Thompson N T, Scrutton M C
Eur J Biochem. 1985 Mar 1;147(2):421-7. doi: 10.1111/j.1432-1033.1985.tb08766.x.
Fluorescence changes and secretory responses have been measured on addition of various excitatory agonists to platelets loaded with the cytosolic Ca2+ probe, Quin 2 or with chlortetracycline as a probe for membrane-associated Ca2+. When extracellular [Ca2+] is decreased to less than 0.1 microM by addition of EGTA a linear correlation is observed between the extent of increase in cytosolic [Ca2+] and the extent of mobilisation of membrane-associated Ca2+ on stimulation by maximal doses of five excitatory agonists. A similar linear correlation between the increase in cytosolic [Ca2+] and the extent of ATP secretion is observed over the thrombin dose/response curve. Similar EC50 values are observed for ATP secretion, the increase in cytosolic [Ca2+] and the decrease in chlortetracycline fluorescence induced by thrombin. However, the decrease in chlortetracycline fluorescence shows a sigmoidal relationship with the increase in cytosolic [Ca2+] and a hyperbolic relationship with ATP secretion over this dose/response curve. Addition of prostaglandin D2 prior to thrombin causes parallel inhibition of the increase in cytosolic [Ca2+] and the decrease in chlortetracycline fluorescence induced by this agonist. However, addition of prostaglandin D2 after thrombin reverses the increase in cytosolic [Ca2+] induced by this agonist but fails to cause a similar reversal of the decrease in chlortetracycline fluorescence. The data provide further evidence supporting the proposal that chlortatracycline can be used as a probe to monitor mobilisation of membrane-associated Ca2+ but suggest that, in platelets stimulated in the effective absence of extracellular Ca2+, both Ca2+ mobilisation and Ca2+ removal can under some conditions involve sites which are not monitored by this probe.
在向加载了胞质Ca2+探针喹哪啶酸或作为膜相关Ca2+探针的金霉素的血小板中添加各种兴奋性激动剂后，测量了荧光变化和分泌反应。当通过添加乙二醇双四乙酸（EGTA）将细胞外[Ca2+]降低至小于0.1微摩尔时，在五种兴奋性激动剂的最大剂量刺激下，观察到胞质[Ca2+]增加的程度与膜相关Ca2+动员的程度之间存在线性相关性。在凝血酶剂量/反应曲线上，观察到胞质[Ca2+]增加与ATP分泌程度之间存在类似的线性相关性。凝血酶诱导的ATP分泌、胞质[Ca2+]增加和金霉素荧光降低的半数有效浓度（EC50）值相似。然而，在该剂量/反应曲线上，金霉素荧光的降低与胞质[Ca2+]的增加呈S形关系，与ATP分泌呈双曲线关系。在凝血酶之前添加前列腺素D2会平行抑制该激动剂诱导的胞质[Ca2+]增加和金霉素荧光降低。然而，在凝血酶之后添加前列腺素D2会逆转该激动剂诱导的胞质[Ca2+]增加，但未能引起金霉素荧光降低的类似逆转。这些数据提供了进一步的证据支持金霉素可作为监测膜相关Ca2+动员的探针的提议，但表明，在有效缺乏细胞外Ca2+刺激的血小板中，在某些情况下，Ca2+动员和Ca2+清除可能涉及该探针未监测的位点。
