Norgauer J, Dobos G, Kownatzki E, Dahinden C, Burger R, Kupper R, Gierschik P
Hautklinik Freiburg, Germany.
Eur J Biochem. 1993 Oct 1;217(1):289-94. doi: 10.1111/j.1432-1033.1993.tb18245.x.
The signal pathways of neutrophils following stimulation with the complement fragment C3a (C3a) were studied in neutrophils and compared to the pathways activated by complement fragment C5a (C5a). Analysis of polyphosphoinositol lipid turnover showed that C5a, but not C3a, activated phosphatidylinositol-bisphosphate-3-kinase (PtdInsP2 3-kinase) indicating that different signal pathways are activated by the two anaphylatoxins. To examine whether C3a stimulated Ca2+ transients, cytosolic free Ca2+ levels were analyzed in Fluo-3-labelled neutrophils by flow cytometry. C3a stimulated a fast and concentration-dependent increase of cytosolic free Ca2+. Comparison of the C3a response with that of C5a revealed a more pronounced C5a-triggered Ca2+ rise. Addition of EGTA to the extracellular buffer prior to stimulation did not significantly alter the initial Ca2+ rise at low C5a concentrations, but reduced the time course of the Ca2+ transients at high concentrations. In marked contrast, EGTA completely blocked the Ca2+ response stimulated by C3a in neutrophils labeled with either Indo-1/AM or Fluo-3. Preincubation of neutrophils with pertussis toxin inhibited both C3a- and C5a-stimulated Ca2+ transients, indicating the involvement of guanine-nucleotide-binding proteins (G proteins) in these processes. In order to examine whether the C3a receptor is coupled to G proteins, binding of guanosine 5'-O-(3-[35S]thiotriphosphate) ([35S]GTP[S]) to purified neutrophil plasma membranes was studied. Both C3a and C5a stimulated high-affinity binding of [35S]GTP[S] up to 1.5-fold and 3-fold, respectively. These data suggest that the two anaphylatoxins activate pertussis-toxin-sensitive G proteins, which then trigger different signal transduction pathways. C3a specifically stimulated Ca2+ influx from the extracellular medium, whereas C5a additionally activated the PtdInsP2 3-kinase and stimulated Ca2+ mobilization from intracellular stores.
在中性粒细胞中研究了补体片段C3a（C3a）刺激后中性粒细胞的信号通路，并与补体片段C5a（C5a）激活的信号通路进行了比较。多磷酸肌醇脂质周转分析表明，C5a而非C3a激活了磷脂酰肌醇-二磷酸-3-激酶（PtdInsP2 3-激酶），这表明这两种过敏毒素激活了不同的信号通路。为了检测C3a是否刺激Ca2+瞬变，通过流式细胞术分析了Fluo-3标记的中性粒细胞中的胞质游离Ca2+水平。C3a刺激了胞质游离Ca2+的快速且浓度依赖性增加。将C3a反应与C5a反应进行比较，发现C5a引发的Ca2+升高更为明显。在刺激前向细胞外缓冲液中加入EGTA，在低C5a浓度下并未显著改变初始Ca2+升高，但在高浓度下缩短了Ca2+瞬变的时间进程。与之形成鲜明对比的是，EGTA完全阻断了用Indo-1/AM或Fluo-3标记的中性粒细胞中C3a刺激的Ca2+反应。用百日咳毒素对中性粒细胞进行预孵育可抑制C3a和C5a刺激的Ca2+瞬变，这表明鸟嘌呤核苷酸结合蛋白（G蛋白）参与了这些过程。为了检测C3a受体是否与G蛋白偶联，研究了鸟苷5'-O-(3-[35S]硫代三磷酸)（[35S]GTP[S]）与纯化的中性粒细胞膜的结合。C3a和C5a分别刺激[35S]GTP[S]的高亲和力结合增加至1.5倍和3倍。这些数据表明，这两种过敏毒素激活了对百日咳毒素敏感的G蛋白，然后触发不同的信号转导通路。C3a特异性刺激Ca2+从细胞外介质流入，而C5a还激活了PtdInsP2 3-激酶并刺激Ca2+从细胞内储存库中释放。
