Fink C C, Slepchenko B, Loew L M
Department of Physiology and Center for Biomedical Imaging Technology, University of Connecticut Health Center, Farmington, Connecticut 06030, USA.
Biophys J. 1999 Jul;77(1):617-28. doi: 10.1016/S0006-3495(99)76918-3.
The level of [InsP3]cyt required for calcium release in A7r5 cells, a smooth muscle cell line, was determined by a new set of procedures using quantitative confocal microscopy to measure release of InsP3 from cells microinjected with caged InsP3. From these experiments, the [InsP3]cyt required to evoke a half-maximal calcium response is 100 nM. Experiments with caged glycerophosphoryl-myo-inositol 4, 5-bisphosphate (GPIP2), a slowly metabolized analogue of InsP3, gave a much slower recovery and a half-maximal response of an order of magnitude greater than InsP3. Experimental data and highly constrained variables were used to construct a mathematical model of the InsP3-dependent [Ca2+]cyt changes; the resulting simulations show high fidelity to experiment. Among the elements considered in constructing this model were the mechanism of the InsP3-receptor, InsP3 degradation, calcium buffering in the cytosol, and refilling of the ER stores via sarcoplasmic endoplasmic reticulum ATPase (SERCA) pumps. The model predicts a time constant of 0.8 s for InsP3 degradation and 13 s for GPIP2. InsP3 degradation was found to be a prerequisite for [Ca2+]cyt recovery to baseline levels and is therefore critical to the pattern of the overall [Ca2+]cyt signal. Analysis of the features of this model provides insights into the individual factors controlling the amplitude and shape of the InsP3-mediated calcium signal.
采用一套新的程序，利用定量共聚焦显微镜测量从显微注射了笼化肌醇三磷酸（InsP3）的细胞中释放的InsP3，从而确定了平滑肌细胞系A7r5细胞中钙释放所需的胞质肌醇三磷酸（[InsP3]cyt）水平。通过这些实验，引发半数最大钙反应所需的[InsP3]cyt为100 nM。使用笼化甘油磷酸肌醇4,5-二磷酸（GPIP2，一种代谢缓慢的InsP3类似物）进行的实验显示，其恢复速度要慢得多，半数最大反应比InsP3大一个数量级。利用实验数据和高度受限的变量构建了InsP3依赖性胞质钙离子浓度（[Ca2+]cyt）变化的数学模型；所得模拟结果与实验高度吻合。构建该模型时考虑的因素包括InsP3受体的机制、InsP3降解、胞质中的钙缓冲以及通过肌浆内质网ATP酶（SERCA）泵对内质网钙库的再填充。该模型预测InsP3降解的时间常数为0.8秒，GPIP2为13秒。发现InsP3降解是[Ca2+]cyt恢复到基线水平的先决条件，因此对整体[Ca2+]cyt信号模式至关重要。对该模型特征的分析有助于深入了解控制InsP3介导的钙信号幅度和形状的各个因素。
