2002 Fiscal Year Final Research Report Summary
Imaging of the signal molecules mediating secretory responses in salivary gland cells
| Project/Area Number |
12470391
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Functional basic dentistry
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| Research Institution | Health Sciences University of Hokkaido |
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Principal Investigator |
TOJYO Yosuke Health Sciences University of Hokkaido, School of Dentistry, Professor, 歯学部, 教授 (90111731)
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| Co-Investigator(Kenkyū-buntansha) |
MORITA Takao Health Sciences University of Hokkaido, School of Dentistry, Instructor, 歯学部, 助手 (20326549)
NEZU Akihiro Health Sciences University of Hokkaido, School of Dentistry, Instructor, 歯学部, 助手 (00305913)
TANIMURA Akihiko Health Sciences University of Hokkaido, School of Dentistry, Associate Professor, 歯学部, 助教授 (70217149)
ARAKAWA Toshiya Health Sciences University of Hokkaido, School of Dentistry, Assistant Professor, 歯学部, 講師 (40306254)
TAKUMA Taishin Health Sciences University of Hokkaido, School of Dentistry, Professor, 歯学部, 教授 (40095336)
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| Project Period (FY) |
2000 – 2002
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| Keywords | salivary cells / calcium ion / inositol 1,4,5-trisphosphateimaging / imaging / Ca^<2+> wave / C-kinase / GFP / SNARE proteins |
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| Research Abstract |
1. The pattern of Ca^<2+> mobilization in rat perotid acinar cells lacking zymogen granules was visualized using a Ca^<2+> imaging system. The pattern of the Ca^<2+> wave was essentially similar to that in control cells.
2. The immunoblotting data did not provide evidence that IP_3 receptors are present in parotid zymogen granules. In addition, ryanodine receptors were not detected in parotid acinar cells.
3. The Ca^<2+> signaling induced by receptor agonists was observed in rat submandibular gland ductal cells using a Ca^<2+> imaging system. Stimulation with adrenergic and muscarinic agonists resulted in significant increases in ductal [Ca^<2+>]I, but substance P had little or no effect on [Ca^<2+>]i.
4. The ATP-induced oscillatory changes in [Ca^<2+>]I were analyzed in HSY cells using a fluorescence ratio imaging system. The obtained results support the hypothesis that the positive and negative feedback effects of Ca^<2+> itself on IP_3 receptor activity play an important role in the generation of oscillations.
5. We constructed a plasmid vector containing full-length rat type 3 IP3R linked to GFP (GFP-IP3R3) for expression in HSY cells. Fluorescence confocal microscopy showed that the fluorescence of GFP-IP3R3 was distributed to an ER-like reticular network.
6. We expressed PKCα fused to GFP (PKCα-GFP) and visualized its translocation in HSY cells. The results suggest a complex interplay between Ca^<2+>, diacylglycerol, and phosphorylation in the regulation of the translocation of PKCα.
7. We expressed the IP3R membrane domain fused to CFP and YFP in HSY cells and analyzed the intracellular formation of IP_3 in HSY cells based on the fluorescence resonance energy transfer (FRET).
8. SNARE proteins, including VAMP-2, syntaxin-4, and SNAP-23, were expressed as GFP-tagged fusion proteins in living cells. We examined the localization, protein-protein interaction, and intracellular trafficking of the SNARE proteins.
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[Publications] Nezu, A., Tanimura, A., Morita, T., Irie, K., Yajima, T. and Tojyo, Y.: "Evidence that zymogen granules do not function as an intracellular Ca^<2+> store for the generation of the Ca^<2+> signal in rat parotid acinar cells"Biochem. J.. 363. 59-66 (2002)
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「研究成果報告書概要(欧文)」より
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[Publications] Tanimura, A., Nezu, A., Morita, T., Hashimoto, N. and Tojyo, Y.: "Interplay between calcium, diacylglycerol, and phosphorylation in the spatial and temporal regulation of PKC α-GFP"J. Biol. Chem.. 277. 29054-29062 (2002)
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[Publications] Takuma, T., Arakawa, T., Okayama, M., Mizoguchi, I., Tanimura, A. and Tajima, Y.: "Trafficking of green fluorescent protein-tagged SNARE proteins in HSY cells"J. Biochem.. 132. 729-735 (2002)
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[Publications] Tanaka, T., Morita, T., Nezu, A., Tanimura, A., Mizoguchi, I. and Tojyo, Y.: "Thrombin-induced Ca^<2+> mobilization in human gingival fibroblasts is mediated by protease-activated receptor-1 (PAR-1)"Life Sciences. (in press). (2003)
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「研究成果報告書概要(欧文)」より
