Coupling mechanisms between fluid secretion and exocytosis

探讨体液分泌与胞吐作用的耦合机制。以大涎腺为模型进行生理和形态观察，产生不同浓度蛋白质的肉眼分泌物。A.分泌物时间进程：分别以腮腺和颌下腺作为浆液性和浆液性分泌物模型。淀粉酶和粘蛋白的分泌时间进程相似，单一毒碱刺激引起蛋白质分泌爆发，随后持续分泌水平较低，超负荷β-肾上腺素能刺激增加蛋白质分泌，然后分泌水平较高。体液分泌主要由M胆碱能刺激引起，少量由β-肾上腺素能刺激引起。在弱毒鼠碱刺激时，β-肾上腺素能刺激可增强体液分泌。这表明，cAMP对转运蛋白的上调可能受到代谢能量供应和胞内过量钙离子的限制。B.M.…更多的形态变化：用电子显微镜观察整个腮腺在分泌时间过程中的形态变化。单次卡巴胆碱刺激可引起细胞间小管(IC)的一过性增大。卡巴胆碱和异丙肾上腺素联合刺激可引起ICs强烈的胞吐作用，a)减少微绒毛数量，b)增大RER，激活Ca~(2+)代谢，c)增加线粒体电子密度，与氧消耗增加有关。激光共聚焦显微镜显示细胞旁分泌途径受分泌刺激控制，分泌初期以跨细胞分泌为主。C.胞吐的分子机制：在腮腺分泌颗粒膜上发现VAMP-2，Synaxin4和SNAP23位于发光膜上。而Synaxin 1A定位于舌腺的腺泡细胞。D.分泌分子的动力学：通过毒鼠碱刺激分泌过程中不同大小的标记葡聚糖的唾液/灌流液的比率来估计跨越细胞旁途径的分子过滤器的大小，如5A。在绵羊的口腔腺体中，准确地检测了液体分泌的阴离子依赖性。因此，该项目揭示了上述新的发现，表明细胞内信号的结合使液体分泌和胞吐在不同方面耦合。较少

To study a coupling mechanism between fluid secretion and exocytosis. the major salivary glands were examined physiologically and morphologically as a model to produce a macroscopic secretion with various concentration of protein.A.Secretory time course : The parotid and submandibular glands were used as models of serous and seromucous secretion, respectively. The secretory time course of amylase and mucin was similar, a single muscarinic stimulation induced an initial burst of protein secretion followed by low sustained level of secretion, Overloading of β-adrenergic stimulation increased the protein secretion followed by high secretion level. Fluid secretion was induced by mainly muscarinic stimulation, but a minimal by β-adrenergic stimulation. During weak muscarinic stimulation, the fluid secretion was potentiated by β-adrenergic stimulation. This suggests that the upregulation of transporters by cyclic AMP could be limited by metabolic energy supply and excess cytosolic Ca^<2+>B.M … More orphology : Morphological change of the perfused whole parotid gland was examined by electron microscope along secretory time course. A single carbachol stimulation caused an initial transient enlargement of intercellular canalliculi (IC). Combined stimulation of carbachol and isoproterenol induced a vigorous exocytosis on IC.Either stimulation a) reduced the number of microvilli, b) enlarge the rER.suggesting activation of Ca^<2+> metabolism, and c) increased electron density of mitochondria, related to the increase of oxygen consumption. Confocal laser microscope revealed that the paracellular route of secretion is controlled by secretory stimulation, and that the transcellular fluid secretion is dominant at initial secretory phase.C.Molecular machinary for exocytosis : In the parotid gland VAMP-2 was identified on the secretory granule membrane, syntaxin4 and SNAP23 were located on the luminar membrane. Whereas, syntaxin 1A was localized at acinar cells of the lingual glands. This suggests that there is some differnce in exocytotic mechanism between mucinous and serous secretion.D.Dynamics of secretory molecules : The size of molecular filter across the paracellular route was estimated from the saliva/perfusate ration of various size of labelled dextran during secretion by muscarinic stimulation, as 5A.Anionic dependency of fluid secretion was examined precisely in the buccal gland of sheep.Thus the project revealed above new findings to show that combination of intracellular signals enables coupling between fluid secretion and exocytosis at various aspects. Less