Wesseling Mauro C, Wagner-Britz Lisa, Nguyen Duc Bach, Asanidze Salome, Mutua Judy, Mohamed Nagla, Hanf Benjamin, Ghashghaeinia Mehrdad, Kaestner Lars, Bernhardt Ingolf
Laboratory of Biophysics, Faculty of Natural and Technical Sciences III, Saarland University, Saarbrücken, Germany.
Cell Physiol Biochem. 2016;39(5):1941-1954. doi: 10.1159/000447891. Epub 2016 Oct 24.
BACKGROUND/AIMS: In previous publications we were able to demonstrate the exposure of phosphatidylserine (PS) in the outer membrane leaflet after activation of red blood cells (RBCs) by lysophosphatidic acid (LPA), phorbol-12 myristate-13acetate (PMA), or 4-bromo-A23187 (A23187). It has been concluded that three different mechanisms are responsible for the PS exposure in human RBCs: (i) Ca2+-stimulated scramblase activation (and flippase inhibition) by A23187, LPA, and PMA; (ii) PKCα activation by LPA and PMA; and (iii) enhanced lipid flip flop caused by LPA. Further studies aimed to elucidate interconnections between the increased Ca2+ content, scramblase- and PKCα-activation. In addition, the role of the Ca2+-activated K+ channel (Gardos channel) activity in the process of PS exposure needs to be investigated.
The intracellular Ca2+ content and the PS exposure of RBCs have been investigated after treatment with LPA (2.5 µM), PMA (6 µM), or A23187 (2 µM). Fluo-4 and annexin V-FITC has been used to detect intracellular Ca2+ content and PS exposure, respectively. Both parameters (Ca2+ content, PS exposure) were studied using flow cytometry. Inhibitors of the scramblase, the PKCα, and the Gardos channel have been applied.
The percentage of RBCs showing PS exposure after activation with LPA, PMA, or A23187 is significantly reduced after inhibition of the scramblase using the specific inhibitor R5421 as well as after the inhibition of the PKCα using chelerythrine chloride or calphostin C. The inhibitory effect is more pronounced when the scramblase and the PKCα are inhibited simultaneously. Additionally, the inhibition of the Gardos channel using charybdotoxin resulted in a significant reduction of the percentage of RBCs showing PS exposure under all conditions measured. Similar results were obtained when the Gardos channel activity was suppressed by increased extracellular K+ content.
PS exposure is mediated by the Ca2+-dependent scramblase but also by PKCα activated by LPA and PMA in a Ca2+-dependent and a Ca2+-independent manner. Furthermore, we hypothesize that a hyperpolarisation of RBCs caused by the opening of the Gardos channel is essential for the scramblase activity as well as for a fraction of the LPA-induced Ca2+ entry.
背景/目的：在之前的出版物中，我们能够证明溶血磷脂酸（LPA）、佛波醇-12-肉豆蔻酸酯-13-乙酸酯（PMA）或4-溴-A23187（A23187）激活红细胞（RBC）后，磷脂酰丝氨酸（PS）在外膜小叶中的暴露情况。已经得出结论，三种不同的机制导致人红细胞中PS的暴露：（i）A23187、LPA和PMA通过Ca2+刺激的翻转酶激活（和翻转酶抑制）；（ii）LPA和PMA激活蛋白激酶Cα（PKCα）；（iii）LPA引起的脂质翻转增强。进一步的研究旨在阐明Ca2+含量增加、翻转酶和PKCα激活之间的相互联系。此外，还需要研究Ca2+激活的钾通道（加尔多斯通道）活性在PS暴露过程中的作用。
在用LPA（2.5 μM）、PMA（6 μM）或A23187（2 μM）处理后，研究了红细胞的细胞内Ca2+含量和PS暴露情况。分别使用Fluo-4和膜联蛋白V-异硫氰酸荧光素（annexin V-FITC）检测细胞内Ca2+含量和PS暴露情况。使用流式细胞术研究了这两个参数（Ca2+含量、PS暴露）。应用了翻转酶、PKCα和加尔多斯通道的抑制剂。
使用特异性抑制剂R5421抑制翻转酶后，以及使用氯化白屈菜红碱或钙泊三醇抑制PKCα后，用LPA、PMA或A23187激活后显示PS暴露的红细胞百分比显著降低。当同时抑制翻转酶和PKCα时，抑制作用更为明显。此外，使用蝎毒素抑制加尔多斯通道导致在所有测量条件下显示PS暴露的红细胞百分比显著降低。当通过增加细胞外K+含量抑制加尔多斯通道活性时，也获得了类似的结果。
PS暴露由Ca2+依赖性翻转酶介导，但也由LPA和PMA以Ca2+依赖性和Ca2+非依赖性方式激活的PKCα介导。此外，我们假设加尔多斯通道开放引起的红细胞超极化对于翻转酶活性以及部分LPA诱导的Ca2+内流至关重要。
