Mechanisms of Renal Na+ Pump Stimulation by Angiotensin

血管紧张素刺激肾钠泵的机制

• 批准号: 6545370
• 负责人: DOUGLAS R YINGST
• 金额: $ 25.29万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6545370
• 关键词: active sites; adenosine triphosphate; angiotensin II; angiotensin receptor; bioluminescence; calcium indicator; enzyme activity; enzyme mechanism; laboratory rat; mass spectrometry; phosphorylation; protein purification; renal tubular transport; site directed mutagenesis; sodium potassium exchanging ATPase; tissue /cell culture

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.

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）活性的分子机制对于任何类型的细胞来说都是未知的。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钠转运影响心血管功能的了解存在严重差距。例如，在近端小管中，ANGII 对钠泵的刺激对于钠和水的跨细胞转运至关重要，此前已证明接触 ANG II 15 分钟可通过激活 AT1 受体适度（约 20%）刺激钠泵活性。在这里，我们展示了在细胞内钠的生理浓度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泵的纯化α亚基的胰蛋白酶消化，我们首次证明ANG II改变多个位点的磷酸化，增加两个位点的磷酸化，减少两个位点的磷酸化，而其他三个位点没有变化。这些数据支持一种分子模型，其中直接刺激是由磷酸化介导的，并表明α-亚基包含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（一种近端肾小管细胞系）中与 AT1A 受体共表达了大鼠 a 亚基。在这些细胞中，ANG II 刺激大鼠钠泵的活性，并以与近端肾小管相同的模式改变多个位点的磷酸化。使用哇巴因亲和柱和磷酸化位点的定点诱变，我们将测试以下假设：由于激活 AT1 受体，ANG II 通过改变先前已知的和一个或多个新位点的 a 亚基的磷酸化/去磷酸化，直接快速（<几分钟）刺激大鼠 Na,K-ATP 酶活性。活性增加是由于泵的内在动力学特性的改变和/或由于其快速募集到质膜。具体目标是：（1）确定ANG II控制钠泵活性的磷酸化调节位点，并确定新的磷酸化位点； (2) 确定 ANG II 通过改变其动力学特性（与其快速募集至质膜相比）来刺激泵活性的程度。这些结果对于了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。