Intracellular calcium homeostasis during hydrogen peroxide injury to cultured P388D1 cells
Intracellular calcium homeostasis during hydrogen peroxide injury to cultured P388D1 cells
复制标题
DOI：
10.1002/jcp.1041290314
复制
发表时间：
1986-12
影响因子：
5.6
通讯作者：
P. Hyslop;D. Hinshaw;I. Schraufstätter;L. Sklar;R. Spragg;C. Cochrane
中科院分区：
文献类型：
--
作者：
P. Hyslop;D. Hinshaw;I. Schraufstätter;L. Sklar;R. Spragg;C. Cochrane
利用细胞内荧光钙螯合剂“Quin 2”研究了培养的 P388D1 细胞暴露于 H2O2 对细胞内游离钙 ([Ca++]i) 的影响。 [Ca++]i 在一段时间内从约 150 nM 上升至 >2 μM，这在很大程度上取决于所用 H2O2 的浓度（5 × 10−5 至 5 × 10−3 M）。将 P388D1 细胞暴露于 5 × 10−3 M H2O2 后，Quin 2 在暴露 15 至 30 分钟之间完全饱和。在此期间，未检测到来自细胞外培养基的 45Ca++ 平衡速率的明显变化，而在预加载 45Ca 的细胞中，净 45Ca 从细胞中的损失率高于对照。通过原子吸收光谱法对细胞总钙的测量证实，在前 30 分钟内细胞中存在钙的净损失。在暴露于 H2O2 后 >45 分钟的时间点，细胞外 45Ca 和净细胞内 Ca++、Na+ 和 K+ 的流入迅速增加。细胞 H2O2 分解代谢的半衰期从 5.0 × 10−4 M H2O2 的约 8 分钟到 5.0 × 10−3 M H2O2 的 14.0 分钟不等。当 Quin 2 池的总 [Ca++]i 缓冲能力通过将细胞内 Quin 2 的负载增加 68 倍而变化时 (1.1 × 102 ‐ 7.6 × 103 amol/细胞），暴露于 5 mM H2O2 后，[Ca++]i 的上升速率仅降低了 1.6 倍。在细胞内[Ca++]i上升期间，通过光学和扫描电子显微镜观察细胞形态，发现在损伤的这一阶段出现了“表面泡”。在通过台盼蓝排斥的丧失或细胞中预加载的 51Cr 的丧失检测到细胞活力的任何损失之前，可以观察到 [Ca++]i 的增加和“起泡”。根据这些结果，我们得出以下结论：（1）H2O2 暴露会引起细胞内钙稳态的剂量依赖性紊乱； (2) [Ca++]i 的升高是由损伤早期暴露于 H2O2 介导的，并不依赖于氧化剂的持续存在； (3) [Ca+++]i 的上升速率在很大程度上与动员到 Quin 2 库的钙量无关； (4)在[Ca++]i上升的早期(<30min)，只有细胞内钙参与反应； (5)这些事件与质膜的总体形态变化同时发生；最后，（6）这些事件发生在质膜作为渗透性屏障的完整性丧失之前。
The effects of exposure of cultured P388D1 cells to H2O2 on intracellular free calcium ([Ca++]i) was investigated utilizing the intracellular fluorescent calcium chelator “Quin 2.” [Ca++]i rose from approximately 150 nM to >2 μM over a time course that was strongly dependent on the concentration of H2O2 used (5 × 10−5 to 5 × 10−3M). After exposure of P388D1 cells to 5 × 10−3 M H2O2, Quin 2 was fully saturated between 15 and 30 min exposure. During this time, no apparent change in the rate of equilibration of 45Ca++ from the extracellular medium could be detected, whereas in cells preloaded with 45Ca, net 45Ca was lost from the cells at a greater rate than controls. Measurements of total cellular calcium by atomic absorption spetcroscopy confirmed that there was a net loss of calcium from the cells during the first 30 min. At time points >45 min after exposure to H2O2 the influx of extracellular 45Ca and net intracellular Ca++, Na+ and K+ rapidly increased. Half times for H2O2 catabolism by the cells varied from about 8 min at 5.0 × 10−4 M H2O2 to 14.0 min at 5.0 × 10−3 M. When the total [Ca++]i‐buffering capacity of the Quin 2 pool was varied by increasing the loading of intracellular Quin 2 by 68‐fold (1.1 × 102 ‐ 7.6 × 103 amol per cell), the rate of rise of [Ca++]i was depressed by only 1.6‐fold following exposure to 5 mM H2O2. During the rise of intracellular [Ca+++]i, cell morphology was observed by both light and scanning electron microscopy and revealed that “surface blebs” appeared during this phase of injury. Both the rise in [Ca+++]i and “blebbing” were observable before any loss in cell viability was detected by either loss of Trypan blue exclusion or loss of preloaded 51Cr from the cells. From these results we conclude the following, (1) H2O2 exposure induces a dose‐dependent disturbance of intracellular calcium homeostatis; (2) the rise in[Ca+++]i is mediated by exposure to H2O2 in the early phase of the injury, and is not dependent on the continuing presence of the oxidant; (3) the rate of rise of [Ca+++]i is largely independent of the quantity of calcium mobilized to the Quin 2 pool; (4) during the early phase (<30 min) of rise of [Ca+++]i, only intracellular calcium is involved in the response; (5) these events occur concomitantly with gross morphological changes to the plasma membrane; and finally, (6) these events precede loss of integrity of the plasma membrane as a permeability barrier.