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

本研究从2005年4月至2006年3月进行，主要研究结果如下：1。cAMP增加剂Forskolin在具有Na^+-K^+-2Cl ^-协同转运活性的MDCK和Calu-3细胞中诱导离子电流。HEK 293细胞被抗人Na^+-K^+-2Cl ^-共转运蛋白染色，但不被抗大鼠Na^+-K^+-2Cl ^-共转运蛋白染色，而在用含有大鼠Na^+-K ^+-2Cl ^-共转运蛋白的质粒转染后，细胞被抗大鼠Na^+-K ^+-2Cl ^-共转运蛋白染色。我们开始了Na^+-K^+-2Cl ^-共转运蛋白的突变实验。2.为了阐明Ca^2+和cAMP信号对Na ^+-K ^+-2Cl ^-共转运体活性的调节机制，我们研究了β-肾上腺素能受体对卡巴胆碱（CCh）诱导的Cl^-电流的影响以及去甲肾上腺素（NA）对α和β受体的影响。 ...更多信息 CCh诱导了布美他尼敏感的振荡性内向Cl^-电流，表明电流载体是Cl^-，它通过Na^+-K^+-2Cl ^-共转运蛋白进入细胞。加入β-肾上腺素能激动剂异丙肾上腺素和毛喉素+ IBMX可抑制CCh诱导的Cl^-电流。这表明cAMP降低Na^+-K^+-2Cl ^-协同转运体活性可能导致电解质转运减少，从而抑制β-肾上腺素能刺激毒蕈碱受体时的流涎。NA诱导的振荡阴离子电流的振幅逐渐下降到稳态水平的下颌下腺腺泡细胞。β-肾上腺素能拮抗剂普萘洛尔不诱导任何电流，但抑制NA诱导电流的逐渐下降，提示电流通过NA的α-肾上腺素能刺激诱导，通过NA的β-肾上腺素能刺激抑制，交感神经刺激诱导的相对小的唾液量可能至少部分是由于β-肾上腺素能刺激减少腺泡细胞中的Cl分泌。肾上腺素能刺激少

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