红花黄素对氧自由基所致的心肌细胞电生理异常的保护作用

背景：氧自由基诱导的功能异常可能使心脏、肝脏、肾脏及脑等组织缺血性疾病的主要致病因素，对损伤有保护作用的药物研究成为当前的热点。目的：研究红花黄索(Safflower yellow pigment，SYP)对氧自由基引起的豚鼠单个心室肌细胞损伤的保护作用。设计：非随机对照的实验研究。地点、材料和干预：本实验在哈尔滨医科大学药学院药理教研室完成。实验选用豚鼠30只，性别不限。以处置前单个心室肌细胞为正常对照。预先应用红花黄索(3．3μg／L)后，给予单个豚鼠心室肌细胞外源性氧自由基(1mmol／L的H2O2)。主要观察指标：采用全细胞膜片钳技术，观察单个豚鼠心室肌细胞动作电位时程(action potential duration APD)和L-广型钙电流、内向整流钾电流。结果：1 mmol／L的H2O2导致豚鼠心室肌细胞损伤，表现为动作电位时程缩短，APD50和APD90分别由正常对照组的(331．2&;#177;31．9)ms和(380．8&;#177;28．2)ms缩短至(169．5&;#177;76．0ms和(238．4&;#177;21．3)ms(n=8，t=3．834，P&;lt;0．01)，红花黄索(3．3μg／L)能明显改善H2O2诱发的动作电位时程缩短；同时氧自由基H2O2抑制L-广型钙电流。+10mV时，L-广型钙电流峰值由对照的(-1023．45&;#177;74．34)pA减少到(-275．21&;#177;38．67)pA(n=6，P&;lt;0．001)；H2O2(1mmol／L)也明显抑制内向整流钾电流(Ik1)，指令电位为-120mV时，Ik1由对照组的(-2133．5&;#177;570．4)pA降低到(-567．0&;#177;218．0)pA。预先应用红花黄索10min后，外源性氧自由基引起单个豚鼠心室肌细胞L-型钙电流的抑制作用得以改善，但不能改变H2O2对Ik的抑制作用。结论：预先应用红花黄索对H2O2损伤有一定的拮抗作用，说明其能够清除氧自由基，为临床应用治疗缺血性心脏病和康复措施的介入提供理论依据。     

卡维地洛对兔心房肌细胞动作电位及离子通道的影响

目的实验研究卡维地洛影响兔心房肌细胞电生理的效果。方法兔心房肌单个细胞的制备采用酶解法,动作电位和离子通道电流的记录应用全细胞膜片钳技术。结果 1长期应用卡维地洛显著延长复极90%的动作电位时程(APD90),APD90从82±10 ms延长至116±14 ms;2卡维地洛对心房肌细胞L-型钙电流(ICa-L)没有影响;3卡维地洛可显著减小心房肌细胞的瞬时外向钾电流(Ito),Ito从13.87±1.26 pA/pF减小到8.62±0.91 pA/pF。结论卡维地洛可减小Ito并明显延长APD90,为β阻断剂的抗心律失常效应提供了证据。     

血管紧张素Ⅱ及其受体拮抗剂对豚鼠心肌细胞电生理及L-型钙电流的作用

目的 探讨血管紧张素Ⅱ及其受体拮抗剂对豚鼠心肌细胞动作电位间期及L-型钙电流的作用。方法 分离豚鼠乳头肌的单个心室肌细胞，采用内充3mol／LKCl的玻璃微电极记录心肌动作电位。采用膜片钳全细胞技术，钳制电位-40mV，保持时间200ms，指令电位为0，并记录L-型钙电流的最大峰电流。结果 灌注血管紧张素Ⅱ可致多种机制的心律失常。灌注1min，动作电位振幅、动作电位复极90％的间期（APD90）、静息膜电位（RMP）较对照状态显著降低或缩短；灌注3min，动作电位复极30％和50％的间期（APD30和APD50）及有效不应期均较对照状态显著缩短。膜片钳研究示血管紧张素Ⅱ灌注5min，L-型钙电流较对照状态显著增加，氯沙坦灌注1min L-型钙电流显著降低，灌注3min较1min进一步降低，电压-电流关系曲线形状均无显著变化。结论 血管紧张素Ⅱ降低动作电位幅度，缩短动作电位时程及有效不应期，电压依赖性增加L-型钙电流最大峰电流，具有致心律失常作用，氯沙坦电压依赖性地降低L-型钙电流。     

高胆固醇血症家兔心脏单相动作电位及钙电流的特征

目的观察高胆固醇血症对家兔心脏单相动作电位及钙电流的影响。方法24只家兔分为高胆固醇饮食组和对照组各12只,分别给予高胆固醇饲料和标准饲料饲养10周后,检测血脂、心电图和室颤阈值,记录离体灌流心脏单相动作电位,并记录心室肌细胞的L型钙通道电流。结果高胆固醇饮食组兔的血脂水平明显高于对照组(P<0.01);室颤阈值(10.2±1.7)V,低于对照组的(13.9±1.3)V(P<0.05);单相动作电位复极90%的时程(MAPD90)较对照组延长并呈更明显的逆频率依赖性,在1500ms起搏时MAPD90为(348±21)ms,而对照组为(271±16)ms;心室肌细胞的L型钙通道电流密度为(14.7±0.8)pA/pF,明显高于对照组的(10.9±1.1)pA/pF(P<0.01)。结论高胆固醇血症家兔的心脏单相动作电位及心肌细胞L型钙通道电流明显改变,复极时程延长,室颤阈值降低。     

盐酸雷诺秦对豚鼠乳头肌动作电位及收缩力的影响

目的通过盐酸雷诺秦对离体豚鼠心室乳头肌动作电位及收缩力的影响,探讨其抗心律失常及心肌缺血的机制。方法健康成年豚鼠18只,随机分为H2O2(200 mmol.L-1)组、雷诺嗪(10 mmol.L-1)+H2O2组和TTX(2 mmol.L-1)+H2O2组,每组6只,采用给药前后自身对照的方法观察雷诺秦对豚鼠乳头肌的作用。结果H2O2可使对照组动作电位APD50、APD90明显增加(P0.001),心肌收缩力降低(P0.05),雷诺嗪可抑制H2O2引起的动作电位时程APD50、APD90的延长(P0.05和P0.01),但作用弱于TTX(P0.05和P0.001),雷诺嗪、TTX可改善H2O2引起的心肌收缩力降低。结论盐酸雷诺嗪可降低H2O2引起的豚鼠乳头肌动作电位动时程的增加和增强心肌收缩力,作用和TTX结果相似。     

养心颗粒含药血清对豚鼠心室肌细胞外向延迟整流K电流的影响

目的观察养心颗粒含药血清对单个豚鼠心室肌细胞动作电位、外向延迟整流K电流的影响,探讨养心颗粒在离子通道水平的药理机制。方法胶原酶急性酶解法分离单个豚鼠心室肌细胞,实验分为空白对照组、养心颗粒含药血清2%、4%、8%组,采用全细胞膜片钳的方法,记录养心颗粒对动作电位、外向延迟整流K电流的影响。结果养心颗粒4%、8%组可延长APD 50和APD 90,其对应峰值钾电流分别为(15.33±1.56)、(14.33±1.11)PA/PF,与对照组比较,差异有统计学意义。养心颗粒2%组则无明显影响。结论养心颗粒通过抑制延迟整流K+通道来延长ADP50,ADP90,从而延长有效不应期,发挥其抗心律失常的作用,且可能有一定的浓度依赖性。     

心力衰竭患者抗β1肾上腺素能受体的自身抗体阳性血清对豚鼠心功能影响的电生理研究

目的探讨心力衰竭患者抗β1肾上腺素能受体自身抗体阳性血清对豚鼠心室肌细胞膜上延迟整流性钾电流及其尾电流的影响.方法实验于2002-10/2003-04在首都医科大学心脏病研究所实验室完成.应用链霉亲和素-酶联免疫吸附测定技术检测188例缺血性心肌病、高血压性心脏病、扩张型心肌病心力衰竭患者及与心力衰竭患者年龄、性别匹配的健康者71人的血清中自身抗体.纳入对象均自愿提供血清用于实验.选用24只健康成年豚鼠,断头处死,分离心肌细胞,置于台氏液中,室温保存备用.应用全细胞膜片钳技术,定量观察并比较β1肾上腺素能受体激动剂异丙肾上腺素和心力衰竭患者抗β1肾上腺素能受体自身抗体的阳性血清对豚鼠单个心室肌细胞的延迟整流性钾电流及其尾电流电流强度的影响.①观察抗β1肾上腺素能受体的自身抗体阴性血清对心肌细胞延迟整流钾电流和尾电流的影响含Cd2+台氏液灌流心肌细胞,记录基础延迟整流钾电流和尾电流→抗β1肾上腺素能受体的自身抗体阴性血清灌流心室肌细胞3～5 min.②异丙肾上腺素对心肌细胞延迟整流钾电流和尾电流电流的影响另取一组含Cd2+台氏液灌流心肌细胞,记录基础延迟整流钾电流和尾电流→异丙肾上腺素1 μmol/L灌流心室肌细胞3～5 min→用β1受体阻滞剂艾司洛尔0.1 μmol/L灌流3～5 min.③抗β1肾上腺素能受体的自身抗体阳性血清对心肌细胞延迟整流钾电流和尾电流的影响另取一组含Cd2+台氏液灌流心肌细胞,记录基础延迟整流钾电流和尾电流→加入抗β1肾上腺素能受体的自身抗体阳性血清灌流心室肌细胞3～5 min→用β1受体阻滞剂艾司洛尔0.1 μmol/L灌流3～5 min.4组间差异用方差分析和两两比较的q检验.结果①β1肾上腺素受体的自身抗体阴性血清对豚鼠心室肌细胞,延迟整流性钾电流和其尾电流峰值电流及标准电流密度无影响.②异丙肾上腺素作用于豚鼠心室肌细胞,延迟整流性钾电峰值电流由加药前的基础电流(对照组)(884.09±346.93) pA增大至异丙肾上腺素组(1 423.39±438.68)pA,标准电流密度由(6.86±1.87)pA/pF上升至(11.04±2.00)pA/pF,差异明显(P＜0.01);尾电流峰值电流由加药前的基础电流(对照组)(229.13±75.76)pA增大至异丙肾上腺素组(449.59±137.38)pA,标准电流密度由(1.84±0.53)pA/pF上升至(3.54±0.78)pA/pF,差异明显(P＜0.01).艾司洛尔后,峰值电流及标准电流密度均下降(P＜0.01).③心力衰竭患者抗β1肾上腺素受体的自身抗体阳性血清作用于豚鼠心室肌细胞,延迟整流性钾电流峰值电流由(864.56±332.67)pA增大到(1 291.52±365.83)pA,标准电流密度由(6.33±1.45)pA/pF上升至(10.13±1.82)pA/pF,与基础电流(对照组)比较差异明显(P＜0.01);尾电流峰值电流由(213.45±81.23)pA增大至(416.18±159.00)pA,标准电流密度由(1.54±0.62)pA/pF上升至(3.30±0.94)pA/pF,再次加入艾司洛尔后,峰值电流及标准电流密度均下降(P＜0.01).结论①不同心脏病心力衰竭患者抗β1受体的自身抗体对自身受体有异丙肾上腺素激动剂样效应,可以使心肌细胞延迟整流性钾电流和尾电流增大.长期钾电流活化,导致心肌细胞损伤,引起心力衰竭时心肌细胞发生电重构,推测抗β1受体的自身抗体对心肌细胞钾电流的影响与心力衰竭时心律失常有关.从离子水平证实人抗β1受体的自身抗体参与心力衰竭发生、发展的病理生理过程.②β1受体阻滞剂可以阻断自身抗体与受体的结合,从离子水平为临床应用β1受体阻滞剂治疗心力衰竭提供有利的实验依据.     

兔心肌梗死后2个月肥大心室肌细胞的电生理研究

背景心肌梗死后电重构与梗死后时间和区域相关.目的研究陈旧性心肌梗死非梗死区肥大心室肌细胞离子通道电流的变化,探讨心肌梗死后肥大心肌发生心律失常的可能的离子机制.设计随机对照实验研究.地点、材料和干预所有实验过程在石家庄市白求恩国际和平医院心内科中心实验室完成.新西兰纯种大耳白兔20只,按随机抽签法分为两组心肌梗死动物模型组和正常对照组.采用结扎兔冠状动脉左前降支的方法建立急性心肌梗死动物模型,应用膜片钳全细胞记录方法.主要观察指标梗死后2个月心外膜远离梗死区组与梗死区组及正常对照组心室肌细胞L-钙通道电流(L-calcium current,ICa-L)、瞬间外向钾电流(transient outward current,Ito)的变化.结果①远离梗死区组细胞电容[(155.7±5.8)pF,n=41]明显大于对照组[(120.3±6.2)pF,n=35]和梗死区组[(130.4±7.8)pF,n=38](t=2.642,2.613,P均＜0.01).②ICa-L远离梗死区组ICa-L电流峰值(0 mV)为[(826.12±121.31)pA,n=21],较对照组[(670.21±183.32)pA,n=10]和梗死区组[(629.43±172.12)pA,n=11]明显增大(t=2.451,2.732,P均＜0.05).但远离梗死区组电流密度峰值为(5.32±0.78)pA/pF,较对照组[(5.58±1.53)pA/pF]略下降,与梗死区组[(4.84±1.48)pA/pF]和对照组比较无显著性意义(P均＞0.05).③Ito远离梗死区组[(13.21±4.13)pA/pF,n=23]和梗死区组[(10.61±4.12)pA/pF,n=18]的Ito电流密度(+60mV时)均明显低于对照组[(17.39±5.24)pA/pF,n=16](t=3.591,2.725,P均＜0.01).但远离梗死区组明显高于梗死区组(t=2.429,P＜0.05).结论心肌梗死后2个月,远离梗死区心室肌细胞电容明显增大,反映心肌细胞发生代偿性肥大.远离梗死区心室肌细胞ICa-L幅值升高,但Ito电流密度明显下降,ICa-L电流密度轻度下降,可导致动作电位平台期延长,复极异常,异常自律性的增加,并且心室不同部位存在电生理异质性,可能是导致陈旧性心肌梗死出现室性心律失常的离子基础.     

兔急性心肌梗死后24小时心室肌细胞离子通道电流的变化

背景急性心肌梗死( acute myocardial infarction ,AMI)后梗死区内部和梗死周边区尚存活的心肌,往往在心律失常的发生上起关键作用. 目的研究 AMI后梗死区心肌细胞钠通道电流( INa)、 L型钙通道电流( ICa-L)、瞬间外向钾电流( Ito)和内向整流性钾电流 (IK1)活性的变化. 设计随机对照研究. 单位白求恩国际和平医院心内科. 材料本实验于 2003- 01/2003- 06在白求恩国际和平医院心内科中心实验室完成.选择新西兰纯种大耳白兔 20只,按随机抽签法分为两组即 AMI组和对照组,每组 10只. 方法结扎兔冠状动脉左前降支建立 AMI动物模型,采用酶解的方法分离心室肌细胞,应用膜片钳全细胞记录方法记录离子电流的变化. 主要观察指标 AMI后 24 h AMI组和对照组心外膜梗死区心肌细胞 INa, ICa-L, Ito和 IK1的变化. 结果 AMI后 24 h, AMI组 INa电流密度峰值 [(28.48± 3.53) pA/pF, n=16]较对照组 [(45.50± 5.33) pA/pF, n=12]明显下降( t=3.026,P< 0.01); AMI组 ICa-L电流密度峰值 [(3.91± 0.95 ) pA/pF, n=12]较对照组 [(5.58± 1.53) pA/pF, n=10]明显下降( t=2.985 ,P< 0.01); AMI组 IK1电流密度峰值 [(26.93± 3.48 ) pA/pF, n=16]较对照组 [(34.12± 4.21) pA/pF, n=10]明显下降( t=2.706 , P< 0.05);两组 Ito差异无显著性意义 (P >0.05). 结论 AMI可引起心室肌细胞 INa 、 ICa-L和 IK1的下降,造成心肌传导速度下降和动作电位时程缩短、复极异常,可能是导致 AMI后出现折返性室性心律失常的离子机制.     

花生四烯酸对L-型钙通道的作用及其抗心律失常机制

目的 研究花生四烯酸(arachidonic acid,AA)对家兔单个心室肌细胞L-广型钙通道的作用及其抗心律失常作用的机制.方法 采用酶解法分离得到家兔单个心室肌细胞,全细胞膜片钳技术记录单个心室肌细胞L-型钙电流(L-type calcium current,Ica-L),用累积给药的方法在灌流液中加入不同浓度的AA,观察给药前后L-型钙电流的变化,统计学方法采用单因素方差分析.结果 不同浓度的从均能明显抑制心室肌细胞,Ica-L.3 μmol/L,μmol/L,20,μmol/L的AA使Ica-L峰电流密度从(10.79±0.93)pA/pF分别减少剑(8.99 ±0.43)pA/pF、(7.60 ±0.35)pA/pF和(5.60±0.30)pA/pF(n=7,P<0.05),经冲洗后Ica-L可部分恢复,并且AA可使Ica-L的I-V关系曲线上移,其形状和峰值电压保持不变;20 μmol/L的AA使Ica-L失活曲线左移,失活后恢复时间明显延长,但对激活曲线无明显影响.结论 花生四烯酸可通过加快L-型钙通道失活,延长其失活后的恢复过程而减少细胞外钙离子的内流,延长有效不应期,从而发挥抗心律失常作用.     

Electrophysiologic Effects of Beta-blocking Agent, Tilisolol, on Isolated Guinea Pig Ventricular Myocytes

BACKGROUND: Electrophysiologic effects of a beta-blocking agent, tilisolol, were studied with isolated guinea pig ventricular myocytes using the whole cell patch clamp technique. METHODS AND RESULTS: Tilisolol at 10 μM or higher concentrations prolonged action potential duration (APD) at 90% repolarization (APD(90)) and at 100 μM or higher concentrations shortened APD at 20% repolarization (APD(20)) without changes in resting membrane potential. At 10 μM concentration tilisolol prolonged APD(90) from 236.6 +/- 55.3 ms in the control to 253.4 +/- 52.4 ms (n = 16; P <.01), while APD(20) was unaffected. At 100 μM tilisolol, APD(20) was shortened from 143.6 +/- 15.7 ms in the control to 133.7 +/- 22.6 ms (n = 8; P <.05). Under voltage clamp, tilisolol decreased the delayed rectifier K(+) current (I(K1)). Applications of 10 μM and 100 μM tilisolol reduced the maximal conductance of I(K) by 35.7 +/- 3.5% and 47.4 +/- 3.5% of the control, respectively, without changes in voltage dependence (n = 10). Tilisolol at 100 μM decreased the L-type Ca(2+) current (I(Ca.L)) by 22.0 +/- 9.8% (n = 6) of the control, and the inactivation curve was shifted to a hyperpolarizing direction. CONCLUSIONS: Tilisolol has a direct membrane action to depress I(K) and I(Ca.L), in addition to its beta-receptor blocking action.     

Effects of exogenous free radicals on electromechanical function and metabolism in isolated rabbit and guinea pig ventricle. Implications for ischemia and reperfusion injury.

Oxygen-derived free radicals have been implicated in the pathogenesis of cardiac dysfunction during ischemia, postischemic myocardial "stunning," and reperfusion injury. We investigated the effects of oxygen-derived free radicals on cardiac function in intact isolated rabbit hearts and single guinea pig ventricular myocytes. In the intact rabbit ventricle, exposure to free radical-generating systems caused increased cellular K+ efflux, shortening of the action potential duration, changes in tension, and depletion of high energy phosphates similar to ischemia and metabolic inhibition. In patch-clamped single ventricular myocytes, free radical-generating systems activated ATP-sensitive K+ channels, decreased the calcium current, and caused cell shortening by irreversibly inhibiting glycolytic and oxidative metabolism. The results suggest that free radicals generated during ischemia and reperfusion may contribute to electrophysiologic abnormalities and contractile dysfunction by inhibiting glycolysis and oxidative phosphorylation. Inhibition of metabolism by free radicals may be an important factor limiting functional recovery from an ischemic insult after reestablishment of effective blood flow.     

苄基四氢帕马汀经蛋白激酶C对豚鼠心肌细胞延迟整流钾电流的作用及其临床应用

目的:研究苄基四氢帕马汀(BTHP)经蛋白激酶C对豚鼠心肌细胞延迟整流钾电流的作用极其在临床中的应用前景。方法:应用膜片钳在全细胞模式下记录延迟整流钾电流(Ik)。结果:PMA10.0μmol/L在细胞内给药可使Ik和Ik,tail增加,电流密度分别从(13.1±1.4)pA/pF和(5.1±0.7)pA/pF增至(19.5±0.7)pA/pF和(7.3±0.4)pA/pF。应用BTHP后上述增加效应被明显减少,BTHP的抑制作用在单独应用时为(38±6)%和(36±6)%,而在预先应用PMA后BTHP的抑制作用增加至(64±7)%和(64±6)%。结论:BTHP对Ik和Ik,tail的阻滞作用可能部分与抑制细胞内蛋白激酶C途径有关。     

常规剂量三氧化二砷对心脏功能影响的临床和实验研究

目的　研究常规剂量的三氧化二砷 (As2 O3)对急性早幼粒细胞白血病 (APL)患者心脏功能的影响。方法　通过对患者基础心率和心电图的动态观察 ,膜片钳技术对豚鼠心肌细胞用药前后动作电位和电流的监测 ,激光共聚焦技术对用药前后豚鼠心肌细胞内钙的测定 ,分析As2 O3对APL患者心脏功能影响的可能机制。结果　常规剂量的As2 O3静脉给药第 1～ 2周 ,5 2 .5 %～ 35 .0 %的APL患者产生不同程度的心脏不良反应 ,心电图上QT间期延长等。膜片钳监测显示 1,2 ,5 μmol LAs2 O3使豚鼠心肌细胞动作电位时程从 (5 6 3.0± 5 5 .8)ms分别延长到 (737.7± 131.7)ms、(84 2 .4± 115 .6 )ms和 (110 3.2± 96 .3)ms (P值分别 <0 .0 5 ,0 .0 1,0 .0 1) ,使L 型钙电流增加。激光共聚焦检测结果显示 ,1,2 ,5 μmol LAs2 O3使心肌细胞内钙增加 ,并可以被钙通道阻滞剂阻断。结论　常规剂量的As2 O3可使部分APL患者出现一过性心动过速、QT间期延长等不良反应。As2 O3对心脏功能的影响可能是通过影响心肌离子通道和细胞内钙来实现的。     

水翁花对神经细胞氧化损伤保护作用的量效值

背景水翁花为中药桃金娘科植物水翁的干燥花蕾,已有报道水翁花提取物能通过抑制Na+/K+-ATP酶的活性加强心脏的收缩功能,同时降低心脏的收缩频率,水翁花提取物是否具有抗氧化方面的生物活性?目的观察水翁花对过氧化氢诱导的PC12神经细胞氧化损伤保护作用的量效关系.设计非随机对照的实验.单位华东理工大学生物工程学院生物反应器国家重点实验室.材料实验于2002-05/11在华东理工大学反应器国家重点实验室鲁华生物技术研究所完成.选择昆明种雄性小鼠8只.PC12神经细胞购自中国科学院上海细胞所.方法制备神经细胞氧化损伤模型.①水翁花细胞毒性测定在96孔微量培养板中加入PC12神经细胞,水翁花以RPMI 1640培养液稀释成0.001,0.01,0.1,0.5,1 g/L 5个浓度,每个浓度3孔,每孔接种2×103个细胞,另设空白对照组,即无药培养液组.标准条件下培养48 h,噻唑蓝比色法测定.②PC12神经细胞预先接种于96孔板中,培养24 h贴壁,分为正常对照组(正常细胞,不加H2O2和水翁花提取物)、0,0.01,0.1,0.5,1g/L水翁花提取物共7个组.除正常对照组外,其余细胞用200 μmol/L的H2O2处理,加入不同浓度的水翁花水提物,作用12 h后用噻唑蓝法测定细胞的存活率.③氧自由基水平测定PC12神经细胞处理同存活率测定法,采用CDCFH染色法测定细胞氧自由基水平.主要观察指标①水翁花提取物对PC12神经细胞的毒性.②水翁花提取物对氧化损伤的PC12神经细胞存活率影响.③水翁花提取物对氧化损伤的PC12神经细胞内、细胞外氧自由基水平的影响.结果①水翁花提取物在浓度低于0.055 g/L时,对神经细胞有保护作用,在0.055～1.00 g/L浓度时,对细胞生长几乎无任何影响.②在低于0.10 g/L的浓度下,水翁花提取物对H2O2诱导的PC12神经细胞的氧化损伤没表现出明显的保护作用.但当浓度为1.00 g/L时,对H2O2诱导的PC12神经细胞的氧化损伤表现出明显的氧化修复能力.③H2O2损伤后,PC12细胞内、外氧自由基水平显著升高,当水翁花提取物浓度为0.01 g/L时,能明显的降低氧化损伤神经细胞内、外的氧自由基水平.结论①水翁花水提物对H2O2诱导的PC12神经细胞的氧化损伤亦有很强的保护作用.②水翁花提取物抗氧化特性与提取液的浓度比有明显的关系,当浓度为1.00 g/L时,具有明显的修复能力;当浓度为0.01 g/L时能够降低细胞内外的氧自由基水平.     

Functional adenylyl cyclase inhibition in murine cardiomyocytes by 2'(3')-O-(N-methylanthraniloyl)-guanosine 5'-[gamma-thio]triphosphate

Beta1-adrenergic receptor activation stimulates cardiac L-type Ca2+ channels via adenylyl cyclases (ACs), with AC5 and AC6 being the most important cardiac isoforms. Recently, we have identified 2'(3')-O-(N-methylanthraniloyl)-guanosine 5'-[gamma-thio-]triphosphate (MANT-GTPgammaS) as a potent competitive AC inhibitor. Intriguingly, MANT-GTPgammaS inhibits AC5 and -6 more potently than other cyclases. These data prompted us to study the effects of MANT-GTPgammaS on L-type Ca2+ currents (ICa,L) in ventricular myocytes of wild-type (WT) and AC5-deficient (AC5-/-) mice by whole-cell recordings. In wild-type myocytes, MANT-GTPgammaS attenuated ICa,L stimulation following isoproterenol application in a concentration-dependent manner (control, +77+/-13%; 100 nM MANT-GTPgammaS, +43+/-6%; 1 microM MANT-GTPgammaS, +21+/-9%; p<0.05). The leftward shift of current-voltage curves was abolished by 1 microM but not by 100 nM MANT-GTPgammaS. In myocytes from AC5-/- mice, the residual stimulation of ICa,L was not further attenuated by the nucleotide, indicating AC5 to be the major AC isoform mediating acute beta-adrenergic stimulation in WT mice. Interestingly, basal ICa,L was lowered by 1 microM but not by 100 nM MANT-GTPgammaS. The decrease was less pronounced in myocytes from AC5-/- mice compared with wild types (-23+/-1 versus -40+/-7%), indicating basal ICa,L to be partly driven by AC5. Collectively, we found a concentration-dependent inhibition of ICa,L by MANT-GTPgammaS, both under basal conditions and following beta-adrenergic stimulation. Comparison of data from wild-type and AC5-deficient mice indicates that AC5 plays a major role in ICa,L activation and that MANT-GTPgammaS predominantly acts via AC5 inhibition.     

Angiotensin II Receptor Regulates Ionic Currents in Guinea Pig Ventricular Myocytes

BACKGROUND: Previous studies have shown that angiotensin II (Ang II) receptors are preset in a wide variety of target tissues and that Ang II regulates the target tissue functions through Ang II receptors. However, the action of Ang II receptors on transsarcolemmal currents in ventricular myocytes has not been elucidated. METHODS AND RESULTS: We performed whole-cell voltage clamp and patch clamp experiments to determine the effects of Ang II-receptor agonists and antagonists on ionic currents in single isolated guinea pig ventricular myocytes. We found that extracellular perfusion of Ang II (30 nM) increased the L-type Ca(2+) current from 581 +/- 27 to 837 +/- 42 pA (n = 5, P <.01). Ang II also prolonged the Ca(2+) current activation and inactivation time constants. These were reversible by losartan (100 nM), a type 1 Ang II receptor (AT(1)) blockade. On the other hand, perfusion of 30 nM Ang II decreased K(+) current (I(K)) from 1543 +/- 28 to 1194 +/- 50 pA (n = 5, P <.05) and K(+) tail current (I(K-tail)) from 275 +/- 24 to 206 +/- 29 pA (n = 5, P <.05). These effects were also abolished by perfusion of losartan. However, perfusion of Ang II resulted in an increase of inward rectified K(+) current (I(K1)) in whole-cell recordings. Single channel recordings showed that the increase in I(K1) was attributed to a burst opening current with a larger unit of amplitude. These effects were reversed by saralasin but not losartan, indicating possible type 2 Ang II receptor (AT(2)) involvement. CONCLUSIONS: Our results provide evidence that Ang II receptors regulate the transsarcolemmal currents in single guinea pig ventricular myocytes. Therefore, Ang II regulation of ionic currents is mediated through the different subtypes of Ang II receptors.     

Enhanced inhibition of L-type Ca2+ current by beta3-adrenergic stimulation in failing rat heart

beta3-adrenergic receptors (AR) have recently been identified in mammalian hearts and shown to be up-regulated in heart failure (HF). beta3-AR stimulation reduces inotropic response associated with an inhibition of L-type Ca2+ channels in normal hearts; however, the effects of beta3-AR activation on Ca2+ channel in HF remain unknown. We compared the effects of beta(3)-AR activation on L-type Ca2+ current (ICa,L) in isolated left ventricular myocytes obtained from normal and age-matched rats with isoproterenol (ISO)-induced HF (4 months after 340 mg/kg s.c. for 2 days). ICa,L was measured using whole-cell voltage clamp and perforated-patch recording techniques. In normal myocytes, superfusion of 4-[-[2-hydroxy-(3-chlorophenyl)ethylamino]propyl]phenoxyacetate (BRL-37,344; BRL), a beta3-AR agonist, caused a dose-dependent decrease in ICa,L with maximal inhibition (21%, 1.1 +/- 0.2 versus 1.4 +/- 0.1 nA) (p < 0.01) at 10(-7) M. In HF myocytes, the same concentration of BRL produced a proportionately greater inhibition (31%) in ICa,L (1.1 +/- 0.2 versus 1.6 +/- 0.2 nA) (p < 0.05). A similar inhibition of ICa,L was also observed with ISO (10(-7) M) in the presence of a beta1- and beta2-AR antagonist, nadolol (10(-5) M). Inhibition was abolished by the beta3-AR antagonist (S)-N-[4-[2-[[3-[3-(acetamidomethyl)phenoxy]-2-hydroxypropyl]amino]ethyl]phenyl]benzenesulfonamide (L-748,337; 10(-6) M), but not by nadolol. The inhibitory effect of BRL was attenuated by a nitric-oxide synthase (NOS) inhibitor, N(G)-nitro-L-arginine methyl ester (10(-4) M), and was prevented by the incubation of myocytes with pertussis toxin (PTX; 2 microg/ml, 36 degrees C, 6 h). In conclusion, beta3-AR activation inhibits L-type Ca2+ channel in both normal and HF myocytes. In HF, beta3-AR stimulation-induced inhibition of Ca2+ channel is enhanced. These effects are likely coupled with PTX-sensitive G-protein and partially mediated through a NOS-dependent pathway.     

Blocking late sodium current reduces hydrogen peroxide-induced arrhythmogenic activity and contractile dysfunction

Reactive oxygen species (ROS), including H2O2, cause intracellular calcium overload and ischemia-reperfusion damage. The objective of this study was to examine the hypothesis that H2O2-induced arrhythmic activity and contractile dysfunction are the results of an effect of H2O2 to increase the magnitude of the late sodium current (late INa). Guinea pig and rabbit isolated ventricular myocytes were exposed to 200 microM H2O2. Transmembrane voltages and currents and twitch shortening were measured using the whole-cell patch-clamp technique and video edge detection, respectively. [Na+]i and [Ca2+]i were determined by fluorescence measurements. H2O2 caused a persistent late INa that was almost completely inhibited by 10 microM tetrodotoxin (TTX). H2O2 prolonged the action potential duration (APD), slowed the relaxation rate of cell contraction, and induced early afterdepolarizations (EADs) and aftercontractions. H2O2 also caused increases of [Na+]i and [Ca2+]i. Ranolazine (10 microM), a novel inhibitor of late INa, attenuated H2O2-induced late INa by 51+/-9%. TTX (2 microM) or 10 microM ranolazine attenuated H2O2-induced APD prolongation and suppressed EADs. Ranolazine accelerated the twitch relaxation rate in the presence of H2O2 and abolished H2O2-induced aftercontractions. Pretreatment of myocytes with ranolazine delayed and reduced the increases of APD, [Na+]i, and [Ca2+]i caused by H2O2. In conclusion, the results confirm the hypothesis that an increase in late INa during exposure of ventricular myocytes to H2O2 contributes to electrical and contractile dysfunction and suggest that inhibition of late INa may offer protection against ROS-induced Na+ and Ca2+ overload.