Lang Philipp A, Kempe Daniela S, Akel Ahmad, Klarl Barbara A, Eisele Kerstin, Podolski Marlies, Hermle Tobias, Niemoeller Olivier M, Attanasio Philipp, Huber Stephan M, Wieder Thomas, Lang Florian, Duranton Christophe
Department of Physiology, University of Tübingen, Tübingen, Germany.
Naunyn Schmiedebergs Arch Pharmacol. 2005 Nov;372(3):228-35. doi: 10.1007/s00210-005-0009-2. Epub 2005 Oct 25.
Osmotic shock, oxidative stress and Cl- removal activate a non-selective Ca2+-permeable cation conductance in human erythrocytes. The entry of Ca2+ leads to activation of a scramblase with subsequent exposure of phosphatidylserine at the cell surface. Phosphatidylserine mediates binding to phosphatidylserine receptors on macrophages which engulf and degrade phosphatidylserine exposing cells. Moreover, phosphatidylserine exposure may lead to adherence of erythrocytes to the vascular wall. In the present study, we explored whether activation of the non-selective cation conductance and subsequent phosphatidylserine exposure might be influenced by catecholamines. Phosphatidylserine exposure has been determined by FITC-annexin V binding while cell volume was estimated from forward scatter in FACS analysis. Removal of Cl- enhanced annexin binding and decreased forward scatter, an effect significantly blunted by the beta agonist isoproterenol (IC50 approx. 1 microM). Fluo-3 fluorescence measurements revealed an increase of cytosolic Ca2+ activity following Cl- removal, an effect again significantly blunted by isoproterenol exposure (10 microM). Whole-cell patch-clamp experiments performed in Cl- free bath solution indeed disclosed a time-dependent inactivation of a non-selective cation conductance following isoproterenol exposure (10 microM). Phenylephrine (IC50<10 microM), dobutamine (IC50 approx. 1 microM) and dopamine (IC50 approx. 3 microM) similarly inhibited the effect of Cl- removal on annexin binding and forward scatter. In conclusion, several catecholamines inhibit the Cl- removal-activated Ca2+ entry into erythrocytes, thus preventing increase of cytosolic Ca2+ activity, subsequent cell shrinkage and activation of erythrocyte scramblase. The catecholamines thus counteract erythrocyte phosphatidylserine exposure and subsequent clearance of erythrocytes from circulating blood.
渗透休克、氧化应激和氯离子去除可激活人红细胞中的一种非选择性钙离子通透阳离子电导。钙离子的进入会激活一种翻转酶，随后磷脂酰丝氨酸在细胞表面暴露。磷脂酰丝氨酸介导与巨噬细胞上的磷脂酰丝氨酸受体结合，巨噬细胞会吞噬并降解暴露磷脂酰丝氨酸的细胞。此外，磷脂酰丝氨酸的暴露可能导致红细胞黏附于血管壁。在本研究中，我们探究了非选择性阳离子电导的激活以及随后磷脂酰丝氨酸的暴露是否会受到儿茶酚胺的影响。通过异硫氰酸荧光素标记的膜联蛋白V结合来测定磷脂酰丝氨酸的暴露，而通过流式细胞术分析中的前向散射来估计细胞体积。去除氯离子会增强膜联蛋白结合并降低前向散射，β激动剂异丙肾上腺素（IC50约为1微摩尔）可显著减弱这种效应。Fluo-3荧光测量显示，去除氯离子后胞质钙离子活性增加，异丙肾上腺素暴露（10微摩尔）同样可显著减弱这种效应。在无氯离子的浴液中进行的全细胞膜片钳实验确实揭示，异丙肾上腺素暴露（10微摩尔）后，非选择性阳离子电导会出现时间依赖性失活。去氧肾上腺素（IC50<10微摩尔）、多巴酚丁胺（IC50约为1微摩尔）和多巴胺（IC50约为3微摩尔）同样抑制了去除氯离子对膜联蛋白结合和前向散射的影响。总之，几种儿茶酚胺抑制了氯离子去除激活的钙离子进入红细胞，从而防止胞质钙离子活性增加、随后的细胞收缩以及红细胞翻转酶的激活。因此，儿茶酚胺可对抗红细胞磷脂酰丝氨酸的暴露以及随后循环血液中红细胞的清除。
