钾通道开放剂对哮喘豚鼠支气管平滑肌电压依赖性钾通道的作用

采用膜片钳技术探讨哮喘豚鼠支气管平滑肌细胞(ＢＳＭＣｓ)电压依赖性钾离子通道 (Ｉｋ)特性的变化和钾通道开放剂对哮喘豚鼠支气管平滑肌细胞膜Ｉｋ的作用。材料与方法　健康豚鼠 2 5只 ,雌雄不限 ,体重约 30 0～40 0ｇ ,分为 (1)哮喘组 5只 (2 5例细胞 ) ;正常对照组 7只 (2 8例细胞 ) ,行外向峰值钾电流测定 ;(2 )哮喘组 6只 (2 4例细胞) ;正常对照组 7只 (2 5例细胞 ) ,应用钾通道开放剂(Ｃｒｏｍａｋａｌｉｍ)前后钾通道动力学特性变化测定。支气管平滑肌细胞分离 :将两肺组织中 3～ 4级支气管剪成碎块 ,加入 0 2 5 %胰蛋白酶 ,洗涤后…     

大鼠冠状动脉平滑肌细胞离子通道研究中的急性酶分离技术

膜片钳技术的应用,为从分子水平了解生物膜离子通道的门控动力学特征及通透性、选择性等膜信息提供了最直接的手段,使人们对细胞膜通道功能的认识进入了一个崭新的阶段,对研究心脑血管离子通道电生理特性具有相当重要的意义。急性分离的单个血管平滑肌细胞对研究生理和病理状态下血管张力及其反应性变化机制极为重要,细胞分离的好坏很大程度上决定着膜片钳离子通道研究的成败。     

二十二碳六烯酸对大鼠冠状动脉平滑肌细胞大电导钙激活性钾通道的影响

目的探讨二十二碳六烯酸(DHA)对大鼠冠状动脉平滑肌细胞(CASMCs)上大电导钙激活性钾通道(BKCa)的影响。方法采用酶消化法获得大鼠CASMCs,用膜片钳技术分别记录0,10,20,40,60和80μmol/LDHA对大鼠CASMCs上BKCa通道动力学的影响。结果在不同浓度DHA作用下,IBKCa和BKCa尾电流均呈浓度依赖性增加。IBKCa和BKCa尾电流I-V曲线均上移,对IBKCa稳态激活曲线无影响。在指令电压+150 mV,不同浓度DHA作用下,IBKCa电流密度分别为68.24±22.75,72.40±24.49,120.44±37.96,237.48±53.22,323.60±74.83和370.61±88.16pA/pF(P<0.05,n=20)。DHA对IBKCa激活的药物半效浓度为36.22±2.17μmol/L。在测试电压+90 mV,不同浓度DHA作用下,BKCa尾电流密度分别为91.02±13.52,100.23±17.34,224.02±38.76,369.19±65.39,511.39±82.77和700.14±96.64 pA/pF(P<0.05,n=20)。结论 DHA对全细胞BKCa有激活作用,对稳态激活曲线无影响。DHA对BKCa通道的激活作用可能是其舒张血管机制之一。     

糖尿病大鼠冠状动脉平滑肌细胞大电导钙激活钾通道特性及开放动力学的变化

目的研究糖尿病时冠状动脉平滑肌细胞大电导钙激活钾通道(BK通道)的电压依赖性、钙敏感性及其开放动力学变化,探讨糖尿病时冠状动脉损伤的细胞电生理机制。方法采用链脲霉素腹腔内注射建立糖尿病大鼠模型(糖尿病组,n=30),对照组相应注射生理盐水(n=30);采用酶消化法急性分离大鼠冠状动脉平滑肌细胞;采用膜内向外型单通道膜片钳实验技术记录BK单通道电流;采用Clampfit 10.4软件分析单通道数据。结果在电极外液钙离子浓度为1mmol/L,刺激电位分别为0,20,40,60,80,100,120和140mV时,随着刺激电位增加,正常组BK通道的NPo逐渐增加,其半数有效激活电压(V1/2)为(88.45±1.72)mV;糖尿病组BK通道NPo亦逐渐增加,V1/2为(104.29±1.09)mV,其电压-NPo曲线较正常组向左下移动。在刺激电位60 mV,电极外液钙离子浓度分别为0,0.01,0.1,0.5,1,5,10和50mmol/L时,随着电极外液钙离子浓度增加,正常组BK通道NPo逐渐增加,其半数有效激活浓度(EC50)为(2.26±0.27)mmol/L;糖尿病组BK通道NPo亦逐渐增加,EC50为(3.15±0.20)mmol/L,其浓度-NPo曲线较正常组向左下移动。在电极外液钙离子浓度为1mmol/L,刺激电位60 mV时,与正常组比较,糖尿病组BK通道NPo明显减少,电流幅度与平均开放时间无明显变化,而平均关闭时间显著延长[(451.35±113.71)ms vs(107.58±15.99)ms,P<0.05]。结论糖尿病时冠状动脉平滑肌细胞BK通道电压依赖性与钙敏感性均受损,开放概率减少,通道平均关闭时间延长,这可能是糖尿病冠状动脉功能损伤的重要原因之一。     

自发性高血压大鼠冠状动脉平滑肌细胞大电导钙激活电压依赖性钾离子通道的研究

目的冠状动脉壁张力升高和冠脉循环阻力上升是诱发心绞痛的重要因素。造成冠脉张力上升的因素有多种,其中血压升高是重要因素之一。升高的血压施加于管壁,使血管平滑肌张力上升,机体通过复杂的自适应机制,启动血管舒张机制,以平衡和维持冠状动脉循环的动态平衡。目前,高血压背景下冠状动脉的适应性舒张机制尚未明确。本研究选取自发性高血压大鼠（SHR）为高血压模型,应用电生理膜片钳技术和分子生物学手段,研究高血压大鼠冠状动脉平滑肌细胞（CASMCs）大电导钙激活电压依赖性钾离子通道（BK）,以及BK通道的表达,并与正常血压组对照,阐明高血压大鼠CASMCs复极电流是否发生适应性增加,并探讨舒张机制在维持冠脉循环中的重要地位。方法 （1）采用"三步"酶消化法,即含0.125%牛血清白蛋白（Bovine serum albumin,BSA）缓冲液室温孵育10分钟,酶液Ⅰ消化10-20分钟,酶液Ⅱ消化10-15分钟。（2）分离大鼠冠状动脉平滑肌细胞后,应用膜片钳全细胞记录模式记录高血压和正常SD大鼠冠状动脉平滑肌细胞钾电流,分析大鼠CASMCs上主要的钾流成分及一般特性。（3）记录并比较SHR和WKY大鼠CASMCs上BK电流以及BK尾电流的大小。（4）提取SHR和WKY大鼠冠状动脉平滑肌细胞的RNA,并进行逆转录,利用相对定量和半定量的方法比较高血压大鼠和正常大鼠的BK通道α和β1亚基的mRNA水平。（5）提取并纯化SHR和WKY大鼠的冠状动脉平滑肌膜蛋白,采用免疫组化和westernblot方法对高血压大鼠和正常血压大鼠BK通道α和β1亚基的蛋白水平进行比较。结果 （1）电生理结果:①WKY大鼠（n=82）平均体重为310±13.2 g,平均尾动脉收缩压为112.39±13.17 mm Hg,SHR（n=61）平均体重270±15.5 g,平均尾动脉收缩压为172.057±20.13 mmHg。SHR大鼠和WKY大鼠的平均静息电位大小分别为-31.7±4.53mV（n=6）和-49.8±5.11 mV?     

高血压大鼠冠状动脉平滑肌细胞大电导钙激活钾通道的变化

目的研究在高血压背景下大电导钙激活钾(BK)通道的功能改变及其机制。方法用酶解消化方法分离12～16周龄自发性高血压大鼠(SHR)和WKY大鼠冠状动脉平滑肌细胞(CASMCS),采用膜片钳全细胞模式记录SHR和WKY大鼠CASMCSBK电流,SHR和WKY大鼠BKα亚基和β1亚基mRNA水平用实时定量取聚合酶链式反应和凝胶电泳测定,蛋白水平表达用免疫组化的方法测定。结果 SHR大鼠CASMCS(n=6)BK电流密度比WKY(n=7)高2.03±0.62(P0.01);在mRNA水平,BKβ1亚基表达SHR组明显高于WKY组5.534±1.03倍(n=4,P0.05),BKα亚基表达则无明显差异(1.266±0.12,n=4,P0.05);BKβ1亚基和α亚基的表达SHR大鼠高于WKY大鼠。结论 SHR大鼠CASMCSBK电流密度比WKY大鼠增大,在mRNA和蛋白水平上BKβ1亚基表达也比WKY大鼠增强,这种变化可能是机体在高血压时自我调节的结果 。     

电压依赖性Kv1.5通道的研究进展

电压依赖性钾离子通道Kv1.5是心房肌超快速激活的延迟整流K+电流的分子基础,具有特有的失活机制,Kv1.5通道的表达和功能受多种因素影响,其电流改变在房颤的发生和维持中起重要作用.特异性Kv1.5通道阻滞剂能通过不同的方式来抑制Kv1.5电流,可能会防止房颤的发生,而无室性心律失常的危险.     

冠状动脉血管平滑肌KATP通道的特性和作用

Ｎｏｍａ于１９８３年首次在心脏中发现ＫＡＴＰ通道,随后相继在血管平滑肌细胞（ＶＳＭＣ）、骨骼肌细胞、神经细胞等组织中发现有这些通道的存在［１］。此通道的共同特征是可以被二磷酸核苷酸（ＮＤＰｓ）例如二磷酸腺苷（ＡＤＰ）、尿苷－５’－二磷酸（ＵＤＰ）、二磷酸鸟苷（ＧＤＰ）等和钾离子通道开放剂（ＫＣＯｓ）所激活,被磺酰腺（ＳＵ）类药物所抑制,例如优降糖和三磷酸腺苷（ＡＴＰ）。由于ＶＳＭＣ膜ＫＡＴＰ通道的研究较其他类型的组织细胞困难,人们对于冠脉ＶＳＭＣ的ＫＡＴＰ通道的认识较局限。随着分子克隆技术和电生…     

血管平滑肌钾离子通道的增龄变化及其研究

21世纪人群的老龄化问题将越来越严重,随之血管病变的发生率也将越来越高,近年人们发现增龄变化对血管平滑肌离子通道有明显影响,从而促进血管硬化.本文对血管平滑肌K+通道的分子结构与电生理特性、调节因素及其增龄变化的研究进展作一综述.     

电压依赖性钾离子通道在肺动脉高压发病过程中的作用

肺动脉高压是一种肺血流受限引起肺血管阻力和压力持续性增高,最终导致右心衰竭甚至死亡的综合征.病理生理学改变主要为肺血管收缩、重塑及原位血栓形成.研究表明,钾离子通道尤其电压依赖性钾离子通道功能与表达水平的降低是引起肺血管平滑肌细胞增殖和凋亡异常、肺血管重塑的关键因素.本文着重论述近年来有关电压依赖性钾离子通道与肺动脉高...     

肺动脉平滑肌细胞钾电流记录方法的建立

目的 在肺动脉平滑肌细胞(pulmonary arterial smooth muscle cells,PASMCs)上建立钙离子激活钾通道(calcium-activated potassium channel,KCa)电流、电压门控钾通道(voltage-gated potassium channel,Ky)电流和内向整流钾通道(Inward rectifier channel,Ku)电流的记录方法 .方法 用急性酶解的方法 分离出单个大鼠PASMC,利用全细胞膜片钳方法 记录钾电流.结果 ①急性酶解分离得到高质量的单个大鼠PASMC,呈梭形,边界清楚,胞浆均匀透亮.②Kv电流可以被5 mmol/L4-AP明显抑制,使电流-电压关系曲线明显下移,在55 mV时,Kv电流密度从(133.86±7.36)pA/pF减少到(59.09±3.35)pA/pF(n=6,P<0.05).③1 mmol/L TEA对KCa电流有明显抑制作用,使电流-电压关系曲线明显下移,在55 mV时,KCa电流密度从(9.03±1.42)pA/pF减少到(2.12±0.52)pA/pF(n=7,P<0.05).④KATP电流可以被10 μmol/L尼可地尔激活,使电流-电压关系曲线明显上移,在50 mV时,Kv电流密度从(29.08±5.90)pA/pF增加到(88.90±7.98)pA/pF(n=10,P<0.05).结论 在肺动脉平滑肌细胞上成功地建立了KCa、Kv和Kir电流的记录方法 ,可以为肺动脉相关疾病的发病机制和治疗方案的探索,提供有效的细胞模型基础.     

Isolation and culture of arterial smooth muscle cells from human placenta

A simple and economical technique was developed to isolate and culture human arterial smooth muscle cells from chorionic plate vessels. Placentas from healthy women were collected at the time of term delivery. Chorionic plate arteries were identified, excised, and cut into small pieces. An explant technique was used to grow cultures of placental arterial smooth muscle (PASM) cells. Small pieces of vessel with lumens down were placed in 100-mm culture plates and grown in Dulbecco modified eagle medium and 10% fetal bovine serum. Cells appeared from explants within 1 week and grew to confluence in approximately 4 weeks. At confluence, PASM cell cultures had a uniform cell morphology that was characterized by elongated cells in parallel rows, typical of smooth muscle cells. Smooth muscle cell phenotype was evaluated by morphology and by immunoblotting and immunofluorescence of smooth muscle myofilament proteins. All PASM cell cultures expressed alpha-smooth muscle actin, beta-tropomyosin, and h-caldesmon. Expression was similar to that of human aortic smooth muscle cells, but not to endothelial cells or fibroblasts. PASM cells stained uniformly for alpha-smooth muscle actin and lacked staining for a fibroblast-specific antigen. PASM cells were evaluated for their response to inflammatory mediators, tumor necrosis factor-alpha, and interleukin-1beta by measurement of interleukin-8 production. Cells cultured for 18 hours showed a progressive increase in interleukin-8 production with time. Treatment with inflammatory mediators increased interleukin-8 production by 3-fold as compared with media control. This technique provides a simple method to obtain normal human arterial smooth muscle cells for in vitro studies of physiology and pathophysiology.     

KATP通道与人肺动脉平滑肌细胞外信号调节激酶磷酸化的关系

目的 研究三磷酸腺苷(ATP)敏感性钾(KATP)通道与人肺动脉平滑肌细胞外信号调节激酶1和2(ERK1/2)磷酸化的关系.方法 原代培养人肺动脉平滑肌细胞,用Western-blot方法 检测磷酸化细胞外信号调节激酶1和2(p-ERK1/2).对照组不予干预,实验组分别加入内皮素-1(ET-1)、ET-1+埃他卡林、吡那地尔或格列本脲等孵育.结果 ①在2～30 min,ET-1呈时间依赖性促进人肺动脉平滑肌细胞ERK1/2磷酸化,10 min时最明显.②埃他卡林和吡那地尔可拮抗ET-1对ERK1/2磷酸化的影响.③特异性KATP通道阻断剂格列本脲可逆转埃他卡林和吡那地尔的作用.结论 KATP通道开放剂可能通过激活KATP通道,抑制ET-1诱导的原代培养人肺动脉平滑肌细胞ERK1/2磷酸化,KATP通道可能是研发新型治疗肺动脉高压药物的重要靶分子.     

Transformed rat arterial smooth muscle cells induce platelet aggregation

Atherogenesis is characterized by a proliferation of arterial smooth muscle cells that may be of transformed nature. Platelets are implicated in the progression of atherosclerotic lesions through thrombosic complications. The present study was designed to investigate whether transformed arterial smooth muscle cells (SMC) could specifically aggregate platelets. We used rat transformed arterial SMC lines, V6- and V8-lines, that we had previously established. Experiments were performed with an in vitro homologous rat system. Suspensions of SMC were added without any other aggregating agent to rat heparinized platelet-rich plasma (PRP) in a coagulo-aggregometer. The effect of transformed V6-line and V8-line SMC was compared to that of their normal parental counterparts, V6- and V8-parent cells. Suspensions of transformed SMC induced, in a dose-dependent manner, an immediate and reversible ADP-like platelet aggregation. The amplitude of platelet aggregation was much higher with addition of transformed cells than of the corresponding control SMC (7.39 ± 0.75 cm vs. 0.85 ± 0.62 cm with 2 × 10⁶ SMC, V6-line vs. V6-parent cells, respectively). ADP-like aggregation did not significantly differ between the two transformed V6- and V8-lines. ADP-like platelet aggregation was also obtained with supernatants of transformed SMC suspensions, the amplitude being higher with supernatants than with cell suspensions (21.0 ± 3.64 cm vs. 6.8 ± 1.22 cm with 1.0 × 10⁶ V8-line cells, supernatant vs. cell suspension, respectively). The transformed SMC-induced aggregation of platelets was inhibited by apyrase (125 μM) and iodoacetate (25mM) and thus was ascribable to ADP released by the SMC. In addition, all suspensions of SMC, normal or transformed, but not their supernatants, induced plasma clotting after variable coagulation times. Coagulation was inhibited by hirudin (25 to 100 U/ml) and phospholipase A2 (10 U/ml) indicating thrombin generation through activity of the SMC membrane tissue factor. The present results show that transformed arterial smooth muscle cells may directly aggregate platelets via a release of ADP and this could be of pathophysiological relevance for thrombosis associated with atherosclerosis.     

马来酸曲美布汀对结肠平滑肌细胞钙激活钾通道的影响

目的研究马来酸曲美布汀对豚鼠结肠平滑肌细胞膜钙激活钾通道的影响。方法酶解法急性分离单个豚鼠结肠平滑肌细胞,利用膜片钳技术在全细胞模式下记录钙激活钾电流[IK（ca）],用含有IK（ca）特异激动剂NS1619的生理盐水灌流细胞,使结肠平滑肌细胞处于超极化状态,检测给马来酸曲美布汀浓度分别为1．19μmol／L、5．95μmol／L、11．91μmol／L后的结肠平滑肌细胞膜IK（ca）。结果浓度为1．19μmol／L、5．95μmol／L、11．91μmol／L的马来酸曲美布汀可抑制豚鼠单个结肠平滑肌细胞膜IK（ca）（P均〈0．01）,当阶跃刺激为＋80mv时,其分别为超极化状态组的（65．87±4．80）％、（48．02±2．39）％、（18．88±2．29）％（P均〈0．01）。结论马来酸曲美布汀能抑制豚鼠结肠平滑肌细胞钙激活钾通道的开放,使细胞兴奋性升高,且呈浓度依赖性,这可能是马来酸曲美布汀治疗肠易激综合征便秘症状的机制之一。     

二十二碳六烯酸增加大鼠冠状动脉平滑肌细胞大电导钙离子激活钾离子流的机制

Objective To investigate the mechanism of enhanced large conductance calciumactivated potassium channel currents (BK) in coronary smooth muscle cells (SMCs) by docosahexaenoic acid (DHA). Methods Coronary SMCs were isolated by enzyme digestion. Potassium channels in coronary SMCs were identified by applications of different potassium blockers. Effects of DHA and its metabolite 16,17-epoxydocosapentaenoic acid (16,17-EDP) on BK channels in the absence and presence of cytochrome P450 epoxygenase inhibitor SKF525A were studied by patch clamp in whole-cell configuration. Results BK channels were widely distributed in SMCs, and BK currents in normal SMCs accounted for (64.2±2.7)%of total potassium currents(n =20). DHA could activate BK channels, and its 50% effective concentration (EC50) was (0.23±0.03)μmol/L, however, the effect of DHA on BK channels was abolished after SMCs were incubated with cytochrome P450 epoxygenase inhibitor SKF525A. 16,17-EDP, a metabolite of DHA, could reproduce the effects of DHA on BK channels, and its EC50 was (19.7± 2.8) nmol/L.Conclusion DHA and metabolites can activate BK channels and dilate coronary arteries through activating cytochrome P450 epoxygenase pathway.     

二十二碳六烯酸增加大鼠冠状动脉平滑肌细胞大电导钙离子激活钾离子流的机制

Objective To investigate the mechanism of enhanced large conductance calciumactivated potassium channel currents (BK) in coronary smooth muscle cells (SMCs) by docosahexaenoic acid (DHA). Methods Coronary SMCs were isolated by enzyme digestion. Potassium channels in coronary SMCs were identified by applications of different potassium blockers. Effects of DHA and its metabolite 16,17-epoxydocosapentaenoic acid (16,17-EDP) on BK channels in the absence and presence of cytochrome P450 epoxygenase inhibitor SKF525A were studied by patch clamp in whole-cell configuration. Results BK channels were widely distributed in SMCs, and BK currents in normal SMCs accounted for (64.2±2.7)%of total potassium currents(n =20). DHA could activate BK channels, and its 50% effective concentration (EC50) was (0.23±0.03)μmol/L, however, the effect of DHA on BK channels was abolished after SMCs were incubated with cytochrome P450 epoxygenase inhibitor SKF525A. 16,17-EDP, a metabolite of DHA, could reproduce the effects of DHA on BK channels, and its EC50 was (19.7± 2.8) nmol/L.Conclusion DHA and metabolites can activate BK channels and dilate coronary arteries through activating cytochrome P450 epoxygenase pathway.     

二十二碳六烯酸增加大鼠冠状动脉平滑肌细胞大电导钙离子激活钾离子流的机制

目的 探讨二十二碳六烯酸(DHA)增加冠状动脉平滑肌细胞(SMC)大电导钙离子激活钾离子流(BK)的机制.方法酶消化法分离正常大鼠冠状动脉SMC.采用不同钾通道阻滞剂,对冠状动脉SMC的钾离子通道进行鉴定.采用全细胞膜片钳实验技术研究DHA及其代谢产物16,17-环氧二十二碳五烯酸(16,17-EDP)对冠状动脉平滑肌细胞BK通道的影响,并观察加入细胞色素P450环氧化酶抑制剂SKF525A孵育SMC后BK通道电流的变化.结果正常冠状动脉平滑肌细胞BK通道电流约占总钾离子流的(64.2±2.7)%(n=20).DHA可激活BK通道,半效浓度为(0.23±0.03)μmoL/L,但当使用细胞色素P450环氧化酶抑制剂SKF525A预孵育SMC后,DHA对BK通道的激活作用消失,而DHA代谢产物16,17-EDP可产生与DHA相同的BK通道激活作用,半效浓度为(19.7±2.8)nmol/L.结论 DHA通过细胞色素P450环氧化酶代谢途径激活平滑肌细胞BK通道,从而扩张冠状动脉.

Abstract：

Objective To investigate the mechanism of enhanced large conductance calciumactivated potassium channel currents (BK) in coronary smooth muscle cells (SMCs) by docosahexaenoic acid (DHA). Methods Coronary SMCs were isolated by enzyme digestion. Potassium channels in coronary SMCs were identified by applications of different potassium blockers. Effects of DHA and its metabolite 16,17-epoxydocosapentaenoic acid (16,17-EDP) on BK channels in the absence and presence of cytochrome P450 epoxygenase inhibitor SKF525A were studied by patch clamp in whole-cell configuration. Results BK channels were widely distributed in SMCs, and BK currents in normal SMCs accounted for (64.2±2.7)%of total potassium currents(n =20). DHA could activate BK channels, and its 50% effective concentration (EC50) was (0.23±0.03)μmol/L, however, the effect of DHA on BK channels was abolished after SMCs were incubated with cytochrome P450 epoxygenase inhibitor SKF525A. 16,17-EDP, a metabolite of DHA, could reproduce the effects of DHA on BK channels, and its EC50 was (19.7± 2.8) nmol/L.Conclusion DHA and metabolites can activate BK channels and dilate coronary arteries through activating cytochrome P450 epoxygenase pathway.