title: Two cAMP-dependent pathways differentially regulate exocytosis of large dense-core and small vesicles in mouse beta-cells.
abstract: It has been reported that cAMP regulates Ca(2+)-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 Ca(2+)-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 Ca(2+)-dependent exocytosis of both LVs and SVs with two-photon imaging in mouse pancreatic beta-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-Ca(2+) compound. The diameter of SVs was identified as approximately 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 beta-cells, and Epac can regulate exocytosis more rapidly than PKA.
authors.affiliation: Division of Biophysics, Center for Disease Biology and Integrative Medicine, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo, 113-0033 Japan.
authors.forename: Hiroyasu
authors.initials: H
authors.lastname: Hatakeyama
authors.forename: Noriko
authors.initials: N
authors.lastname: Takahashi
authors.forename: Takuya
authors.initials: T
authors.lastname: Kishimoto
authors.forename: Tomomi
authors.initials: T
authors.lastname: Nemoto
authors.forename: Haruo
authors.initials: H
authors.lastname: Kasai
mesh_terms: D000818:Animals; D002118:Calcium / Q000502:physiology; D002460:Cell Line; D000242:Cyclic AMP / Q000502:physiology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D022162:Cytoplasmic Vesicles / Q000502:physiology; D005089:Exocytosis / Q000502:physiology*; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000502:physiology* / Q000648:ultrastructure; D007700:Kinetics; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D008854:Microscopy, Electron
chemical_list: D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D002118:Calcium