Molecular mechanism of stimulus-dependent exocytosis in parotid acinar cells.

淀粉酶的分泌是由细胞内cAMP的积累引起的。VAMP2被证明定位于分泌颗粒（SGs），对腮腺腺泡细胞camp依赖性胞吐至关重要。然而，由于外分泌腺泡细胞在细胞系或原代培养中难以维持其功能，对外分泌细胞的分子研究一直不足。我们试图建立一个原代培养和转染外源基因的系统。大多数培养细胞具有含有淀粉酶的SGs，表明它们来源于腺泡细胞。即使在分散后48小时，培养细胞仍保留颗粒和释放淀粉酶的能力，以响应b-肾上腺素能和胆碱能激动剂。这些细胞也有能力产生新的SGs。因此，该培养体系可用于SG生成和信号依赖性胞吐的分子研究。为了研究SG的生物发生，我们采用percol -density梯度离心分离法将腮腺SGs分为低、中、高密度颗粒，因为密度与成熟程度相对应。电镜观察表明，三组分的颗粒结构非常相似。高密度颗粒的平均直径略大于低密度颗粒，但明显大于低密度颗粒。虽然这三个组分都含有淀粉酶，表明它们都是SGs，但膜蛋白的分布有明显差异。Syntaxin6和VAMP4集中在低密度颗粒段，VAMP2集中在高密度颗粒段。这些结果表明，中密度颗粒是低密度颗粒到高密度颗粒的中间产物，在成熟过程中，SGs膜组分通过出芽和融合发生动态变化。

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.