单个海马神经元,海马锥体细胞具有锥形胞体穴10～20μm雪和厚墩的顶树突特征。活性较好的海马神经元熏胞体呈三角形或椭圆形,表面光亮,细胞膜完整、清晰的,立体感较强。一般有1个较长的顶树突和2个以上较短的基树突。②利用膜片钳技术内向电流被诱发,用1μmol/L河豚毒素完全阻断该电流,说明该内向电流为钠电流。结论:该法可获得形态和生理特性良好的单个海马神经元,证实该方法适用于海马神经元膜片钳研究,对深入探讨药物和毒物对海马离子通道及信号转导机制的作用有重要价值。     

O2 deprivation inhibits Ca2+-activated K+ channels via cytosolic factors in mice neocortical neurons

O(2) deprivation induces membrane depolarization in mammalian central neurons. It is possible that this anoxia-induced depolarization is partly mediated by an inhibition of K(+) channels. We therefore performed experiments using patch-clamp techniques and dissociated neurons from mice neocortex. Three types of K(+) channels were observed in both cell-attached and inside-out configurations, but only one of them was sensitive to lack of O(2). This O(2)-sensitive K(+) channel was identified as a large-conductance Ca(2+)-activated K(+) channel (BK(Ca)), as it exhibited a large conductance of 210 pS under symmetrical K(+) (140 mM) conditions, a strong voltage-dependence of activation, and a marked sensitivity to Ca(2+). A low-O(2) medium (PO(2) = 10-20 mmHg) markedly inhibited this BK(Ca) channel open probability in a voltage-dependent manner in cell-attached patches, but not in inside-out patches, indicating that the effect of O(2) deprivation on BK(Ca) channels of mice neocortical neurons was mediated via cytosol-dependent processes. Lowering intracellular pH (pH(i)), or cytosolic addition of the catalytic subunit of a cAMP-dependent protein kinase A in the presence of Mg-ATP, caused a decrease in BK(Ca) channel activity by reducing the sensitivity of this channel to Ca(2+). In contrast, the reducing agents glutathione and DTT increased single BK(Ca) channel open probability without affecting unitary conductance. We suggest that in neocortical neurons, (a) BK(Ca) is modulated by O(2) deprivation via cytosolic factors and cytosol-dependent processes, and (b) the reduction in channel activity during hypoxia is likely due to reduced Ca(2+) sensitivity resulting from cytosolic alternations such as in pH(i) and phosphorylation. Because of their large conductance and prevalence in the neocortex, BK(Ca) channels may be considered as a target for pharmacological intervention in conditions of acute anoxia or ischemia.     

Urocortin induces endothelium-dependent vasodilatation and hyperpolarization of rat mesenteric arteries by activating Ca2+-activated K+ channels

Urocortin, a member of corticotropin releasing factor (CRF) peptide family, has positive chronotropic and inotropic effects on heart and also shows a vasodilatory effect. However, the mechanism underlying its vasodilatory effect has yet to be elucidated. Endothelium-dependent relaxation of resistance arteries is mainly achieved by activation of K+ channels. Therefore, we investigated possible role of K+ channels and hyperpolarization for the vasodilatory effect of urocortin using the isolated perfused rat mesenteric arteries. Urocortin (0.2 nM) produced a slow-onset decrease in the perfusion pressure of the mesenteric vascular bed, which was elevated by an alpha1-adrenoceptor agonist, phenylephrine (2-4 microM). Urocortin also hyperpolarized the main mesenteric artery. Removal of endothelium with saponin treatment considerably inhibited the relaxation and hyperpolarization induced by urocortin. In contrast, the hyperpolarization was not significantly changed by cyclooxygenase inhibitor, indomethacin (1 microM) and/or nitric oxide synthase inhibitor, N(omega)-nitro-L-arginine (100 microM). Urocortin-induced relaxation was not affected by the combination of a guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 1 microM), indomethacin and N(omega)-nitro-L-arginine. However, the relaxation and hyperpolarization were abolished by high extracellular potassium concentration (40 mM) or by a large conductance Ca(2+)-activated K+ channel blocker, charybdotoxin (1 nM). Glibenclamide (1 microM), an ATP-dependent K+ channel inhibitor, did not affect the relaxation and hyperpolarization. These results suggest that urocortin causes endothelium-dependent relaxation and hyperpolarization of rat mesenteric arteries, probably through the activation of charybdotoxin sensitive Ca2+-activated K+ channels. These findings also indicate an essential role of the endothelium for the urocortin-elicited vascular relaxation and hyperpolarization.     

Ca2+-activated K+ channels underlying the impaired acetylcholine-induced vasodilation in 2K-1C hypertensive rats

We tested the hypothesis that an abnormal function of K(+) channels in vascular smooth muscle cells plays a key role in the impaired acetylcholine (ACh) vasodilation in aortas from two kidney-one clip (2K-1C) hypertensive rats and further investigated the K(+) channel subtype involved in this altered response. ACh-induced endothelium-dependent relaxation was assessed in aortic rings from 2K-1C and normotensive two kidney (2K) rats. Glibenclamide, an ATP-sensitive K(+) channel blocker, did not inhibit ACh-induced relaxation in aortic rings from 2K or 2K-1C rats. The voltage-dependent K(+) channels inhibitor 4-aminopyridine attenuated ACh-induced relaxation in both groups. Charybdotoxin and iberiotoxin, blockers of Ca(2+)-sensitive (K(Ca)) and large-conductance K(Ca) (BK(Ca)) channels, respectively, reduced ACh-induced relaxation in aortic rings from 2K rats without affecting this response in those from 2K-1C rats, abolishing the differences between groups. ACh-induced relaxation in vessels from both 2K and 2K-1C rats was unaffected by apamin, a small-conductance K(Ca) blocker. NS1619 [1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one], an activator of K(Ca), induced a smaller vasodilation in endothelium-denuded aortic rings from 2K-1C rats compared with those from 2K rats. Iberiotoxin reduced sodium nitroprusside-induced relaxation in endothelium-denuded aortic rings from 2K without affecting this response in those from 2K-1C rats. The inhibition of Na(+),K(+)-ATPase with ouabain had no effects on ACh-induced relaxation in aortic rings from 2K-1C or 2K rats. These data indicate that a deficient functional activity of BK(Ca) channels plays a key role in the impaired ACh vasodilation in aortas from 2K-1C rats.     

The endogenous brain constituent N-arachidonoyl L-serine is an activator of large conductance Ca2+-activated K+ channels

The novel endocannabinoid-like lipid N-arachidonoyl L-serine (ARA-S) causes vasodilation through both endothelium-dependent and -independent mechanisms. We have analyzed the vasorelaxant effect of ARA-S in isolated vascular preparations and its effects on Ca(2+)-activated K(+) currents in human embryonic kidney cells stably transfected with the alpha-subunit of the human, large conductance Ca(+)-activated K(+) (BK(Ca)) channel [human embryonic kidney (HEK) 293hSlo cells]. ARA-S caused relaxation of rat isolated, intact and denuded, small mesenteric arteries preconstricted with (R)-(-)-1-(3-hydroxyphenyl)-2-methylaminoethanol hydrochloride (pEC(50), 5.49 and 5.14, respectively), whereas it caused further contraction of vessels preconstricted with KCl (pEC(50), 5.48 and 4.82, respectively). Vasorelaxation by ARA-S was inhibited by 100 nM iberiotoxin. In human embryonic kidney cells stably transfected with the alpha-subunit of the human BK(Ca) channel cells, ARA-S and its enantiomer, N-arachidonoyl-D-serine, enhanced the whole-cell outward K(+) current with similar potency (pEC(50), 5.63 and 5.32, respectively). The potentiation was not altered by the beta(1) subunit or mediated by ARA-S metabolites, stimulation of known cannabinoid receptors, G proteins, protein kinases, or Ca(2+)-dependent processes; it was lost after patch excision or after membrane cholesterol depletion but was restored after cholesterol reconstitution. BK(Ca) currents were also enhanced by N-arachidonoyl ethanolamide (pEC(50), 5.27) but inhibited by another endocannabinoid, O-arachidonoyl ethanolamine (pIC(50), 6.35), or by the synthetic cannabinoid O-1918 [(-)-1,3-dimethoxy-2-(3-3,4-trans-p-menthadien-(1,8)-yl)-orcinol] (pIC(50), 6.59), which blocks ARA-S-induced vasodilation. We conclude the following. 1) ARA-S directly activates BK(Ca) channels. 2) This interaction does not involve cannabinoid receptors or cytosolic factors but is dependent on the presence of membrane cholesterol. 3) Direct BK(Ca) channel activation probably contributes to the endothelium-independent component of ARA-S-induced mesenteric vasorelaxation. 4) O-1918 is a BK(Ca) channel inhibitor.     

Opioid receptor antagonists modulate Ca2+-activated K+ channels in mesenteric arterial smooth muscle cells of rats in hemorrhagic shock

Previous study has indicated a significant enhancement of activity of large-conductance Ca2+-activated K+ channel (BKCa) in mesenteric arterial vascular smooth muscle cells isolated from rats in vascular hyporesponsive stage of hemorrhagic shock. In the present study, the effect of opioid receptor antagonism on BKCa activity in the vascular smooth muscle cells of rats in the hyporesponse stage of hemorrhagic shock was investigated by using inside-out configuration of the patch-clamp technique. The results showed that naloxone (10 microM) down-regulated the activity of BKCa by reducing open probability (Po) and open frequency of the channels. The reduction of Po resulted from a decrease of mean open time and an increase of the slow closed time constant. Naltrindole and nor-binaltorphimine (100 nM) had the similar effects to that of naloxone, but no significant effect of beta-funaltrexamine (100 nM) on the activity of the channels could be found. These results suggest that delta- and kappa-opioid receptors, but not mu-receptors, may be involved in the regulation of BKCa in vascular hyporesponse stage, and that inhibition of BKCa may be one of the mechanisms of the opioid receptor antagonists improving the response of resistance arteries to vasoactive stimulants during the decompensatory stage of hemorrhagic shock.     

Acute- and long-term glutamate-mediated regulation of [Ca++]i in rat hippocampal pyramidal neurons in vitro

We used a fura 2-based digital imaging technique to analyze the effects of glutamate (GLU) and GLU agonists on intracellular free calcium ([Ca++]i) in cultures of rat hippocampal pyramidal neurons. Depolarization of cells with 50 mM K+ raised [Ca++]i in all parts of the cell (e.g., soma and dendrites). [Ca++]i was also increased in these cells by GLU, kainate, quisqualate, N-methyl-D-aspartate (NMDA) and caffeine (CAF). Multiple challenges of a neuron with GLU gave rise to high "plateau" levels of [Ca++]i that were maintained over the entire length of an experiment (up to 1 hr). In the presence of the NMDA receptor antagonist 2-amino-5-phosphonovalerate multiple applications of GLU only produced multiple transient increases in [Ca++]i. Multiple challenges of a cell with NMDA (0 Mg++, 1 microM glycine) also produced maintained plateau responses in [Ca++]i. Multiple challenges with kainate or quisqualate only produced multiple transient responses in [Ca++]i. Plateau responses induced by GLU or NMDA could be reversibly reduced by removal of extracellular Ca++. Co++ and Ni++ (500 microM) also reduced the magnitude of the plateau, but nitrendipine and tetrodotoxin were generally ineffective. The kinase inhibitor staurosporine also reversibly reduced the magnitude of the plateau. The initiation of a [Ca++]i plateau could be blocked by 2-amino-5-phosphonovalerate although this compound was ineffective at reducing a plateau once it had formed. Thus, activation of NMDA receptors in these neurons leads to a maintained influx of Ca++ that could be responsible for certain long-term effects of GLU.     

Mechanisms of decreased bradykinin- induced vasodilation in experimental hyperlipemia-hyperglycemia: contribution of nitric oxide and Ca2+-activated K+ channels

Common complications of diabetes are accelerated atherosclerosis and vascular disturbances. We investigated whether the simultaneous insult of hyperlipemia-hyperglycemia affects the reactivity of the resistance arteries to bradykinin (BK), and if so, what are the mechanisms responsible for this disturbance. Experiments were conducted on male Golden Syrian hamsters rendered hyperlipemic (H) by a fat-rich diet, diabetic (D) by streptozotocin injection, or simultaneously hyperlipemic-diabetic (HD). Normal age-matched animals were used as controls (C). At 24 weeks after the induction of disease(s) the vascular reactivity of the mesenteric resistance arteries to BK (10(-8)-10(-4) M) was assayed by the myograph technique. To explore the role of nitric oxide (NO) in modulating the endothelium-dependent BK-induced relaxation, two experimental approaches were employed: (i) in vivo administration of L-arginine (622.14 mg/kg bw) to H, D, and HD hamsters (for 12 weeks); (ii) in vitro blockage of nitric oxide synthase by N(omega)-nitro- L-arginine methyl ester (10(-4) M). To evaluate the contribution of Ca2+-activated K+ channel(s) to BK-induced relaxation, the resistance arteries were exposed to 10(-3) M tetraethylammonium. Comparatively, the endothelium-independent relaxation was assayed using sodium nitroprusside (10(-8)-10(-4) M). The results showed that compared to the H and D groups, the HD hamsters exhibited the most reduced vasodilation of the resistance arteries to BK (34.09 +/- 1.5%). The diminished vasodilation was found to be due to a dual mechanism: an L-arginine:NO pathway and a NO-independent process, mediated via Ca2+-activated K+ channels. In vivo administration of L-arginine had favourable effects especially in the HD group, which manifested (i) an; 30% improvement of attenuated BK relaxation, (ii) an increase in sensitivity of the response to BK, (iii) a 3-fold diminishment of plasma hyperglycemia. Collectively, these data explain in part, the mechanisms and possible ways to correct the arterial endothelial dysfunction when diabetes is complicated with hyperlipemia.     

Iberiotoxin-induced block of Ca2+-activated K+ channels induces dihydropyridine sensitivity of ACh release from mammalian motor nerve terminals

The role which Ca(2+)-activated K(+) (K(Ca)) channels play in regulating acetylcholine (ACh) release was examined at mouse motor nerve terminals. In particular, the ability of the antagonist iberiotoxin to recruit normally silent L-type Ca(2+) channels to participate in nerve-evoked release was examined using conventional intracellular electrophysiological techniques. Incubation of cut hemidiaphragm preparations with 10 microM nimodipine, a dihydropyridine L-type Ca(2+) channel antagonist, had no significant effect on quantal content of end-plate potentials. Nevertheless, 1 microM S-(-)-1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-[trifluoromethyl]phenyl)-3-pyridine carboxylic acid methyl ester (Bay K 8644) enhanced quantal content to 134.7 +/- 3.5% of control. Iberiotoxin (150 nM) increased quantal content to 177.5 +/- 9.9% of control, whereas iberiotoxin plus nimodipine increased quantal content to only 145.7 +/- 10.4% of control. Coapplication of 1 microM Bay K 8644 with iberiotoxin did not significantly increase quantal content further than did treatment with iberiotoxin alone. The effects of iberiotoxin and nimodipine alone or in combination on the miniature end-plate potential (MEPP) frequency following KCl-induced depolarization were examined using uncut hemidiaphragm preparations. Nimodipine alone had no effect on MEPP frequency from preparations incubated in physiological saline containing 5 to 20 mM KCl. Moreover, iberiotoxin alone or combined with nimodipine also had no effect on MEPP frequency in physiological salines containing 5 to 15 mM KCl. At 20 mM KCl, however, iberiotoxin significantly increased MEPP frequency to 125.6% of iberiotoxin-free values; combined treatment with nimodipine and iberiotoxin prevented this increase in MEPP frequency. Thus, loss of functional K(Ca) channels unmasks normally silent L-type Ca(2+) channels to participate in ACh release from motor nerve terminals, particularly under conditions of intense nerve terminal depolarization.     

不同浓度利多卡因对大鼠脑海马锥体神经元钠、钙电流的影响

目的:观察不同浓度利多卡因对大鼠海马锥体神经元N-甲基-D-天冬氨酸受体介导钙电流和钠电流的影响,初步探讨低浓度利多卡因对神经元缺氧保护的电生理基础。方法:实验于2001-06/2002-08在北大医院麻醉生理实验室进行。将已培养12～14d的Wistar大鼠海马神经元按利多卡因浓度(10-5～10-1mol/L)分成5组(n=6),以不含利多卡因组做对照,应用全细胞膜片钳方法,采用无间隙模式,采集记录各组大鼠海马神经元N-甲基-D-天冬氨酸介导钙电流、电压依赖性钠电流的变化及各组静息电位的情况。结果:①同对照组相比,利多卡因浓度为10-3,10-2,10-1mol/L时明显抑制N-甲基-D-天冬氨酸介导钙电流,电流密度依次为5.2±1.9,3.0±0.5,3.3±1.0(P<0.05或0.01),其余浓度对N-甲基天冬氨酸介导钙电流无明显作用。②利多卡因浓度为10-4,10-3mol/L时,电压依赖性钠通道电流密度依次为160.9±19.2,68.2±6.5,同对照组相比明显减少,利多卡因浓度为10-2,10-1mol/L时钠电流被完全抑制(P<0.05或0.01)。结论:利多卡因可浓度依赖性抑制N-甲基-D-天冬氨酸介导钙电流和钠电流,低浓度利多卡因的脑保护作用可能与此有关。