Brüne B, Ullrich V
University of Konstanz, Faculty of Biology, Federal Republic of Germany.
J Biol Chem. 1991 Oct 15;266(29):19232-7.
Activation of human platelets by diverse receptor-transduced signals is followed by an intracellular calcium increase. Calcium liberation from an inositol 1,4,5-trisphosphate-sensitive compartment is recognized to be one of the prime events, followed by further mechanisms to amplify the signal. Among these, the formation of prostaglandin endoperoxides and thromboxane A2 are part of the self-amplificating activation system. Two inhibitors of intracellular Ca(2+)-ATPases, thapsigargin and 2,5-di-(tert-butyl)-1,4-benzohydroquinone have been reported to deplete the intracellular inositol 1,4,5-trisphosphate-responsive stores. In human platelets with EGTA present, we found that these inhibitors of the microsomal Ca2+ sequestration generate quite different Ca2+ transients due to an inherent cyclooxygenase inhibition by the benzohydroquinone derivative compared to thapsigargin, and, therefore, only one-half of the fura-2 signal is generated. For a maximal calcium release, Ca(2+)-ATPase inhibitors depend on the self-amplification system involving thromboxane formation. Following the thapsigargin-induced [Ca2+]i transient, thrombin was unable to raise [Ca2+]i, indicating that thapsigargin mobilizes calcium from the thrombin-responsive store, as long as the self-amplifying system of platelets is intact. With the thromboxane receptor blocked, thapsigargin releases only one-half of the calcium, and, hence, thrombin was able to release additional calcium. Interestingly, in the converse experiment, thrombin did not prevent a raise of [Ca2+]i by thapsigargin at all, although applying thrombin a second time was without any effect. Therefore, we propose two calcium pools in human platelets: receptor activation transiently releases calcium from an inositol-sensitive pool including the thapsigargin-sensitive compartment, followed by reuptake within minutes. Sequestration occurs into the thapsigargin-sensitive compartment from where it can be released even when the endoperoxide/thromboxane receptor is blocked. Calcium release from both compartments allows the formation of thromboxane B2, but not if only the Ca(2+)-ATPase inhibitor-sensitive pool is emptied. In the presence of a protonophor, a calcium accumulation in the Ca(2+)-ATPase-sensitive pool could be observed.
多种受体转导信号激活人血小板后，细胞内钙离子浓度会升高。从肌醇1,4,5 -三磷酸敏感区释放钙离子被认为是主要事件之一，随后还有进一步放大信号的机制。其中，前列腺素内过氧化物和血栓素A2的形成是自我放大激活系统的一部分。据报道，两种细胞内Ca(2 +)-ATP酶抑制剂，毒胡萝卜素和2,5 -二-(叔丁基)-1,4 -苯氢醌可耗尽细胞内肌醇1,4,5 -三磷酸反应性储存库。在存在乙二醇双四乙酸(EGTA)的人血小板中，我们发现，与毒胡萝卜素相比，由于苯氢醌衍生物对环氧化酶有内在抑制作用，这些微粒体Ca2 +螯合抑制剂产生的Ca2 +瞬变有很大不同，因此，仅产生一半的fura - 2信号。为实现最大程度的钙释放，Ca(2 +)-ATP酶抑制剂依赖于涉及血栓素形成的自我放大系统。在毒胡萝卜素诱导的[Ca2 +]i瞬变后，凝血酶无法升高[Ca2 +]i，这表明只要血小板的自我放大系统完整，毒胡萝卜素就能从凝血酶反应性储存库中动员钙离子。当血栓素受体被阻断时，毒胡萝卜素仅释放一半的钙，因此，凝血酶能够释放额外的钙。有趣的是，在相反的实验中，凝血酶根本无法阻止毒胡萝卜素引起的[Ca2 +]i升高，尽管再次应用凝血酶没有任何效果。因此，我们提出人血小板中有两个钙池：受体激活会暂时从包括毒胡萝卜素敏感区的肌醇敏感池中释放钙离子，随后在数分钟内重新摄取。钙离子被螯合到毒胡萝卜素敏感区，即使内过氧化物/血栓素受体被阻断，钙离子也能从该区域释放。两个区域释放钙离子都能形成血栓素B2，但如果仅清空Ca(2 +)-ATP酶抑制剂敏感池则不能。在存在质子载体的情况下，可以观察到Ca(2 +)-ATP酶敏感池中钙离子的积累。
