Wesseling Mauro C, Wagner-Britz Lisa, Huppert Henri, Hanf Benjamin, Hertz Laura, Nguyen Duc Bach, Bernhardt Ingolf
Cell Physiol Biochem. 2016;38(4):1376-90. doi: 10.1159/000443081. Epub 2016 Mar 24.
BACKGROUND/AIMS: The exposure of phosphatidylserine (PS) on the outer membrane leaflet of red blood cells (RBCs) serves as a signal for suicidal erythrocyte death or eryptosis, which may be of importance for cell clearance from blood circulation. PS externalisation is realised by the scramblase activated by an increase of intracellular Ca2+ content. It has been described in literature that RBCs show an increased intracellular Ca2+ content as well as PS exposure when becoming aged up to 120 days (which is their life span). However, these investigations were carried out after incubation of the RBCs for 48 h. The aim of this study was to investigate this effect after short-time incubation using a variety of stimulating substances for Ca2+ uptake and PS exposure.
We separated RBCs by age in five different fractions by centrifugation using Percoll density gradient. The intracellular Ca2+ content and the PS exposure of RBCs with different age has been investigated after treatment with lysophosphatidic acid (LPA) as well as after activation of protein kinase C (PKC) using phorbol-12 myristate-13 acetate (PMA). For positive control RBCs were treated with 4-bromo-A23187. Measurement techniques included flow cytometry and live cell imaging (fluorescence microscopy).
The percentage of RBCs showing increased Ca2+ content as well as the PS exposure did not change significantly in dependence on cell age after short-time incubation in control experiments (without stimulating substances) or using LPA or PMA. However, we confirm findings reported that Ca2+ content and the PS exposure of RBCs increased after 48 h incubation.
No significant differences of intracellular Ca2+ content and PS exposure can be seen for RBCs of different age in resting state or after stimulation of Ca2+ uptake at short-time incubation.
背景/目的：红细胞（RBC）外膜小叶上磷脂酰丝氨酸（PS）的暴露是红细胞自杀性死亡或红细胞凋亡的信号，这对于从血液循环中清除细胞可能具有重要意义。PS外化是通过细胞内Ca2+含量增加激活的磷脂翻转酶实现的。文献报道，当红细胞老化至120天（其寿命）时，细胞内Ca2+含量以及PS暴露会增加。然而，这些研究是在红细胞孵育48小时后进行的。本研究的目的是在短时间孵育后，使用多种促进Ca2+摄取和PS暴露的刺激物质来研究这种效应。
我们使用Percoll密度梯度离心法将红细胞按年龄分为五个不同组分。在用溶血磷脂酸（LPA）处理后，以及使用佛波醇-12-肉豆蔻酸酯-13-乙酸酯（PMA）激活蛋白激酶C（PKC）后，研究了不同年龄红细胞的细胞内Ca2+含量和PS暴露情况。作为阳性对照，红细胞用4-溴-A23187处理。测量技术包括流式细胞术和活细胞成像（荧光显微镜）。
在对照实验（未使用刺激物质）、使用LPA或PMA进行短时间孵育后，显示Ca2+含量增加以及PS暴露的红细胞百分比并未随细胞年龄而显著变化。然而，我们证实了文献报道的结果，即红细胞在孵育48小时后Ca2+含量和PS暴露增加。
在静息状态下或短时间孵育刺激Ca2+摄取后，不同年龄的红细胞在细胞内Ca2+含量和PS暴露方面未见显著差异。
