| Co-Investigator(Kenkyū-buntansha) |
AKAMATSU Tetsuya University of Tokushima School of Dentistry, Department of Physiology and Oral Physiology, Research Associate, 歯学部, 助手 (80294700)
MATSUURA Sachiko Matsumoto Dental University School of Dentistry Department of Histology, Assistant Professor, 歯学部, 講師 (50139854)
KANAMORI Norio University of Tokushima School of Dentistry, Department of Physiology and Oral Physiology, Associate Professor, 歯学部, 助教授 (90064865)
TSUMURA Keiko University of Tokushima School of Dentistry, Department of Physiology and Oral Physiology, Technical Official, 歯学部, 教務員 (50127841)
TADA Jun University of Tokushima School of Dentistry, Department of Physiology and Oral Physiology, Research Associate, 歯学部, 助手 (00314865)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2000: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1999: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Research Abstract |
AQPs are family of water channel proteins expressed in various tissues ; ten isoforms of the aquaporin family have been identified in mammals. AQP5 is an exocrine type water channel strongly expressed in the salivary gland, lacrimal gland, and lung, and is thought to play a fundamental role in water movement in formation of the saliva, tears, and other secretions. On the other hand, there are only few reports about its trafficking, water transport, or gene regulation . The purpose of the present study is therefore to understand the mechanism of AQP5 trafficking from the cytosol to the plasma membrane. Here we established AQP5-gene-transfected HSG cells since no AQP5-expressing cell line that has originated from the salivary glands was available.
A cDNA of rat aquaporin 5 (AQP5) was used to transfect to HSG (human salivary gland cells), and the trafficking mechanism was studied in vitro by confocal laser microscopy. The trafficking of AQP5 to the plasma membrane was induced by stimulatio
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n of AQP5-gene-transfected human salivary gland cells with thapsigargin, an inhibitor of endoplasmic Ca^<2+>-ATPase, or with A-23187, a calcium ionophore. Pretreatment of these cells with colchicine or vinblastine, microtubule inhibitors, prevented the trafficking induced by thapsigargin or A-23187. The trafficking event was not completely inhibited by cytochalasin B, a microfilament inhibitor. These results demonstrate that the trafficking of AQP5 vesicles to the plasma membrane is triggered by an increase in intracellular Ca^<2+> and that the interaction of AQP5-containing vesicles with the cytoskeleton is involved in this trafficking.
We also investigated the expression and localization of AQP5 in the gastrointestinal tract of the rat, since this tract transports a large amount of water and is mainly composed of many exocrine tissues, we tried to detect the expression and localization of AQP5 in this tract.
Aquaporin 5 (AQP5) mRNA was detected in the lower stomach and duodenum by reverse transcriptase-polymerase chain reaction (RT-PCR). RT-PCR Southern blotting demonstrated the presence of AQP5 in other tissues of the gastrointestinal tract i.e., upper stomach, lower stomach, duodenum, ileum, caecum, and colon but not in jejunum, and rectum. Other AQPs i.e., AQP1, AQP3, and AQP4 mRNAs were also detected in the lower stomach and duodenum by RT-PCR.Western blot analysis detected the AQP5 protein in the lower stomach and duodenum. Immunohistochemical analysis demonstrated that AQP5 was localized in the apical and lateral membrane of the Brunner's gland in the duodenum. These results suggest that AQPs are functional in the fluid secretion or absorption in the gastrointestinal tract. Less
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