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

外分泌腺细胞水通道蛋白水通道蛋白表达与调控的分子机制

• 批准号: 13671941
• 负责人: HOSOI Keiko
• 金额: $ 1.98万
• 依托单位: Tokushima University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13671941/
• 关键词: aquaporin2; polydipsia; kidney; STR; N mice; aquaporin 5; fetal; submandibular gland; aquaporin 1; アクアポリン; トラフィッキング; 外分泌腺; プロテインキナーゼA

The purpose of this study focussed on the study of the trafficking mechanism and function of one of aquaporins (AQPs), AQP5, an exocrine type water channel.1. In order to understand the molecular mechanism of water transport and its regulation, we set the experiment to understand the PKA-target motif in loop-D of AQP5. For this purpose we prepared mutant cDNAs, lacking either seine, threonine or both amino acids by using in vitro mutagenesis techniques. The sequence for green fluorescence protein (GFP) was also introduced into either 5'-or 3'- end of these cDNAs to prepare GFP-AQP5 and AQP5-GFP proteins. These AQPs will be introduced to MDCK cells and HSG cells to assess the effects of inhibitors or activators of signaling pathway.2. Aquaporin 2 (AQP2) is responsible for the concentration of urine in the kidney collecting tubule under the regulation of vasopressin. In polydipsic STR/N mice, the reduction in the AQP2 Mrna level was not evident at 3 weeks of age, at which time the water … More intake was not increased at all. At 10 weeks of age, however, the AQP2 Mrna level was reduced to 10% of that of the control mice, whereas the water intake was increased by 36%. When STR/N mice were 44 weeks old, the water intake became 5 times of that of the control ICR mice, and the AQP2 Mrna level in these polydipsic mice was only approx. 5% of the control one. These data imply that elevated water intake due to polydipsia may have affected tha AQP2 Mrna transcription in the kidney, resultiong in reduced AQP2 expression, which would contribute to a reduction in over-retention of water.3. The expression and localozation of aquaporins (AQP1-AQP5), members of the water channel family in the developing rat submandibular gland were analyzed by RT-PCR, Northen blotting, and immunohistochemistry to explore their relation to the development of this salivary gland. RT-PCR analysis revealed uniqui expression patterns of each AQP. AQP1 was constitutively expressed during prenatal development, whereas the expression of AQP5 became more intense in the course of development were not detected by Northern blotting, although they were detected by RT-PCR. During postnatal development, AQP5 and AQP1 mRNAs were continuously expressed, but no message for AQP3 or AQP4 was detected. AQP2mRNA was not detected during either prenatal or postnatal development in this tissue. An immunohistichemical experiment was also done to explore the precise localozation of these AQPs. Less

本研究的目的是研究水通道蛋白AQP5的转运机制和功能，AQP5是一种外分泌型水通道。为了了解水运输的分子机制及其调控，本实验对AQP5环-D中的PKA靶基序进行了研究。为此，我们利用体外诱变技术制备了缺失Seine、苏氨酸或两者兼有的突变型cDNA。将绿色荧光蛋白(GFP)序列分别插入这些cDNA的5‘端和3’端，制备GFP-AQP5和AQP5-GFP蛋白。这些水通道蛋白将被引入MDCK细胞和HSG细胞，以评估信号通路的抑制剂或激活剂的作用。水通道蛋白2(Aquaporin 2，AQP2)在加压素的调节下，负责尿液在肾脏集合管的浓度。在多发性STR/N小鼠中，AQP2mRNA水平在3周龄时下降不明显，此时水…更多的摄入量根本没有增加。然而，在10周龄时，AQP2mRNA水平下降到对照组的10%，而摄水量增加了36%。在44周龄时，STR/N小鼠的摄水量是对照组ICR小鼠的5倍，而这些多饮小鼠的AQP2基因表达水平仅约为ICR小鼠的5倍。对照组的5%。这些数据表明，多饮导致的水摄入量增加可能影响了肾脏中AQP2的转录，导致AQP2表达减少，这将有助于减少水的过度滞留。为探讨水通道蛋白AQP1-AQP5在发育中的大鼠颌下腺发育中的作用，采用RT-PCR、Norten印迹和免疫组织化学方法分析水通道蛋白(AQP1-AQP5)在发育中的大鼠颌下腺中的表达和定位。RT-PCR分析显示，每种AQP的表达模式都是独特的。AQP1在胚胎发育过程中呈结构性表达，而AQP5在胚胎发育过程中表达增强，RT-PCR检测到AQP5的表达，但Northern杂交未检测到。在出生后发育过程中，AQP5和AQP1mRNAs持续表达，但未检测到AQP3或AQP4的表达。AQP2mRNA在出生前和出生后的发育过程中均未检测到。还进行了免疫组织化学实验，以探索这些水通道蛋白的精确定位。较少

项目成果

Kurabuchi.S, K.Hosoi, E.W.Gresik: "Multihormonal regulation of granular convoluted tubular cells expressing Mk1, a true tissue kallikrein, in the mouse submandibular gland"Proccedings of the 21 Conference of European Comparative Endocrinologi sts, edited

Kurabuchi.S、K.Hosoi、E.W.Gresik：“小鼠颌下腺中表达 Mk1（一种真正的组织激肽释放酶）的颗粒曲管细胞的多激素调节”第 21 届欧洲比较内分泌学家会议记录，编辑

Hosoi.K, Edited by Barrett A.J. Rawlings N.D., and Woessner, Jr.,J.F.: "Mouse kallikreins Mk13 and Mk26, in "Handbook of proteolytic enzymes, 2nd edition""Elsevier Science. (2003)

Hosoi.K，Barrett A.J. 编辑

Yao, C.et al.: "Acute phase protein induction by experimental inflammation in the salivary"Proceedings for THE 1st International Congress on Mastication and Health-Oral Health for Healthy Life. (in press). (2003)

Yao, C.等人：“唾液中实验性炎症诱导急性期蛋白”第一届国际咀嚼与健康大会论文集——口腔健康促进健康生活。

Kurabuchi, S.et al.: "Developmental and androgenic regulation of the immunocytochemical distribution of mK1, a true tissue kallikrein, in the granular convoluted tubule of the mouse submandibular gland"Journal of Histochemistry and Cytochemistry. 50(2). 1

Kurabuchi, S.等人：“mK1（一种真正的组织激肽释放酶，在小鼠颌下腺颗粒回管中）免疫细胞化学分布的发育和雄激素调节”组织化学和细胞化学杂志。

Kurabuchi, S. et al.: "Developmental and androgenic regulation of the immunocytochemical distribution of mK1, a true tissue kallikrein, in the granular convoluted tubule of the mouse submandibular gland"Journal of Histochemistry and Cytochemistry. (in pre

Kurabuchi, S. 等人：“mK1（一种真正的组织激肽释放酶，在小鼠下颌下腺颗粒曲管中）免疫细胞化学分布的发育和雄激素调节”组织化学和细胞化学杂志。