Ryanodine as a Tool to Determine the Contributions of Calcium Entry and Calcium Release to the Calcium Transient and Contraction of Cardiac Purkinje Fibers
Ryanodine as a Tool to Determine the Contributions of Calcium Entry and Calcium Release to the Calcium Transient and Contraction of Cardiac Purkinje Fibers
复制标题
Ryanodine 作为确定钙进入和钙释放对心脏浦肯野纤维钙瞬变和收缩贡献的工具
作者：
E. Marbán;W. Wier
我们的目的是评估心肌浦肯野纤维收缩中跨肌膜钙进入和细胞内钙释放的相对作用。我们观察到细胞内钙瞬变，膜电位，和收缩的水母发光蛋白注射犬心脏浦肯野纤维暴露于高度选择性的药理学修饰剂的兴奋-收缩偶联。为了选择性地影响肌浆网中钙的释放，我们使用了植物生物碱ryanodine。为了影响钙离子进入，我们选择性地使用钙通道拮抗剂尼群地平或钙通道激动剂Bay K 8644。单独Ryanodine（1μM）可降低细胞内水母发光蛋白发光信号的两种成分（L1和L2）。在三块肌肉中，发光信号的幅度为对照的3%（平均值的标准误差为2%），没有两个不同的成分，抽搐张力为对照的2%（平均值的标准误差为3%），而动作电位延长。钙拮抗剂尼群地平（10 μM）可消除ryanodine中的水母发光蛋白信号和抽搐，这也降低了动作电位平台，与功能性钙通道阻滞一致。在两个实验中，钙通道激动剂，湾K 8644，在兰尼碱的存在下，增加水母发光蛋白的发光和收缩，但只有一个非常小的一部分，他们的控制值。在不含钾、含ryanodine的溶液中，钠的戒断引起钙和张力的大幅度缓慢增加，表明张力仍能产生，水母发光蛋白仍有功能，钠/钙交换不受ryanodine抑制。咖啡因增加细胞内钙，表明钙储存没有耗尽。总之，兰尼定的负性肌力作用是由于从内部储存释放的钙减少，钙进入通常只占总激活剂钙的很小一部分。
Our object was to assess the relative roles of transsarcolemmal calcium entry and intracellular calcium release in the contraction of cardiac Purkinje fibers. We observed intracellular calcium transients, membrane potential, and contraction in aequorin-injected canine cardiac Purkinje fibers exposed to highly selective pharmacological modifiers of excitation-contraction coupling. To influence selectively the release of calcium from the sarcoplasmic reticulum, we used the plant alkaloid, ryanodine. To influence calcium entry, selectively, we used either the calcium channel antagonist, nitrendipine, or the calcium channel agonist, Bay k 8644. Ryanodine alone (1μM) reduced both components of the intracellular aequorin luminescence signal (L1 and L2). In three muscles, the luminescence signals were 3% of control in amplitude (standard error of the mean, 2%) without two distinct components and the twitch tension was 2% of control (standard error of the mean, 3%), whereas the action potential was prolonged. The aequorin signal and twitch remaining in ryanodine were abolished by the calcium antagonist nitrendipine (10 μM), which also lowered the action potential plateau, consistent with the block of functional calcium channels. In two experiments, the calcium-channel agonist, Bay k 8644, in the presence of ryanodine, increased the aequorin luminescence and the contraction, but only to a very small fraction of their control values. Sodium withdrawal in potassium-free, ryanodine-containing solution produced large slow increases in calcium and tension, showing that tension could still be produced, that aequorin remained functional, and that sodium/calcium exchange was not inhibited by ryanodine. Caffeine increased intracellular calcium, showing that calcium stores were not depleted. In summary, the negative inotropic effect of ryanodine is due to a decrease in the calcium released from internal stores, and calcium entry normally contributes only a very small fraction of the total activator calcium.