Analyses of Regulatory Mechanism of Na+-K+-Cl- Cotransporter Function in Wild Type and Mutant

野生型和突变体Na-K-Cl-协同转运蛋白功能调控机制分析

基本信息

批准号：
17591939
负责人：
HIRONO Chikara
金额：
$ 2.11万
依托单位：
HIROSHIMA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

项目摘要

The results obtained in the research from April in 2005 to March in 2006 are as follows :1. Establishment of culture cell system for analyses of Cl transportForskolin, a cAMP-increasing agent, induced an ionic current in MDCK and Calu-3 cells, which have an intrinsic Na^+-K^+-2Cl^- cotransporter activity. HEK293 cells were stained by anti-human Na^+-K^+-2Cl^- cotransporter but not by anti-rat Na^+-K^+-2Cl^- cotransporter, while the cells were stained by anti-rat Na^+-K^+-2Cl^- cotransporter after transfection with rat Na^+-K^+-2Cl^- cotransporter-containing plasmid. We started mutation experiments of the Na^+-K^+-2Cl^- cotransporter.2. Analyses of the rat Na^+-K^+-2Cl^- cotransporter activity in salivary glandsTo clarify the regulation mechanism of Na^+-K^+-2Cl^- cotransporter activity by Ca^<2+> and cAMP signals, we investigated the effects of β-adrenergic receptor stimulation on carbachol (CCh)-induced Cl^- current and the effect of α and β receptor stimulation by noradrenaline (NA). … More CCh induced a bumetanide-sensitive oscillatory inward Cl^- current, suggesting that the current carrier is Cl^- which moves into the cell through the Na^+-K^+-2Cl^- cotransporter. Addition of the β-adrenergic agonist, isoproterenol, and forskolin + IBMX suppressed the CCh-induced Cl^- current. This suggests that the reduction of Na^+-K^+-2Cl^- cotransporter activity by cAMP may results in decrease in electrolyte transport and then suppression of salivation during muscarinic receptor stimulation by β-adrenergic stimulation. NA induced an oscillatory anion current of which amplitude gradually decreased to a steady-state level in submandibular acinar cells. The β-adrenergic antagonist, propranolol, did not induce any current, but inhibited the gradual decrease in the NA-induced current, suggesting that the current is induced via the α-adrenergic stimulation by NA and suppressed via the β-adrenergic stimulation by NA and that the relatively small volume of saliva induced by sympathetic stimulation may be at least partially due to the reduction of Cl secretion in the acinar cells by the β-adrenergic stimulation. Less

2005年4月至2006年3月研究取得的结果如下： 1．Cl转运分析培养细胞体系的建立cAMP增强剂Forskolin在MDCK和Calu-3细胞中诱导离子电流，使细胞内产生离子电流。具有内在的Na^+-K^+-2Cl^-协同转运蛋白活性的HEK293细胞被抗人染色。 Na^+-K^+-2Cl^- 协同转运蛋白，但不被抗大鼠 Na^+-K^+-2Cl^- 协同转运蛋白染色，而细胞被抗大鼠 Na^+-K^+-2Cl^ 染色- 转染大鼠 Na^+-K^+-2Cl^- 共转运蛋白后的共转运蛋白我们开始了 Na^+-K^+-2Cl^- 的突变实验。 2.大鼠唾液腺Na^+-K^+-2Cl^-协同转运蛋白活性分析阐明Ca^<2+>和Na^+-K^+-2Cl^-协同转运蛋白活性的调节机制cAMP信号，我们研究了β-肾上腺素受体刺激对卡巴胆碱（CCh）诱导的Cl^-电流的影响以及去甲肾上腺素（NA）刺激α和β受体的影响。 … 更多 CCh 诱导布美他尼敏感的振荡向内 Cl^- 电流，表明电流载体是 Cl^-，通过添加 β-肾上腺素能协同转运蛋白进入细胞。激动剂、异丙肾上腺素和毛喉素 + IBMX 抑制了 CCh 诱导的 Cl^- 电流。 cAMP 的 Na^+-K^+-2Cl^- 协同转运蛋白活性可能导致电解质转运减少，然后在 NA 刺激毒蕈碱受体期间抑制唾液分泌，诱导振荡阴离子电流，其幅度逐渐降低至下颌下腺泡细胞中的β-肾上腺素能拮抗剂普萘洛尔不诱导任何电流，但抑制NA诱导的电流的逐渐降低。表明电流是通过 NA 的 α-肾上腺素能刺激诱导的，并通过 NA 的 β-肾上腺素能刺激抑制，并且交感神经刺激诱导的唾液量相对较小可能至少部分是由于腺泡细胞受β-肾上腺素能刺激较少。