Genewsky Andreas, Jost Ingmar, Busch Catharina, Huber Christian, Stindl Julia, Skerka Christine, Zipfel Peter F, Rohrer Bärbel, Strauß Olaf
Max-Planck Institute of Psychiatry, Munich, Germany.
Experimental Ophthalmology, Eye Clinic, University Medical Center Regensburg, Regensburg, Germany.
Pflugers Arch. 2015 Oct;467(10):2179-91. doi: 10.1007/s00424-014-1656-2. Epub 2014 Nov 27.
Defective regulation of the alternative pathway of the complement system is believed to contribute to damage of retinal pigment epithelial (RPE) cells in age-related macular degeneration. Thus we investigated the effect of complement activation on the RPE cell membrane by analyzing changes in membrane conductance via patch-clamp techniques and Ca(2+) imaging. Exposure of human ARPE-19 cells to complement-sufficient normal human serum (NHS) (25 %) resulted in a biphasic increase in intracellular free Ca(2+) ([Ca(2+)]i); an initial peak followed by sustained Ca(2+) increase. C5- or C7-depleted sera did not fully reproduce the signal generated by NHS. The initial peak of the Ca(2+) response was reduced by sarcoplasmic Ca(2+)-ATPase inhibitor thapsigargin, L-type channel blockers (R)-(+)-BayK8644 and isradipine, transient-receptor-potential (TRP) channel blocker ruthenium-red and ryanodine receptor blocker dantrolene. The sustained phase was carried by CaV1.3 L-type channels via tyrosine-phosphorylation. Changes in [Ca(2+)]I were accompanied by an abrupt hyperpolarization, resulting from a transient increase in membrane conductance, which was absent under extracellular Ca(2+)- or K(+)-free conditions and blocked by (R)-(+)-BayK8644 or paxilline, a maxiK channel inhibitor. Single-channel recordings confirmed the contribution of maxiK channels. Primary porcine RPE cells responded to NHS in a comparable manner. Pre-incubation with NHS reduced H2O2-induced cell death. In summary, in a concerted manner, C3a, C5a and sC5b-9 increased [Ca(2+)]i by ryanodine-receptor-dependent activation of L-type channels in addition to maxi-K channels and TRP channels absent from any insertion of a lytic pore.
补体系统替代途径的调节缺陷被认为与年龄相关性黄斑变性中视网膜色素上皮（RPE）细胞的损伤有关。因此，我们通过膜片钳技术和Ca(2+)成像分析膜电导的变化，研究了补体激活对RPE细胞膜的影响。将人ARPE - 19细胞暴露于补体充足的正常人血清（NHS）（25%）中，导致细胞内游离Ca(2+)（[Ca(2+)]i）呈双相增加；最初出现一个峰值，随后Ca(2+)持续增加。C5或C7缺失的血清不能完全重现NHS产生的信号。Ca(2+)反应的初始峰值可被肌浆网Ca(2+)-ATP酶抑制剂毒胡萝卜素、L型通道阻滞剂（R）-(+)-BayK8644和异搏定、瞬时受体电位（TRP）通道阻滞剂钌红以及兰尼碱受体阻滞剂丹曲林降低。持续阶段由CaV1.3 L型通道通过酪氨酸磷酸化介导。[Ca(2+)]I的变化伴随着突然的超极化，这是由膜电导的短暂增加引起的，在无细胞外Ca(2+)或K(+)的条件下这种超极化不存在，并被（R）-(+)-BayK8644或大电导钙激活钾通道抑制剂哌克昔林阻断。单通道记录证实了大电导钙激活钾通道的作用。原代猪RPE细胞对NHS的反应方式类似。用NHS预孵育可减少H2O2诱导的细胞死亡。总之，C3a、C5a和sC5b - 9协同作用，除了通过任何溶细胞孔插入所不存在的大电导钾通道和TRP通道外，还通过兰尼碱受体依赖性激活L型通道增加[Ca(2+)]i。
