Zahradníková Alexandra, Poláková Eva, Zahradník Ivan, Zahradníková Alexandra
Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Vlárska 5, Bratislava, Slovak Republic.
J Physiol. 2007 Feb 1;578(Pt 3):677-91. doi: 10.1113/jphysiol.2006.117796. Epub 2006 Nov 23.
The local calcium release flux signals (calcium spikes) evoked by membrane depolarization were recorded at high temporal resolution (2000 lines s(-1)) in isolated ventricular myocytes of male rats, using combination of scanning confocal microscopy and the patch-clamp technique. The kinetic properties of calcium spikes were investigated. The time course of calcium spike activation could be described reliably by a model with higher-order (n = 3) kinetics, but not by a first-order exponential process. A model of calcium spike with calcium release termination coupled to its activation was preferential to a model with the release termination independent of its activation. Three fluorescent calcium dyes (OG-5N, fluo-3, and fluo-4) were compared for calcium spike measurements. Experimental measurements as well as simulations showed that the occurrence and latency of calcium spikes could be measured faithfully with all indicators, while the kinetics of calcium spikes was reliably traced only with OG-5N. Calcium spikes evoked by a step depolarization from -50 to 0 mV commenced with a mean latency of 4.1 +/- 0.2 ms and peaked 6.7 +/- 0.2 ms later. Their full amplitudes were normally distributed. The activation time constant of calcium spikes was 3.1 +/- 0.1 ms, and the time constant of termination was 5.5 +/- 0.2 ms. A negative correlation was observed between the observed amplitude of calcium spikes and their time constant of activation, but there was no correlation between their observed amplitude and time constant of termination, in agreement with the concept of steep calcium-dependent activation and fateful inactivation of calcium release flux.
利用扫描共聚焦显微镜和膜片钳技术相结合的方法，在雄性大鼠离体心室肌细胞中，以高时间分辨率（2000线每秒）记录了膜去极化诱发的局部钙释放通量信号（钙瞬变）。研究了钙瞬变的动力学特性。钙瞬变激活的时间进程可以用高阶（n = 3）动力学模型可靠地描述，而不能用一阶指数过程描述。与释放终止与其激活无关的模型相比，具有释放终止与其激活相耦合的钙瞬变模型更具优势。比较了三种荧光钙染料（OG-5N、Fluo-3和Fluo-4）用于钙瞬变测量。实验测量和模拟结果表明，所有指示剂都能忠实地测量钙瞬变的发生和潜伏期，而只有OG-5N能可靠地追踪钙瞬变的动力学。从-50 mV到0 mV的阶跃去极化诱发的钙瞬变开始时的平均潜伏期为4.1±0.2 ms，6.7±0.2 ms后达到峰值。它们的全振幅呈正态分布。钙瞬变的激活时间常数为3.1±0.1 ms，终止时间常数为5.5±0.2 ms。观察到的钙瞬变振幅与其激活时间常数之间呈负相关，但观察到的振幅与其终止时间常数之间没有相关性，这与钙依赖性激活陡峭和钙释放通量致命失活的概念一致。
