| Co-Investigator(Kenkyū-buntansha) |
MURAMATSU Takashi TOKYO DENTAL COLLEGE, DEPARTMENT OF DENTISTRY, LECTURER, 歯学部, 講師 (00276982)
SHIMA Kaori TOKYO DENTAL COLLEGE, DEPARTMENT OF DENTISTRY, LECTURER, 歯学部, 講師 (10343526)
SHIMONO Masaki TOKYO DENTAL COLLEGE, DEPARTMENT OF DENTISTRY, PROFESSOR, 歯学部, 教授 (00085771)
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| Research Abstract |
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
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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
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