Ng J, Gustavsson J, Jondal M, Andersson T
Department of Immunology, Karolinska Institute, Stockholm, Sweden.
Biochim Biophys Acta. 1990 Jun 12;1053(1):97-105. doi: 10.1016/0167-4889(90)90031-8.
A rise in the cytosolic free Ca2+ concentration due to both mobilization of Ca2+ from internal stores and influx of extracellular Ca2+ across the plasma membrane through 'second messenger-operated Ca2+ channels' is one of the first transmembrane signals detected following activation of CD2 or CD3 receptors on T-cells. In this study, we have further elucidated the regulation of these channels in the human T-leukemic cell line, JURKAT. Stimulation with either OKT3 or PHA induced a prompt influx of Ca2+ as assessed by MN2+ quenching of intracellular fura-2 fluorescence. When cytosolic free Ca2+ transient was partially buffered by loading the cells with BAPTA, neither agonist could induce Ca2+ entry into the cells as depicted by the lack of quenching of the fluorescence signal by Mn2+. This is in good agreement with our previous data on agonist-induced 45Ca2+ influx demonstrating that a rise in cytosolic free Ca2+ due to agonist-induced mobilization of Ca2+ from intracellular stores, could, directly or indirectly via the inositol cycle, initiate Ca2+ influx in these cells. Further support of this idea comes from the data demonstrating that agonist-induced mobilization of Ca2+ precedes the influx of Ca2+ across the plasma membrane. The present findings show that agonist-stimulation significantly increased the levels of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 after only 5 s, indicating that one or both of these substances could play a role in the regulation of Ca2+ influx. However, when agonist-induced Mn2+ influx was totally abolished, by partially buffering the cytosolic free Ca2+ rise, the formation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 was not affected. Consequently, the dependence of an initial rise in cytosolic free Ca2+ for the subsequent regulation of Ca2+ influx across the plasma membrane, can be dissociated from the formation of both Ins(1,4,5)P3 and Ins(1,3,4,5)P4.
由于细胞内钙库中Ca2+的释放以及细胞外Ca2+通过“第二信使操纵的Ca2+通道”跨质膜内流，导致胞质游离Ca2+浓度升高，这是T细胞上CD2或CD3受体激活后检测到的最早的跨膜信号之一。在本研究中，我们进一步阐明了人类T白血病细胞系JURKAT中这些通道的调控机制。用OKT3或PHA刺激可诱导Ca2+迅速内流，这通过细胞内fura-2荧光的Mn2+淬灭来评估。当用BAPTA加载细胞使胞质游离Ca2+瞬变部分缓冲时，两种激动剂均不能诱导Ca2+进入细胞，这表现为荧光信号未被Mn2+淬灭。这与我们之前关于激动剂诱导的45Ca2+内流的数据非常一致，表明激动剂诱导细胞内钙库释放Ca2+导致的胞质游离Ca2+升高，可直接或间接通过肌醇循环引发这些细胞中的Ca2+内流。这一观点的进一步支持来自于表明激动剂诱导的Ca2+释放先于Ca2+跨质膜内流的数据。目前的研究结果表明，激动剂刺激仅5秒后就显著增加了Ins(1,4,5)P3和Ins(1,3,4,5)P4的水平，表明这两种物质中的一种或两种可能在Ca2+内流的调控中起作用。然而，当通过部分缓冲胞质游离Ca2+升高使激动剂诱导的Mn2+内流完全消除时，Ins(1,4,5)P3和Ins(1,3,4,5)P4的形成不受影响。因此，胞质游离Ca2+的初始升高对随后跨质膜Ca2+内流调控的依赖性，可以与Ins(1,4,5)P3和Ins(1,3,4,5)P4的形成相分离。
