Functional Studies of the Na,K-ATPase

Na,K-ATP酶的功能研究

• 批准号: 7437306
• 负责人: JERRY B LINGREL
• 金额: $ 36.39万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-01-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7437306
• 关键词: Affinity; Animals; Area; Binding Sites; Biological; Biological Process; Blood Pressure; Cardiac; Cardiac Glycosides; Cardiovascular Physiology; Cardiovascular system; Class; Condition; Congestive Heart Failure; Corticotropin; Digoxin; Enzymes; Exhibits; Follow-Up Studies; Gene Targeting; Genes; Genetically Engineered Mouse; Goals; Grant; Heart; Hypertension; Knock-out; Laboratories; Measurement; Mediating; Mus; Na(+)-K(+)-Exchanging ATPase; Ouabain; Partner in relationship; Phenotype; Physiological; Play; Protein Isoforms; Regulation; Resistance; Rodent; Role; Site; Smooth Muscle; Sodium Chloride; Spermatogonia; Systems Analysis; Tail; Telemetry; Testing; Tissues; Vascular Smooth Muscle; Wild Type Mouse; insight; interest; knockout animal; promoter; receptor; recombinase; sperm cell

DESCRIPTION (provided by applicant): This application proposes to continue our studies of the Na,K-ATPase and is directed toward several ongoing goals. One of these is to test whether the cardiac glycoside binding site of the alpha2 or alpha3 isoforms of this enzyme, which is highly conserved among species, plays a biological role. Such studies will provide insight as to whether endogenous cardiac glycosides such as endogenous ouabain observed by many laboratories is physiologically significant. Another objective is to define the role of the alpha2 isoform in heart and vascular smooth muscle by analyzing tissue-specific alpha2 isoform knockout animals. The specific aims of our grant are (1) To determine whether the cardiac glycoside binding site of the Na,K-ATPase has biological significance and whether endogenous cardiac glycosides play a physiological role. To accomplish this, genetically engineered mice expressing either ouabain-resistant alpha2 or alpha3 isoforms will be analyzed under conditions known to increase the levels of endogenous cardiac glycosides such as ACTH-induced hypertension. If the ouabain-resistant mice respond differently from wild type animals, this will suggest a physiological role for endogenous ouabain. However, if mice with ouabain resistant alpha2 or alpha3 isoforms respond similarly to wild type animals, this will detract from the hypothesis that endogenous cardiac glycosides have physiological significance. (2) To define the specific role of the alpha2 isoform using tissuespecific knockouts. We have already developed mice where the alpha2 isoform gene is flanked by loxP sites and we have mated these to animals carrying Cre-recombinase under the control of a cardiac-specific promoter. We will now mate the alpha2 isoform loxP animals with those expressing Cre-recombinase under the control of a smooth muscle promoter. These matings will provide animals where the alpha2 isoform is missing only in heart or smooth muscle and this will allow us to study the specific role of this isoform in cardiovascular function.

描述（由申请人提供）：本申请建议继续我们对 Na,K-ATP 酶的研究，并针对几个正在进行的目标。其中之一是测试该酶的α2或α3亚型的强心苷结合位点（在物种间高度保守）是否发挥生物学作用。此类研究将深入了解许多实验室观察到的内源性强心苷（例如内源性哇巴因）是否具有生理意义。另一个目标是通过分析组织特异性 α2 同工型敲除动物来确定 α2 同工型在心脏和血管平滑肌中的作用。我们资助的具体目的是（1）确定Na,K-ATP酶的强心苷结合位点是否具有生物学意义以及内源性强心苷是否发挥生理作用。为了实现这一目标，将在已知会增加内源性强心苷（例如 ACTH 诱导的高血压）水平的条件下对表达哇巴因抗性 α2 或 α3 亚型的基因工程小鼠进行分析。如果耐哇巴因小鼠的反应与野生型动物不同，这将表明内源性哇巴因具有生理作用。然而，如果具有哇巴因抗性α2或α3亚型的小鼠与野生型动物的反应相似，这将偏离内源性强心苷具有生理意义的假设。 (2) 使用组织特异性敲除来定义 alpha2 同工型的具体作用。我们已经培育出 α2 同工型基因侧翼为 loxP 位点的小鼠，并将这些小鼠与携带受心脏特异性启动子控制的 Cre 重组酶的动物交配。我们现在将 alpha2 同工型 loxP 动物与在平滑肌启动子控制下表达 Cre 重组酶的动物交配。这些交配将提供仅在心脏或平滑肌中缺失α2亚型的动物，这将使我们能够研究该亚型在心血管功能中的具体作用。