NA-PUMP ISOFORM-SPECIFIC REGULATION OF VASCULAR FUNCTION

描述（改编自应用程序）：Na+-K+ ATP酶的表达 (NKA)a亚基同种型（a-同种型）是组织特异性的，发育上 调节，并在激素和神经控制下，但生理 NXA α-同种型的基础在很大程度上是未知的。我们的总体假设是 NKA亚型的相对分布和亚细胞位置以及 总NKA活性对于调节血管平滑肌是关键的 功能我们建议阐明NIKA α-同种型在调节细胞凋亡中的作用。 血管平滑肌收缩和新陈代谢的影响。我们的长期目标是 为了将其生理功能的知识扩展到疾病状态， 如高血压和糖尿病，其中NKA的相对表达 α-同种型被改变。我们的整体战略采用最近开发的A1和A2 同种型敲除（KO）小鼠。我们建议开发新的小鼠模型，使用 SMP-8平滑肌特异性启动子。这些老鼠可以杂交繁殖， 科斯提供额外的重要模型，具有广泛的总NKA 活性和α-同种型分布。我们将把a-异构体与血管 在细胞、血管和整个动物水平上发挥作用， NKA活性、α-亚型表达和血管功能之间的关系。 具体目标1。为了确定NKA α-亚型及其相对的 表达对血管收缩性和心血管功能的影响。我们 假设收缩性受到α 2同种型的强烈影响。 将比较主动脉、门静脉和阻力血管的收缩性， 整个动物的心血管参数。 具体目标2。为了定量NKA α-同种型的亚细胞定位， 相关表达与Ca 2 t和Na+-离子处理血管 平滑肌细胞我们将使用免疫组织学技术， 用于NKA α-亚型的亚细胞定位的3D显微成像 与特定的房室标志物相比。胞浆和亚细胞[Ca 2 +]i 和[Na+]i将使用荧光染料技术进行评估。申请人 将测试离子稳态和收缩性改变是由于 特异性NKA同种型表达和定位。 具体目标3。为了确定NKA α-同种型及其 血管代谢和异构体特异性能量学的相对表达。 耗氧量和乳酸盐产生的测量将用于 基于其在体内的ATP利用率， 对哇巴因抑制的不同敏感性。申请人将测试 假设这些异构体是独立调节的， 观察到的血管平滑肌激活的葡萄糖敏感性是 NKA α亚型分布的结果。

DESCRIPTION (Adapted from the application): The expression of the Na+-K+ ATPase (NKA) a subunit isoforms (a-isoforms) is tissue specific, developmentally regulated, and under hormonal and neurogenic control, but the physiological basis for NXA a-isoforms is largely unknown. Our overall hypothesis is that the relative distribution and subcellular location of NKA isoforms as well as the total NKA activity are critical to regulation of vascular smooth muscle function. We propose to elucidate the role of NIKA a-isoforms in the regulation of vascular smooth muscle contractility and metabolism. Our long-term goal is to extend this knowledge of their physiological function to disease states, such as hypertension and diabetes, in which the relative expression of NKA a-isoforms is altered. Our overall strategy employs recently developed a 1 & a2 isoform knockout (KO) mice. We propose to develop new mouse models, using the SMP-8 smooth muscle specific promoter. These mice can be crossbred and bred to the KOs to provide additional important models with wide ranges of total NKA activity and a-isoform distribution. We will correlate a-isoform with vascular function at cellular, vessel and whole animal levels to quantify the relations between NKA activity, a-isoform expression and vascular function. Specific Aim 1. To determine the efftcts of NKA a-isoforms and their relative expression on vascular contractility and cardiovascular function. Our hypothesis is that contractility is strongly affected by the a2 isoform. Contractility in aorta, portal vein and resistance vessels will be compared to cardiovascular parameters in the whole animal. Specific Aim 2. To quantify the subcellular localization of NKA a-isoforms and correlate the relative expression with Ca2t and Na+-ion handling in vascular smooth muscle cells. We will use, immunohistological techniques coupled with 3D-microscopic imaging for the subcellular localization of NKA a-isoforms compared to specific compartmental markers. Cytosolic and subcellular [Ca2+]i and [Na+]i will be assessed using fluorescent dye technology. The applicant will test the hypothesis that altered ion homeostasis and contractility are due to specific NKA isoform expression and localization. Specific Aim 3. To determine the effect of NKA alpha-isoforms and their relative expression on vascular metabolism and isoform-specific energetics. Measurements of oxygen consumption and lactate production will be used to access the in vivo ATP utilization of each alpha-isoform based on their differential sensitivity to inhibition by ouabain. The applicant will test the hypotheses that these isoforms are independently regulated and that the observed glucose-sensitivity of activation of vascular smooth muscle is a consequence of NKA alpha-isoform distribution.