Iseki R, Kudo Y, Iwata M
Department of Cellular Recognition Research, Mitsubishi Kasei Institute of Life Sciences, Tokyo, Japan.
J Immunol. 1993 Nov 15;151(10):5198-207.
Contradictory results have been reported on the question of the role of Ca2+ in glucocorticoid-induced apoptosis in thymocytes. To resolve this problem, we investigated the effect of dexamethasone, a synthetic glucocorticoid, on intracellular Ca2+ concentration ([Ca2+]i), by microscopic fluorometry that enables us to monitor real-time [Ca2+]i of cells loaded with fura-2, a fluorescent Ca2+ indicator, on a single cell basis. The results indicated that dexamethasone does not induce an increase in [Ca2+]i above control level both in murine and rat thymocytes at least for 1 h after the start of the culture. We also investigated whether the depletion of extracellular Ca2+ with EGTA or buffering intracellular Ca2+ with quin-2/AM inhibited glucocorticoid-induced apoptosis as reported on rat thymocytes. Dexamethasone-induced apoptosis in both murine and rat thymocytes, however, was not inhibited by EGTA. High concentrations (25 microM and over) of quin-2/AM inhibited DNA fragmentation, but failed to inhibit cytolysis. Calmodulin inhibitors, trifluoperazine and calmidazolium, also inhibited DNA fragmentation as reported, although they markedly enhanced cytolysis. Therefore, glucocorticoid-induced death is not inhibited by quin-2/AM or calmodulin inhibitors. Furthermore, we have previously found that a proper combination of the calcium ionophore, ionomycin, and the protein kinase activator, PMA, inhibits corticosterone-induced apoptosis. These results suggest that an early increase in [Ca2+]i is neither induced by glucocorticoids nor responsible for glucocorticoid-induced apoptosis in thymocytes.
关于Ca2+在糖皮质激素诱导胸腺细胞凋亡中所起作用的问题，已有相互矛盾的报道。为解决这一问题，我们通过显微荧光测定法研究了合成糖皮质激素地塞米松对细胞内Ca2+浓度（[Ca2+]i）的影响，该方法能让我们在单细胞水平上监测加载了荧光Ca2+指示剂fura-2的细胞的实时[Ca2+]i。结果表明，在培养开始后的至少1小时内，地塞米松在小鼠和大鼠胸腺细胞中均未诱导[Ca2+]i升高至对照水平以上。我们还研究了用EGTA耗尽细胞外Ca2+或用quin-2/AM缓冲细胞内Ca2+是否如对大鼠胸腺细胞的报道那样抑制糖皮质激素诱导的凋亡。然而，EGTA并未抑制地塞米松在小鼠和大鼠胸腺细胞中诱导的凋亡。高浓度（25 microM及以上）的quin-2/AM抑制了DNA片段化，但未能抑制细胞溶解。如报道的那样，钙调蛋白抑制剂三氟拉嗪和平他米松也抑制了DNA片段化，尽管它们显著增强了细胞溶解。因此，quin-2/AM或钙调蛋白抑制剂并未抑制糖皮质激素诱导的死亡。此外，我们之前发现钙离子载体离子霉素和蛋白激酶激活剂PMA的适当组合可抑制皮质酮诱导的凋亡。这些结果表明，[Ca2+]i的早期升高既不是由糖皮质激素诱导的，也与糖皮质激素诱导的胸腺细胞凋亡无关。
