【摘要】目的探讨腺苷拮抗异丙基肾上腺素对豚鼠乳头肌动作电位和心室肌细胞ATP敏感性钾通道(K_ATP)的作用,K_ATP在儿茶酚胺增高性室性心动过速的作用机制。方法采用玻璃微电极及全细胞膜片钳方法,分别记录异丙基肾上腺素、异丙基肾上腺素加腺苷对豚鼠乳头肌动作电位及心室肌细胞K_ATP的影响。结果(1)异丙基肾上腺素可延长动作电位的时程（APD）、诱发早期后除极(EAD)，延迟后除极(DAD)及触发激动,增加L-型钙电流(I_Ca-L)及ATP敏感性钾电流(I_K.ATP)。(2)腺苷抑制异丙基肾上腺素所致的APD延长和离子流的变化。(3)腺苷A₁受体阻断剂(DPCPX)可拮抗腺苷对异丙基肾上腺素应激下 APD和I_K.ATP的作用。结论腺苷可以减弱异丙基肾上腺素的作用，使APD缩短，对心肌细胞具有保护作用。腺苷的作用与抑制L-型钙电流和I_K.ATP有关。

Effects of isoproterenol on action potentials and ATP-sensitive channel currents in guinea pig papillary muscles and ventricular myocytes: antagonism by adenosine

ZHANG Hongyan·, LI Xiaomei, HU Dayi, et al.

Department of Pediatrics, First Hospital, Beijing Medical University, Beijing 100034, China

【Abstract】ObjectiveIt has been indicated by other study groups that the adenosine and ATP-sensitive potassium channel current (K_ATP) play roles to protect the cardiac muscle by inhibiting membrane excitability and shorting action potential duration (APD) of the cardiac muscle, which suggests a possible therapeutic function of adenosine in the various models of ischemic preconditioning. However, there is little report on the treatment mechanism of adenosine and K_ATP current in idiopathic ventricular tachycardiac (IVT). The present study investigated the antagonize effect of the adenosine to isoproterenol (ISO) on the action potentials and K_ATP channel in isolated guinea pig papillary muscles and ventricular myocytes. This study aimed to explore the mechanism of K_ATP and adenosine treatments in IVT and to prove the hypothesis that adenosine could mediate cardioprotection. MethodsThe whole-cell patch clamp recording technique was used to detect the K_ATP current alterations during the stimulated triggered activity. The cell dissociation was carried on. Adult guinea pigs weighing 320-350g were killed by cervical dislocation. Hearts were immediately removed and mounted on a modified Langendorff perfusion system for retrograde perfusion of the aortic circulation. Myocytes were isolated from guinea pig ventricular by enzyme digestion. The experiment was divided into four groups, (1) baseline (BL); (2) ISO; (3) ISO plus adenosine (ADO); (4) ISO, ADO, 8-cyclopentyl-1 and 3-dipropylxathine (DPCPX). The effects could be blocked by glibenclamide (GLB). The action potentials of guinea pig papillary muscles were measured by using standard microelectrode. The parameters in the experiment included the amplitude of action potentials (APA), action potentials duration (APD), resting potentials (RP), as well as the maximum rise rate of the action potential (Vmax). ResultsWhen guinea pig ventricular myocytes were pretreated with ISO (10nml/L) for 30 minutes, APD prolonged significantly (P＜0.01), especially APD₂₀, APD₅₀ and APD₉₀. There were no changes of APA , RP and Vmax. EAD, DAD and triggered activity (TA) were elicited. Pacing 200-300 ms stimulated ventricular myocytes of guinea pigs. DAD and TA were induced by ISO. Pacing 500 ms stimulated in the guinea pigs ventricular myocytes and papillary muscles, EAD was induced by ISO. The perfusion with ADO of 10, 50, 100 and 200 μmol/L in ventricular myocytes could shorten APD in a concentration-dependent manner and completely abolish EAD and TA in all experiment. Treating ventricular myocytes with ISO (1 μmol/L) increased K_ATP. The outward K_ATP current was increased from 525±90 pA to 2 440±408 pA (n=10, P＜0.001). I_Ca- was increased by ISO. ISO group was significantly increased compared with BL group. GLB (10 μmol/L) pretreated the ventricular myocytes to antagonize the suppressive effect of K_ATP, and EAD, DAD as well as TA increased. The effects of ISO were completely antagonized by adenosine. ADO antagonized the K_ATP currents in a concentration-related fashion. K_ATP current decreased and APD recovered to normal level. The effects of adenosine were prevented by DPCPX (0.6 μmol/L) which suggested that adenosine was mediated by A₁ receptor. Adenosine had no significant effect on ventricular myocytes. ConclusionISO might induce the opening of K_ATP channel, which results in shortening of APD，indirect inhibiting I_Ca-L in ventricular myocytes. The effect of ISO could be effectively antagonized by adenosine, which associated with the inhibition of I_Ca-L and K_ATP current. Adenosine was mediated by A₁ receptor. The ATP-sensitive potassium channel activation may suppress rhythm abnormalities related to DAD and EAD, and might provide a novel and useful intervention method in the clinical occurrence of the acquired long QT syndrome and IVT.

【Key words】Adenosine;Potassium channels;Isoproterenol;Heart ventricle;Action potentials

腺苷(adenosine, ADO)是机体的生物活性物质，参与调节人体的许多重要生理功能，同时又作为一种抗心律失常药物用于治疗运动或儿茶酚胺增高性室性心动过速。本研究采用标准玻璃微电极和全细胞膜片钳技术观察腺苷对异丙基肾上腺素(isoproterenol,ISO)应激下豚鼠心室肌细胞ATP敏感性钾通道(K_ATP)作用的电生理机制及对触发激动的作用，并应用腺苷A₁受体阻断剂8-环戊基-1,3 二丙基黄嘌呤(8-cyclopentyl,3-dipropylxathine,DPCPX), 探讨其作用机制。

材料及方法

一、动作电位记录

取成年豚鼠10只，雌雄不拘，体重320～350 g。断头处死后迅速取出心脏，取右心室乳头肌，按标准微电极技术记录心室乳头肌动作电位(AP)，测其振幅(APA)、动作电位超射(OS)、时程(APD)、静息膜电位(RP)、零相最大除极化速率(Vmax)^［1,2］。

二、全细胞膜片钳方法

全细胞膜片钳记录方法^［1,2］：取酶解分离的豚鼠心室肌细胞，采用标准全细胞膜片钳记录方法，将细胞悬液滴于三维倒置显微镜工作台上的灌流槽内，待细胞沉淀10 min后，以95% O₂+5% CO₂混和气体饱和的细胞外液灌流，流速1～1.5 ml/min，室温22℃,电极阻抗为3～5 MΩ，内充以细胞内液。利用三维操纵器(Narishige,日本)移动电极，并轻压细胞表面，用负压使电极尖端与细胞膜表面形成10～100 GΩ水平的高阻抗封接，再以较大的负压吸破细胞膜，补偿电容电流及电极串联阻抗，形成全细胞记录。用计算机连续发放2 mV，脉宽10 mV的刺激脉冲，观察破膜情况。电流信号经EPC-9型膜片钳放大器(HEKA Electronic,德国)放大，滤波后储存于计算机（Macintosh,Quadra 650,德国)硬盘。脉冲信号的控制、数据的采样和分析均由Pulse+Pulsefit 7.89软件完成，在同一细胞上获得，将之阻抗封接的细胞维持电压-80 mV,以除极化-(100±70) mV 持续200 ms的阶跃脉冲(10 mV为一阶跃)，连续除极化18次，记录ATP敏感性钾电流(I_K.ATP)的电流-电压（I-V）曲线。钳制电压-40 mV,以除极化至0 mV,持续200 ms,再以除极化200 ms阶跃＋10 mV的除极化电压,记录L-型钙电流的I-V曲线。

三、实验药物

测动作电位用台氏液(mmol/L)：NaCl 137, KCl 3, CaCl₂ 2.7, NaH₂PO₄ 1.8, NaHCO₃ 12, MgCl₂ 0.5, 葡萄糖 11。用NaOH或HCl调至pH为7.35。测I_K.ATP的电极内液(mmol/L)：KCl 150,MgCl₂ 1, 依地酸钠(EGTA) 10。HEPES 10, pH 7.2。测I_K.ATP 的台氏液(mmol/L)：NaCl 150, KCl 5.4, MgCl₂ 2, 葡萄糖10, HEPES 5, CaCl₂ 1.8。pH 7.3～7.4。氯化镉(CdCl₂) 0.1,加入台氏液中。无钙液：除不含CaCl₂外，其余与台氏液相同。腺苷储存液制备：将腺苷溶于注射用水配成浓度20 mmol/L的贮存液1 ml，冷藏8℃使用。DPCPX储存液制备：将DPCPX