Föller Michael, Mahmud Hasan, Koka Saisudha, Lang Florian
Department of Physiology, University of Tuebingen, Gmelinstr. 5, 72076 Tübingen, Germany.
Pflugers Arch. 2008 Feb;455(5):939-49. doi: 10.1007/s00424-007-0336-x. Epub 2007 Sep 25.
The phosphoinositide-dependent kinase PDK1 is a key element in the phosphoinositol-3-kinase signalling pathway, which is involved in the regulation of ion channels, transporters, cell volume and cell survival. Eryptosis, the suicidal death of erythrocytes, is characterized by decrease in cell volume, cell membrane blebbing and phospholipids scrambling with phosphatidylserine exposure at the cell surface. Oxidative stress, osmotic shock or Cl- removal trigger eryptosis by activation of Ca2+-permeable cation channels and subsequent increase in cytosolic Ca2+ activity. To explore the impact of PDK1 for erythrocyte survival, eryptosis was analysed in hypomorphic mice (pdk1hm) expressing only some 25% of PDK1 and in their wild-type littermates (pdk1wt). Cell volume was estimated from forward scatter and phosphatidylserine exposure from annexin-V binding in fluorescence activated cell sorter analysis. Forward scatter was smaller in pdk1hm than in pdk1wt erythrocytes. Oxidative stress (100 microM tert-butylhydroperoxide), osmotic shock (+300 mM sucrose) and Cl- removal (replacement of Cl- with gluconate) all decreased forward scatter and increased the percentage of annexin-V-binding erythrocytes from both pdk1hm and pdk1wt mice. After treatment, the forward scatter was similar in both genotypes, but the percentage of annexin-V binding was significantly smaller in pdk1hm than in pdk1wt erythrocytes. According to Fluo-3 fluorescence, cytosolic Ca2+ activity was significantly smaller in pdk1hm than in pdk1wt erythrocytes. Treatment with Ca2+-ionophore ionomycin (1 microM) was followed by an increase in annexin-V binding to similar levels in pdk1hm and pdk1wt erythrocytes. The experiments reveal that PDK1 deficiency is associated with decreased Ca2+ entry into erythrocytes and thus with blunted eryptotic effects of oxidative stress, osmotic shock and Cl- removal.
磷酸肌醇依赖性激酶PDK1是磷酸肌醇-3-激酶信号通路中的关键元件，该信号通路参与离子通道、转运体、细胞体积和细胞存活的调节。红细胞凋亡，即红细胞的自杀性死亡，其特征是细胞体积减小、细胞膜起泡以及磷脂紊乱，导致磷脂酰丝氨酸暴露于细胞表面。氧化应激、渗透压休克或氯离子去除通过激活钙离子通透阳离子通道并随后增加胞质钙离子活性来触发红细胞凋亡。为了探究PDK1对红细胞存活的影响，我们在仅表达约25%PDK1的低表达小鼠（pdk1hm）及其野生型同窝小鼠（pdk1wt）中分析了红细胞凋亡。在荧光激活细胞分选分析中，通过前向散射估计细胞体积，通过膜联蛋白-V结合估计磷脂酰丝氨酸暴露情况。pdk1hm红细胞的前向散射比pdk1wt红细胞小。氧化应激（100微摩尔叔丁基过氧化氢）、渗透压休克（+300毫摩尔蔗糖）和氯离子去除（用葡萄糖酸盐替代氯离子）均降低了pdk1hm和pdk1wt小鼠红细胞的前向散射，并增加了膜联蛋白-V结合红细胞的百分比。处理后，两种基因型的前向散射相似，但pdk1hm红细胞中膜联蛋白-V结合的百分比显著低于pdk1wt红细胞。根据Fluo-3荧光，pdk1hm红细胞中的胞质钙离子活性显著低于pdk1wt红细胞。用钙离子载体离子霉素（1微摩尔）处理后，pdk1hm和pdk1wt红细胞中膜联蛋白-V结合增加至相似水平。实验表明，PDK1缺乏与红细胞钙离子内流减少有关，因此与氧化应激、渗透压休克和氯离子去除的凋亡效应减弱有关。
