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

我已经阐明，钙调素依赖的、cADPR触发的CICR对于海胆卵受精过程中的钙振荡是必不可少的。在这个项目中，我试图阐明哺乳动物激素分泌细胞是否以及如何参与类似的机制，并阐明其在几种疾病机制中的相关性。(1)在第一步中，我进一步分析了cADPR、钙调蛋白和钙之间的关系，阐明了钙单独可以触发cADPR相关的CICR，进一步支持了工作假说。(2)在初步实验中，有效的钙调蛋白抑制剂W7抑制了海胆卵和某些大鼠细胞的钙信号。基于这一观察，我使用了更具特异性的钙调蛋白抑制剂(特异性抑制肽)。然而，出乎意料的是，这些特定的抑制剂并没有抑制哺乳动物激素分泌细胞的钙信号。失败的原因尚不清楚，但很可能与钙调蛋白cADPR相关的CICR不是I…W7的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生物体不释放钙对cADPR的反应，但添加海胆卵提取物可以恢复其反应，因此我们努力纯化这种增强部分。然而，经过反复的实验，这些活性也已经丧失。由于所用物种的差异可能解释了这种差异，我从哈佛大学获得了相同物种的海胆，我在那里获得了最初的观察，并试图重建最初的观察。然而，至少目前在海胆卵提取液中还没有发现增强组分。最初的提取物可能含有一些微粒组分的污染。该项目的目标之一被认为是对哺乳动物cADPR反应通道的分子克隆。为此，本研究以人小脑cDNAs为起始材料，采用基因表达克隆技术。现在，由于上述原因，它被迫停止。较少

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