| Budget Amount *help |
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2005: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2004: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Research Abstract |
Amylase secretion is induced by accumulation of intracellular cAMP. VAMP2 was shown to be localized at secretory granules (SGs) and essential for cAMP-dependent exocytosis in parotid acinar cells. However, molecular studies about exocrine cells has been insufficient becuase exocrine acinar cells are difficult to maintain their functions in cell lines or in primary cultures. We attempted to establish a system for primary culture and transfection of exogenous genes. Most of the cultured cells have SGs that contain amylase, suggesting that they are derived from acinar cells. The cultured cells retained granules and the ability to release amylase in response to b-adrenergic and cholinergic agonists even at 48 h after dispersion. The cells also has an ability to generate new SGs. Therefore, the culture system can be used for molecular study of generation of SG and signal-dependent exocytosis. To study biogenesis of SG, SGs of parotid gland were separated into low-, medium-, and high-density granule fractions by Percoll-density gradient centrifugation, since it was proposed that the density corresponds to the degree of maturation. The observation with electron microscopy showed that granules in the three fractions were very similar. The average diameter of high-density granules were a little but significantly larger than that of low-density granules. Although the three fractions contained amylase, suggesting that they are all SGs, distribution of membrane proteins were markedly different. Syntaxin6 and VAMP4 were localized in the low-density granule fraction, while VAMP2 was concentrated in the high-density granule fraction. These results suggest that the medium-density granules are intermediates from low- to high-density granules, and that the membrane components of SGs dynamically change by budding and fusion during maturation.
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