Mechanisms of Renal Na+ Pump Stimulation by Angiotensin

描述(申请人提供)：血管紧张素II(Ang II)直接刺激钠泵(Na，K-ATPase)活性的分子机制在任何类型的细胞中都是未知的。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影响来影响心血管功能的了解存在严重差距。例如，在近端小管上，血管紧张素Ⅱ对泵的刺激是钠和水跨细胞运输的基础，先前已证明，暴露于血管紧张素转换酶II 15分钟，可通过激活AT1受体适度(约20%)刺激钠泵活动。在这里，我们显示了一个快速(&lt；1分钟)强劲(5至10倍)和瞬时直接刺激生理和限速浓度的细胞内钠。使用创新的哇巴因亲和柱和纯化的泵α亚基的胰酶消化，我们首次表明Ang II改变了多个位点的磷酸化，在两个位点增加了磷酸化，在两个位点降低了磷酸化，而在另外三个位点没有变化。这些数据支持一个分子模型，在该模型中，直接刺激是由磷酸化介导的，并表明a-亚单位至少包含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，我们在负鼠肾细胞中共表达了大鼠的a-亚单位，这是一种近端小管细胞系，与AT1a受体一起。在这些细胞中，血管紧张素II刺激大鼠钠泵的活性，并改变其在多个位置的磷酸化，其模式与近端小管相同。使用哇巴因亲和柱和磷酸化位点的定点突变，我们将检验这一假设，即Ang II通过激活AT1受体导致的已知和一个或多个新位点的a亚基的磷酸化/去磷酸化的变化，直接和快速地(几分钟)刺激大鼠Na，K-ATPase活性。活性的增加要么是由于泵的固有动力学性质的改变，要么是因为它快速地重新聚集到质膜上。其具体目的是：(1)确定Ang II控制泵活性的钠泵上的磷酸化调节位点，并确定新的磷酸化位点；(2)确定Ang II通过改变其动力学性质在多大程度上刺激泵活性，而不是迅速募集到质膜上。这些结果对于了解正常的肾功能，以及高血压和心力衰竭的发展具有重要的意义。

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.