TY - JOUR
T1 - Two cAMP-dependent pathways differentially regulate exocytosis of large dense-core and small vesicles in mouse β-cells
AU - Hatakeyama, Hiroyasu
AU - Takahashi, Noriko
AU - Kishimoto, Takuya
AU - Nemoto, Tomomi
AU - Kasai, Haruo
PY - 2007/8/1
Y1 - 2007/8/1
N2 - 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.
AB - 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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U2 - 10.1113/jphysiol.2007.135228
DO - 10.1113/jphysiol.2007.135228
M3 - Article
C2 - 17510178
AN - SCOPUS:34547114234
VL - 582
SP - 1087
EP - 1098
JO - Journal of Physiology
JF - Journal of Physiology
SN - 0022-3751
IS - 3
ER -