Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK.
J Thromb Haemost. 2011 Mar;9(3):540-51. doi: 10.1111/j.1538-7836.2010.04159.x.
Most Ca(2+) signaling research in platelets has relied solely on monitoring the cytosolic Ca(2+) concentration (Ca(2+)). Changes in Ca(2+) constitute the net effect of Ca(2+) fluxes into the cytosol across the plasma membrane (PM) and from intracellular stores, and Ca(2+) sequestration into the stores and Ca(2+) removal across the PM. This makes interpretation of the effects of pharmacologic or genetic interventions on Ca(2+) signaling difficult and subject to error.
To validate the use of the low-affinity Ca(2+) indicator Fluo-5N to monitor the concentration of Ca(2+) in the intracellular stores (Ca(2+)) of human platelets as a first step in developing assays for a systems-level analysis of platelet Ca(2+) signaling.
Fluo-5N-loaded and Fura-2-loaded human platelets were used to observe the effects of agonist stimulation and other manipulations on Ca(2+) and Ca(2+).
Fluo-5N fluorescence changed appropriately in response to compounds that induce passive depletion of intracellular Ca(2+) stores and to physiologic agonists. Ca(2+) reuptake inhibitors and blockers of Ca(2+) release channels had the expected effects on Fura-2 and Fluo-5N fluorescence. Agonist-evoked Ca(2+) release was reversed by Ca(2+) addition to the medium, and required intact Ca(2+) reuptake mechanisms. Store refilling was observed in the presence of sarcoplasmic/endoplasmic reticulum Ca(2+) -ATPase (SERCA) inhibitors and ionomycin, suggesting the presence of a non-SERCA Ca(2+) reuptake mechanism. Evidence for a role for Ca(2+) -induced Ca(2+) release in agonist-evoked responses was obtained.
Our data provide a validation of the use of Fluo-5N as a method for monitoring changes in Ca(2+) in human platelets.
大多数血小板中的 Ca(2+)信号研究仅依赖于监测细胞浆 Ca(2+)浓度(Ca(2+))。Ca(2+)的变化构成了 Ca(2+)通过质膜 (PM) 流入细胞质以及从细胞内储存库中流出、Ca(2+)储存到储存库中和 Ca(2+)通过 PM 去除的净效应。这使得解释药物或遗传干预对 Ca(2+)信号的影响变得困难且容易出错。
验证使用低亲和力 Ca(2+)指示剂 Fluo-5N 来监测人血小板细胞内储存库中 Ca(2+)浓度(Ca(2+))的方法，作为开发用于血小板 Ca(2+)信号系统水平分析的测定法的第一步。
使用负载 Fluo-5N 和 Fura-2 的血小板观察激动剂刺激和其他操作对 Ca(2+)和 Ca(2+)的影响。
Fluo-5N 荧光在响应诱导细胞内 Ca(2+)储存库被动耗竭的化合物以及生理激动剂时发生适当变化。Ca(2+)再摄取抑制剂和 Ca(2+)释放通道阻断剂对 Fura-2 和 Fluo-5N 荧光具有预期的影响。激动剂诱导的 Ca(2+)释放可被向培养基中添加 Ca(2+)逆转，并且需要完整的 Ca(2+)再摄取机制。在 SERCA 抑制剂和离子霉素存在下观察到储存库再填充，提示存在非 SERCA Ca(2+)再摄取机制。获得了 Ca(2+)诱导的 Ca(2+)释放在激动剂诱导的反应中起作用的证据。
我们的数据为 Fluo-5N 作为监测人血小板中 Ca(2+)变化的方法提供了验证。
