ROS production as a common mechanism of ENaC regulation by EGF, insulin, and IGF-1
ROS production as a common mechanism of ENaC regulation by EGF, insulin, and IGF-1
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DOI：
10.1152/ajpcell.00231.2012
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发表时间：
2013-01-01
影响因子：
5.5
通讯作者：
Staruschenko, Alexander
中科院分区：
文献类型：
--
作者：
Ilatovskaya, Daria V.;Pavlov, Tengis S.;Staruschenko, Alexander
放大图片作者：Zhang Jiang，Zhang Jiang，Zhang Jiang. ROS的产生是EGF、胰岛素和IGF-1调节ENaC的共同机制。美国生理学杂志细胞生理学304：C102-C111，2013年。首次发表于2012年11月7日; doi：10.1152/ajpcell.00231.2012.-上皮Na+通道（ENaC）是参与肾单位Na+重吸收微调的关键转运蛋白。ENaC活性被表皮生长因子（EGF）、胰岛素和胰岛素样生长因子-1（IGF-1）急性上调。还提出活性氧（ROS）对ENaC具有刺激作用。在这里，我们研究了EGF，胰岛素和IGF-1的作用是否与小鼠皮质集合管（mpkCCD（c14））细胞中的ROS产生相关。Western blotting证实了NADPH氧化酶复合物亚单位在这些细胞中的表达。用EGF、胰岛素或IGF-1处理mpkCCD（c14）细胞引起ROS产生的增加，如通过CM-H2 DCF-DA荧光测量的。由黄嘌呤-黄嘌呤氧化酶反应引起的ROS产生也导致通过mpkCCD（c14）单层的短路电流显著升高。跨上皮电流测量表明，急性增加阿米洛利敏感电流通过mpkCCD（c14）单层的EGF，胰岛素，或IGF-1。预处理与非选择性NADPH氧化酶活性抑制剂夹竹桃麻素钝化ROS的产生和ENaC介导的电流在这些药物的响应增加。为了进一步测试NADPH氧化酶亚基是否参与EGF的作用，我们使用了一个稳定的M-1细胞系与Rac 1，这是NADPH氧化酶复合物的关键亚基之一，敲低，并测量阿米洛利敏感电流响应EGF。与对照细胞相比，EGF在Rac 1敲除细胞中没有作用。我们假设EGF、胰岛素和IGF-1对ROS产生介导的ENaC具有共同的刺激作用。
Ilatovskaya DV, Pavlov TS, Levchenko V, Staruschenko A. ROS production as a common mechanism of ENaC regulation by EGF, insulin, and IGF-1. Am J Physiol Cell Physiol 304: C102-C111, 2013. First published November 7, 2012; doi:10.1152/ajpcell.00231.2012.-The epithelial Na+ channel (ENaC) is a key transporter participating in the fine tuning of Na+ reabsorption in the nephron. ENaC activity is acutely upregulated by epidermal growth factor (EGF), insulin, and insulin-like growth factor-1 (IGF-1). It was also proposed that reactive oxygen species (ROS) have a stimulatory effect on ENaC. Here we studied whether effects of EGF, insulin, and IGF-1 correlate with ROS production in the mouse cortical collecting duct (mpkCCD(c14)) cells. Western blotting confirmed the expression of the NADPH oxidase complex subunits in these cells. Treatment of mpkCCD(c14) cells with EGF, insulin, or IGF-1 evoked an increase in ROS production as measured by CM-H2DCF-DA fluorescence. ROS production caused by a xanthine-xanthine oxidase reaction also resulted in a significant elevation in short-circuit current through the mpkCCD(c14) monolayer. Transepithelial current measurements showed an acute increase of amiloride-sensitive current through the mpkCCD(c14) monolayer in response to EGF, insulin, or IGF-1. Pretreatment with the nonselective NADPH oxidase activity inhibitor apocynin blunted both ROS production and increase in ENaC-mediated current in response to these drugs. To further test whether NADPH oxidase subunits are involved in the effect of EGF, we used a stable M-1 cell line with a knockdown of Rac1, which is one of the key subunits of the NADPH oxidase complex, and measured amiloride-sensitive currents in response to EGF. In contrast to control cells, EGF had no effect in Rac1 knockdown cells. We hypothesize that EGF, insulin, and IGF-1 have a common stimulatory effect on ENaC mediated by ROS production.