牛磺酸对大鼠离体气管平滑肌舒张作用及机制的研究

目的研究牛磺酸（Tau）对大鼠离体气管平滑肌的作用及可能机制。方法观察牛磺酸对高钾预收缩大鼠离体气管环的作用,通过工具药观察牛磺酸对气管环作用的可能机制。经Powedab生物信号测定系统记录气管环张力。结果①牛磺酸【（10～80）mmol／L】对高钾（30mmol／L）诱发的大鼠离体气管环收缩有舒张作用,且呈浓度依赖性,最大舒张率为（50．22±18．30）％。②牛磺酸舒张大鼠气管平滑肌的作用可被K、Ca通道抑制剂四乙胺（10mmol／L）和KATP通道抑制剂格列苯脲（10μmol／L）显著减弱,而KIR通道抑制剂氯化钡（1mmol／L）和KV通道抑制剂4-氨基吡啶（1mmol／L）对此则无显著影响。（3）L—NAME（1mmol／L）、Pino（10μmol／L）、Indo（10μmol／L）对牛磺酸舒张大鼠离体气管环无明显影响。结论牛磺酸可舒张大鼠离体气管平滑肌．且舒张作用呈浓度依赖性。其舒张作用与一氧化氮、前列环素和β受体无关,可能与激活KATP通道和K、Ca通道有关。     

大电导钙激活钾通道对子宫平滑肌作用的分析

大电导钙激活钾(large conductance Ca2+-activated K+-channel,BKCa)通道在未妊娠和妊娠子宫平滑肌细胞中是最主要的钾通道。BKCa通道的激活可导致细胞膜超极化,从而抑制电压依赖性钙通道的激活及钙离子的内流,引起平滑肌舒张。近年来的研究结果证实,子宫平滑肌细胞中BKca通道的激活、失活和变异与产科多种生理现象及疾病的发生有关,如可调控妊娠维持、分娩启动、产后出血、早产等。本文主要分析BKCa通道的基本结构,并总结分析近年来关于BKCa通道在生理妊娠及病理妊娠中相关作用的研究进展。     

大电导钙激活钾通道在平滑肌细胞中的调节机制

大电导钙激活钾通道由形成孔道的α亚基及具有调节作用的β亚基组成,因其电导大,对调节平滑肌细胞功能起重要作用,从而成为近年来研究的热点。大电导钙激活钾通道的调节机制多种多样,现对其近年来调节机制的研究进展作一综述。     

利多卡因对大鼠离体气管平滑肌的作用及机制

目的:研究利多卡因对气管平滑肌的作用及机制.方法:观察利多卡因对乙酰胆碱(ACh)和高钾预收缩大鼠离体气管的作用,对ACh、高钾和CaCl2诱发气管平滑肌收缩作用剂量反应曲线和ACh致两种收缩组分的影响并与维拉帕米进行比较.结果:利多卡因对ACh和高钾诱发的大鼠离体气管平滑肌标本收缩有舒张作用,并不被L-NAME和普萘洛尔阻断.利多卡因浓度依赖性地使ACh、KCl和CaCl2对大鼠离体气管条标本的量效反应曲线右移,最大反应降低.利多卡因0.69和3.45 mmol/L对ACh所致依赖细胞内钙收缩的抑制率及依赖细胞外钙收缩的抑制率差别均十分显著(P＜0.01).结论:利多卡因对离体大鼠气管平滑肌的舒张作用可能是通过非特异性地抑制细胞外钙经电压调节和受体调节钙通道向内流动及抑制细胞内钙释放来实现的.     

SK3表达在雌二醇介导的大鼠结肠平滑肌收缩中的作用

目的：探讨小电导钙激活钾通道3（SK3）表达在17β?雌二醇（E2）介导的大鼠结肠平滑肌收缩中的作用及机制。方法：将24只雄性SD大鼠随机分为4组。30 mm硅胶管皮下植入大鼠背部,内含不同溶液。分为对照组（C）,内含溶剂;生理剂量组（EP）内含E2 0.3 mg/mL,使大鼠血清雌激素在生理浓度;超剂量组（ES）内含E2 0.9 mg/mL,使大鼠血清雌激素超生理浓度;ER抑制剂+E2组（EI）内含相同摩尔浓度E2和雌激素受体抑制剂（ICI 182780）。各组干预14 d后处理,免疫荧光及Western blot检测结肠平滑肌组织SK3分布及表达;MPA分析系统记录肌条收缩张力及对卡巴胆碱（Cch）刺激反应。E2孵育结肠平滑肌细胞（SMC）24 h后及过表达SK3后,激光共聚焦显微镜下动态观察Cch刺激后SMC内Ca2+变化。结果：Cch刺激后,EP组及ES组肌条收缩明显低于其他各组（P < 0.05）。 EP组及ES组平滑肌组织SK3表达高于C组及EI组,其中ES组有统计学意义（P < 0.05）。Cch刺激后,E2孵育组及过表达SK3组胞内Ca2+上升幅度较相应对照组显著降低（P < 0.05）。结论：E2可能通过促进SK3表达,减少Ca2+内流从而抑制结肠收缩。     

小鼠脑动脉平滑肌钙激活钾通道基本特性和动力学调控研究

目的采用膜片钳技术记录小鼠脑动脉平滑肌细胞上的大电导钙激活钾通道(BKCa)和自发瞬时外向电流(STOCs),研究了其基本特性以及钙离子对通道动力学的调控。方法采用两步法急性酶分离小鼠脑动脉,得到单个平滑肌细胞。使用200μg/mL两性霉素B为穿孔剂进行穿孔全细胞膜片钳实验,包括全细胞宏观电流记录和STOCs记录,采用inside-out和outside-out构型进行单通道电流记录。结果 BKCa宏观电流和STOCs能够被BKCa的特异性阻断剂IbTX(200nmol)阻断。BKCa单通道具有明显电压依赖性(半数最大激活电压V1/2=78.09mV)、K+选择性和钙敏感性(Ca2+解离常数Kd=0.881μmol),能够被胞外IbTX阻断。胞内Ca2+调控通道动力学特性主要表现为减少了开放时间常数,促进了通道由关闭向开放的转变。结论 BKCa宏观电流和STOCs非常容易记录,单通道活性好,具有BKCa的电生理学特性。小鼠脑动脉平滑肌是研究BKCa功能活动和血管活性药物作用机制的良好实验标本。     

氟灭酸对豚鼠微动脉平滑肌细胞BKCa通道的增强作用

目的 观察氟灭酸对微动脉平滑肌细胞电生理学特性的影响.方法 应用全细胞膜片钳技术在离体豚鼠脑动脉(BA)和肠系膜动脉(MA)的平滑肌细胞上,观察氟灭酸(100～1 000 μ mol/L)对微动脉平滑肌细胞膜电流的影响.结果 4-氨基吡啶(1 mmol/L)和四乙胺(1mmol/L)都可以部分抑制微动脉平滑肌细胞膜电流.氟灭酸可以浓度依赖的增强微动脉平滑肌细胞膜电流,100、300和1000μmol/L氟灭酸分别使BA平滑肌细胞膜电流幅度(+40 mV)增强了(36±14)％、(136±26)％和(223±24)％,使MA平滑肌细胞膜电流幅度(+40mV)分别增强了(45±11)％、(166±24)％和(278±24)％.氟灭酸增强平滑肌细胞膜电流具有电压依赖性,氟灭酸主要增强0～+40 mV电压区间的激活电流,其中对+40 mV激活电流的增强作用最强.背景灌流四乙胺(1mmol/L)能显著抑制氟灭酸对微动脉平滑肌细胞膜电流的增强作用.结论 氟灭酸可以浓度和电压依赖的激活微动脉平滑肌细胞膜上BKCa通道.     

川芎嗪对正常人体肠系膜血管平滑肌细胞大电导钙激活钾通道的作用

目的：研究川芎嗪（（tetramethylpyrazine,TMP））对正常人体肠系膜血管平滑肌细胞大电导钙激活钾通道宏观电流（large-conductance Ca^2＋-activated potassium channels,BKCa）的作用,探讨TMP对BKCa的作用机制。方法：用急性酶分离法分离人体肠系膜动脉平滑肌细胞,采用全细胞穿孔膜片钳技术记录该细胞上的大电导钙激活钾通道宏观电流,并测试TMP对该电流的作用。结果：TMP在0.73、3.67mmol/L时可明显地可逆性地激活正常人体肠系膜血管平滑肌细胞上的BKCa,在0.73mmol/L浓度时,测试电压从-50～＋60mV时差异都有统计学意义,其中测试电压在-50mV时,电流密度从0.25±0.03pA/pF增加到0.34±0.039pA/pF（P〈0.01,n=12）,测试电压在＋60mV时,电流密度从8.37±1.75pA/pF增加到14.12±2.66pA/pF（P〈0.01,n=12）;TMP在3.67mmol/L时可明显地可逆性地激活正常人体肠系膜血管平滑肌细胞上的BKCa,测试电压从0～＋60mV时差异都有统计学意义,其中测试电压在0mV时,电流密度从1.25±0.23pA/pF增加到1.90±0.31pA/pF（P〈0.05,n=7）,测试电压在＋60mV时,电流密度从10.57±3.61pA/pF增加到20.32±4.68pA/pF（P〈0.05,n=7）。但TMP在8.07mmol/L时可明显地可逆性地抑制正常人体肠系膜血管平滑肌细胞上的BKCa,测试电压从-60～＋60mV时差异都有统计学意义,其中测试电压在-60mV时,电流密度从0.28±0.02pA/pF降低到0.15±0.04pA/pF（P〈0.01,n=6）,测试电压在＋60mV时,电流密度从10.49±0.44pA/pF降低到1.83±0.68pA/pF（P〈0.01,n=6）。结论：TMP在0.73、3.67mmol/L下,可激活正常人体肠系膜血管平滑肌细胞大电导钙激活钾通道,而在8.07mmol/L时可明显地可逆性地抑制正常人体肠系膜血管平滑肌细胞上的BKCa。在低浓度和较高浓度下TMP对正常人体肠系膜血管平滑肌细胞上的BKCa有不同效应的实验结果,为TMP应用于临床提供了实验依据     

侧柏叶乙酸乙酯提取物对豚鼠离体气管平滑肌的作用

用离体豚鼠气管条,探讨侧柏叶乙酸乙酯提取物对气管平滑肌作用。结果表明,侧柏叶乙酸乙酯提取物能抑制乙酰胆碱,氯化钾所致气管平滑肌收缩,而且能使乙酰胆碱收缩气管平没肌的量效曲线右移,并抑制最大效应,其作用为剂量依赖性心肌肥厚提示侧柏叶乙酸乙酯提取物松驰气管平滑肌作用机制可能与影响Ｃａ＾２＋的跨膜转运有关。     

Potassium channels in airway smooth muscle and airway hyperreactivity in asthma

Our knowledge of the physiology of ion channels has increased tremendously during the past 20 years because of the advances of the single-channel recording and molecular cloning techniques. More than 50 different identified potassium channels have already been found. 1,2 They are distributed ubiquitously in wide variety of cells including airway smooth muscle (ASM) cells and inflammatory cells in airway such as eosinophils,     

大鼠膀胱平滑肌细胞自发性钙瞬变及其机制研究

目的观察大鼠离体活膀胱肌条铺片中平滑肌细胞自发性钙瞬变,并探讨平滑肌细胞自发性钙瞬变产生的机制。方法制作大鼠离体膀胱肌条铺片,生理液灌流保持肌条铺片活性。采用Fluo-4负载肌条铺片,激光共聚焦显微镜法观察其中平滑肌细胞自发性钙瞬变,并观察尼莫地平(Nimodipine)、2-氨基乙基二苯硼酸盐(2-aminoethoxydiphenylbo-rate,2-APB)、雷诺丁(Ryanodine)、毒胡萝卜素(Thapsigargin)对平滑肌细胞自发性钙瞬变的影响,探讨平滑肌细胞自发性钙瞬变产生的机制。结果可见大鼠离体活膀胱肌条铺片中平滑肌细胞产生节律性自发性钙瞬变,自发性钙瞬变幅度(F/F0)和频率分别为(2.20±0.13)和(10.31±2.74)次/min。L型钙离子通道阻断剂Nimodipine能完全抑制平滑肌细胞的自发性钙瞬变,2-APB、Ryanodine可将钙瞬变幅度分别降低至(1.98±0.14)、(1.81±0.11),而相应的钙瞬变频率则被减少为(8.18±1.97)(P<0.05,n=18)、(7.59±2.16)次/min(P<0.01,n=17)。Thapsigargin可抑制平滑...     

哇巴因对大鼠血管平滑肌细胞内钙离子浓度的影响

目的探讨哇巴因对大鼠胸主动脉血管平滑肌细胞（vascular smooth muscle cells, VSMCs）内Ca2+浓度的影响。方法用组织
块贴壁法进行大鼠胸主动脉VSMCs原代培养,用免疫组织化学方法进行细胞鉴定,采用放射自显影术检测哇巴因对大鼠胸主动
脉VSMCs的结合能力,采用Fluo 3-AM（钙离子荧光探针）法研究哇巴因在短时间内对VSMCs细胞内Ca2+浓度的影响,探讨不同
浓度的钠泵α2亚单位截断性片段的拮抗作用。结果（1）放射自显影术检测结果显示,哇巴因与VSMCs间有一定的结合能力。
在0.1~100 nmol/L浓度范围,随着哇巴因浓度的增加,VSMCs与哇巴因的结合能力增加;（2）Fluo 3-AM检测VSMCs内Ca2+浓度
结果显示：不同浓度的哇巴因在短时间内能够引起VSMCs内Ca2+浓度的变化。在0~3200 nmol/L浓度范围,细胞内Ca2+离子浓
度会随着哇巴因浓度的增加呈现出逐渐升高的趋势;（3）不同浓度的钠泵α2亚单位截断性片段可以拮抗哇巴因引起的VSMCs内
Ca2+浓度的升高作用。结论哇巴因引起的细胞内高钙是高哇巴因高血压发生的细胞学基础,钠泵α2亚单位截断性片段可以拮
抗哇巴因引起的VSMCs内Ca2+浓度的升高作用,为进一步探讨哇巴因的作用机制结高血压的治疗提供了实验依据。
     

Role of protein kinase C in the activation of store-operated Ca2+ entry in airway smooth muscle cells

Store-operated Ca2+ channels (SOCs) are plasma membrane Ca2+ permeable channels activated by depletion of intracellular Ca2+ store. Ca2+ entry through SOCs is known as store-operated Ca2+ entry (SOCE), which plays an important role in the functional regulation of airway smooth muscle cells (ASMCs). Protein kinase C (PKC) has been shown to have an activating or inhibiting effect on SOCE, depending on cell types and PKC isoforms that are involved. In ASMCs, the effect of PKC on SOCE has not been elucidated so far. In this study, the role of PKC in the activation of SOCE in rat ASMCs was examined by using Ca2+ fluorescence imaging technique. The results showed that acute application of PKC activators PMA and PDBu did not affect SOCE induced by the sarcoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor thapsigargin. The non-selective PKC inhibitor chelerythrine significantly inhibited thapsigargin- and bradykinin-induced SOCE. RT-PCR assay identified PKCα, δ and ε isoforms in rat ASMCs. PKCα-selective inhibitor G6976 and PKCε-inhibiting peptide Epsilon-V1-2 had no effect on SOCE; by contrast, PKCδ-selective inhibitor rottlerin attenuated SOCE dramatically, suggesting that PKCδ was the major PKC isoform involved in the activation of SOCE in ASMCs. Moreover, PKC down-regulation by extended exposure to high doses of PMA or PDBu also reduced SOCE, confirming the essential role of PKC in the activation of SOCE in ASMCs. In addition, PKC down-regulation did not influence the expression of stromal interaction molecule 1 (STIM1) and Orai1, two elementary molecules in the regulation and activation of SOCs. These results identified PKCδ as an essential PKC isoform involved in the activation of SOCE, and confirmed that PKC regulates the function of ASMCs in a SOCE-dependent manner.     

大鼠气管平滑肌细胞的分离及L-钙通道的记录

目的建立一种大鼠气管平滑肌细胞急性酶分离法并在分离的细胞上研究其L型钙通道(L-Ca)特性。方法采用链酶蛋白酶分离大鼠气管平滑肌细胞,用膜片钳单通道记录法记录L-Ca通道电流。结果该方法分离过程简单,所获细胞数量多,形态正常,在这些细胞上容易记录到正常的L-Ca通道电流。结论应用本方法易得到单个活性正常的、形态完整的大鼠支气管平滑肌细胞。     

罗哌卡因对乙酰胆碱诱发兔离体气管平滑肌收缩的作用

目的:探讨罗哌卡因对乙酰胆碱(ACH)诱发兔离体气管平滑肌收缩的影响及其作用机制。方法:取兔气管环,浸浴在克-亨氏营养液(K-H液)中,气管一端固定张力换能器,每个标本先用10-4mol/LACH收缩,然后使其回到基线,加入药物测定其对ACH诱发的离体气管平滑肌收缩的影响。结果:10-3mol/L罗哌卡因对ACH诱发的收缩可产生明显的抑制作用(P<0.05或P<0.01)。在2min左右张力是对照组的70%,10min左右张力降至基线,β阻滞剂普奈洛尔对罗哌卡因的作用没有明显影响。而钙通道阻滞剂地尔硫、佩尔地平可加强罗哌卡因的气管平滑肌舒张效应。结论:10-3mol/L罗哌卡因对兔离体气管平滑肌具有较强的舒张作用,其作用机制可能和阻滞钙通道有一定的关系。     

川芎嗪对抗原诱发致敏豚鼠气管平滑肌环的舒张作用

目的:观察川芎嗪对过敏原诱发的哮喘豚鼠离体气管平滑肌环收缩的影响及其相关机制。方法:采用卵蛋白致敏法建立豚鼠哮喘动物模型,每只豚鼠制备5～7个气管平滑肌环,悬挂于恒温浴槽中,并将其与力位移换能器相连来纪录气管环张力的变化,所有标本分为正常KH液和无钙KH液2大组。正常KH液组又分为卵蛋白组、10mmol/L川芎嗪+卵蛋白组、30mmol/L川芎嗪+卵蛋白组、100mmol/L川芎嗪+卵蛋白组、300mmol/L川芎嗪+卵蛋白组、thapsigargin(TSG)+卵蛋白组、TSG+川芎嗪(100mmol/L)+卵蛋白组。无钙KH液组分为卵蛋白组和川芎嗪(100mmol/L)+卵蛋白组。结果:①卵蛋白组平滑肌环收缩产生的张力为(59.7±8.5)×10-6N,分别预先加入30mmol/L、100mmol/L、300mmol/L的川芎嗪20min后,再加入卵蛋白,平滑肌环产生的张力分别降低至(44.1±5.5)×10-6N、(20.7±20.4)×10-6N、(10.0±1.1)×10-6N(P均<0.05),10mmol/L的川芎嗪对卵蛋白的作用无明显影响(P>0.05);②去除溶液中的Ca2+后,再加入卵蛋白,平滑肌环收缩产生的张力为(10.5±1.9)×10-6N,预先加入100mmol/L的川芎嗪后,卵蛋白诱导产生的平滑肌环的收缩力无明显改变(P>0.05);③预先加入TSG(10mmol/L)40min后,再加入卵蛋白,平滑肌环收缩产生的张力为(27.3±3.4)×10-6N,分别预先加入TSG和100mmol/L的川芎嗪后,再加入卵蛋白诱导,平滑肌环的收缩力减少至(6.5±1.1)×10-6N(P<0.05)。结论:①川芎嗪可能通过抑制细胞膜上的钙通道来抑制抗原引起的哮喘豚鼠离体气管平滑肌的收缩作用。②抗原可能通过细胞外的Ca2+内流和细胞内肌浆网对Ca2+的释放来促进哮喘豚鼠离体气管平滑肌的收缩。     

Effect of Nitric Oxide on Potassium Channels of Rat Airway Smooth Muscle Cells

Summary: The effect of nitric oxide donor sodium nitroprusside (SNP) on resting membrane potential (Em) and potassium currents of the bronchial smooth muscle cells from rats was investigated. All experiments were conducted in conventional whole-cell configuration. The changes of Em and potassium currents after addition of 0. 1 mmol/L SNP were measured under the current-clamp mode and the voltage-clamp mode respectively. Results showed that (1) SNP could decrease the Em from --33. 8±7.4 mV to -43. 7±6. 7mV (n=10, P＜0. 01); (2) SNP could increase the Ca2+-activated K+ channel peak currents under ramp protocol from 466.9±180. 1 pA to 597. 7±237. 6 pA (n= 7, P＜0. 01), and the currents under pulse protocol at +50 mV were increased from 544.2±145.4 pA to 678.1±206. 2 pA (n=6, P＜0.05); (3) SNP also could increase voltage-gated K+ channel peak currents under ramp protocol from 389. 6±84. 1 pA to 526. 7±98. 7 pA (n=7, P＜0. 01), the currents under pulse protocol at +50 mV were increased from 275.7±85.2 pA to 444.3±128.5 pA(n=6,P＜0. 01). It was concluded that SNP increases the activities of Ca2+-activated K+ channels and voltage-gated K+ channels and leads to K+ efflux and hyperpolarization of the cell membrane, resulting in a decrease of the cell excitement.     

Effects and its possible mechanism of Radix Saposhnikoviae on rat colonic smooth muscle in vitro

Objective: To determine the effect of different concentrations of Radix Saposhnikoviae (RS) on the contraction of smooth muscle strips and the Ca2+ mobilization of cultured smooth muscle cells of rat colon and its possible mechanism of action. Methods: Strips of rat colon longitudinal muscle were prepared and smooth muscle cells from rat colon were isolated and cultured. In the experiments, in vitro muscle strips were suspended in an organ bath and the contraction of the strips was recorded. In the cell-experiments, intracellular Ca2+ was assessed using fluorescent intensity (FI) of smooth muscle cells loaded with Fluo-4/AM, measured with a laser scanning confocal microscope and related software. Results: In the in vitro experiment, RS (0.02, 0.2, 2, 20 g/L) inhibited contraction of muscle strips in a concentration-dependent manner, and this inhibition was significant for the three higher RS concentrations (P < 0.01) for both Peak (the maximal contraction amplitude) and Area (the area under curves). Similarly, RS inhibited Ach-induced contraction. In these experiments the inhibition of the Peak values in the RS 2 and 20 g/L groups was significant (P < 0.01), as was the inhibition of the Area values in all RS groups (P < 0.05). Naloxone and propranolol did not significantly affect the inhibitory effect of RS on smooth muscle contractility, while phentolamine significantly reduced the inhibitory effect (P < 0.01). In experiments using primary smooth muscle cell cultures in Ca2+ - containing buffer, the post-treatment fluorescence of cells in the RS 0.2, 2 and 20 g/L groups differed significantly from pre-treatment values (P < 0.05), and the percent inhibition of fluorescence in the RS 2 g/L and 20 g/L groups was significant (P < 0.01). However, in Ca2+-free buffer, FS had no significant effect on cell fluorescence. Conclusion: RS inhibited both the spontaneous and Ach-stimulated contraction of rat colonic smooth muscle strips. This RS effect appeared to involve α-adrenoceptors, but not β-adrenoceptors or opioid receptors. In cultured primary smooth muscle cells, RS reduced the mobilization of Ca2+ from extracellular sources, but may had no effect on the release of Ca2+ from sarcoplasmic reticulum and endoplasmic reticulum.