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首页> 外文期刊>American Journal of Physiology >Impaired intestinal NHE3 activity in the PDK1 hypomorphic mouse.
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Impaired intestinal NHE3 activity in the PDK1 hypomorphic mouse.

机译：PDK1亚型小鼠的肠道NHE3活性受损。

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In vitro experiments have demonstrated the stimulating effect of serum- and glucocorticoid-inducible kinase (SGK)1 on the activity of the Na+/H+ exchanger (NHE3). SGK1 requires activation by phosphoinositide-dependent kinase (PDK)1, which may thus similarly play a role in the regulation of NHE3-dependent epithelial electrolyte transport. The present study was performed to explore the role of PDK1 in the regulation of NHE3 activity. Because mice completely lacking functional PDK1 are not viable, hypomorphic mice expressing approximately 20% of PDK1 (pdk1(hm)) were compared with their wild-type littermates (pdk1(wt)). NHE3 activity in the intestine and PDK1-overexpressing HEK-293 cells was estimated by utilizing 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein fluorescence for the determination of intracellular pH. NHE activity was reflected by the Na+-dependent pH recovery from an ammonium prepulse (DeltapH(NHE)). The pH changes after an ammonium pulse allowed the calculation of cellular buffer capacity, which was not significantly different between pdk1(hm) and pdk1(wt) mice. DeltapH(NHE) was in pdk1(hm) mice, only 30 +/- 6% of the value obtained in pdk1(wt) mice. Conversely, DeltapH(NHE) was 32 +/- 7% larger in PDK1-overexpressing HEK-293 cells than in HEK-293 cells expressing the empty vector. The difference between pdk1(hm) and pdk1(wt) mice and between PDK1-overexpressing and empty vector-transfected HEK cells, respectively, was completely abolished in the presence of the NHE3 inhibitor S3226 (10 microM). In conclusion, defective PDK1 expression leads to significant impairment of NHE3 activity in the intestine, pointing to a role of PDK1-dependent signaling in the regulation of NHE-mediated electrolyte transport.
机译：体外实验表明，血清和糖皮质激素诱导激酶（SGK）1对Na + / H +交换子（NHE3）的活性具有刺激作用。 SGK1需要磷酸肌醇依赖性激酶（PDK）1激活，因此它可能在NHE3依赖性上皮电解质运输的调节中同样发挥作用。进行本研究以探索PDK1在调节NHE3活性中的作用。由于完全缺乏功能性PDK1的小鼠不可行，因此将表达约20％PDK1（pdk1（hm））的亚型小鼠与其野生型同窝仔（pdk1（wt））进行了比较。通过利用2'，7'-双（2-羧乙基）-5（6）-羧基荧光素荧光测定细胞内pH值，估计肠和PDK1过表达的HEK-293细胞中的NHE3活性。 NHE活性通过从铵预脉冲（DeltapH（NHE））中Na +依赖的pH回收反映出来。铵脉冲后pH的变化允许计算细胞缓冲能力，这在pdk1（hm）和pdk1（wt）小鼠之间没有显着差异。 DeltapH（NHE）在pdk1（hm）小鼠中，仅为在pdk1（wt）小鼠中获得的值的30 +/- 6％。相反，过表达PDK1的HEK-293细胞中的DeltapH（NHE）比表达空载体的HEK-293细胞大32 +/- 7％。在NHE3抑制剂S3226（10 microM）存在的情况下，分别消除了pdk1（hm）和pdk1（wt）小鼠之间以及过表达PDK1和空载体转染的HEK细胞之间的差异。总之，有缺陷的PDK1表达导致肠道NHE3活性显着受损，这表明PDK1依赖性信号传导在NHE介导的电解质运输调节中发挥了作用。

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