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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Phorbol ester stimulation of RasGRPI regulates the sodium-chloride cotransporter by a PKC-independent pathway
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Phorbol ester stimulation of RasGRPI regulates the sodium-chloride cotransporter by a PKC-independent pathway

机译：RasGRPI的佛波酯刺激通过不依赖PKC的途径调节氯化钠共转运蛋白

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摘要

The sodium-chloride cotransporter (NCC) is the principal salt-absorptive pathway in the mammalian distal convoluted tubule (DCT) and is the site of action of one of the most effective classes of antihypertensive medications, thiazide diuretics. We developed a cell model system to assess NCC function in a mammalian cell line that natively expresses NCC, the mouse DCT (mDCT) cell line. We used this system to study the complex regulation of NCC by the phorbol ester (PE) 12-O-tetradecanoylphorbol-13-acetate (TPA), a diacylglycerol (DAG) analog. It has generally been thought that PEs mediate their effects on transporters through the activation of PKC. However, there are at least five other DAG/PE targets. Here we describe how one of those alternate targets of DAG/PE effects, Ras guanyl-releasing protein 1 (RasGRPI), mediates the PE-induced suppression of function and the surface expression of NCC. Functional assessment of NCC by using thiazide-sensitive ~(22)Na~+ uptakes revealed that TPA completely suppresses NCC function. Biotinyla-tion experiments demonstrated that this result was primarily because of decreased surface expression of NCC. Although inhibitors of PKC had no effect on this suppression, MAPK inhibitors completely prevented the TPA effect. RasGRPI activates the MAPK pathway through activation of the small G protein Ras. Gene silencing of RasGRPI prevented the PE-mediated suppression of NCC activity, the activation of the H-Ras isoform of Ras, and the activation of ERK1/2 MAPK. This finding confirmed the critical role of RasGRPI in mediating the PE-induced suppression of NCC activity through the stimulation of the MAPK pathway.
机译：氯化钠共转运蛋白（NCC）是哺乳动物远端旋回小管（DCT）中的主要盐吸收途径，并且是最有效的降压药物之一，噻嗪类利尿剂的作用部位。我们开发了一种细胞模型系统来评估哺乳动物细胞系中的NCC功能，该哺乳动物细胞系天然表达NCC（小鼠DCT（mDCT）细胞系）。我们使用该系统研究了佛波酯（PE）12-O-十四烷酰佛波13-乙酸酯（TPA）（一种二酰基甘油（DAG）类似物）对NCC的复杂调控。通常认为，PE通过PKC的活化介导其对转运蛋白的作用。但是，至少还有五个其他DAG / PE目标。在这里，我们描述了DAG / PE效应的替代目标之一，鸟嘌呤释放蛋白1（RasGRPI）如何介导PE诱导的功能抑制和NCC表面表达。通过对噻嗪类敏感的〜（22）Na〜+摄入量评估NCC的功能，发现TPA完全抑制NCC的功能。生物素化实验表明，该结果主要是由于NCC的表面表达下降所致。尽管PKC抑制剂对此抑制作用没有影响，但MAPK抑制剂完全阻止了TPA的作用。 RasGRPI通过激活小G蛋白Ras激活MAPK途径。 RasGRPI的基因沉默阻止了PE介导的NCC活性的抑制，Ras的H-Ras亚型的激活以及ERK1 / 2 MAPK的激活。这一发现证实了RasGRPI在通过MAPK途径的刺激介导PE诱导的NCC活性抑制中的关键作用。

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