Ko Benjamin, Joshi Leena M, Cooke Leslie L, Vazquez Norma, Musch Mark W, Hebert Steven C, Gamba Gerardo, Hoover Robert S
Department of Medicine, University of Chicago, Chicago, IL 60637, USA.
Proc Natl Acad Sci U S A. 2007 Dec 11;104(50):20120-5. doi: 10.1073/pnas.0709506104. Epub 2007 Dec 5.
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 (RasGRP1), 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. Biotinylation 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. RasGRP1 activates the MAPK pathway through activation of the small G protein Ras. Gene silencing of RasGRP1 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 RasGRP1 in mediating the PE-induced suppression of NCC activity through the stimulation of the MAPK pathway.
氯化钠协同转运蛋白（NCC）是哺乳动物远曲小管（DCT）中主要的盐吸收途径，也是最有效的一类抗高血压药物噻嗪类利尿剂的作用位点。我们开发了一种细胞模型系统，以评估在天然表达NCC的哺乳动物细胞系——小鼠DCT（mDCT）细胞系中的NCC功能。我们使用该系统研究佛波酯（PE）12 - O - 十四酰佛波醇 - 13 - 乙酸酯（TPA，一种二酰基甘油（DAG）类似物）对NCC的复杂调控。一般认为，PEs通过激活蛋白激酶C（PKC）来介导其对转运蛋白的作用。然而，DAG/PE至少还有其他五个靶点。在此我们描述DAG/PE作用的这些替代靶点之一，Ras鸟苷酸释放蛋白1（RasGRP1）如何介导PE诱导的NCC功能抑制和表面表达下调。通过使用对噻嗪敏感的（22）Na（+）摄取对NCC进行功能评估，结果显示TPA完全抑制NCC功能。生物素化实验表明，这一结果主要是由于NCC表面表达降低所致。尽管PKC抑制剂对这种抑制作用没有影响，但丝裂原活化蛋白激酶（MAPK）抑制剂完全阻止了TPA的作用。RasGRP1通过激活小G蛋白Ras来激活MAPK途径。RasGRP1的基因沉默阻止了PE介导的NCC活性抑制、Ras的H - Ras亚型的激活以及细胞外信号调节激酶1/2（ERK1/2）MAPK的激活。这一发现证实了RasGRP1在通过刺激MAPK途径介导PE诱导的NCC活性抑制中起关键作用。
