Dynamic analysis by confocal microscopy of Salivary water secretion

用激光共聚焦扫描显微镜分析活体细胞内唾液腺分泌水分的机制。将组织切片或游离的腺泡注入含有各种荧光探针的培养液，并加入氨基甲胆碱刺激液体分泌。1)膜动力学：腺泡细胞腔内膜表现为长时间(30min以上)的胞吐-内吞耦合。扫描和透射电子显微镜显示，该体系为非介导性包膜囊泡。2)紧密连接：液相示踪剂DXTRAN-FITC和膜标记物FM1-43显示分泌刺激后管腔内出现高达10K的右旋糖核酸，而FM1-43没有对管腔膜染色，表明在液体分泌过程中，紧密连接打开了它的‘门’，但保持了它的‘栅栏’功能不变。3)IP3受体和钙信号在共聚焦显微镜下的实时钙成像显示，某些导管细胞的顶端胞浆中有钙释放。免疫电子显微镜显示IP3反应部位位于顶端小泡，这被认为是参与钙信号转导的新定义的IP3受体部位。

Secretion mechanism of water by saliary glands was analyzed in living cells by confocal laser scanning microscopy. Tissue slices or dissociated acini were perfused with medium containing various fluorescent probes, and stimulated fluid secretion by adding carbachol. 1) membrane dynamics : The luminal membrane of acinar cells exhibited the long-term (more than 30 min) exocytosis-endocytosis coupling. Scanning and transmission electron microscopy revealed this system to be coated vesicle non-mediated. 2) Tight junction : Fluid-phase tracer dextrans-FITC and membrane marker FM1-43 revealed that dextrans up to MW 10K appeared in the lumen after the secretory stimulation whereas FM1-43 did not stain the luminal membrane, indicating that during fluid secretion tight junction opens its 'gate' but keeps its 'fence' function to remain unchanged. 3) IP3 receptors and Ca signaling Real-time Ca imaging by confocal microscopy exhibited Ca release from the apical cytoplasm of certain duct cells. Immunoelectron microscopy showed the IP3-reactive sites to be localized at the apical vesicles, which are considered newly defined IP3-receptor sites involved in Ca signaling.