Functional Studies of the Na,K-ATPase

描述(由申请人提供)：本申请建议继续我们对Na，K-ATPase的研究，并针对几个正在进行的目标。其中之一是测试这种酶的α2或α3亚型的心脏糖苷结合部位是否发挥生物学作用，这种酶在物种之间高度保守。这些研究将为许多实验室观察到的内源性心脏糖苷(如内源性哇巴因)是否具有生理学意义提供洞察力。另一个目标是通过分析组织特异性的α2亚型敲除动物来确定α2亚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-ATPase的心脏糖苷结合部位是否具有生物学意义，以及内源性心脏糖苷是否发挥生理作用。为了做到这一点，表达哇巴因耐药α2或α3亚型的基因工程小鼠将在已知增加内源性心脏糖苷水平的条件下进行分析，例如ACTH诱导的高血压。如果哇巴因抗性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，如果携带哇巴因耐药α2或α3亚型的小鼠的反应类似于野生型动物，这将削弱内源性心脏糖苷具有生理意义的假设。(2)利用组织特异性基因敲除来确定α2亚型的特定作用。我们已经培育出了alpha2亚型基因两侧有loxP位点的小鼠，并在心脏特异性启动子的控制下将这些基因与携带Cre重组酶的动物交配。现在，我们将在平滑肌启动子的控制下，将α2亚型loxP动物与那些表达Cre重组酶的动物配对。这些配对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。

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.