Two cAMP-dependent pathways differentially regulate exocytosis of large dense-core and small vesicles in mouse β-cells

Authors

  • Hiroyasu Hatakeyama,

    1. 1Division of Biophysics, Center for Disease Biology and Integrative Medicine, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo, 113-0033 Japan
    2. 2Center for NanoBio Integration, University of Tokyo, Bunkyo-ku, Tokyo, 113-0033 Japan
    3. 3Department of Cell Physiology, National Institute for Physiological Sciences, and Graduate University of Advanced Studies (SOKENDAI), Myodaiji, Okazaki, 444-8585 Japan
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  • Noriko Takahashi,

    1. 1Division of Biophysics, Center for Disease Biology and Integrative Medicine, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo, 113-0033 Japan
    2. 2Center for NanoBio Integration, University of Tokyo, Bunkyo-ku, Tokyo, 113-0033 Japan
    3. 3Department of Cell Physiology, National Institute for Physiological Sciences, and Graduate University of Advanced Studies (SOKENDAI), Myodaiji, Okazaki, 444-8585 Japan
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  • Takuya Kishimoto,

    1. 1Division of Biophysics, Center for Disease Biology and Integrative Medicine, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo, 113-0033 Japan
    2. 2Center for NanoBio Integration, University of Tokyo, Bunkyo-ku, Tokyo, 113-0033 Japan
    3. 3Department of Cell Physiology, National Institute for Physiological Sciences, and Graduate University of Advanced Studies (SOKENDAI), Myodaiji, Okazaki, 444-8585 Japan
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  • Tomomi Nemoto,

    1. 3Department of Cell Physiology, National Institute for Physiological Sciences, and Graduate University of Advanced Studies (SOKENDAI), Myodaiji, Okazaki, 444-8585 Japan
    2. 4Section of Information Processing, Center for Brain Experiment, National Institute for Physiological Sciences, and Graduate University of Advanced Studies (SOKENDAI), Myodaiji, Okazaki, 444-8585 Japan
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  • Haruo Kasai

    1. 1Division of Biophysics, Center for Disease Biology and Integrative Medicine, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo, 113-0033 Japan
    2. 2Center for NanoBio Integration, University of Tokyo, Bunkyo-ku, Tokyo, 113-0033 Japan
    3. 3Department of Cell Physiology, National Institute for Physiological Sciences, and Graduate University of Advanced Studies (SOKENDAI), Myodaiji, Okazaki, 444-8585 Japan
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Corresponding author H. Kasai: Division of Biophysics, Center for Disease Biology and Integrative Medicine, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo, 113-0033 Japan. Email: hkasai@m.u-tokyo.ac.jp

Abstract

It has been reported that cAMP regulates Ca2+-dependent exocytosis via protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac) in neurons and secretory cells. It has, however, never been clarified how regulation of Ca2+-dependent exocytosis by cAMP differs depending on the involvement of PKA and Epac, and depending on two types of secretory vesicles, large dense-core vesicles (LVs) and small vesicles (SVs). In this study, we have directly visualized Ca2+-dependent exocytosis of both LVs and SVs with two-photon imaging in mouse pancreatic β-cells. We found that marked exocytosis of SVs occurred with a time constant of 0.3 s, more than three times as fast as LV exocytosis, on stimulation by photolysis of a caged-Ca2+ compound. The diameter of SVs was identified as ∼80 nm with two-photon imaging, which was confirmed by electron-microscopic investigation with photoconversion of diaminobenzidine. Calcium-dependent exocytosis of SVs was potentiated by the cAMP-elevating agent forskolin, and the potentiating effect was unaffected by antagonists of PKA and was mimicked by the Epac-selective agonist 8-(4-chlorophenylthio)-2′-O-methyl cAMP, unlike that on LVs. Moreover, high-glucose stimulation induced massive exocytosis of SVs in addition to LVs, and photolysis of caged cAMP during glucose stimulation caused potentiation of exocytosis with little delay for SVs but with a latency of 5 s for LVs. Thus, Epac and PKA selectively regulate exocytosis of SVs and LVs, respectively, in β-cells, and Epac can regulate exocytosis more rapidly than PKA.

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