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AQPexpression on intestinal epithelial cells, relation to intestinal hormone and cytekine.

肠上皮细胞AQP的表达与肠激素和细胞因子的关系。

基本信息

批准号：
13670508
负责人：
TSUJIKAWA Tomoyuki
金额：
$ 2.3万
依托单位：
Shiga University of Medical Science
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-13670508/
关键词：
intestinal epithelial cell aquaporin VIP cAMP permeability CRBB PKA

项目摘要

Vasoactive intestinal polypeptide (VIP) plays an important role in water transport in the intestine. Several specialized channels termed aquaporins (AQPs) facilitate water transport in the gastrointestinal tract. AQP3 localizes to epithelial cells in the human small intestine and colon. However, the regulatory mechanisms underlying the functions of AQP3 remain unclear. To characterize the regulation of AQP3 expression by VIP, we studied mRNA expression, protein expression and DNA binding activity in a human colonic epithelial cell line HT-29. Human colonic epithelial cell, HT-29 was incubated with VIP (10^<-12> to 10^<-7> M). The cells were treated with the protein kinase-A (PK-A) inhibitors (H-89, H-9) or the Cl channel blockers (DPC, NPPB). The expression of AQP3 mRNA and protein was determined by Northern blot and Western blot, respectively. The DNA binding activities of cyclic-AMP response element (CRE/ATF) in the nuclear extract were determined by electrophoretic mobility shift assay (EMSA). AQP3 mRNA was upregulated at a concentration of 10^<-10> M VIP. The expression of AQP3 protein was enhanced at 3 hr after addition of VIP. The protein kinase-A (PK-A) inhibitohs (H-89, H-9) inhibited the expression of AQP3 mRNA enhanced by VIP and cAMP. The gel shift assay of CRE/ATF in HT-29 cells revealed a single band. These results indicate that VIP up-regulated the expression of AQP3 mRNA and protein, and a cAMP dependent pathway mediated this effect in a human colonic epithelial cell line HT-29.

血管活性肠肽（VIP）在肠道水分转运中起重要作用。水通道蛋白（aquaporins，AQP）是胃肠道中的一种重要的水通道。AQP 3定位于人小肠和结肠的上皮细胞。然而，AQP 3功能的调节机制仍不清楚。为了表征VIP对AQP 3表达的调节，我们研究了人结肠上皮细胞系HT-29中的mRNA表达、蛋白表达和DNA结合活性。将人结肠上皮细胞HT-29与VIP（10 μ M<-12>至10 μ <-7>M）孵育。用蛋白激酶-A（PK-A）抑制剂（H-89、H-9）或Cl通道阻断剂（DPC、NPPB）处理细胞。分别采用北方印迹法和Western印迹法检测AQP 3 mRNA和蛋白的表达。用电泳迁移率变动分析法（EMSA）测定细胞核提取液中环腺苷酸反应元件（CRE/ATF）的DNA结合活性。当VIP浓度为10 μ M时，AQP 3 mRNA表达上调<-10>。加入VIP后3 h，AQP 3蛋白表达增强。蛋白激酶A（PK-A）抑制剂H-89、H-9可抑制VIP和cAMP诱导的AQP 3 mRNA表达。CRE/ATF在HT-29细胞中的凝胶迁移试验显示单一条带。这些结果表明，VIP上调AQP 3的mRNA和蛋白的表达，和cAMP依赖性途径介导的人结肠上皮细胞系HT-29的这种作用。