Molecular and functional relations and intracellular polarized movement of the functional molecules in stomach and intestine.

胃和肠功能分子的分子和功能关系以及细胞内极化运动。

基本信息

批准号：
12144205
负责人：
SAKAI Hideki
金额：
$ 21.63万
依托单位：
Toyama Medical and Pharmaceutical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research on Priority Areas
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2004
项目状态：
已结题

项目摘要

We investigated vectorial-transporting mechanisms of gastric acid secretion and intestinal ion-secretion.1. We established the stable cell lines expressing the gastric proton a-and/or B-subunits. The a-subunit was retained in intracellular compartment and was unstable in the absence of β-subunit, but it was stabilized and reached the cell surface in the presence of β-subunit. We found that the extracellular three disulfide bonds of β-subunit are essential for assembly with a-subunit and expression of the pump activity as well as for cell surface delivery of a-subunit. The ubiquitin/proteasome system was involved in degradation of unassembled α-subunits in ER.2. We found that Glu-345 in the fourth transmembrane (M4) segment of the proton pump is involved in cation-induced conformational change in the pump. We also found that Leu-819 and Glu-822 in the M6 segment are involved in K+-dependent dephosphorylation, and that Asp-826, Ile-827 and Leu-833 in the M6 are involved in phosphorylation of the pump.3. We clarified that the binding site of SCH 28080, an acid pump antagonist, is a cavity in the E2 form of the proton pump a-subunit, and Tyr-801 faces the cavity.4. We found that the phospholipid flippase activity is part of the proton pump reaction.5. We confirmed that ClC-2 Cl^-channel is not a Cl^-transporting protein for gastric acid secretion in parietal cells.6. We established new gastric epithelial cell lines expressing proton a-subunit mRNA from mice transgenic for temperature-sensitive simian virus 40 large T antigen.7. We found that thromboxane A2 is involved in the mechanism of secretory diarrhea in colon, and that thromboxane A2 is released only in pathophysiological condition.8. We found that a decrease in Na^+,K^+-ATPase al-isoform expression and an increase in Na^+,K^+-ATPase a3-isoform expression are associated with human colorectal cancer.

我们研究了胃酸分泌和肠离子分泌的矢量传输机制1。我们建立了表达胃质子A和/或b-subunit的稳定细胞系。 A-亚基被保留在细胞内室中，在没有β-subunit的情况下是不稳定的，但在存在β-亚基的情况下稳定并到达细胞表面。我们发现，β-亚基的细胞外三硫化物键对于具有a-亚基和泵活性的表达以及A-亚基的细胞表面递送至关重要。泛素/蛋白酶体系统参与ER.2中未组装的α-亚基的降解。我们发现，质子泵的第四个跨膜（M4）段中的GLU-345参与了阳离子诱导的泵的构象变化。我们还发现，M6段中的LEU-819和GLU-822参与K+依赖性去磷酸化，并且M6中的ASP-826，Ile-827和Leu-833参与泵的磷酸化3。我们澄清说，酸泵拮抗剂SCH 28080的结合位点是质子泵A-Subunit的E2形式的空腔，而Tyr-801则面向腔。4。我们发现磷脂氟脂酶活性是质子泵反应的一部分。5。我们确认Clc-2 Cl^ - 通道不是顶细胞中胃酸分泌的Cl^转运蛋白。6。我们建立了新的胃皮细胞系，从小鼠转基因中表达质子A-亚基mRNA，用于温度敏感的邻肌病毒40大抗原7。我们发现血栓烷A2参与结肠秘密腹泻的机理，而血栓烷A2仅在病理生理状态下释放。8。我们发现Na^+，K^+ - ATPase al-Isoform表达和Na^+，K^+ - ATPase A3-异型表达的降低与人类结直肠癌有关。