Malik Abaid, Bissinger Rosi, Liu Guoxing, Liu Guilai, Lang Florian
Department of Physiology, University of Tübingen, Gmelinstr. 5, 72076 Tuebingen, Germany.
Toxins (Basel). 2015 Apr 23;7(5):1396-410. doi: 10.3390/toxins7051396.
The peptide antibiotic and ionophore gramicidin has previously been shown to trigger apoptosis of nucleated cells. In analogy to apoptosis, the suicidal death of erythrocytes or eryptosis involves cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include oxidative stress, increase of cytosolic Ca2+ activity ([Ca2+]i), and ceramide. The present study explored, whether gramicidin triggers eryptosis. To this end phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, red blood cell distribution width (RDW) from electronic particle counting, reactive oxidant species (ROS) from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, [Ca2+]i from Fluo3- and Fluo4 fluorescence, and ceramide abundance from binding of specific antibodies. As a result, a 24 h exposure of human erythrocytes to gramicidin significantly increased the percentage of annexin-V-binding cells (≥1 µg/mL), forward scatter (≥0.5 µg/mL) and hemolysis. Gramicidin enhanced ROS activity, [Ca2+]i and ceramide abundance at the erythrocyte surface. The stimulation of annexin-V-binding by gramicidin was significantly blunted but not abolished by removal of extracellular Ca2+. In conclusion, gramicidin stimulates phospholipid scrambling of the erythrocyte cell membrane, an effect at least partially due to induction of oxidative stress, increase of [Ca2+]i and up-regulation of ceramide abundance. Despite increase of [Ca2+]i, gramicidin increases cell volume and slightly reduces RWD.
肽抗生素和离子载体短杆菌肽先前已被证明可引发有核细胞凋亡。与凋亡类似，红细胞的自杀性死亡或红细胞凋亡涉及细胞收缩以及细胞膜磷脂酰丝氨酸易位至红细胞表面。红细胞凋亡的触发因素包括氧化应激、胞质Ca2+活性（[Ca2+]i）增加以及神经酰胺。本研究探讨了短杆菌肽是否会引发红细胞凋亡。为此，通过膜联蛋白V结合来估计细胞表面磷脂酰丝氨酸的暴露情况，通过前向散射来估计细胞体积，通过电子颗粒计数来估计红细胞分布宽度（RDW），通过2',7'-二氯二氢荧光素二乙酸酯（DCFDA）荧光来估计活性氧（ROS），通过Fluo3和Fluo4荧光来估计[Ca2+]i，并通过特异性抗体的结合来估计神经酰胺丰度。结果显示，将人红细胞暴露于短杆菌肽24小时后，膜联蛋白V结合细胞的百分比（≥1μg/mL）、前向散射（≥0.5μg/mL）和溶血率均显著增加。短杆菌肽增强了红细胞表面的ROS活性、[Ca2+]i和神经酰胺丰度。去除细胞外Ca2+后，短杆菌肽对膜联蛋白V结合的刺激作用显著减弱但并未消除。总之，短杆菌肽刺激红细胞细胞膜的磷脂易位，这种作用至少部分归因于氧化应激的诱导、[Ca2+]i的增加以及神经酰胺丰度的上调。尽管[Ca2+]i增加，但短杆菌肽会增加细胞体积并略微降低RDW。
