MECHANISMS OF PROTEIN/FLUID SECRETION AND INTRACELLULAR SIGNAL TRANSDUCTION THROUGH THE TIGHT JUNCTION IN SALIVARY GLAND

由于水的标志物很难在形态上被检测到，体液分泌的类细胞途径仍然是一个长期存在的问题。本课题研究唾液的分泌机制，重点是通过紧密连接(TJ)的细胞旁途径的蛋白质分泌和液体分泌的调节途径。本研究从旁细胞途径和跨细胞途径两个方面进行了研究，(1)唾液腺TJ途径的调节机制：1)研究了毒扁豆碱(卡巴胆碱/CCH)和β-肾上腺素能(异丙肾上腺素/isp)刺激大鼠唾液腺TJ结构的结构变化；2)采用快速冷冻深蚀刻冷冻骨折复制法进行分析；(2)腺泡细胞跨细胞途径的机制：I)水通道蛋白在细胞膜和…中的表达和功能分析分泌颗粒膜较多。本项目取得了以下成果。1)在分泌过程中，小管扩张，ZO_1、ZO_2、闭塞和Claudin的荧光定位发生显著变化。细胞间小管的紧密连接结构变得更加收缩、曲折和间歇性。ZO-1的分布范围越来越广，在腺泡细胞的胞浆中也可见到ZO-1。2)在液氦、快速冷冻、深刻蚀复制法的三维分析中，紧密连接的链条也形成了网状结构，由膜内颗粒链和在P面处熔融的脊线组成，在E面处被识别为浅沟和少量膜颗粒。紧密连接的链状颗粒排列得更粗糙，在刺激过程中变得更加散乱和中断。TJ结构可能由亚细胞结构，特别是膜下肌动蛋白细丝网络的持续存在而维持。紧密连接的结构可以通过分泌刺激来改变，从而允许细胞旁运输。3)水通道蛋白-5不仅在腮腺腔膜上有明显的免疫定位，而且在分泌颗粒膜上也有明显的免疫定位。IPR和卡巴考尔刺激后，这种定位改变，分泌颗粒融合到管腔膜上。免疫印迹法检测到大鼠腮腺分泌颗粒的纯化膜组分中有水通道蛋白-5的表达。通过敏感的超薄冰冻切片的免疫金标记法，水通道蛋白-5在分泌颗粒膜上明显实现了免疫定位。综上所述，我们证明在分泌过程中，紧密连接结构和紧密连接相关蛋白的定位发生了显著变化，并导致了旁细胞途径的改变，水通道蛋白5在分泌颗粒膜上的定位可能在跨细胞途径中起到渗透调节的作用。较少

The para-cellular route for fluid secretion was still long standing question, because of the marker for water was difficult to be detected morphologically. This project examining the secretion mechanism of saliva focused on the regulated pathway of protein secretion and fluid secretion of the para-cellular pathway through the tight junction(TJ). In this study, both of the para-cellular pathway and trans-cellular pathway were examined, (1)Regulation mechanisms of para-cellular pathway via the TJ in the salivary gland : i)The structural change on TJ structure caused by stimulation with muscarinic (carbachol/CCh) and β-adrenergic (isoproterenol/ISP) in the isolated perfused rat SMG, ii)Analysis by the quick freezing deep etching freeze fracture replica(FF) method, iii)Analysis by the immuno replica method using quick freezing FF, were examined. (2)Mechanisms of trans-cellular pathway in acinar cell : i)Analysis of expression and function of the aquaporin water channel in cell membrane and … More secretory granule membrane. Followings results were obtained in this project. 1)During the secretion, canaliculi became dilated, and the fluorescence localization of ZO_1,ZO_2,occluding, and claudin changed significantly. The tight junction structure in the intercellular canaliculi became more contracted, meandering, and intermittent. ZO-1 distribution was becoming wider and was also recognized in the cytoplasm of acinar cells. 2)In the three-dimensional analysis by the liquid helium, quick freezing, deep etching replica method, the tight junction strands also formed a meshwork structure, consisting of chains of intramembrane particles and fused ridges at the P-face, and were recognized as shallow furrows with some few membrane particles at the E-face. The tight junction strand particles were arranged more roughly and became more straggled and interrupted during stimulation. The TJ structure may be maintained by the persistence of subcellular structures, especially of the submembranous actin filament network. The structure of tight junction can be changed by secretory stimulation to allow paracellular transport. 3)Immuno-localization of aquapolin-5 was distinctly recognized not only at the luminal membrane of parotid gland but also secretory granules membrane. After IPR and carbacol stimulation, this localization changed with secretary granule fusion to the luminal membrane. In the purified membrane fraction of the secretory granules, which was obtained from rat parotid gland, aquaporin-5 was detected by western blotting. Immuno-localization of aquaporin-5 was clearly achieved on the secretory granules membrane by immuno-gold labeling of sensitive, ultra-thin frozen sections. In conclusion, we demonstrated that, during the secretion, tight junction structure and localization of tight junction associated protein were changed significantly, and caused to alteration of para-cellular pathway, and localization of the aquaporin 5 on the secretory granules membrane, may play role for osmoregulation in trans cellular path way. Less