Analysis of exocytosis with adenovirus coexpression vector in salivary gland cells

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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.