NA-PUMP ISOFORM-SPECIFIC REGULATION OF VASCULAR FUNCTION

描述(改编自本申请)：Na+-K+ATPase的表达 (Nka)亚基异构体(a-异构体)在发育过程中具有组织特异性。 调节的，在荷尔蒙和神经性控制下，但生理 NXAα-亚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定位 NKA总活性对血管平滑肌的调节至关重要 功能。我们建议阐明Nika a-异构体在调节中的作用。 血管平滑肌的收缩能力和新陈代谢。我们的长期目标是 为了将这种关于它们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 A-异构体发生改变。我们的整体战略采用了最近开发的1&a2 同型基因敲除(KO)小鼠。我们建议开发新的鼠标模型，使用 SMP-8为平滑肌特异性启动子。这些小鼠可以被杂交和饲养来 KO将提供更多具有广泛NKA总量的重要型号 活度和α-异构体分布。我们将a-亚型与维管束联系起来 在细胞、血管和整个动物水平上的功能，以量化关系 NKA活性、α-亚型表达与血管功能之间的关系。 具体目的1.测定NKAα-亚型及其相关亚型的作用 血管收缩功能和心血管功能的表达。我们的 假设收缩能力受到a2亚型的强烈影响。 主动脉、门静脉和阻力血管的收缩能力将与 整个动物的心血管参数。 具体目标2.量化NKAα-亚型亚细胞定位和 血管内钙离子和钠离子处理与相关基因表达的关系 平滑的肌肉细胞。我们将使用免疫组织学技术结合 NKAα亚型亚细胞定位的三维显微成像研究 与特定的隔间标记进行比较。胞浆和亚细胞内[Ca~(2+)]i 和[Na+]i将使用荧光染料技术进行评估。申请人 将检验离子动态平衡和收缩能力改变的假设 特异性NKA异构体的表达和定位。 具体目的3.确定NKAα亚型及其亚型的作用 血管代谢和异构体特异性能量学的相对表达。 耗氧量和乳酸产量的测量将用于 基于每个α-异构体的体内ATP利用情况 哇巴因抑制的差异敏感性。申请者将测试 假设这些异构体是独立调节的，并且 观察到的血管平滑肌激活的葡萄糖敏感性是一种 NKAα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.