【作者】 赵桂玲；

【导师】 赵克森；

【作者基本信息】 中国人民解放军第一军医大学 ， 病理生理学， 2003， 博士

【摘要】 本文应用膜片钳和激光共聚焦显微镜成像技术对失血性休克后大鼠肠系膜细动脉血管平滑肌细胞（arterial smooth muscle cell,ASMC）大电导钙激活钾通道(large-conductance calcium activated potassium channel,BK_Ca)的变化及其机制进行了研究，结果如下： 一、ASMC中BK_Ca的特性 用膜片钳的内面向外式记录法，在36只正常大鼠ASMC膜片上记录的BK_Ca电流幅度随电压的增加而增加，通道的反转电位为0.9mV，BK_Ca电流幅度和电压呈现良好的线性关系，无整流现象，在电极液和浴槽液均为140mM高钾溶液时，通道Ⅰ-Ⅴ曲线的斜率即单通道电导为221±6pS（R²=0.9965，p＜0.01）。当细胞外钙离子浓度为0.1μM时，使NPo为最大开放概率（NPomax）一半时的电压(V_1／2)为22±2mV，电压每增加17±1mV，NPo增加e倍。BK_Ca单通道开放和关闭均符合二级指数拟合特点，在细胞内自由钙浓度为0.1μM，钳制电压为40mV时，通道开放时间常数为0.7674±0.1359和6.0946±0.8998，关闭时间常数为0.5964±0.0929和10.8426±2.9037。我们所记录到的BK_Ca具有高度的钙敏感性：通道在细胞内钙浓度很低时(10^-9M)即可被激活，在钳制电压为40mV时，通道开放概率与细胞内钙离子浓度对数的关系用Boltzman方程拟合显示使NPo达到最大开放概率一半时的钙离子浓度（Kd）为3×10^-7M。在细胞内钙离子浓度为1μM时，可记录到长时间的开放伴以长时间的关闭或失活现象。细胞外给予低浓度的TEA（200μM）和ChTX（100nM）可抑制通道的开放。说明我们所记录的通道为低浓度TEA和ChTX依赖的钙激活钾通道。 二、重症失血性休克后BK_Ca活动的变化 失血性休克后，ASMC膜电位由假手术组的-41.90±4.25mV（n=10）变化为休克时的-66.21±2.88mV（n=8p＜0.01）。在细胞贴附式记录中发现：失血性休克2小时后，在钳制电压为20，40，60mV时BK_Ca的电流幅度显著增加（n=52，p＜0.05，独立样本t检验）。失血性休克后BK_Ca通道的钙麟矛污绷通逍与玲血趁粼‘兑.单通道电导没有改变（分别为145月1 ps和145士15 ps），而反转电位则发生了明显的位移，由假手术组的0.9 mV变化为休克后的一13.0 mV，表明休克后钾离子平衡电位发生了变化，同时进一步证明休克后细胞膜发生了超极化。 为了进一步阐明BKc。的通道特性是否改变，我们进一步采用膜片钳的内面向外式记录法记录通道特性的变化。结果表明:在浴槽液和电极液均为140 mM高钾状态下，通道的电导和电压依赖性在失血性休克后没有改变，而通道的钙离子敏感性发生了改变。统计每个膜片连续记录205的实验结果表明:在失血性休克组，B狡。通道钙离子敏感特性，尤其是通道的钙依赖性失活特性发生了重要改变。在假手术组，当细胞内钙离子浓度为l件M时即可记录到继通道开放后的长时间失活现象;而在休克组，这种长时间的失活现象在细胞内钙浓度为1林M时记录不到，而在细胞内钙浓度为10林M时，方可记录到。说明使通道失活的钙离子浓度增加。 失血性休克2小时后，细胞内钙离子浓度由假手术组的130.5士巧.4nM变化为202.6士23.6nM（P<0.05），提示细胞内钙离子浓度增加。三、ASMC钙火花和一过性外向电流（S TOC）的特性 钙火花为细胞内肌浆网上一个或数个Ryanodine受体（RyR）所介导的钙释放的基本单位。在血管平滑肌细胞上，钙火花能够激活细胞膜上的数个或数十个B从a通道，形成自发性外向电流（sToc），从而引起细胞膜超极化，介导血管平滑肌的舒张。本实验用共聚焦显微镜观察细胞内钙火花的自发性发放频率、持续时间和空间分布，发现:ASMC中的钙火花多分布于靠近细胞膜的部位。静息状态下，83.12士1.83%的细胞有钙火花的出现。其发放频率为0.045士0.003恤m·S。钙火花的峰值(amplitude，F/F（，）为1.32士。一4，最大持续时l石J（duration。f;naxinit;m，Dmax）为132.84士6.72 Ins，乎均持续[I寸l石J（dt，ration of avel·age，nave）为85.34士5一8 ms，时l泊J半高宽（till一duration half maximum，FDHM）为46.5（）士3.51 ms;最大空l泊J持续宽度（width of maximum，wmax）为4.66士（）一3林m，平均持续宽度（width ofaverage，wave）为2.91士0.10卜:m，空I‘HJ半高宽（full duration halfmaximum，FwHM）为1 .98士0.05卜Lm。Nifedipine 10卜、M，Ryanodinelo林M匀能够抑制钙火花的产生，使产生钙火花的细胞比率分别降低为22.22川.88%和满要，27.27士7.87%，提示钙火花是通过细胞膜上的电压依赖性钙通道（vOC）触发的，同时它的释放依赖于肌浆网上的Ryanodine受体。 在钳制电压为0 mV时，STOC的活动频率和幅度分别为1.23士O.19Hz和25.87士0.31 pA;持续时间为65.5牡o.52ms，其中上升时间为16.39士0.11ms，下降时间为49.15土0.47 ms，时间半高宽（FDHM）为19.81士0.22 ms。STOC的上升时间远远短于其衰减时间，这与钙火花的时间分布相对应。CTXloo nM，Ryanodinelo林M均可抑制sToe的频率和幅度，而咖啡因-mM能够使STOC的开放频率增加，且呈规律性的簇状（duster）开放，说明sToC的活动与肌更多还原

【Abstract】 Patch clamp and laser confocal scanning microscopy techniques were used in the experiments to investigate the change of BKca in vascular hyporeactivity in hemorrhagic shock, and the results were as follows:1. Properties of BKca in ASMC. The current recorded was voltage dependent, high potassium selective and intracellular calcium sensitive. The reversal potential of BKca was 0.9 mV, slope conductance was 221? pS. [Ca2+]_i was 3 X10-7 M when the channel was half maximum activated with 140 mM K^- bathing and pipette solutions in inside-out configuration. NPo increased e-fold with 17mV positive change in membrane potential at 0.1 uM [Ca2+]j. Two exponentials were required to describe the observed open and closed time distribution of BKca, with open time constant of 0.7674?.1359, 6.0946?.8998 and closed time constant of 0.5964?.0929, 10.8426?.9037 respectively at 40 mV. At 1#M [Ca²⁺ ];, long term activation of BKCa was followed by long time inactivation, which was known as calcium dependent inactivation. BKca could be inhibited by extracellular amplication of TEA （200 uM） and ChTX （100 nM）, indicating that BKca in ASMC was a TEA and ChTX sensitive potassium channel.2. Change of BKca in ASMC in hemorrhagic shock. The resting membrane potential of ASMC changed from -41.90?.25 mV in sham （n=10） to -66.21?.88 mV in hemorrhagic shock （n=8, p<0.01）. The current amplitude of BKca increased significantly at holding potential of 20, 40, 60 mV respectively with cell attached patch clamp recordings, whereas the single channel conductance （145?1 and 145?5 pS respectively） had no alteration in shock group. The reversal potential of BKCa shifted negatively from 0.9?.9 mV in sham group to -13.0?.2 mV in shock group （p<0.01）, resulting in the shift of equilibrium potential of potassium, which was consistent with theASMC hyperpolarization in hemorrhagic shock.An excised patch clamp configuration with symmetric high potassium （140 mM） solutions was used to further investigate if the property of BKca changed in hemorrhagic shock and the result showed that neither slope conductance （221 ? and 216? pS respectively） of the channel nor reversal potential was altered in shock group. The voltage dependence of BKca didn’t change either, whereas the calcium dependence property of the channel greatly altered. [Ca2+]_i causing inactivation of BKca shifted from 10⁶ M in sham group to 10-5 M in shock group. Cytosolic calcium concentration ([Ca⁺]_i) was further detected and the result showed that [Ca2+]_i increased apparently from 130.5?5.4 nM in sham group to 202.6?3.6 nM in shock group, indicating that the calcium overload might be involved in the activation of BK_Ca in hemorrhagic shock.3. The properties of calcium spark and spontaneous outward current （STOC） and their change in hemorrhagic shock. Laser confocal scanning microscopy was used to detect the calcium spark in ASMC and the results showed that calcium spark localized mainly to the submembrane. 83.12?.83% of the cells tested had spontaneous calcium spark in resting conditions. When nifedipine （10 nM）, a blocker of L-type voltage dependent calcium channel （L-VOC） and ryanodine（10 #M）, an inhibitor of ryanodine receptor （RyR） were applied, only 22.22?.88% and 27.27?.87% of the cells detected had calcium spark respectively, suggesting that the calcium spark was closely related with L-VOC and RyR. The amplitude （F/F0）, Dmax, Dave, FDHM, Wmax, Wave and FWHM of calcium spark all increased significantly in hemorrhagic shock （p<0.05, compared with sham group）, indicating the elevation of local calcium release from RyR.With perforated whole cell patch clamp techniques, STOC in ASMC was recorded at holding potential of 0 mV, and the results demonstrated that the amplitude, rise time, decay time, duration, and FDHM of STOC increased apparently in hemorrhagic shock （p<0.01, compared with sham group）,suggesting the activation of STOC during shock.4. Experimental treatment. To further confi更多还原