Mechanisms of Renal Na+ Pump Stimulation by Angiotensin

血管紧张素刺激肾钠泵的机制

• 批准号: 6888969
• 负责人: DOUGLAS R YINGST
• 金额: $ 22.16万
• 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 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6888969
• 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影响钠转运影响心血管功能的认识存在严重差距。例如，在近端小管中，ANGII对钠泵的刺激是钠和水跨细胞运输的基础，先前的研究表明，暴露于ANGII 15分钟可以通过激活AT1受体来适度（约20%）刺激钠泵的活性。在这里，我们展示了在生理和限速浓度的细胞内钠下快速（< 1分钟）强劲（5至10倍）和短暂的直接刺激。利用创新的瓦阿巴因亲和柱和胰蛋白酶消化纯化的泵的a-亚基，我们首次表明ANG II改变了多个位点的磷酸化，两个位点的磷酸化增加，两个位点的磷酸化减少，其他三个位点没有变化。这些数据支持一个分子模型，其中直接刺激是由磷酸化介导的，并表明a-亚基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（近端小管细胞系）中与AT1A受体共同表达了大鼠a-亚基。在这些细胞中，ANG II刺激大鼠钠泵的活性，并以与近端小管相同的模式在多个位点改变其磷酸化。使用瓦阿巴因亲和柱和磷酸化位点定向突变，我们将验证ANG II直接和快速（<几分钟）刺激大鼠Na， k - atp酶活性的假设，通过在先前已知的和一个或多个新位点上改变a亚基的磷酸化/去磷酸化，作为激活AT1受体的结果。活性的增加是由于泵的内在动力学性质的改变和/或其快速聚集到质膜上。具体目的是：(1)确定ANG II通过其控制钠泵活性的磷酸化调控位点，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来刺激泵活性的程度，与其快速募集到质膜相比。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。

项目成果

Decreased renal perfusion rapidly increases plasma membrane Na-K-ATPase in rat cortex by an angiotensin II-dependent mechanism.

肾灌注减少通过血管紧张素 II 依赖性机制迅速增加大鼠皮质中的质膜 Na-K-ATP 酶。

• DOI: 10.1152/ajprenal.90363.2008
• 发表时间: 2009
• 期刊: American journal of physiology. Renal physiology
• 作者: Yingst,DouglasR;Araghi,Ali;Doci,TabithaM;Mattingly,Raymond;Beierwaltes,WilliamH
• 通讯作者: Beierwaltes,WilliamH

Angiotensin II stimulates elution of Na-K-ATPase from a digoxin-affinity column by increasing the kinetic response to ligands that trigger the decay of E2-P.

血管紧张素 II 通过增加对触发 E2-P 衰变的配体的动力学响应，刺激 Na-K-ATP 酶从地高辛亲和柱中洗脱。

• DOI: 10.1152/ajprenal.00492.2007
• 发表时间: 2008
• 期刊: American journal of physiology. Renal physiology
• 作者: Yingst,DouglasR;Doci,TabithaM;Massey,KatherineJ;Rossi,NoreenF;Rucker,Ebony;Mattingly,RaymondR
• 通讯作者: Mattingly,RaymondR