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

我已经阐明了钙调素依赖性，cadpr触发的CICR对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)第一步，我进一步分析了cADPR、calmodulin和Ca三者之间的关系，明确了Ca单独可以触发cADPR相关的CICR，进一步支持了工作假设。(2)在初步实验中，强效钙调素抑制剂W7抑制海胆卵和某些大鼠细胞的钙信号。基于这一观察，我使用了更多的特异性钙调素抑制剂（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激素分泌细胞的Ca^<2+>信号。失败的原因尚不清楚，但很可能钙调蛋白cADPR相关的CICR不是它更多地参与哺乳动物激素分泌，上述W7的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+>，但添加海胆卵提取物可以恢复其反应，因此已经努力纯化这一增强部分。然而，经过反复实验，这种活性也已经丧失。因为所用物种的不同可以解释这种差异，所以我从哈佛大学获得了相同种类的海胆，我在那里进行了最初的观察，并试图重建最初的观察结果。然而，至少目前在海胆卵提取物中还不能再发现增强作用的成分。最初的萃取物可能含有一些微粒体的污染。这个项目的目标之一应该是哺乳动物cadpr反应通道的分子克隆。为此，以人小脑cDNA为起始材料，最初认为可以采用表达克隆技术。现在，由于上述原因，它被迫停止。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