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

淀粉酶分泌是由细胞内cAMP的积累诱导的。VAMP 2定位于分泌颗粒（SGs），是腮腺腺泡细胞cAMP依赖性胞吐的关键。然而，由于外分泌腺泡细胞在细胞系或原代培养中很难维持其功能，因此对外分泌腺泡细胞的分子生物学研究还很不够。我们尝试建立一个原代培养和外源基因转染的系统。大多数培养的细胞具有含有淀粉酶的SG，表明它们来源于腺泡细胞。培养的细胞保留颗粒和释放淀粉酶的能力，以响应β-肾上腺素能和胆碱能激动剂，即使在分散后48小时。这些细胞还具有产生新SG的能力。因此，该培养体系可用于SG产生和信号依赖性胞吐作用的分子研究。为了研究SG的生物发生，通过Percoll密度梯度离心将腮腺的SG分离成低密度、中密度和高密度颗粒组分，因为有人提出密度对应于成熟程度。电镜观察表明，三个组分中的颗粒非常相似。高密度颗粒的平均直径略大于低密度颗粒，但差异显著。虽然这三个组分都含有淀粉酶，表明它们都是SG，但膜蛋白的分布明显不同。Syntaxin 6和VAMP 4定位于低密度颗粒部分，而VAMP 2集中在高密度颗粒部分。这些结果表明，中密度颗粒是从低密度到高密度颗粒的中间体，并且在成熟过程中，SGs的膜组分通过出芽和融合而动态变化。

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.