Babich M, Choi H, Johnson R M, King K L, Alford G E, Nissenson R A
Endocrine Section, Veterans Affairs Medical Center, San Francisco, California 94121.
Endocrinology. 1991 Sep;129(3):1463-70. doi: 10.1210/endo-129-3-1463.
The mechanisms by which PTH and thrombin mobilize intracellular Ca2+ (Cai2+) were examined in UMR 106-H5 rat osteosarcoma cells. Bovine PTH-(1-34) (24 pM to 240 nM) produced a dose-dependent increase in Cai2+ (EC50, 3 nM), which returned to baseline within 75 sec. Human alpha-thrombin produced an increase in Cai2+ (ECmax, 10 U/ml) which was similar to that of PTH with respect to both magnitude and time course. Chelation of extracellular calcium with 5.0 mM EGTA did not alter the Cai2+ response to either PTH or thrombin. When added together at maximally effective concentrations, PTH and thrombin produced additive effects on Cai2+ in the presence and absence of EGTA. The additive effects of PTH and thrombin on Cai2+ were confirmed at the single cell level, using laser-based image analysis. Bradykinin (1 microM) produced a significant increase in Cai2+ in UMR 106-H5 cells which was of lesser magnitude than the peak 2- to 3-fold increase elicited by PTH or thrombin. Preexposure of cells to 10 U/ml thrombin for 2 min abolished the Cai2+ response to bradykinin, whereas preexposure to 240 nM PTH had no effect on the Cai2+ response to bradykinin. Thrombin elicited a rapid increase in the accumulation of 3H-labeled inositol phosphates (IP2 and IP3) in UMR 106-H5 cells, with increases in [3H]1,4,5-IP3 detectable as early as 15 sec after the addition of thrombin. Bradykinin increased [3H]IP production to a lesser extent than thrombin, whereas PTH neither increased [3H]IP accumulation nor potentiated the [3H]IP response to thrombin. The results suggest that thrombin and bradykinin mobilize Cai2+ from a shared IP3-responsive calcium pool, whereas PTH may use signals in addition to 1,4,5-IP3 to mobilize calcium from a distinct cellular calcium pool. Alternatively, specific calcium compartmentalization exists, and there is differential coupling of these agonists to the 1,4,5-IP3/Cai2+ pathway.
在UMR 106-H5大鼠骨肉瘤细胞中研究了甲状旁腺激素（PTH）和凝血酶动员细胞内钙离子（Cai2+）的机制。牛PTH-（1-34）（24 pM至240 nM）使Cai2+呈剂量依赖性增加（半数有效浓度[EC50]为3 nM），并在75秒内恢复至基线水平。人α-凝血酶使Cai2+增加（最大效应浓度[ECmax]为10 U/ml），其在幅度和时间进程方面与PTH相似。用5.0 mM乙二醇双四乙酸（EGTA）螯合细胞外钙不会改变对PTH或凝血酶的Cai2+反应。当以最大有效浓度一起添加时，无论有无EGTA，PTH和凝血酶对Cai2+均产生相加作用。使用基于激光的图像分析在单细胞水平证实了PTH和凝血酶对Cai2+的相加作用。缓激肽（1 μM）使UMR 106-H5细胞中的Cai2+显著增加，但其幅度小于PTH或凝血酶引起的峰值2至3倍增加。细胞预先暴露于10 U/ml凝血酶2分钟可消除对缓激肽的Cai2+反应，而预先暴露于240 nM PTH对缓激肽的Cai2+反应无影响。凝血酶使UMR 106-H5细胞中3H标记的肌醇磷酸（IP2和IP3）积累迅速增加，早在添加凝血酶后15秒就能检测到[3H]1,4,5-IP3增加。缓激肽使[3H]IP产生的增加幅度小于凝血酶，而PTH既不增加[3H]IP积累，也不增强对凝血酶的[3H]IP反应。结果表明，凝血酶和缓激肽从共享的IP3反应性钙库中动员Cai2+，而PTH可能除了1,4,5-IP3外还利用其他信号从不同的细胞钙库中动员钙。或者，存在特定的钙区室化，并且这些激动剂与1,4,5-IP3/Cai2+途径存在差异偶联。
