Analysis of exocytosis with adenovirus coexpression vector in salivary gland cells

• Project/Area Number: 15591973
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Functional basic dentistry
• Research Institution: Health Sciences University of Hokkaido
• Principal Investigator: TAKUMA Taishin Health Sciences University of Hokkaido, School of Dentistry, Professor, 歯学部, 教授 (40095336)
• Co-Investigator(Kenkyū-buntansha): ARAKAWA Toshiya Health Sciences University of Hokkaido, School of Dentistry, Assistant Professor, 歯学部, 講師 (40306254)
• Project Period (FY): 2003 – 2004
• Project Status: Completed (Fiscal Year 2004)
• Budget Amount: ¥3,500,000 (Direct Cost: ¥3,500,000); Fiscal Year 2004: ¥1,100,000 (Direct Cost: ¥1,100,000); Fiscal Year 2003: ¥2,400,000 (Direct Cost: ¥2,400,000)
• Keywords: SNARE hypothesis / VAMP-2 / VAMP-7 / human growth hormone / GFP / VAMP-8 / PC12 / exocytosis

Research Abstract

Various combinations of SNARE proteins play an essential role in diverse intracellular membrane fusion processes, including exocytosis. Tetanus neurotoxin-insensitive VAMP (TI-VAMPNAMP-7) has been reported to function in some types of regulated and constitutive exocytosis, although VAMP-7 is also supposed to act in the vesicle trafficking from the late endosomes to lysosomes. In addition, the role of VAMP-2 in constitutive exocytosis remains to be elucidated. Here we evaluated the role of VAMP-2 and VAMP-7 in the exocytic pathways of PC12 cells and HSY cells by using human growth hormone (hGH) tagged (hGH-GFP) or not with C-terminal GFP for visualization and measurement of exocytosis. Exocytosis of hGH and hGH-GFP from PC12 cells, but not from HSY cells, was stimulated by 1 itM ionomycin, indicating that the exocytosis from PC12 and HSY cells was regulated and constitutive, respectively. In PC12 cells hGH-GFP was mainly transported to the tips of neural processes and was colocalized with chromogranin A, a marker of large dense-score granules. The granule containing hGH well overlapped with VAMP-2-GFP, but did so scarcely with VAMP-7-GFP. On the other hand, cytoplasmic vesicles containing hGH clearly overlapped with VAMP-7-GFP, but not with VAMP-2-GFP, in HSY cells. However, when the vesicle transport from the trans Golgi network to the plasma membrane was arrested by incubation at 20 ーC for 2 h and then released by warming up to 37 ーC, VAMP-2-GFP and hGH were clearly colocalized together in small cytoplasmic vesicles. Neither VAMP-7-GFP nor hGH-GFP was colocalized with LAMP-1, a marker of the late endosome and lysosome, in HSY cells. These results suggest that 1) VAMP-2 can be a major v-SNARE in both regulated and constitutive exocytosis ; and 2) VAMP-7 is involved in the constitutive exocytosis from HSY cells by acting as a slow v-SNARE.