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N-甲基小檗胺对大鼠肠系膜阻力血管平滑肌细胞钙激活钾电流的作用
Effects of N-methyl Berbamine on Calcium-activated Potassium Currents in Rat Mesenteric Resistance Vascular Smooth Muscle Cells
【作者】 韩冬云;
【导师】 李金鸣;
【作者基本信息】 中国医科大学 , 药理学, 2003, 硕士
【摘要】 前言 高血压是危害人类健康的重要疾病,其发病因素是多方面的,然而,由于高血压的主要表现是外周阻力血管张力持续升高,同时血管对加压物质的反应性增高,因此,高血压发病的血管机制受到高度重视。 血管张力持续升高是血管舒缩活动异常所致,血管舒缩活动则取决于平滑肌的收缩与舒张状态。细胞膜离子通道的正常活动是血管平滑肌兴奋舒缩过程的关键,也是维持细胞的兴奋性与膜稳定性的保证。其中,钙通道与钾通道的作用至为重要。钾通道通过影响细胞膜电位引起电压依赖性钙通道的开放与关闭,改变胞内游离Ca2+浓度进而影响血管的舒缩状态。近年来应用膜片钳技术发现血管平滑肌细胞膜上至少存在四种不同类型的钾离子通道,钙激活钾(KCa)通道在调节血管的舒缩中起着重要作用。 NMB是从小檗属植物中提取的一种生物碱。动物实验表明小檗胺具有降压、抗心律失常、抗心肌缺血等作用。本实验室以前的研究表明NMB可抑制豚鼠心室肌细胞Ca2+电流和ATP敏感钾电流,从离子转运角度阐明其抗心律失常作用的机理。但有关NMB降压机制的研究尚未见报道。本实验应用膜片钳全细胞记录模式观察了NMB对大鼠肠系膜阻力血管平滑肌细胞IKca的影响,以阐明其降压的离子机制。 实验方法 1.大鼠肠系膜动脉平滑肌细胞的制备 Wistar大鼠,体重(300±20)g,由中国医科大学实验动物部提供,95兼用。m%水含氯醛4价他 .麻醉,开腹分离肠系膜动脉A。-A。分支并迅速移至冰冷的含钙生理盐溶液中,经修饰后用小剪子将动脉剪碎,用二步酶解法分离出单个血管平滑肌细胞,当细胞数量最多、质量最好时,收集细胞。将细胞悬液保存于4℃冰箱中,8小时内使用。 2.I。c。的电生理记录 选取胞膜光滑完整,胞浆均匀,长梭形舒展的细胞进行实验。全细胞状态形成后,用 pCLAMP 5.5.1软件程序发放刺激并收集记录信号。采用保持电位为一60 mV,刺激电位为一60 mV-+60mV,步阶脉冲 10 mV,波宽 500 ms,刺激间隔 3 s的方波钳制方案进行刺激。 3.数据分析 记录的电流在 pCLAMP 5.5.1程序中进行分析和电流大小的测量/。。。幅度为刺激结束时相对于零电流水平的值,数据用 k。S表示,将同一细胞用药前后的电流值进行配对资料的 t检验,P<0.05具有统计学意义。 实验 结 果 三种浓度的 NMB(0.1**0卜mol·L叫)可使I卜。幅值分别由给药前的(33.62 t 2.00)pA/nF增至给药后的(35.68土互.87)PA/PF、(42.90 i 2·73)PA/PF和(59·36。二.39)PA/PF(n=6f<0.01)。其增加后分别为对照的1.06。o.02、1.28。O.03和1·77土0·09(n=6,P<0.01)倍。其它指令电位下1。m的改变也符合此趋势。 ·2· 讨 论 1.膜片钳技术及其在心血管研究中的应用 80年代初发展起来的膜片钳技术已成为研究细胞膜离子通 道和药物作用机制不可缺少的重要手段。膜片钳技术由离子单通 道记录和全细胞记录两部分组.成。近年来研究离子通道的变化对 细胞功能的影响以及在药物作用机制的研究中越来越多地采用全 细胞记录方式。 近年来,随着人们对高血压发病机制认识的不断深人,各类抗 高血压药物应运而生。其中开发作用于血管平滑肌钾通道的药物 成为研究的热点。本实验应用膜片钳技术全细胞记录模式观察了 NMB对血管平滑肌细胞Ih的作用,阐明其降压的离子机制。 2.大鼠肠系膜动脉平滑肌细胞的制备 内脏血管床含有大量的阻力血管,承担着相当部分循环外周 阻力。另外内脏血管对循环中活性物质及药物十分敏感,所以,内 脏血管的舒缩在血压的变化以及高血压发病中的意义更为突出。 故本实验利用大鼠内脏肠系膜动脉A4-A。分支阻力血管平滑肌 细胞来研究NMB的降压作用正是从其对阻力血管的影响来阐明 其机制的。 本实验分离细胞的方法用较少的酶在二h内即能分得舒展的 长梭形细胞,具有较高的成活率(>70助人所测电流均以电流密 度表示,以消除细胞大小不同所带来的实验误差,使结果具有更好 的可比性。 3.NMB对大鼠肠系膜动脉平滑肌细胞I加的作用 钙激活钾通道在调节血管的舒缩活动中起着十分重要的作 用,其功能的正常与否直接影响着血管平滑肌的张力。已证明动 物血管平滑肌细胞主要存在大电导钙激活钾通道。细胞外Ca卜 。3·浓度升高及细胞膜去极化均可促进通道开放,钾离子顺电一化学梯度外流,导致细胞膜超极化,阻止细胞对C/”的摄取、储存和释放,使细胞内Ca卜浓度降低而引起血管舒张。研究表明,血管平滑肌细胞Kc。通道功能减弱是高血压发生和维持的主要原因之一。因此,近年来作用于Kc。通道的血管舒张剂受到广泛关注。 本实验条件下引出的外向钾电流主要包
【Abstract】 IntroductionHypertension is a threatening disease to humans health, whose causes are complicated. High blood pressure results from the increased peripheral vascular resistance and the hypersensitivity to he-mopiesic substances of vessels, so vascular mechanism of hypertension has been given high attention.The increased vascular resistance is caused by abnormal activity of vessels. Ion channels in cell membrane, especial calcium channels and potassium channels, play an important role in the regulation of vascular tone. Potassium channels can alter the status of voltage - dependent calcium channels by influencing membrane potential, which induces changes of the intracellular Ca + concentration, ultimately affecting vascular tone. Vascular smooth muscle cells generally exhibit four major types of potassium channels. Calcium - activated potassium ( KCa) channel plays a significant role in regulating vascular tone.N - methyl berbamine ( NMB) is a dibezyliso - crinoline alkaloid isolated from a Chinese plant, Berberis poireii Schneid. Its chemical structure is similar to berbamine. The previous studies on animals showed that berbamine had effects of lowering blood pressure, antiar-rhythmic action and prevention of cardiac muscle ischemia. The studies on pharmacological mechanism of NMB have deepened into ionic level since patch - clamp techniques appeared. The previous studiesin our lab revealed that NMB could inhibit Ca2 + channels and ATP -sensitive potassium ( KATP) channels in single isolated myocardiocyte of guinea pigs to show antiarrhythmic effect. However, the ionic mechanism on lowering blood pressure has not been elucidated. The present study was to observe the effect of NMB on / Kca in rat mesenter-ic resistance vascular smooth muscle ceils using patch - clamp techniques under the standard whole - cell configuration.Methods1. Isolation of single arterial smooth muscle cellMale or female Wistar rats ( 300 ?20g ) , supplied by Laboratory Animal Center of China Medical University, were anesthetized by ip. injection of a dose ( 4 ml kg ) of 10% Chloral Hydrate. The A4 ~ A5 branches of mesenteric artery were dissected out and placed in Physiologic Salt Solution ( PSS). The connective tissues were carefully removed and the cleaned vessels were cut into small pieces. Single myocyte was isolated with enzymatic dissociation techniques and the cells with best quality were collected and stored in Ca2 + - free PSS at 4 for at least 1 h before use.2. Electrophysiological recordingOnly relaxed, spindle - shaped cells were used for experiments. Single cell was voltage - clamped, and membrane currents were measured using the whole - cell configuration of the patch - clamp technique. For the current measurement, membranes were held at -60 mV, the step pulse was 10 mV, the test potential was -60 mV to + 60 mV and the duration was 200 ms.3. Data analysisAll data analysis was performed with pCLAMP 5.5.1 software. / Kca was measured as the amplitude of the current remaining at the end of the test pulse relative to the zero current level. Data were presented as x s. Statistical significance was assessed using a paired t - test and P <0.05 was considered significant.ResultsWhen the cells were perfused with extracellular solution containing NMB 0. 1, 1 and 10 (junol L-1, the amplitude of I Kca increased from control ( 33.62 2.00 ) PA/pF to ( 35.68 1. 87 ) pA/pF, ( 42.9 2.73 ) pA/pF and (59.36 1.39 ) pA/PF ( n =6 cells from 6 rats,P<0.01 ) , respectively, at this time cells were kept at -60 mV and commanding potential was + 60 mV. The activated currents decreased after washout with PSS. The amplitude of/ Kca was (1. 06 0. 02 ) , (1. 28 0. 03 ) and ( 1. 77 0. 09 ) ( n = 6 ceUs from 6 rats, P< 0.01 ) times that of control, respectively. The opening effect of NMB on I Kca was recognized clearly in the I - V curve.Discussion1. Patch - clamp technique and its application in cardiovascular studiesPatch - clamp technique developed in 1980s has become an indispensable m
【Key words】 hypertension; vascular smooth muscle; N -methyl berbamine; patch- clamp technique; potassium channels;
- 【网络出版投稿人】 中国医科大学 【网络出版年期】2003年 03期
- 【分类号】R965
- 【下载频次】90