Vasopressin V2 Receptor, Tolvaptan, and ERK1/2 Phosphorylation in the Renal Collecting Duct II. Vasopressin V2 Receptor, Tolvaptan, and ERK1/2 Phosphorylation in the Renal Collecting Duct II

Activation of ERK1 and ERK2 is essential in regulation of a wide variety of cellular and physiological processes. In native inner medullary collecting ducts, vasopressin (AVP) working through the V2 subtype vasopressin receptor (V2R)-mediated activation of Gαs, inhibits ERK1 and ERK2 activity. However, it has been reported that V2R can signal independently of Gαs through the activation of β-arrestin, which activates ERK1 and ERK2. Vaptans, V2R antagonists that function as so-called “inverse agonists”, have the potential of promoting cell proliferation via β-arrestin-dependent ERK activation. Here we use the mpkCCD cell line which natively expresses V2R to investigate the effects of AVP, the V2-selective analog dDAVP, and tolvaptan on ERK1 and ERK2 phosphorylation and activation. We demonstrated that ERK1 and ERK2 phosphorylation in mpkCCD cells was significantly reduced by either AVP or dDAVP, in contrast to the increases seen in non-collecting duct cells overexpressing V2R. We also found that tolvaptan has a strong effect to increase ERK1 and ERK2 phosphorylation in the presence of dDAVP and that the tolvaptan effect to increase ERK1 and ERK2 phosphorylation is absent in PKA-null mpkCCD cells. Thus, it appears that the tolvaptan effect to increase ERK activation is PKA-dependent and, therefore, not mediated by the β-arrestin pathway. Overall, the studies show that AVP decreases and that tolvaptan increases ERK1 and ERK2 activation in cells expressing V2R at endogenous levels, and provide no evidence for a role for β-arrestin in the regulation of ERK1 and ERK2 activity. Overall design: Here we use the mpkCCD cell line to address the following questions regarding the endogenously expressed V2 vasopressin receptor: 1) What are the effects of AVP and dDAVP on ERK1 and ERK2 phosphorylation/activation? 2) What is the effect of tolvaptan on ERK1 and ERK2 phosphorylation/activation in the presence and absence of dDAVP? 3) What is the role of PKA in vasopressin-mediated regulation of ERK1 and ERK2 phosphorylation/activation and on the effect of tolvaptan?

细胞外调节蛋白激酶1（ERK1）和细胞外调节蛋白激酶2（ERK2）的激活，对于调控多种多样的细胞及生理过程至关重要。在天然内髓集合管中，血管加压素（vasopressin, AVP）通过V2亚型血管加压素受体（V2 subtype vasopressin receptor, V2R）介导Gαs的激活，进而抑制ERK1与ERK2的活性。然而已有研究表明，V2R还可通过激活β-阻遏蛋白（β-arrestin）实现不依赖Gαs的信号转导，从而激活ERK1和ERK2。
伐普坦类（Vaptans）是一类被称为"反向激动剂"的V2R拮抗剂，其具备通过β-阻遏蛋白依赖的ERK激活途径促进细胞增殖的潜力。本研究采用天然表达V2R的mpkCCD细胞系，探究血管加压素、V2R选择性类似物去氨加压素（desmopressin, dDAVP）以及托伐普坦（tolvaptan）对ERK1和ERK2磷酸化与激活的影响。研究发现，AVP或dDAVP均可显著降低mpkCCD细胞中的ERK1和ERK2磷酸化水平，这与过表达V2R的非集合管细胞中观察到的磷酸化水平升高效应恰好相反。此外，我们还发现，在dDAVP存在的情况下，托伐普坦可强力提升ERK1和ERK2的磷酸化水平，而这一效应在蛋白激酶A敲除（PKA-null）的mpkCCD细胞中并未出现。由此可见，托伐普坦促进ERK激活的效应依赖于PKA，并非通过β-阻遏蛋白通路介导。
综上，本研究显示，在内源表达V2R的细胞中，AVP会降低ERK1和ERK2的激活水平，而托伐普坦则会提升其激活水平，且未发现β-阻遏蛋白在调控ERK1和ERK2活性中发挥作用的证据。
整体实验设计：本研究利用mpkCCD细胞系，针对内源表达的V2血管加压素受体解答以下三个问题：1）AVP与dDAVP对ERK1和ERK2的磷酸化/激活具有何种影响？2）在存在或不存在dDAVP的情况下，托伐普坦对ERK1和ERK2的磷酸化/激活具有何种影响？3）PKA在血管加压素介导的ERK1和ERK2磷酸化/激活调控以及托伐普坦的效应中发挥何种作用？