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

水通道蛋白是一类在多种组织中表达的水通道蛋白家族，目前已在哺乳动物中鉴定出水通道蛋白家族的10种亚型。AQP5是一种外分泌型水通道，在唾液腺、泪腺和肺中强烈表达，被认为在唾液、泪液和其他分泌物的形成中对水的运动起着基础性的作用。另一方面，关于它的贩运、水运或基因调控的报道很少。因此，本研究的目的是了解AQP5从胞浆向质膜转运的机制。由于目前尚无唾液腺来源的表达AQP5的细胞系，因此我们建立了AQP5基因转导的HSG细胞。利用大鼠水通道蛋白5(AQP5)的基因片段，将其转入人唾液腺细胞，并用激光共聚焦显微镜研究其体外转运机制。刺激…诱导水通道蛋白5向质膜转运AQP5基因转染人唾液腺细胞中加入内质钙离子酶抑制剂thapsigargin或钙离子载体A-23187的作用更强。用微管抑制剂秋水仙碱或长春花碱对这些细胞进行预处理，可阻止由thapsigargin或A-23187诱导的转运。微丝抑制剂细胞松弛素B不能完全抑制转运事件。这些结果表明，AQP5囊泡向质膜的运输是由细胞内Ca~(2+)和Gt~(2+)的增加触发的，并参与了AQP5囊泡与细胞骨架的相互作用。此外，我们还研究了AQP5在大鼠胃肠道中的表达和定位。由于AQP5主要由许多外分泌组织组成，因此我们试图检测AQP5在该区域的表达和定位。用逆转录聚合酶链式反应(RT-PCR)检测了AQP5在下胃和十二指肠的表达和定位。RT-PCRSouthern印迹显示AQP5在胃肠道的其他组织中表达，即上胃、下胃、十二指肠、回肠、盲肠和结肠，而空肠和直肠则不表达。RT-PCR法检测到AQP1、AQP3和AQP4mRNAs，Western印迹分析检测到AQP5蛋白在下胃和十二指肠中的表达。免疫组织化学分析表明，AQP5定位于十二指肠布伦纳氏腺的顶膜和侧膜。这些结果表明，水通道蛋白在胃肠道的液体分泌或吸收中具有功能。较少

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