氟康唑对豚鼠心室肌细胞I_K和在HEK-293细胞中表达的HERG钾通道的抑制作用

目的研究氟康唑对豚鼠心室肌细胞延迟整流钾电流(IK)和在HEK-293细胞中表达的HERG钾通道的抑制作用。方法应用酶解法消化豚鼠单个心室肌细胞,观察氟康唑对IK的影响;采用磷酸钙沉淀瞬时转染的方法将HERG基因表达于HEK-293细胞上,观察氟康唑对野生型HERG钾通道电流、激活和失活曲线的影响,以及氟康唑对Y652A和F656C突变型HERG钾通道的作用;IK和HERG电流的记录均采用全细胞膜片钳技术。结果氟康唑(0.01、0.1、1、3、10、30、100、300和1 000μmol.L-1)浓度依赖性地抑制IK和HERG钾电流,其IC50值分别为(68.1±21.6)μmol.L-1和(48.2±9.4)μmol.L-1,对HERG钾通道的电压依赖性激活和失活曲线无影响;与野生型(WT)比较,Y652A和F656C突变型可减弱氟康唑对HERG通道的阻断作用。结论氟康唑能阻断IK和HERG通道,Y652和F656是氟康唑与HERG通道结合的关键位点。     

N-甲基小檗胺对人心房肌细胞瞬时外向钾电流和延迟整流钾电流的作用(英文)

目的　动物实验表明N 甲基小檗胺 (NMB)通过抑制豚鼠心室肌细胞ATP敏感性钾电流和钙电流来发挥抗心律失常和抗心肌缺血作用 ,故进一步研究NMB对人心肌细胞电流的作用。方法　膜片钳制技术全细胞记录模式研究NMB对人心房肌细胞瞬时外向钾电流 (Ito)和延迟整流钾电流 (IK)的作用。结果　指令电位为 +60mV时 ,NMB 0 .1 ,1 ,1 0 μmol·L-1 分别使Ito幅值下降 (1 6± 4) % ,(2 5±4) %和 (49± 3) % ,使IK 幅值下降 (42± 6) % ,(47±7) %和(65± 3) %。结论　NMB对人心房肌细胞Ito和IK 均有抑制作用。     

氯胺酮对豚鼠心室肌细胞中延迟整流钾电流的作用

目的验证并研究局麻药氯胺酮对豚鼠心肌延迟整流钾电流（IK）的作用,并探讨其对心脏功能的影响。方法采用II型胶原酶急性分离豚鼠心室肌细胞,利用全细胞膜片钳技术观察氯胺酮对豚鼠心室肌细胞中IK的作用。结果氯胺酮对心室肌IK有较强的抑制作用,但不影响该类型电流的整流特征。结论临床剂量的氯胺酮可以抑制心肌细胞中的IK,显示氯胺酮有致心律失常之作用,或者具有抗心律失常作用。     

豚鼠Ⅱ型前庭毛细胞ACh-敏感性BK电流

目的研究豚鼠球囊和椭圆囊Ⅱ型前庭毛细胞乙酰胆碱(ACh)-敏感性钾电流的特性。方法应用全细胞膜片钳技术检测新鲜单离的豚鼠球囊和椭圆囊Ⅱ型前庭毛细胞ACh-敏感性钾电流的离子特性及其药理学特点。结果①细胞外ACh激活一缓慢持久的外向性电流,其效应呈浓度依赖性,半数激活浓度为45μmol.L-1。②ACh-敏感性钾电流对钾通道阻断剂TEA(tetraethylammon ium)、IBTX(ibe-riotoxin)和Cs+敏感,而对4-AP(4-am inopyride)和apam in不敏感。③细胞内应用钙离子螯合剂BAPTA[b is(2-am inophe-noxy)ethane-N,N,N,′N-t′etra-acetic ac id],ACh-敏感性钾电流的幅值迅速被抑制。④ACh-敏感性钾电流对细胞外液中钙离子浓度变化敏感,0.2 mmol.L-1低钙外液中ACh-敏感性钾电流被抑制,电流最大值由0~10 mV向-20 mV~-30 mV超极化方向移动。结论细胞外ACh激活豚鼠球囊和椭圆囊Ⅱ型前庭毛细胞产生大电导钙依赖性钾电流(BK)。ACh-敏感性BK电流在豚鼠Ⅱ型前庭毛细胞超极化保护机制中具有重要的作用。     

白花前胡甲素对豚鼠单一心室肌细胞Ⅰ_K通道作用机制的初步探讨

目的通过比较白花前胡甲素（Pd－Ia）与异丙肾上腺素对豚鼠单一心室肌细胞迟发性外向钾电流（IK）的影响，初步探讨Pd－Ia开放钾通道作用的可能机制。方法；应用全细胞膜片钳制技术。结果与结论：Pd－Ia对IK通道作用的最大效应与异丙肾上腺素相近，均使IK增加2～2．5倍，对IK的激活动力学过程均无明显影响，而且两药的作用不具有相加性。另外，乙酰胆碱对Pd－Ia增加IK作用的影响也与对异丙肾上腺素的影响相似，提示Pd－Ia对IK的作用可能是通过激活PKA而产生的。其确切机制还有待进一步实验研究。     

黄连素对结肠平滑肌细胞膜钙激活钾通道和延迟整流钾通道的影响

目的　研究黄连素对结肠平滑肌细胞膜钙离子激活钾通道 (IK(Ca) )和延迟整流钾通道 (IK(V) )的影响以初步探讨其治疗运动性腹泻的机制。方法　酶解法急性分离单个豚鼠结肠平滑肌细胞 ,运用膜片钳方法检测 10、5 0、10 0 μmol·L-1的黄连素对结肠平滑肌细胞膜IK(Ca) 和IK(V) 的影响。结果　 10、5 0、10 0 μmol·L-1的黄连素可抑制豚鼠单个结肠平滑肌细胞膜IK(Ca) (P <0 0 1) ,当阶跃刺激为 +80mV时 ,其IK(Ca) 分别为生理盐水对照组的 (6 8 2 0± 5 17) % ,(5 5 89± 1 6 1) % ,(4 8 0 8± 2 4 5 ) % (P <0 0 1) ;10、5 0、10 0 μmol·L-1的黄连素可抑制豚鼠单个结肠平滑肌细胞膜IK(V) (P <0 0 1) ,当阶跃刺激为 +80mV时 ,其IK(V) 分别为生理盐水对照组的 (77 0 6± 6 4 2 ) % ,(6 8 6 7± 6 79) % ,(6 1 0 7±7 72 ) % (P <0 0 1)。结论　Ber能抑制豚鼠结肠平滑肌钙离子激活钾通道和延迟整流钾通道的开放 ,这可能是其治疗运动性腹泻的机制之一。     

延胡索乙素对豚鼠单个心室肌细胞钾离子通道的影响

目的研究延胡索乙素(dltetrahydropalmatine,THP)对正常豚鼠心室肌细胞钾电流的影响,旨在探讨延胡索乙素抗心律失常作用机制。方法酶解法分离豚鼠单个心室肌细胞,应用全细胞膜片钳技术记录延胡索乙素对豚鼠单个心室肌细胞钾电流的影响。结果延胡索乙素可明显抑制延迟整流钾电流(IK)和内向整流钾电流(IK1),并呈剂量依赖性。结论延胡索乙素抗心律失常作用机制可能与它对心肌细胞钾通道作用有关,THP可抑制IK和IK1,使动作电位时程(APD)和有效不应期(ERP)延长从而发挥其抗心律失常作用。     

2,3-二甲基-2-丁胺新衍生物对脑神经细胞钾通道的影响

目的　研究 2, 3 二甲基 2 丁胺衍生物对大脑皮层神经细胞钾通道的影响。方法　在原代培养的新生大鼠大脑皮层神经元细胞上,用细胞膜片钳全细胞记录技术,评价新衍生物对神经元IA及IK电流的影响。结果　在评价的 9个新化合物中,化合物(5) (6) (7) (9)不影响皮层神经元细胞上钾通道的活性, 化合物 ( 4 ) ( 8 )能降低皮层神经元IK电流, 这 6个化合物均可激活血管平滑肌钾通道; 另一方面,其余 3个化合物对血管平滑肌钾通道功能无明显影响,但其中的化合物 (3)可降低神经元上的IK电流。结论　2, 3 二甲基 2 丁胺衍生物对脑神经细胞上钾通道的作用不同于其在血管平滑肌上钾通道的作用特点。     

乙酰胆碱对心脏脱敏的离子机制

脱敏是指靶细胞对激素、药物或神经递质的反应逐渐降低现象.已有研究表明无论是直接刺激迷走神经还是施加乙酰胆碱(ACh)到心脏,均可见脱敏的情况存在.心脏对ACh脱敏本身在生理条件下就可能具有重要意义,因为处于迷走紧张性支配的条件下,心脏的窦房结持续暴露在ACh作用下,而脱敏正是在这种情况下发生的,亦可能是一种防止ACh的过度作用的保护、适应及负反馈调节.我们采用膜片钳全细胞记录的方法发现在给豚鼠单个心房肌细胞灌注ACh时,乙酰胆碱可以激活一种经毒蕈碱受体激活的钾通道(即iK.ACh).     

ATP敏感性钾通道的心肌保护机制研究进展

自心肌细胞膜片钳技术应用以来,Nome 1 983年首先在豚鼠心室肌细胞发现ATP敏感性钾通道(KATP channel)[1],随后证实KATP在缺血再灌注中对心肌细胞具有保护作用,为细胞离子通道的研究开创了崭新的局面,成为临床及基础研究的热点.迄今,对心血管系统ATP敏感性钾通道的调节作用缺乏系统研究,本文就KATP的生理作用、在缺血再灌注及在触发性心律失常中对心室肌细胞的保护机制加以综述,为KATP通道开放的临床应用与基础研究提供理论依据.     

Effects of mitoxantrone on action potential and membrane currents in isolated cardiac myocytes.

1. The effects of mitoxantrone (MTO), an anticancer drug, on the membrane electrical properties of cardiac myocytes were investigated using the whole-cell clamp technique. 2. In isolated guinea-pig ventricular myocytes, 30 microM MTO induced a time-dependent prolongation of action potential duration (APD) which was occasionally accompanied by early afterdepolarizations. APD prolongation was preserved in the presence of 10 microM tetrodotoxin and showed reverse rate-dependence. 3. Both the inward rectifier K+ current (I(KI)) and the delayed rectifier K+ current (I(K)) of guinea-pig ventricular myocytes were significantly depressed by 30 microM MTO. The rapidly activating component of I(k) (I(Kr)) seemed to be preferentially blocked by MTO. The transient outward current was not affected by MTO in rat ventricular myocytes. 4. Thirty microM MTO had no direct effect on the L-type Ca2+ current (I(Ca(L))), but reversed the inhibitory effect of 1 microM carbamylcholine but not the A1-adenosine receptor agonist (-)-N6-phenylisopropyladenosine (1 microM) on I(Ca(L)) enhanced by 50 nM isoprenaline in guinea-pig ventricular myocytes. In guinea-pig atrial mycotyes, 30 microM MTO inhibited by 93% the muscarinic receptor gated K+ current (I(K,ACh)) evoked by 1 microM carbamylcholine, whereas I(K,ACh) elicited by 100 microM GTPgammaS, a nonhydrolysable GTP analogue, was only decreased by 12%. 5. The specific binding of [3H]QNB, a muscarinic receptor ligand, to human atrial membranes was concentration-dependently displaced by MTO (1-1000 microM). 6. In conclusion, MTO blocks cardiac muscarinic receptors and prolongs APD by inhibition of I(KI) and I(Kr). The occasionally observed early afterdepolarizations may signify a potential cardiac hazard of the drug.     

Effects of azimilide on the muscarinic acetylcholine receptor-operated K+ current and experimental atrial fibrillation in guinea-pig hearts

Effects of azimilide, a class III antiarrhythmic drug, on the acetylcholine (ACh) receptor-operated K+ current (I K.ACh) and the delayed rectifier K+ current (IK) were examined in guinea-pig atrial cells using patch-clamp techniques. Effects of azimilide on experimental atrial fibrillation (AF) were also examined in isolated guinea-pig hearts. In single atrial myocytes, azimilide inhibited both the rapid (IKr) and slow component of IK (IKs). Azimilide inhibited the I K.ACh induced by carbachol (CCh, 1 microM), adenosine (10 microM), and intracellular loading of GTPgammaS (100 microM) in a concentration-dependent manner. The IC50 values of azimilide for inhibiting the CCh-, adenosine-, and GTPgammaS-induced I K.ACh were 1.25, 29.1, and 20.9 microM, respectively, suggesting that azimilide inhibits I K.ACh mainly by blocking the muscarinic receptors. Azimilide concentration-dependently (0.3 - 10 microM) prolonged the action potential duration (APD) in the absence and presence of muscarinic stimulation. In isolated hearts, perfusion of CCh shortened the duration of the monophasic action potential (MAP) and effective refractory period (ERP) of the left atrium and lowered the atrial fibrillation threshold (AFT). Addition of azimilide inhibited the induction of AF by prolonging the duration of MAP and ERP. The I K.ACh inhibition by azimilide may at least in part contribute to the effectiveness to prevent parasympathetic-type AF.     

碘化N-正丁基氟哌啶醇对大鼠心室肌细胞L型钙通道的阻断作用

目的研究碘化N-正丁基氟哌啶醇(F2)对大鼠心室肌细胞L-型钙通道(ICa)的影响。方法采用酶急性分离的单个大鼠心室肌细胞,应用膜片钳全细胞记录技术,观察F2对ICa的影响。结果F20.1,1,10×10-6mol.L-1可剂量依赖地抑制ICa,抑制率分别为40%,72%,84%,IC50为1.19×10-6mol.L-1。F2上移ICa的I-V曲线,但不改变ICa的最大锋电位和翻转电位;F2对ICa稳态激活曲线无明显改变;F2可使得ICa稳态失活曲线左移;延长ICa失活恢复时间。结论F2对心肌细胞ICa具有阻断作用。     

Regulation of the muscarinic K+ channel by extracellular ATP through membrane phosphatidylinositol 4,5-bisphosphate in guinea-pig atrial myocytes

1 The present study was designed to examine the functional role of membrane phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) in the regulation of the muscarinic K(+) channel (I(K,ACh)) by extracellular ATP and adenosine in guinea-pig atrial myocytes, using the whole-cell patch-clamp method. 2 Bath application of ATP in micromolar concentrations typically evoked a transient activation of I(K,ACh); a rapid activation phase was consistently followed by a progressive decline even to the baseline level despite the continued presence of ATP. This progressive decline of I(K,ACh) was significantly attenuated either by blockade of phospholipase C (PLC) with compound 48/80 (100 microM) or by addition of PtdIns(4,5)P(2) (50 microM) to the cell inside, suggesting that depletion of membrane PtdIns(4,5)P(2) via PLC activation is mainly, if not totally, responsible for the progressive decline of I(K,ACh) during the presence of ATP. 3 When atrial myocytes were exposed to wortmannin (50 microM) following ATP (50 microM) application to impair the resynthesis of PtdIns(4,5)P(2), the activation of I(K,ACh) evoked by subsequently applied ATP (50 microM) was greatly reduced. Activation of I(K,ACh) by adenosine (100 microM) was partially reduced by pretreatment of atrial myocytes with ATP (100 microM) and was largely abolished by a further addition of wortmannin (50 microM) in the presence of ATP (100 microM). These results support the view that the activation of I(K,ACh) by ATP and adenosine depends on membrane PtdIns(4,5)P(2) that is subject to reduction by extracellular ATP. 4 The present study thus provides functional evidence to suggest that extracellular ATP activates PLC and thereby depletes membrane PtdIns(4,5)P(2) that is critically involved in the activation process of I(K,ACh) by its agonists ATP and adenosine in guinea-pig atrial myocytes.     

苦参碱对心房颤动犬心房肌I_(KM3)电流的作用

目的探讨正常和持续性心房颤动时犬心房肌M3受体介导IKM3电流的变化及苦参碱对IKM3的作用,为筛选抗心律失常药物作用靶点提供理论和实验依据。方法建立快速心房起搏致心房颤动犬模型,通过膜片钳技术比较犬心房肌正常和模型组电流变化及药物作用的不同。结果持续性心房颤动犬心房肌IKM3电流密度明显增高(实验电压+50mV时,232±81pAvs198±80pA,n=4,P<0.05),苦参碱对IKM3电流有抑制作用,且对正常组抑制作用明显高于模型组。结论M3受体及其介导的IKM3电流可能是治疗房颤的新靶点。     

Antiarrhythmic agents act differently on the activation phase of the ACh-response in guinea-pig atrial myocytes.

1. Anti-acetylcholine effects of pilsicainide, flecainide, disopyramide and propafenone on the acetylcholine (ACh)-induced K+ current (IK.ACh) were examined in dissociated guinea-pig atrial myocytes under whole-cell voltage clamp by the use of the 'concentration-clamp' technique. 2. The IK.ACh was activated with a latency of about 100 ms after 1 microM ACh application and desensitized to a steady-state level. The latent period and the time to peak response were shortened with increasing ACh concentration. 3. The values of half-maximal inhibition (IC50) on the peak and steady state responses were 25 and 25 microM for pilsicainide, 1.7 and 2.0 microM for disopyramide, 19 and 2.0 microM for flecainide and 0.7 and 0.2 microM for propafenone, respectively. 4. Pilsicainide and disopyramide increased the latent period and the time to peak of IK.ACh in a concentration-dependent manner. Flecainide and propafenone did not change the latent period, but shortened the time to peak and hastened the decay of IK.ACh in a voltage-independent manner. 5. The results suggest that the mechanisms underlying the anti-acetylcholine effect of antiarrhythmic drugs are different among these drugs: i.e., pilsicainide and disopyramide mainly block the muscarinic ACh receptors while flecainide and propafenone inhibit the K+ channel itself as open channel blockers.     

Cholinergic prejunctional inhibition of nitrergic neurotransmission in the guinea-pig isolated basilar artery

1. The effects of endogenous and exogenous acetylcholine (ACh) on nitrergic relaxations elicited by electrical field stimulation (EFS) were studied in guinea-pig endothelium-denuded basilar artery preparations precontracted with 1 mumol/L prostaglandin F2 alpha and a possible role of K+ channels in mediating the effects was investigated. 2. Acetylcholine (3 mmol/L) and physostigmine (10 mumol/L) produced small, yet statistically significant, inhibitions of EFS-induced nitrergic relaxations, while atropine (1 mumol/L) slightly enhanced the nitrergic response. The ACh-induced inhibition was atropine sensitive. Acetylcholine or atropine did not affect relaxations induced by sodium nitroprusside. 3. The inhibition of nitrergic relaxations by 3 mumol/L ACh was prevented by the K+ channel blockers tetraethylammonium and 4-aminopyridine, but was not changed by iberiotoxin, apamin or glibenclamide. 4. Neither vasoactive intestinal polypeptide nor the alpha 2-adrenoceptor agonists noradrenaline and clonidine modulated nitrergic neurotransmission in the guinea-pig basilar artery. 5. The findings show that ACh acts on prejunctional muscarinic receptors of nitrergic nerves to inhibit nitrergic neurotransmission. It is suggested that endogenous ACh may have this effect; however, the physiological significance of this prejunctional modulation is not clear due to the relatively small effect produced. The prejunctional inhibitory action of ACh may involve opening of neuronal K+ channels.     

Electrophysiological evidence for a gradient of G protein-gated K+ current in adult mouse atria

Whole cell current and voltage clamp techniques were used to examine the properties of acetylcholine-sensitive K+ current (IKACh) in myocytes from adult mouse atrium. Superfusion of a maximal dose of carbachol (CCh; 10 microM) caused a substantial increase in K+ current in all myocytes examined. The current-voltage (I-V) relation of maximally activated IKACh exhibited weak inward rectification. Consequently, CCh increased the amount of depolarising current necessary to evoke action potentials (APs), and APs evoked in CCh had significantly shorter durations than control APs (P<0.05). The effects of CCh on K+ current and on AP properties were blocked by the muscarinic receptor antagonist methoctramine (1 microM). ACh (10 microM) activated a K+ current with identical properties to that activated by CCh, as did the A1 receptor agonist adenosine (100 microM). Right atrial myocytes had significantly more IKACh than left atrial myocytes (P<0.05), regardless of whether IKACh was evoked by superfusion of muscarinic or A1 receptor agonists. IKACh current density was significantly higher in SA node myocytes than either right or left atrial myocytes. These data identify a gradient of IKACh current density across the supraventricular structures of mouse heart. This gradient, combined with the heterogeneous distribution of parasympathetic innervation of the atria, may contribute to the proarrhythmic ability of vagal nerve stimulation to augment dispersion of atrial refractoriness.     

Effects of anticancer chemotherapeutic drugs on the acetylcholine receptor-operated potassium current in guinea pig atrial myocytes

The effects of 7 anticancer chemotherapeutic drugs on the muscarinic acetylcholine receptor-operated potassium current (I(K.ACh)) in guinea pig atrial myocytes were investigated using the whole cell patch clamp technique. Doxorubicin, pirarubicin, and mitoxantrone inhibited the carbachol-induced I(K.ACh) in a concentration-dependent manner in atrial cells at a holding potential of -40 mV. IC50 values of doxorubicin, pirarubicin, and mitoxantrone for the carbachol-induced I(K.ACh) were 7.7 microM, 3.7 microM, and 9.1 microM, respectively. Pirarubicin inhibited the adenosine-induced and the GTPgammaS-induced I(K.ACh) in a concentration-dependent manner (IC50=6.0 and 5.1 microM, respectively). Doxorubicin and mitoxantrone up to 100 microM did not have an influence on the adenosine-induced I(K.ACh). Doxorubicin did not affect the GTPgammaS-induced I(K.ACh). Mitoxantrone 100 microM inhibited the current only by 25%. For concentrations up to 100 microM, anticancer drugs that have chemical structures entirely different from that of doxorubicin, i.e., 5-fluorouracil, 6-mercaptopurine, cyclophosphamide, and actinomycin D, did not have an influence on the carbachol-induced I(K.ACh). Doxorubicin and chemically related compounds possess anticholinergic effects mediated via an inhibitory action on I(K.ACh) by different underlying molecular mechanisms. Doxorubicin and mitoxantrone may inhibit I(K.ACh) by the blockade of muscarinic receptors, whereas pirarubicin may inhibit the current not only via blocking the muscarinic receptors but also by depressing the functions of the K+ channel itself and/or GTP-binding proteins.