Mechanism of the Release of Acetylcholine from Presynaptic Nerve Terminals Isolated from Electric Organ of Japanese Electric Ray

日本电射线电器官突触前神经末梢释放乙酰胆碱的机制

基本信息

批准号：
04454526
负责人：
KIRINO Yutaka
金额：
$ 4.35万
依托单位：
The University of Tokyo (1993)Kyushu University (1992)
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

项目摘要

1. Action and binding of various Ca channel blockers were examined on the calcium channels triggering the release of acetylcholine (ACh) in the synaptosomes prepared form the electric organ of Japanese electric ray, Narke japonica. The data obtained suggests that the Ca channels subserving the ACh release are mainly of N- and P-types and the L-type also has a small contribution. 2. A monoclonal antibody, MCC-1, that recognizes the alpha _2 delta subunit of rabbit skeletal muscle, inhibited partially the ACh release. 3. Immunoaffinity chromatography using MCC-1 was employed to solubilized plasma membrane of the synaptosomes in order to purify proteins with affinity to MCC-1. The SDS-PAGE analysis and Western blotting of the purified fraction identified a 170 kDa polypeptide as an MCC-1 binding protein. 4. The purified fraction also contained syntaxin, which is known to be an essential protein for the membrane fusion. 5. Incorporation of the synaptosomes into a planar lipid membrane revealed the presence of a K channel but failed to show the presence of Ca channels. 6. A novel radioactive probes were developed to measure phospholipid translocation across the biomembranes. With this probe, it was found that synaptosomal plasma membrane contains a phoshatidylserine-specific translocase. On the other hand, no specific translocase was found in synaptic vesicles. 7. A simultaneous measurement of the degranulation and intracellular Ca change in rat peritoneal mast cells implicated that intracellular Ca rise is a prerequisite for degranulation. 8. Membranes of the secretory granules of mast cells contain a cation-channel which may be responsible for the formation of so-called fusion pore.

1.在由日本电鳐Narke japonica的电器官制备的突触体中，检查了各种Ca通道阻滞剂对触发乙酰胆碱（ACh）释放的钙通道的作用和结合。获得的数据表明，促进ACh释放的Ca通道主要是N型和P型，L型也有少量贡献。 2.识别兔骨骼肌α_2δ亚基的单克隆抗体MCC-1，部分抑制ACh释放。 3.采用MCC-1的免疫亲和层析来溶解突触体的质膜，以纯化与MCC-1具有亲和力的蛋白质。纯化级分的 SDS-PAGE 分析和蛋白质印迹鉴定出 170 kDa 的多肽为 MCC-1 结合蛋白。 4.纯化的级分还含有突触融合蛋白，已知它是膜融合的必需蛋白质。 5. 将突触体掺入平面脂质膜显示出 K 通道的存在，但未能显示 Ca 通道的存在。 6. 开发了一种新型放射性探针来测量跨生物膜的磷脂易位。利用该探针，发现突触体质膜含有磷脂酰丝氨酸特异性转位酶。另一方面，在突触小泡中没有发现特异性转位酶。 7. 同时测量大鼠腹膜肥大细胞的脱颗粒和细胞内 Ca 变化表明细胞内 Ca 升高是脱颗粒的先决条件。 8. 肥大细胞分泌颗粒的膜含有阳离子通道，可能是所谓融合孔形成的原因。