Callewaert G, Carmeliet E, Vereecke J
J Physiol. 1984 Apr;349:643-61. doi: 10.1113/jphysiol.1984.sp015179.
Single Purkinje cells from dog, sheep and cow hearts were isolated by injecting a Ca-free collagenase containing Tyrode solution in the space between the connective tissue sheath and the Purkinje cells. A small proportion of these cells survived the isolation procedure and these cells were used for further investigation. The cells showed electrophysiological properties similar to intact Purkinje fibres as indicated by the following results. Maximum diastolic potentials between -70 and -85 mV and specific membrane resistances of 21-32 k omega cm2 indicated that the single cells were not leaky or hyperpermeable . The action potential showed a rapid upstroke, with a maximum rate of rise, Vmax' between 150 and 750 V/s, and two phases of fast repolarization separated by a plateau phase with a duration of about 200 ms. Each action potential was followed by a spontaneous depolarization with an amplitude between 1 and 10 mV. The upstroke of the action potential could be blocked by tetrodotoxin (TTX) in a dose-dependent manner. The rate of depolarization of the action potential was sensitive to changes in membrane potential; the resulting S-shaped curve showed a half-maximum potential of -65 mV and a steepness of 0.46 mV-1. The duration of the action potential was sensitive to external K concentrations, catecholamines and TTX in a way similar to intact Purkinje fibres. Both application of catecholamines and lowering the external K concentration induced spontaneous activity. The cells were used to study the ionic nature of the pace-maker current under voltage-clamp conditions using the two-micro-electrode technique. This pace-maker current was blocked in a voltage-dependent manner by 1 mM-Cs, and was not affected by 1 mM-Ba. The steady-state activation curve was shifted in the depolarizing direction by application of adrenaline. In contrast to voltage-clamp data obtained on the pace-maker current of intact Purkinje fibres, the pace-maker current in a single cell did not reverse near the presumed equilibrium potential for K ions; no reversal could be seen in the voltage range negative to -50 mV. These observations together with preliminary results on the Na and K dependence of the pace-maker current are strong arguments in favour of the hypothesis that the pace-maker current in cardiac Purkinje fibres is an inward current carried by Na and K ions and activates upon hyperpolarization.
通过在结缔组织鞘与浦肯野细胞之间的间隙中注入含无钙胶原酶的台氏液，分离出狗、绵羊和牛心脏的单个浦肯野细胞。这些细胞中有一小部分在分离过程中存活下来，并用于进一步研究。以下结果表明，这些细胞表现出与完整浦肯野纤维相似的电生理特性。最大舒张电位在-70至-85 mV之间，比膜电阻为21 - 32 kΩ·cm²，这表明单个细胞不存在漏电或高渗透性。动作电位表现为快速上升，最大上升速率Vmax'在150至750 V/s之间，快速复极化有两个阶段，中间由持续约200 ms的平台期分隔。每个动作电位之后是幅度在1至10 mV之间的自发去极化。动作电位的上升支可被河豚毒素（TTX）以剂量依赖的方式阻断。动作电位的去极化速率对膜电位变化敏感；得到的S形曲线显示半最大电位为-65 mV，斜率为0.46 mV⁻¹。动作电位的持续时间对细胞外钾浓度、儿茶酚胺和TTX敏感，其方式与完整浦肯野纤维相似。应用儿茶酚胺和降低细胞外钾浓度均可诱导自发活动。利用双微电极技术，在电压钳条件下，这些细胞被用于研究起搏电流的离子性质。该起搏电流被1 mM - Cs以电压依赖的方式阻断，且不受1 mM - Ba的影响。应用肾上腺素后，稳态激活曲线向去极化方向移动。与在完整浦肯野纤维起搏电流上获得的电压钳数据相反，单个细胞中的起搏电流在假定的钾离子平衡电位附近不会反转；在负于-50 mV的电压范围内未观察到反转。这些观察结果以及关于起搏电流对钠和钾依赖性的初步结果，有力地支持了以下假设：心脏浦肯野纤维中的起搏电流是由钠和钾离子携带的内向电流，在超极化时激活。
