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Morphological and functional studies of IP3 receptors underneath the plasma membrane in adrenal medullary cells

肾上腺髓质细胞质膜下IP3受体的形态学和功能研究

基本信息

批准号：
15590200
负责人：
INOUE M.
金额：
$ 2.24万
依托单位：
University of Occupational and Environmental Health
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

Rat adrenal medullnry cells mainly contain the IP3 receptor type 2 (IP3R2). Thus, immunocytochemical studies were done to identify in which organelles the IP3R2 is present. We used two different anti-IP3R2 Abs, the mAb KM1083 and a polyclonal Ab made by Chemicon. Materials immunoreactive to the Chemicon Ab were distributed in the vicinity of the nucleus and at the cell periphery, and most of these immunoreactive materials were bound by BODIPY-IP3, IP3 conjugated with a fluorescent moiety. On the other hand, materials immunoreactive to KM1083 were also present in the vicinity of the nucleus and at the cell periphery, and those near the nucleus were bound by BODIPY IP3. However, the KM1083-immunoreactive materials that were located in a band-like fushion along the cell periphery were devoid of BODIPY-IP3 binding, and were stained with an anti-dopamine-3-hydoxylase Ab, but not with an anti-calnexin Ab. These results indicate that Chemicon Ab-immunoreactive materials were present in the endoplasmic reticulum and KM1083-immunoreactive materials were located not only in the endoplasmic reticulum, but also in secretory granules. The Chemicon Ab recognized IP3R2 of about 250 kDa in immunoblots of adrenal medulla homogenates, whereas KM1083 detected not only the 250 kDa protein, but also an about 200 kDa protein. Intracellular mobilization of Ca^<2+> ions in response to muscarinic receptor stimulation was abolished by a prior exposure to thapsigargin, a SERCA pump inhibitor. Moreover, most of BODIPY thapsigargin binding sites agreed with the distribution of materials immunoreactive to the anti-calnexin Ab. Base on these results, we conclude that the IP3R2 is distributed in the endoplasmic reticulum. KM1083 may recognize not only the IP3R2, but also IP3R2-like proteins in secretory granules.

大鼠肾上腺髓质细胞主要含有IP3受体2型(IP3R2)。因此，进行了免疫细胞化学研究，以确定IP3R2存在于哪些细胞器中。我们使用了两种不同的抗IP3R2单抗，即单抗KM1083和一种多克隆抗体。化学粒子抗体的免疫反应物质主要分布在细胞核附近和细胞周围，这些免疫反应物质大多与BODIPY-IP3结合，IP3与荧光部分结合。另一方面，KM1083免疫反应阳性的物质也存在于细胞核附近和细胞周围，靠近细胞核的物质被BODIPY IP3结合。然而，KM1083免疫反应阳性物质沿细胞外围呈带状融合，不与BODIPY-IP3结合，并被抗多巴胺-3-羟基酶抗体染色，但不被抗钙粘蛋白抗体染色。这些结果表明，化学试剂抗体阳性物质存在于内质网中，KM1083阳性物质不仅存在于内质网中，也存在于分泌颗粒中。化学试剂盒在肾上腺髓质匀浆免疫印迹中识别约250 kDa的IP3R2，而KM1083不仅可识别约250 kDa的IP3R2蛋白，还可识别约200 kDa的蛋白。预先暴露于SERCA泵抑制剂thapsigargin后，细胞内Ca~(2+)~(2+)和Gt~(2+)离子对M受体刺激的反应被取消。此外，大多数BODIPY thapsigarin结合位点的分布与抗Calnexin Ab免疫反应物质的分布一致。根据这些结果，我们推测IP3R2分布在内质网中。KM1083可能不仅识别IP3R2，而且可能识别分泌颗粒中的IP3R2样蛋白。