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

我已经阐明了钙调蛋白依赖性、cADPR 触发的 CICR 对于海胆卵受精过程中 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，并阐明其与几种疾病机制的相关性。(1)第一步，我进一步分析了cADPR、钙调蛋白和Ca之间的关系，并阐明了Ca单独可以触发cADPR相关的CICR，这进一步支持了工作假设。(2)在初步实验中，强效钙调蛋白抑制剂W7抑制了海洋中的Ca信号。海胆卵和某些大鼠细胞。根据这一观察，我使用了更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，那些特异性抑制剂不抑制哺乳动物激素分泌细胞的Ca 2 信号传导。失败的原因尚不清楚，但钙调蛋白 cADPR 相关的 CICR 很可能并不参与哺乳动物激素分泌，并且 W7 的上述作用可能至少部分是非特异性的。(3)因为我已经观察到哺乳动物微粒体不会释放 Ca^<2> 来响应 cADPR，但添加海胆卵提取物可以恢复其响应，因此已努力纯化这种增强作用分数。然而，经过反复实验，这些活性也消失了。因为所用物种的差异可以解释这种差异，所以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结果，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找不到这种增效成分。最初的提取物可能含有一些微粒体部分的污染。该项目的目标之一应该是哺乳动物 cADPR 响应通道的分子克隆。为此，使用人小脑 cDNA 作为起始材料，最初认为采用表达克隆技术。现在由于上述原因被迫叫停。较少的

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