Fujita H, Ishizaki Y, Yanagisawa A, Morita I, Murota S I, Ishikawa K
Second Department of Internal Medicine, Kyorin University School of Medicine, 6-20-2, Shinkawa, Mitaka, Tokyo, 181, Japan.
Cell Biol Int. 1999;23(4):241-9. doi: 10.1006/cbir.1999.0342.
We examined the role of ion movement in staurosporine-induced apoptosis of vascular endothelial cells. Cultured vascular endothelial cells from bovine carotid arteries were used. Apoptosis was determined by propidium iodide assay. Treatment of the endothelial cells with staurosporine (10 nmol/l-1 micromol/l) for 6 h induced nuclear fragmentation in a dose-dependent manner. Staurosporine (1 micromol/l) elicited apoptosis in 70.5+/-1.5% of cells. Concomitant treatment of endothelial cells with 1 mmol/l of 4, 4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS), a chloride-bicarbonate exchange blocker, completely inhibited staurosporine-induced apoptosis. Other ion transporter inhibitors such as dimethyl amiloride and anthracene-9 carboxylic acid were less effective inhibitors of staurosporine-induced apoptosis of endothelial cells. DIDS prevented staurosporine-induced apoptosis of endothelial cells as well as cardiomyocytes. Next, we determined whether chloride ions or bicarbonate are involved in apoptosis. Incubation with a chloride ion removal buffer did not inhibit staurosporine-induced apoptosis of endothelial cells. However, endothelial cell apoptosis was completely suppressed by an inhibitor of caspase, benzyloxycarbonyl-Asp-CH(2)-O(C)O-dichlorobenzene (zD-dcb, 50 micromol/l). Staurosporine (1 micromol/l) increased the intracellular pH of endothelial cells, and DIDS (1 mmol/l), but not a caspase inhibitor, inhibited this increase in pH caused by staurosporine. Our findings suggest that endothelial cell apoptosis induced by staurosporine may be associated with the Cl(-)and bicarbonate (HCO-3) ions. Thus, Cl(-)efflux from cells or HCO-3 influx to cells (which increases pH) may play an important role in signal transduction leading events such as activation of caspase in staurosporine-induced apoptosis.
我们研究了离子运动在星形孢菌素诱导的血管内皮细胞凋亡中的作用。使用了从牛颈动脉分离培养的血管内皮细胞。通过碘化丙啶检测法测定细胞凋亡。用星形孢菌素（10 nmol/L - 1 μmol/L）处理内皮细胞6小时，可剂量依赖性地诱导细胞核碎裂。星形孢菌素（1 μmol/L）可使70.5±1.5%的细胞发生凋亡。同时用1 mmol/L的4,4 - 二异硫氰酸根合芪 - 2,2 - 二磺酸（DIDS，一种氯 - 碳酸氢根交换阻滞剂）处理内皮细胞，可完全抑制星形孢菌素诱导的凋亡。其他离子转运体抑制剂，如二甲基氨氯吡脒和蒽 - 9 - 羧酸，对星形孢菌素诱导的内皮细胞凋亡的抑制作用较弱。DIDS可预防星形孢菌素诱导的内皮细胞和心肌细胞凋亡。接下来，我们确定氯离子或碳酸氢根是否参与凋亡过程。用去除氯离子的缓冲液孵育并未抑制星形孢菌素诱导的内皮细胞凋亡。然而，半胱天冬酶抑制剂苄氧羰基 - 天冬氨酸 - CH(2)-O(C)O - 二氯苯（zD - dcb，50 μmol/L）可完全抑制内皮细胞凋亡。星形孢菌素（1 μmol/L）可使内皮细胞内pH值升高，而DIDS（1 mmol/L）而非半胱天冬酶抑制剂可抑制星形孢菌素引起的pH值升高。我们的研究结果表明，星形孢菌素诱导的内皮细胞凋亡可能与氯离子和碳酸氢根离子有关。因此，氯离子从细胞外流或碳酸氢根离子流入细胞（这会使pH值升高）可能在星形孢菌素诱导的凋亡中信号转导导致的事件（如半胱天冬酶激活）中起重要作用。
