Polla B S, Bonventre J V, Krane S M
Department of Medicine, Harvard Medical School, Boston, Massachusetts.
J Cell Biol. 1988 Jul;107(1):373-80. doi: 10.1083/jcb.107.1.373.
1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) increases synthesis of heat shock proteins in monocytes and U937 cells and protects these cells from thermal injury. We examined whether 1,25-(OH)2D3 would also modulate the susceptibility of U937 cells to H2O2-induced oxidative stress. Cell viability was assessed by trypan blue exclusion and [3H]thymidine incorporation into DNA. Prior incubation for 24 h with 1,25-(OH)2D3 (25 pM or higher) unexpectedly increased H2O2 toxicity. Since cellular Ca2+ may be a mediator of cell injury we investigated effects of altering extracellular Ca2+ ([Ca2+]e) on 1,25-(OH)2D3-enhanced H2O2 toxicity as well as effects of 1,25-(OH)2D3 and H2O2 on cytosolic free Ca2+ concentration ([Ca2+]f). Basal [Ca2+]f in medium containing 1.5 mM Ca as determined by fura-2 fluorescence was higher in 1,25-(OH)2D3-pretreated cells than control cells (137 versus 112 nM, P less than 0.005). H2O2 induced a rapid increase in [Ca2+]f (to greater than 300 nM) in both 1,25-(OH)2D3-treated and control cells, which was prevented by a reduction in [Ca2+]e to less than basal [Ca2+]f. The 1,25(OH)2D3-induced increase in H2O2 toxicity was also prevented by preincubation with 1,25-(OH)2D3 in Ca2+-free medium or by exposing the cells to H2O2 in the presence of EGTA. Preexposure of cells to 45 degrees C for 20 min, 4 h earlier, partially prevented the toxic effects of H2O2 particularly in 1,25-(OH)2D3-treated cells, even in the presence of physiological levels of [Ca2+]e. Thus 1,25-(OH)2D3 potentiates H2O2-induced injury probably by increasing cellular Ca2+ stores. The 1,25-(OH)2D3-induced amplification of the heat shock response likely represents a mechanism for counteracting the Ca2+-associated enhanced susceptibility to oxidative injury due to 1,25-(OH)2D3.
1,25 - 二羟基维生素D3（1,25 - (OH)2D3）可增加单核细胞和U937细胞中热休克蛋白的合成，并保护这些细胞免受热损伤。我们研究了1,25 - (OH)2D3是否也会调节U937细胞对H2O2诱导的氧化应激的敏感性。通过台盼蓝排斥法和[3H]胸苷掺入DNA来评估细胞活力。用1,25 - (OH)2D3（25 pM或更高）预先孵育24小时意外地增加了H2O2的毒性。由于细胞内Ca2+可能是细胞损伤的介质，我们研究了改变细胞外Ca2+（[Ca2+]e）对1,25 - (OH)2D3增强的H2O2毒性的影响，以及1,25 - (OH)2D3和H2O2对胞质游离Ca2+浓度（[Ca2+]f）的影响。通过fura - 2荧光测定，在含有1.5 mM Ca的培养基中，1,25 - (OH)2D3预处理的细胞的基础[Ca2+]f高于对照细胞（137对112 nM，P小于0.005）。H2O2在1,25 - (OH)2D3处理的细胞和对照细胞中均诱导[Ca2+]f迅速增加（至大于300 nM），这可通过将[Ca2+]e降低至低于基础[Ca2+]f来预防。在无Ca2+培养基中用1,25 - (OH)2D3预孵育或在EGTA存在下将细胞暴露于H2O2也可预防1,25(OH)2D3诱导的H2O2毒性增加。在4小时前将细胞预先暴露于45摄氏度20分钟，可部分预防H2O2的毒性作用，特别是在1,25 - (OH)2D3处理的细胞中，即使在生理水平的[Ca2+]e存在下也是如此。因此，1,25 - (OH)2D3可能通过增加细胞内Ca2+储存来增强H2O2诱导的损伤。1,25 - (OH)2D3诱导的热休克反应放大可能代表一种机制，用于抵消由于1,25 - (OH)2D3导致的与Ca2+相关的对氧化损伤易感性增加。
