Summary of Research Project Results Under the JSPS FY2000
"Research for the future Program"



1.Research Institution Okazaki National Research Institutes
 
2.Research Area Life Science
 
3.Research Field Cellular Signaling
 
4.Term of Project FY1996〜FY2000
 
5.Project Number 96L00301
 
6.Title of Project Calcium Signaling in Secretion

7.Projetct Leader
Name Institution,Department Title of Position
Kasai, Haruo National Institute for Physiological Sciences Professor

8.Core Members

9.Cooperating Researchers

Names Institution,Department Title of Position
Kukita, Fumio National Institute for Physiological Sciences Research Associate
Nemoto, Kazuki National Institute for Physiological Sciences Research Associate
Takahashi, Noriko National Institute for Physiological Sciences Research Associate

10.Summary of Research Results

We have systematically investigated the Ca2+ dependent exocytosis from neuronal, endocrine and exocrine cells in a quantitative manner, using caged Ca2+ compounds, amperometry and 2-photon excitation imaging.
1. The results of neuronal PC12 cell line indicate that the rate of exocytosis are markedly different between the dense-core vesicles (LVs) and the small synaptic vesicles (SVs), even though they are stimulated by the same amplitudes of Ca2+ increases. Removal of neuronal SNAREs resulted in further slowing of SVs in PC12 cells, while the amplitude of exocytosis remained unchanged, suggesting that the neuronal SNAREs controls the rate of exocytosis. Natures of Ca2+ sensors were investigated by using different divalent cations. The results is consistent with the role of synaptotamin in the LV exocytosis, while Ca2+ sensors for SVs likely involve another Ca2+ sensing mechanisms.
2. Exocytosis of insulin vesicles were investigated with amperometry and with 2-photon excitation imaging. Our results indicate the presence of two distinct populations of insulin vesicles, one undergoes exocytosis rapidly upon stimulation by Ca2+ and cAMP, while the other slowly by Ca2+. The two populations of vesicles can account for the classical two phases of insulin exocytoses from the islet of Langerhans under high-glucose stimulation.
3. Exocytosis of exocrine glands is found to require micromolar increases in Ca2+, as in the case of neuronal and endocrine cells, suggesting the involvement of synaptotagmin in the exocrine secretion. We found sequential progresses of exocytosis into the cytosol of acinar cells with physiological agonist stimulation. These results indicate that the plasma membrane factor is necessary for the fusion of LVs, and that the Ca2+ release from internal Ca2+ stores is optimized to maintain such sequential exocytosis.

11.Key Words

(1)Secretion、(2)Calcium、(3)Synapse
(4)Exocrine gland、(5)Pancreatic Beta-cells、(6)Insulin

12.References

[Reference Articles]
Author Title of Article
Kasai, H Two components of exocytosis and endocytosis in phaeochromocytoma cells studied using caged-Ca2+ compounds
Journal Volume Year Pages Concerned
Journal of Physiology 496 1996 53-65

Author Title of Article
Ninomiya, Y. Ca2+-dependent exocytotic pathways in Chinese hamster ovary fibroblast revealed by capacitace measurement and caged-Ca2+ compound
Journal Volume Year Pages Concerned
Journal of Biological Chemistry 271 1996 17751-17754

Author Title of Article
Kasai, H. Neuronal Ca2+ signalling
Journal Volume Year Pages Concerned
Seminar in Neuroscience 8 1996 259

Author Title of Article
Petersen, O. H. P. Intracellular calcium. in Text book of Receptor Pharmacology, ed. J. C. Foreman and T. Johansen
Journal Volume Year Pages Concerned
CRC Press, England   1996 213-226

Author Title of Article
Sjoedin, L. Cytoplasmic Ca2+ gradients evoked by acetylcholine and peptides in pancreatic acinar cells of the guinea pig
Journal Volume Year Pages Concerned
Pflugers Archiv 433 1997 397-402

Author Title of Article
Ito, K. Micromolar and submicromolar Ca2+ spikes regulating distinct cellular functions in pancreatic acinar cells
Journal Volume Year Pages Concerned
EMBO Journal 16 1997 242-251

Author Title of Article
Ninomiya, Y. Kinetic diversity in the fusion of exocytotic vesicles
Journal Volume Year Pages Concerned
EMBO Journal 16 1997 929-934

Author Title of Article
Takahashi, N. Multiple exocytotic pathways in pancreatic β-cells
Journal Volume Year Pages Concerned
Journal of Cell Biology 138 1997 55-64

Author Title of Article
Kasai, H. Ca2+ dependent exocytosis in endocrine, exocrine and non-secretory cells
Journal Volume Year Pages Concerned
Korean Journal of Physiology and Pharmacology 2 1998 1-7

Author Title of Article
Kasai, H. Comparative biology of Ca2+-dependent exocytosis: implications of kinetic diversity for secretory function
Journal Volume Year Pages Concerned
Trends in Neurosciences 22 1999 88-93

Author Title of Article
Takahashi, N. Post-priming actions of ATP on Ca2+-dependent exocytosis in pancreatic beta cells
Journal Volume Year Pages Concerned
Proceedings of the National Academy of Sciences of the U. S. A. 96 1999 760-765

Author Title of Article
Kasai, H. Multiple components of exocytosis and Ca2+ requirements.
Journal Volume Year Pages Concerned
Philosophical Transactions of the Royal Society of London- Series B:Biological Sciences 354 1999 331-335

Author Title of Article
Kasai, H. Mutiple and diverse forms of regulated exocytosis revealed in wild-type and defective PC12 cells
Journal Volume Year Pages Concerned
Proceedings of the National Academy of Sciences of the U. S. A. 96 1999 945-949

Author Title of Article
Eto, K. Role of NADH shuttle system in glucose-induced activation of mitochondrial metabolism and insulin secretion
Journal Volume Year Pages Concerned
Science 283 1999 981-985

Author Title of Article
Ito, K. Kinetic control of multiple forms of Ca2+ spikes by inositol trisphosphate in pancreatic acinar cells
Journal Volume Year Pages Concerned
Journal of Cell Bioogy 146 1999 405-414

Author Title of Article
Eto, K. NADH shuttle system regulates KATP channel-dependent pathway and steps distal to cytosolic Ca2+ concentration elevation in glucose-induced insulin secretion
Journal Volume Year Pages Concerned
Journal of Biological Chemistry 274(36) 1999 25386-25393

Author Title of Article
Maeda, H. Supralinear Ca2+ signaling by cooperative and mobile Ca2+ buffering in Purkinje neurons
Journal Volume Year Pages Concerned
Neuron 24 1999 989-1002

Author Title of Article
Tsubamoto Y. Hexamminecobalt (III) Chloride Inhibits Glucose-Induced Insulin Secretion at the Exocytotic Process
Journal Volume Year Pages Concerned
Journal of Biological Chemistry 276, 2001 2979-2985

Author Title of Article
Nemoto, T. Sequential-replenishment mechanism of exocytosis in pancreatic acini.
Journal Volume Year Pages Concerned
Nature Cell Biology 3 2001 253-258

Author Title of Article
Kishimoto, T. Ion selectivities of the Ca2+ sensors for exocytosis in rat phaeochromocytoma cells
Journal Volume Year Pages Concerned
Journal of Physiology(Lonon) in press 2001  


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