权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Molecular mechanisms of expression and regulation of function of water channel proteins aquaporins in the salivary gland cells

唾液腺细胞水通道蛋白水通道蛋白表达及功能调节的分子机制

基本信息

批准号：
11671844
负责人：
KAZUO Hosoi
金额：
$ 2.24万
依托单位：
University of Tokushima
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

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 … More 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

水通道蛋白（AQP）是一种在多种组织中表达的水通道蛋白家族，在哺乳动物中已发现10种亚型。AQP 5是在唾液腺、泪腺和肺中强烈表达的外分泌型水通道，并且被认为在形成唾液、眼泪和其他分泌物的水运动中起基础作用。另一方面，关于其贩运、水运输或基因调控的报道很少。因此，本研究的目的是了解水通道蛋白5从胞质运输到质膜的机制。由于目前尚无来源于唾液腺的AQP 5表达细胞株，本研究构建了AQP 5基因转染的HSG细胞，并将大鼠水通道蛋白5（aquaporin 5，AQP 5）的cDNA转染到人唾液腺细胞（human salivary gland cells，HSG）中，通过激光共聚焦显微镜观察其转运机制。AQP 5向细胞膜的运输是由刺激引起的。 ...更多信息用毒胡萝卜素（一种内质网Ca^2+-ATP酶抑制剂）或A-23187（一种钙离子载体）处理AQP 5基因转染的人唾液腺细胞。用微管抑制剂秋水仙碱或长春碱预处理这些细胞，可防止毒胡萝卜素或A-23187诱导的贩运。细胞松弛素B（一种微丝抑制剂）不能完全抑制运输事件。这些结果表明，AQP 5囊泡向质膜的运输是由细胞内Ca^<2+>增加触发的，并且含有AQP 5的囊泡与细胞骨架的相互作用参与了这种运输。我们还研究了AQP 5在大鼠胃肠道中的表达和定位，由于该管道运输大量的水并且主要由许多外分泌组织组成，本研究试图检测AQP 5在该束中的表达和定位。RT-PCR检测胃下部和十二指肠中的mRNA。RT-PCR Southern印迹证实了AQP 5存在于胃肠道的其他组织中，上胃、下胃、十二指肠、回肠、盲肠和结肠，但空肠和直肠中没有。其他AQP，即，RT-PCR检测到AQP 1、AQP 3和AQP 4 mRNA在胃下部和十二指肠中的表达，Western blot检测到AQP 5蛋白在胃下部和十二指肠中的表达。免疫组织化学分析表明，AQP 5定位于十二指肠Brunner腺的顶膜和侧膜。这些结果表明，水通道蛋白在胃肠道的液体分泌或吸收功能。少