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The mechanism of intracellular Ca signaling in hormone action/secretion and its physiolojical relevance

细胞内 Ca 信号传导在激素作用/分泌中的机制及其生理相关性

基本信息

批准号：
08671138
负责人：
TANAKA Yuji
金额：
$ 1.34万
依托单位：
University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

I have already clarified that calmodulin-dependent, cADPR-triggered CICR is essential for Ca^<2+> oscillation during fertilization of sea urchin eggs. In this project, I attempted to clarify whether and how a similar mechanism is involved in mammalian hormone-secreting cells, and to clarify its relevance in several disease mechanims.(1)In the first step, I have further analyzed the relationship among cADPR,calmodulin and Ca, and have clarified that Ca alone could trigger cADPR-related CICR,Which further supports the working hypothesis.(2)In a preliminary experiment, W7, a potent calmodulin inhibitor innibited Ca signal in sea urchin egg and certain rat cells. Based on this observations, I have used more specific calmodulin inhibitors(specific inhibitory peptides). Unexpectedly, however, those specific inhibitors did not inhibit Ca^<2+> signaling of mammalian hormone-secreting cells. The reason for the failure is not yet clear, but it is likey that calmodulin cADPR related CICR is not i … More nvolved in mammalian hormone secretion and that aforementioned effect of W7 might have been, at least in part, non-specific.(3)Because I have already observed that mammalian microsome does not release Ca^<2+> in resnponse to cADPR but addition of sea urchin egg extract could resume its response, efforts have been made to purify this potentiating fraction. However, thes activity has also been lost after repeated experiments. Because differences in species used could have explained this discrepancy, I obtained the same species of sea urchin from Harvard University, where I got the initial obvservation, and have tried to reconstruct the initial observation. At least at present, however, the potentiating fraction cannlt be found in sea urchin egg extract again. The initial extract might have contained some contamination of microsome fraction.One of the goal of this project was supposed to be a molecular cloning of mammalian cADPR-responsive channel. For this purpose, human cerebellum cDNA was used as a starting material and expression colning technique was originally thought to be employed. Now it is forced to be stopped because of reasons mentioned above. Less

我已经阐明，钙调素依赖性、cADPR触发的CICR对于海胆卵受精过程中的Ca^2+振荡至关重要。在这个项目中，我试图阐明是否以及如何在哺乳动物的前列腺分泌细胞中参与类似的机制，并阐明其在几种疾病机制中的相关性。(1)In第一步，进一步分析了cADPR、钙调素和Ca之间的关系，阐明了单独Ca可以触发cADPR相关的CICR，进一步支持了工作假设。(2)In一个初步的实验，W7，一种有效的钙调蛋白抑制剂，抑制了海胆卵和某些大鼠细胞中的钙信号。基于这一观察，我使用了更特异的钙调素抑制剂（特异性抑制肽）。然而，出乎意料的是，这些特异性抑制剂并没有抑制哺乳动物细胞分泌钙离子的细胞信号。失败的原因尚不清楚，但可能与钙调素cADPR相关的CICR不存在。 ...更多信息参与哺乳动物激素分泌，且W7上述作用可能至少部分是非特异性的。（3）由于我已经观察到哺乳动物微粒体对cADPR不释放Ca^<2+>，但加入海胆卵提取物可以恢复其反应，因此已经努力纯化这种增强作用的组分。然而，经过反复实验，这些活性也已经丧失。由于所用物种的差异可以解释这种差异，我从哈佛大学获得了相同物种的海胆，在那里我得到了最初的观察，并试图重建最初的观察。然而，至少在目前，在海胆卵提取物中不能再次发现增强级分。本项目的目标之一是克隆哺乳动物cADPR反应通道的分子。为此目的，使用人小脑cDNA作为起始材料，并且最初认为采用表达克隆技术。现在由于上述原因，它被迫停止。少