Coupling mechanisms between fluid secretion and exocytosis

目的：探讨体液分泌与胞吐作用的耦合机制。对大唾液腺进行生理学和形态学检查，作为产生具有不同浓度蛋白质的肉眼可见分泌物的模型。A.分泌时间过程：腮腺和下颌下腺分别用作浆液和血清粘液分泌物的模型。淀粉酶和粘蛋白的分泌时程相似，单次M刺激引起蛋白质分泌的爆发，然后是低水平的持续分泌，超负荷β-肾上腺素能刺激引起蛋白质分泌的增加，然后是高水平的分泌。液体分泌主要由毒蕈碱刺激引起，但由β-肾上腺素能刺激引起的最小。在弱M刺激时，β-肾上腺素能刺激可增强液体分泌。这表明环腺苷酸对转运蛋白的上调可能受到代谢能供应和过量胞浆Ca ^<2 +> B的限制。 关于我们 形态学：电镜观察灌流后腮腺沿着分泌时间的形态学变化。一个单一的卡巴胆碱刺激引起了最初的短暂扩大细胞间小管（IC）。卡巴胆碱和异丙肾上腺素联合刺激可引起IC的强烈胞吐作用。两种刺激均使微绒毛数量减少，rER增大，提示Ca ^2+代谢激活，线粒体电子密度增加，与氧消耗增加有关。共聚焦激光显微镜显示分泌的细胞旁途径受分泌刺激控制，并且在分泌初期跨细胞液体分泌占主导地位。C.胞吐作用的分子机制：在腮腺中，VAMP-2被鉴定为位于分泌颗粒膜上，突触融合蛋白4和SNAP 23位于腔膜上。而syntaxin 1A则定位于舌腺的腺泡细胞。这表明粘液分泌和浆液分泌之间的胞吐机制存在一些差异。D.分泌分子的动力学：通过毒蕈碱刺激分泌过程中不同大小的标记葡聚糖的唾液/灌流液比例估计穿过细胞旁途径的分子过滤器的大小，如5A所示，在绵羊的颊腺中精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，表明细胞内信号使得流体分泌和胞吐作用在各个方面能够耦合。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