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

液体分泌的细胞旁途径仍然是一个长期存在的问题，因为水的标记物难以在形态学上检测到。本项目对唾液分泌机制的研究重点是通过紧密连接（tight junction， TJ）的细胞旁通路中蛋白分泌和液体分泌的调控途径。本研究对细胞旁通路和跨细胞通路进行了研究，(1)唾液腺TJ对细胞旁通路的调控机制：i)观察毒碱（carbachol/CCh）和β-肾上腺素（isoproterenol/ISP）刺激对离体大鼠SMG TJ结构的影响，ii)速冻深刻蚀冻裂复制法（FF）分析，iii)速冻FF免疫复制法分析。(2)腺泡细胞跨细胞通路的机制：1)胞膜及分泌颗粒膜水通道水通道的表达及功能分析。在本项目中获得了以下结果：1)分泌过程中小管扩张，ZO_1、ZO_2、occludin的荧光定位发生明显变化。细胞间小管的紧密连接结构变得更加收缩、弯曲和间断。ZO-1的分布越来越广，并且在腺泡细胞的细胞质中也被发现。2)在液氦快速冷冻深蚀刻复制法三维分析中，紧密结链在p面也形成了由膜内颗粒链和融合脊组成的网状结构，在e面被识别为浅沟，有少量膜颗粒。在刺激作用下，紧密连接链颗粒排列更加粗糙，变得更加杂乱和中断。TJ结构可以通过亚细胞结构，特别是膜下肌动蛋白丝网络的持久性来维持。分泌刺激可以改变紧密连接的结构，使其能够进行细胞旁运输。3)水通道蛋白-5的免疫定位不仅在腮腺腔膜，而且在分泌颗粒膜上都有明显的识别。在IPR和carbacol刺激后，这种定位随着秘书颗粒与腔膜融合而改变。在大鼠腮腺分泌颗粒的纯化膜组分中，用western blotting检测水通道蛋白-5。通过对敏感的超薄冷冻切片进行免疫金标，可以清楚地在分泌颗粒膜上实现水通道蛋白-5的免疫定位。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，导致细胞旁通路的改变，而水通道蛋白5在分泌颗粒膜上的定位可能在跨细胞通路上发挥渗透调节作用。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