【作者】 柳强妮；

【导师】 龚培力；

【作者基本信息】 华中科技大学 ， 药理学， 2007， 硕士

【摘要】 目的:蝙蝠葛碱(dauricine,Dau)和蝙蝠葛苏林碱(daurisoline,DS)是从中药蝙蝠葛(Menisperum dauricum,DC)根茎中提取的双苄基异喹林类生物碱。近年来,研究发现Dau和DS均具有显著的抗实验性心律失常作用。长QT综合征(long QT syndrome,LQTS)的基础是动作电位(AP)复极延迟。目前,普遍认为AP过度延长及其所导致的早后除极(EADs)是LQTS发生的重要电生理机制之一。在本研究中,拟采用标准微电极技术和膜片钳技术,以快速激活的延迟整流钾电流(IKr)阻断剂多非利特(dofetilide,Dof)作为工具药,模拟LQT2模型,研究Dau和DS对获得性LQTS的作用,并在细胞和离子水平探讨Dau和DS抗心律失常的作用机制。方法:1.采用腹主动脉缩窄法制备家兔心肌肥厚模型;采用标准微电极技术,记录正常及肥厚家兔右心室乳头肌动作电位,在正常以及细胞外低钾情况下,研究Dau和DS对家兔乳头肌APD和EADs的影响。2.采用酶消化法分离家兔左心室单个心肌细胞;利用全细胞膜片钳技术,在电流钳制模式下记录单个细胞动作电位,观察Dau和DS对单个心肌细胞APD和EADs的影响。3.采用酶消化法分离家兔左心室单个心肌细胞,用膜片钳技术,在电压钳制模式下记录单个细胞L-型钙电流(ICa-L),研究Dau和DS对心室肌细胞ICa-L动力学的影响。结果:1.蝙蝠葛碱和蝙蝠葛苏林碱对家兔乳头肌早后除极的影响(1)Dau:在正常家兔乳头肌标本,未发生EADs。将正常台氏液换成低钾液并加入Dof后,与给药前相比,50%动作电位时程(APD50)及90%APD(APD90)分别延长了77±6 ms和72±7 ms, EADs发生率为16.7%(1/6)。心肌肥厚家兔乳头肌标本在正常台氏液中EADs发生率为33.3%(2/6);将正常台氏液换成低钾液并加入Dof 1μmol·L-1后,APD50和APD90分别延长了197±9 ms和249±14 ms,APD90-APD30的差值延长了122±5.4 ms,EADs发生率为66.7%(4/6)。EADs发生后,给予终浓度为30μmol·L-1的Dau,作用10 min,发现Dau可缩短APD,APD50和APD90分别缩短了100±8 ms和111±8 ms,APD90-APD30的差值缩短了79±14 ms;抑制EADs的发生,发生率为0%(0/4)(P<0.05)。(2)DS:在正常家兔乳头肌标本,未发生EADs。将正常台氏液换成低钾液并加入Dof后,与给药前相比,50%动作电位时程(APD50)及90%APD(APD90)分别延长了58±9 ms和92±3 ms,EADs发生率为16.7%(1/6)。心肌肥厚家兔乳头肌标本在正常台氏液中EADs发生率为33.3%(2/6);将正常台氏液换成低钾液并加入Dof 1μmol·L-1后,APD50和APD90分别延长了174±20 ms和218±19 ms,APD90-APD30的差值延长了129±8.9 ms, EADs发生率为83.3%(5/6)。EADs发生后,给予终浓度为15μmol·L-1的DS,作用10 min,发现DS可缩短APD,APD50和APD90分别缩短了95±10 ms和113±8 ms,APD90-APD30的差值缩短了56±3.8 ms;抑制EADs的发生,发生率为0%(0/5)(P<0.05)。2.蝙蝠葛碱和蝙蝠葛苏林碱对家兔单个心肌细胞早后除极的影响(1)Dau:在正常台氏液中,未观察到EADs,给予Dof 0.1μmol·L-1后,APD50和APD90分别延长了118±9.3 ms和145±13 ms,APD90-APD30的差值延长了73±7.6 ms, EADs发生率为16.7(1/6);在低钾低镁台氏液中, EADs发生率为33.3%(2/6),给予Dof 0.1μmol·L-1后,APD50和APD90分别延长了145±13 ms和196±4.6 ms,APD90-APD30的差值延长了59±7.5 ms, EADs发生率为83.3%(5/6)。在低钾、低镁台氏液中,给予Dof,诱发EADs后,给予终浓度分别为1,3,10μmol·L-1的Dau发现,Dau能抑制EADs的发生(P<0.05),缩短APD(P<0.05),使APD90-APD30的差值缩短,分别为201±22 ms、187±32 ms和174±15 ms(P<0.05)。(2)DS:在正常台氏液中,未观察到EADs,给予Dof 0.1μmol·L-1后,APD50和APD90分别延长了91±7.9 ms和132±6.5 ms,APD90-APD30的差值延长了77±7.6 ms,EADs发生率为16.7(1/6);在低钾、低镁台氏液中,EADs发生率为33.3%(2/6),给予Dof 0.1μmol·L-1后,APD50和APD90分别延长了95±2.3 ms和139±9.9 ms,APD90-APD30的差值延长了74±6.4 ms, EADs发生率为83.3%(5/6)。在低钾、低镁台氏液中,给予Dof,诱发EADs后,给予终浓度分别为0.5,1.5,5μmol·L-1的DS发现,DS抑制EADs的发生(P<0.05),缩短APD(P<0.05),并可使APD90-APD30差值缩短,分别为243±12 ms、206±17 ms和184±12 ms(P<0.05)。3.蝙蝠葛碱和蝙蝠葛苏林碱对家兔单个心肌细胞L型钙通道的影响(1)Dau:Dau 1,3,10μmol·L-1组均可使ICa-L峰值电流减小。Control组和Dau各剂量组ICa-L分别为-1.8±0.3 nA、-1.3±0.3 nA、-1.1±0.2 nA和-1.0±0.2 nA。与control组相比,Dau各剂量组差异均有统计学意义(P<0.05)。对照组以及给药后各组细胞最大激活电压均为0 mV,Dau使各测试电压水平ICa-L减小,I-V曲线上移。Dau浓度为1,3,10μmol·L-1时,半激活电压(V1/2)分别从药前的-20.9±1.5 mV增至-16.4±0.6 mV(P<0.05)、-20.8±1.6 mV增至-15.6±1.7 mV(P<0.05)和-20.5±1.4 mV增至-12.8±1.2 mV(P<0.01);激活曲线斜率κ值分别从药前的2.2±0.5增至3.2±0.8、2.2±0.5增至3.2±0.1(P<0.05)和1.8±0.5增至3.7±0.2(P<0.05);半失活电压(V1/2)分别从药前的-23.0±5.0 mV减小至-27.7±4.0 mV、-20.5±5.3 mV减小至-25.6±4.3 mV和-21.8±5.4 mV减小至-32.3±2.5 mV(P<0.05);失活曲线斜率κ值分别从药前的-6.4±0.8减小至-8.1±1.0、-5.8±0.7减小至-7.7±1.2和-6.6±0.7减小至-10.2±1.6(P<0.05);ICa-L恢复曲线的时间常数(τ)分别由给药前的150±35 ms延长至216±36 ms、209±29 ms延长至327±61 ms(P<0.05)和275±16 ms延长至361±11 ms(P<0.05)。(2)DS:DS 0.5,1.5,5μmol·L-1组均可使ICa-L峰值电流减小。Control组和DS各剂量组ICa-L分别为-1.6±0.4 nA、-1.1±0.1 nA、-1.0±0.2 nA和-0.9±0.1 nA。与control组相比,DS各剂量组差异均有统计学意义(P<0.05)。对照组以及给药后各组细胞最大激活电压均为0 mV,DS使各测试电压水平ICa-L减小,I-V曲线上移。DS在1.5, 5μmol·L-1时,半激活电压(V1/2)分别从药前的-12.8±1.7 mV增至-10.4±1.7 mV和-12.8±1.3 mV增至-9.7±1.1 mV(P<0.05);激活曲线斜率κ值分别从药前的3.5±0.7增至5.1±0.8和5.0±0.6增至6.9±0.8(P<0.05);半失活电压(V1/2)分别从药前的-23.5±2.0 mV减小至-25.3±1.2 mV(P<0.05)和-24.3±1.8 mV减小至-27.8±3.4 mV(P<0.05);失活曲线斜率κ值分别从药前的-5.6±0.7减小至-7.1±0.9(P<0.05)和-5.9±0.8减小至-8.1±1.3(P<0.05);ICa-L恢复曲线的时间常数(τ)分别由给药前的245±49 ms延长至290±38 ms和222±60 ms延长至363±46 ms(P<0.05)。结论:1.在正常、低钾低镁以及心肌肥厚状态下,Dau和DS能抑制Dof诱发的家兔乳头肌和家兔单个心肌细胞EADs,缩短EAD发生后的APD50和APD90,并能缩短APD90-APD30,后者可能与其抗心律失常机制有关。2. Dau和DS能减小ICa-L峰值电流,使I-V曲线上移;稳态激活曲线右移, ICa-L激活减慢,稳态失活曲线左移,ICa-L失活加快;失活后恢复时间延长,ICa-L恢复至能再被激活状态的时间延长;半激活电压增大,半失活电压减小,使通道的“窗流”减小,以上结果可能为Dau和DS抗心律失常机制之一。更多还原

【Abstract】 Objective: Daurincine(Dau) and daurinsoline(DS), the bisbenzyl isoquinoline alkaloids, are extracted from the root of menispermum dauricum(DC). Their antiarrhythmic effects have been demonstrated in various experimental models. The excessive prolonged repolarization of action potential duration (APD) is the electrophysiological basis of long QT syndrome (LQTS). It is generally accepted that early afterdepolarizations (EADs) induced by excessive prolonged APD are the electrophysiological mechanisms of LQTS. In this study, using standard microelectrode technique and patch-clamp technique, we developed an in vitro model of LQT2 with dofetilide (Dof), a selective blocker of rapid activating delayed rectifier potassium current, to study the effects of Dau and DS on acquired LQTS, and to explore the antiarrhythmic mechanisms of Dau and DS in celluar and ionic levels.Methods: (1) Coarctation of abdominal aorta were used to induce hypertrophy in rabbits; using standard microelectrode technique, we recorded the transmembrane action potential of papillary muscles of right ventricle in normal and hypotrophied rabbits and studied the effects of Dau and DS on APD and EADs in normal and extracellular hypokalemia. (2) Single left ventricular myocardial cells of rabbits were isolated by enzymic method; using patch-clamp technique, we recorded the transmembrane potential of single cell to study the effects of Dau and DS on APD and EADs; (3) ICa-L of single cell were recored to study the effects of Dau and DS on its kinetics.Results:1. The effects of Dau and DS on early afterdepolarizations of papillary muscles of rabbits(1) Dau: EADs did not occur in papillary muscles of normal rabbits,but in hypokalemia Tyrode’s, Dof 1μmol·L-1 prolonged the APD50 and APD90 by 77±6 ms and 72±7 ms, respectively, and the incidence of EADs was 16.7%(1/6). In normal Tyrode’s, the incidence of EADs of papillary muscles of hypertrophied rabbits was 33.3%(2/6), Dof 1μmol·L-1 respectively prolonged the APD50、APD90 and APD90-APD30 by 197±9 ms、249±14 ms and 122±5.4 ms and the incidence of EADs was 66.7 %(4/6) in hypokalemia Tyrode’s. After EADs induced by Dof, Dau 30μmol·L-1 was administered. We found that Dau 30μmol·L-1 shortened the prolonged APD in EADs and the APD50、APD90 and APD90-APD30 were shortened by 100±8 ms、111±8 ms and 79±14 ms, respectively. Dau 30μmol·L-1 also suppressed the incidence of EAD and the incidence was 0%(0/4) (P<0.05).(2) DS: EADs did not occur in papillary muscles of normal rabbits,but in hypokalemia Tyrode’s, Dof 1μmol·L-1 prolonged the APD50 and APD90 by 58±9 ms and 92±3 ms, respectively, and the incidence of EADs was 16.7%(1/6)(P<0.05).In normal Tyrode’s, the incidence of EADs of papillary muscles of hypertrophied rabbits was 33.3%(2/6), Dof 1μmol·L-1 respectively prolonged the APD50、APD90 and APD90-APD30 by 174±20 ms、218±19 ms and 129±8.9 ms and the incidence of EADs was 83.3 %(5/6) in hypokalemia Tyrode’s. After EADs induced by Dof, DS 15μmol·L-1 was administered. We found that Dau 30μmol·L-1 shortened the prolonged APD in EADs and the APD50、APD90 and APD90-APD30 were shortened by 95±10 ms、113±8 ms and 56±3.8 ms, respectively. DS 15μmol·L-1 also suppressed the incidence of EAD and the incidence was 0%(0/5) (P<0.05). 2. The effects of Dau and DS on early afterdepolarizations of single myocardial cells of left ventrical of rabbits(1) Dau: EADs did not occur in myocardial cells of rabbits, Dof 0.1μmol·L-1 prolonged the APD50、APD90 and APD90-APD30 by 118±9.3 ms、145±13 ms and 73±7.6 ms, respectively, and the incidence of EADs was 16.7%(1/6). In hypokalemia and hypomagnesemia Tyrode’s, the incidence of EADs of myocardial cells was 33.3%(2/6), Dof 0.1μmol·L-1 respectively prolonged the APD50、APD90 and APD90-APD30 by 143±13 ms、196±4.6 ms and 59±7.5 ms and the incidence of EADs was 83.3 %(5/6). After EADs induced by Dof, Dau 1, 3, 10μmol·L-1 were administered. We found that Dau suppressed the incidence of EADs(P<0.05), and shortened the prolonged APD in EADs and the APD50、APD90 and APD90-APD30were shortened by 201±22 ms、187±32 ms and 174±15 ms (P<0.05), respectively.(2) DS: EADs did not occur in myocardial cells of rabbits, Dof 0.1μmol·L-1 prolonged the APD50、APD90 and APD90-APD30 by 91±7.9 ms、132±6.5 ms and 77±7.6 ms, respectively, and the incidence of EADs was 16.7%(1/6). In hypokalemia and hypomagnesemia Tyrode’s, the incidence of EADs of myocardial cells was 33.3%(2/6), Dof 0.1μmol·L-1 respectively prolonged the APD50、APD90 and APD90-APD30 by 95±2.3 ms、139±9.9 ms and 74±6.4 ms and the incidence of EADs was 83.3 % (5/6). After EADs induced by Dof, DS 1.5, 1.5, 5μmol·L-1 were administered. We found that DS suppressed the incidence of EADs(P<0.05), and shortened the prolonged APD in EADs and the APD50、APD90 and APD90-APD30were shortened by 243±12 ms、206±17 ms and 184±12 ms (P<0.05), respectively.3. The effects of Dau and DS on L-type calcium current(1) Dau: Dau could inhibit the ICa-L. The peak current of ICa-L of control and Dau 1, 3, 10μmol·L-1 were -1.8±0.3 nA、-1.3±0.3 nA、-1.1±0.2 nA and -1.0±0.2 nA,respectively. The maximal activation voltage in control and Dau groups all were 0 mV. Dau made the I-V curve shift upward. Dau 1, 3, 10μmol·L-1 increased the half activation voltage (V1/2) from the predrug of -20.9±1.5 mV to -16.4±0.6 mV (P<0.05)、from -20.8±1.6 mV to -15.6±1.7 mV (P<0.05) and from -20.5±1.4 mV to -12.8±1.2 mV (P<0.01), respectively. The slopes of activation curve was increased from the predrug of 2.2±0.5 to 3.2±0.8、2.2±0.5 to 3.2±0.1 (P<0.05) and 1.8±0.5 to 3.7±0.2 (P<0.05) , respectively. The half inactivation voltage were decreased (V1/2) from the predrug of -23.0±5.0 mV to -27.7±4.0 mV、-20.5±5.3 mV to -25.6±4.3 mV and -21.8±5.4 mV to -32.3±2.5 mV(P<0.05), respectively. The slopes of inactivation curve were decreased from -6.4±0.8 to -8.1±1.0、-5.8±0.7 to -7.7±1.2 and -6.6±0.7 to -10.2±1.6(P<0.05), respectively. The recovery time from inactivation (τ)of ICa-L were prolonged from the predrug of 150±35 ms to 216±36 ms、209±29 ms to 327±61 ms(P<0.05) and 275±16 ms to 361±11 ms(P<0.05) , respectively.(2) DS: DS could inhibit the ICa-L. The peak current of ICa-L of control and DS 0.5, 1.5, 5μmol·L-1 were -1.6±0.4 nA、-1.1±0.1 nA、-1.0±0.2 nA and -0.9±0.1 nA, respectively. The maximal activation voltage in control and Dau groups all were 0 mV. Dau made the I-V curve shift upward. DS 1.5, 5μmol·L-1 increased the half activation voltage (V1/2) from the predrug of–12.8±1.7 mV to -10.4±1.7 mV and from–12.8±1.3 mV to–9.7±1.1 mV (P<0.05), respectively. The slope of activation curve were increased from the predrug of 3.5±0.7 to 5.1±0.8 and 5.0±0.6 to 6.9±0.8 (P<0.05), respectively. The half inactivation voltage were decreased (V1/2) from the predrug of -23.5±2.0 mV to–25.3±1.2 mV (P<0.05) and–24.3±1.8 mV to–27.8±3.4 mV(P<0.05), respectively. The slope of inactivation curve was decreased from–5.6±0.7 to -7.1±0.9(P<0.05) and–5.9±0.8 to–8.1±1.3(P<0.05), respectively. The recovery time from inactivation (τ) of ICa-L were prolonged from the predrug of 245±49 ms to 290±38 ms and 222±60 ms to 363±46 ms(P<0.05), respectively.Conclusion:1. In normal、hypokalemia-hypomagnesemia and hypertrophy conditions, Dau and DS could inhibit the EADs induced by Dof in papillary muscles and single myocardial cells of rabbits, shorten the APD50 and APD90 prolonged by Dof after EADs occurred, and decreased the APD90-APD30, which may be associated with the antiarrhythmic mechanisms of Dau and DS.2. Dau and DS could reduce the peak current of ICa-L, make the I-V curve shift upward, the steady state activation curve shift right and the steady state inactivation curve shift left. The half -activation voltage was increased and the half-inactivation voltage was decreased, and the effects of both make the“window current”of ICa-L decreas. Dau and DS could also prolong the recovery time of ICa-L. These results showed that the activation of ICa-L was stepped down, the inactivation was accelerated and the recovery time from inactivation was prolonged, which demonstrated that Dau and DS exerted inhibitory effects on ICa-L. These effects of Dau and DS on ICa-L may be one of their antiarrythmic mechanisms.更多还原