| Research Abstract |
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
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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
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