| Project/Area Number |
16590172
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
General physiology
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| Research Institution | National institute for physiological sciences |
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Principal Investigator |
MURAKAMI Masataka National Institute for Physiological Sciences, Department of Cell Physiology, Associate Professor, 細胞器官研究系, 助教授 (10104275)
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| Co-Investigator(Kenkyū-buntansha) |
OZAKI Tsuyoshi National Institute for Physiological Sciences, Okazaki Research Facilities, Center for Experimental Animals, Associate Professor, 動物実験センター, 助教授 (20045694)
HASHIMOTO Sadamitsu Tokyo Dental College, School of Dentistry, Associate Professor, 歯学部, 助教授 (10201708)
SUGIYA Hiroshi Nihon University, School of Dentistry at Matsudo, Professor, 松戸歯学部, 教授 (20050114)
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| Project Period (FY) |
2004 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2005: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2004: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Keywords | salivary gland / paracellular transport / intracellular cAMP / Intracellular Ca / fluoroescent dye / cytoskelton / aquaporin / albumin / aquaprin |
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| Research Abstract |
The primary saliva is a mixture of secretes both via transcellular and paracellular pathways. This project was designed to clarify the control mechanisms for the paracellular transport. The following results were obtained during the project term.
i)Osmolarity changes created by sucrose during the perfusion of isolated rat submandibular glands (SMG) in vitro show that secretion rates alter much more than predicted by the theory of osmotic fluid production. However, these are in accord with a theory involving AQP5 control of paracellular fluid transfer. The changes in transport rate can be predicted with parameters determined earlier for this gland and a quantitative model of the SMG system is presented involving an osmosensor function for AQP5 at the apical membrane. Experiments were performed with SMG from genetically selected rats that have very low levels of AQP5 as determined by Western blotting. The fluid secretion and behavior after osmolarity changes were similar to normal. We sug
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gest that the feedback control involving AQP5 still can operate even at low levels and may probably involve a greater amplification by other elements of the signalling system. While, retrograde injection of Hg ions into the duct to partially inhibit AQP5 lead to a concentration-dependent reduction in flow rates but reduction of fluid secretion after hyper osmotic shock was to that expected for an osmotically equilibrating system. The results indicate that fluid production in the SMG is not osmotic in the steady-state but involves a feedback loop under control of AQP5 acting as an osmosensor.
ii)Morphological basis for control of paracellular transport was examined by electron microscope observation of the deep-etching of the rapid frozen samples from isolated perfused rat submandibular gland both in the resting and stimulated perfusion with carbachol/isoproterenol. The integrated proteins which form the tight junction, were connected with deeper actin fibers via the short and fine fibers. Simultaneous stimulation of muscarinic and alpha adrenergic receptors increases both cytosolic Ca and camp and increases the permeability of paracellular route, as revealed by increased permeation of fluorescent dye. During such stimulation the strand of junctional particles changed their order and the strand was expanded toward the basal direction. The submembranous bundles of actin filaments beneath the basolateral membrane and tight junction became dense. The observation indicates that the dynamic structural change of actin filaments could vibrate the distribution of junctional particles, thus may increase the paracellular permeability.
iii)Mercurial inhibition of aquaporin was examined functionally and morphologically by retrograde-injection of merculic chloride. From 1 microM to 10 microM of Hg the fluid secretion started to be inhibited in the isolated perfused rat submandibular gland, but no morphological change was observed by High resolution SEM. Whereas further concentration induced fine wrinkles on the surface of intercellular canaluculi.
iv)To examine the molecular modulation of large molecule during the passage of paracellular route, the experimental system was set using bovine serum albumine. Less
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