Receptor-mediated restoration of exocytotic capacity

我们证明了嗜碱性白血病(RBL-2H3)细胞具有防止胞吐作用的机制，直到细胞接收到适当的信号，细胞骨架，特别是肌动蛋白网络的意义在于它防止胞吐作用。在RBL-M3细胞的制备中，我们还证实了Rho在多种受体介导的胞吐作用中发挥重要作用，其中包括高亲和力IgE受体(FcepsilonRI)和M3型M受体(MAChRs)，并提示Rho通过调节肌动蛋白重组而发挥胞吐作用的整合点。这种受体介导的肌动蛋白网络的重组是一个不依赖于钙离子的过程，可能揭示了诱导胞吐的能力。通过将编码人m3mAChRs的基因转染RBL-2H3细胞来研究分泌的脱敏作用。Rbl-M3细胞在无钙培养液中暴露于100um氨基甲胆碱30min可抑制两种分泌诱导的…在不改变m3mAChR或受体与磷脂酶C之间的功能解偶联的情况下，通过随后加入卡巴胆碱和交联剂FcepsilonRI.使RBL-M3细胞脱敏。我们的新发现是，RBL-M3细胞的异源脱敏发生在细胞内钙浓度升高的远端。在这项研究中，RBL-M3细胞的脱敏处理是在没有细胞外钙离子的情况下进行的，这表明异源和同源的分泌脱敏是如前所述的肌动蛋白相关‘启动’信号持续刺激的结果。在无钙的培养液中，RBL-M3细胞与10um氨基甲胆碱孵育产生了膜褶皱，而脱敏细胞在随后加入氨基甲胆碱的情况下没有出现这种褶皱。这些发现表明，卡巴胆碱诱导的异源和同源脱敏分泌物涉及信号通路的负调控，导致膜皱折。总之，上调和下调激动剂激活的肌动蛋白重组导致膜褶皱可能是调节胞吐反应能力的一种方式。较少

We demonstrated that basophilic leukemia (RBL-2H3) cells possess a mechanism for preventing exocytosis until an appropriate signal is received by the cell, and that the significance of the cytoskeleton, particularly the actin network, lies in its preventing exocytosis. We also demonstrated in the preparation of RBL-m3 cells that Rho plays an essential role in the exocytosis mediated by a multiple type of receptor including high affinity IgE receptors (FcepsilonRI) and m3 muscarinic acetylcholine receptors (mAChRs) and suggested that Rho functions as an integration point for exocytosis by regulating actin reorganization. Such a receptor-mediated reorganization of the actin network, a process independent of Ca^<2+>, may reveal the capacity to induce exocytosis.The desensitization of secretion was investigated by transfecting RBL-2H3 cells with cDNA encoding the human m3 mAChRs. Exposure of RBL-m3 cells for 30 min to 100 muM carbachol in Ca^<2+>-free medium inhibited both secretion induce … More d by subsequent addition of carbachol and by cross-linking FcepsilonRI.RBL-m3 cells were desensitized without any modification of m3 mAChR or functional uncoupling between the receptor and phospholipase C.Our novel finding was that the heterologous desensitization of RBL-m3 cells occurred at the steps distal to the rise in concentration of intracellular calcium. Desensitizing treatment of RBL-m3 cells in this study was performed in the absence of extracellular Ca^<2+>, indicating that heterologous as well as homologous desensitization of secretion occurred as a consequence of persistent stimulation of the actin-related 'priming' signaling as described earlier. Incubation of RBL-m3 cells with 10 muM carbachol in Ca^<2+>-free medium developed membrane ruffling, while desensitizad cells failed to develop such ruffling with the subsequent addition of carbachol. These findings indicate that carbachol-induced heterologous as well as homologous desensitization of secretion involves negative regulation of the signaling pathway leading to membrane ruffling.In conclusion, it is likely that up- and down-regulation of agonist-activated actin reorganization leading to membrane ruffling is one way in which the capacity of exocytotic responses is regulated. Less