Mechanisms of Renal Na+ Pump Stimulation by Angiotensin

描述（由申请人提供）：血管紧张素II（ANG II）直接刺激钠泵（Na，K-ATP酶）活性的分子机制在任何类型的细胞中均未知。因此，我们对血管紧张素转换酶（ACE）抑制剂和ANG II受体阻滞剂如何通过其对钠转运的作用影响心血管功能的认识存在严重差距。例如，在近端小管中，ANG II对泵的刺激是钠和水的跨细胞转运的基础，先前已显示暴露于ANG II 15分钟通过激活AT 1受体适度（约20%）刺激钠泵活性。在这里，我们显示了在细胞内钠的生理和限速浓度下的快速（< 1分钟）稳健（5至10倍）和瞬时直接刺激。使用创新的哇巴因亲和柱和胰蛋白酶消化纯化的泵的a-亚基，我们已经首次表明，ANG II改变磷酸化在多个网站，增加磷酸化在两个网站，减少它在两个，在其他三个没有变化。这些数据支持直接刺激是由磷酸化介导的分子模型，并表明α-亚基含有至少一个未表征的磷酸化位点。为了测试磷酸化在调节泵活性中的作用，我们在负鼠肾细胞（一种近曲小管细胞系）中共表达大鼠α-亚基，沿着AT 1A受体。在这些细胞中，ANG II刺激大鼠钠泵的活性，并以从近端小管看到的相同模式在多个位点改变其磷酸化。使用哇巴因亲和柱和磷酸化位点的定点突变，我们将测试以下假设：ANG II直接和快速（<几分钟）刺激大鼠Na，K-ATP酶活性，通过改变先前已知和一个或多个新位点的α-亚基的磷酸化/去磷酸化，作为激活AT 1受体的结果。增加的活性是由于泵的内在动力学性质的改变和/或其快速募集到质膜。具体目标是：（1）确定ANG II通过其控制泵活性的钠泵上的磷酸化的调节位点，并鉴定新的磷酸化位点;（2）确定ANG II通过改变其动力学性质与其快速募集到质膜相比刺激泵活性的程度。这些结果对于理解正常肾功能以及高血压和心力衰竭的发展具有重要意义。

DESCRIPTION (provided by applicant): The molecular mechanisms by which angiotensin II (ANG II) directly stimulates the activity of the sodium pump (Na,K-ATPase) are unknown any type of cell. Thus, there is a serious gap in our knowledge of how angiotensin-converting enzyme (ACE) inhibitors and ANG II receptor blockers affect cardiovascular function via their effects on sodium transport. For instance, in the proximal tubule, where stimulation of the pump by ANGII is fundamental to the transcellular transport of sodium and water, a 15 min exposure to ANG II has been previously shown to modestly (approximately 20%) stimulate sodium pump activity via activation of the AT1 receptor. Here we show a rapid (< 1 min) robust (5 to 10 fold) and transient direct stimulation at physiological and rate-limiting concentrations of intracellular sodium. Using an innovative ouabain-affinity column and a trypsin digest of the purified a-subunit of the pump, we have shown for the first time that ANG II alters phosphorylation at multiple sites, increasing phosphorylation at two sites, decreasing it at two, with no change at three others. These data support a molecular model in which direct stimulation is mediated by phosphorylation and suggest that the a-subunit contains at least one uncharacterized site of phosphorylation. To test the role of phosphorylation in regulating pump activity we have co-expressed the rat a-subunit in opossum kidney cells, a proximal tubule cell line, along with the AT1A receptor. In these cells ANG II stimulates the activity of the rat sodium pump and alters its phosphorylation at multiple sites in the same pattern seen from the proximal tubule. Using the ouabain-affinity column and site-directed mutagenesis of phosphorylation sites, we will test the hypothesis that ANG II directly and rapidly (<few min) stimulates rat Na,K-ATPase activity through changes in the phosphorylation/dephosphorylation of the a-subunit at both previously known and one or more novel sites as a result of activating the AT1 receptor. Increased activity is due either to alterations in the intrinsic kinetic properties of the pump and/or to its rapid recruitment to the plasma membrane. The specific aims are to: (1) determine the regulatory sites of phosphorylation on the sodium pump through which ANG II controls pump activity and to identify new phosphorylation sites; (2) determine to what extent ANG II stimulates pump activity by altering its kinetic properties compared to its rapid recruitment to the plasma membrane. These results have important implications for understanding normal kidney function, as well as the development of hypertension and heart failure.