Molecular mechanisms of salivary exocytosis-endocytosis coupling

本研究旨在通过形态学、功能学和生物化学的方法阐明唾液腺胞吐-内吞耦合的分子机制。今年，我们对13 -半乳糖苷酶敲除小鼠的全腺体灌注系统、钙信号和腮腺进行了分析。1)全腺灌注：用异丙肾上腺素（β-激动剂）或恰巴哥醇（毒蕈碱激动剂）动脉灌注离体大鼠整个腮腺，并依次收集分泌唾液和组织；β刺激引起胞吐，随后融合颗粒膜被逐一去除。毒蕈碱刺激抑制了这一去除过程。由于毒蕈碱刺激引起液体分泌，而β-刺激则没有，提示膜恢复过程在液体分泌调节中起重要作用。2)钙信号：活腮腺的高速共聚焦显微镜显示腺泡的同步反应和导管的非同步反应。腺泡细胞间可见间隙连接，而导管细胞间未见间隙连接。腺泡可能起着syncium的作用。3) β-半乳糖苷酶敲除小鼠：β-半乳糖苷酶参与溶酶体膜组分的降解。缺乏这种酶的小鼠腮腺腺泡细胞在细胞质中显示空泡轮廓。电镜观察显示，内质网区出现液泡。出乎意料的是，这种酶被认为在生物合成过程中发挥作用，而不是降解。

This research project is aimed to clarify the molecular mechanisms involved in exocytosis-endocytosis coupling in salivary glands by morphological/functional/biochemical approaches. This year, analyses were performed on whole gland perfusion system, calcium signaling and parotid glands in l3-galactosidase knock-out mice.1) Whole gland perfusion: Isolated whole parotid glands of rats were perfused arterially with isoproterenol (β-agonist) or carbachol (muscarinic agonist), and secreted saliva and tissues were collected sequentially; β-stimulation evoked exocytosis following which the fused granule membranes were removed one by one. Muscarinic stimulation inhibited this removal process. Since muscarinic stimulation causes fluid secretion but β-stimulation does not, it is suggested that membrane retrieval process plays significant roles in regulation of fluid secretion.2) Calcium signaling: High speed cofocal microscopy of living parotid glands showed the synchronized response in acini and the non-synchronized response in ducts. Gap junction was detected between acinar cells but not duct cells. Acini are likely to function as a syncitium.3) β-galactosidase knock-out mice: β-galactosidase is involved in the lysosomal degradation of membrane components. Parotid acinar cells in mice deficient in this enzyme revealed vacuolar profiles in the cytoplasm. Electron microscopic observation demonstrated that the vacuoles appeared in the RER region. Unexpectedly, this enzyme is suggested to play roles in the biosynthetic processes rather than the degradation.