| Budget Amount *help |
¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 1996: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1995: ¥800,000 (Direct Cost: ¥800,000)
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| Research Abstract |
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
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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
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