Ouabain, Na+ Pumps, and Ca2+ Signaling in Human Arteries

描述（由申请人提供）：盐潴留通常被认为是人类高血压（BP）的最常见原因。三分之一的美国成年人患有高血压（高血压），这可能导致更严重的并发症，如心力衰竭和中风。因此，重要的是要了解盐潴留与人类高血压之间的联系机制，以更好地控制甚至预防这种疾病。 最近的研究已经确定了盐与啮齿动物高血压之间联系的途径中的几个关键步骤。首先，盐潴留促进肾上腺皮质激素，内源性哇巴因（EO）的分泌。然后，EO特异性结合并抑制动脉平滑肌细胞（ASMC）哇巴因敏感的α 2 Na+泵，而更丰富的哇巴因耐药的α 1泵不受影响。α 2 Na+泵特异性地定位于覆盖肌浆网（SR）的质膜（PM）微区;因此哇巴因仅增加PM和SR之间的微小“连接空间”中的局部Na+浓度。Na+/Ca 2+交换器-1（NCX 1）将细胞Na+交换为细胞外Ca 2+，其也位于PM-SR连接处，增加Ca 2+保留。这反过来又提高了血管张力和外周血管阻力，从而升高了血压。 在人类身上也可以看到类似的啮齿动物机制。然而，将啮齿动物机制转化为人类的一个主要问题是，人类中的主要（“管家”）a1 Na+泵是哇巴因敏感的。因此，尚不清楚a1和/或a2 Na+泵是否参与了人体哇巴因依赖性BP升高。我的目标是检验这样一个假设，即在人类ASMCs（hASMCs）中，纳摩尔哇巴因抑制α 2 Na+泵而不是α 1 Na+泵会增强Ca 2+信号传导，就像在啮齿动物ASMCs中一样。我将通过首先确定什么样的Na+泵a（催化）亚基亚型在hASMCs中表达以及它们的相对丰度来检验这一假设（目的1）。接下来，我将通过对原代hASMC进行高分辨率共聚焦免疫细胞化学来确定Na+泵a亚型如何在hASMC PM中定位/分布（目的2）。最后，我将确定是否低剂量（1-10 nM）哇巴因增加Ca 2+信号响应血管收缩剂（5-羟色胺，ATP，和高K+）使用Fura-2（目标3）。如果观察到增强，我将通过脂质体转染具有哇巴因抗性（OR）或显性阴性（DN）大鼠α 2 Na+泵构建体的hASMCs来确定这种效应是否是由于α 1或α 2 Na+泵。通过这些实验验证我的假设将提供第一个直接证据，证明啮齿动物中盐依赖性高血压的关键分子机制在人类中存在并发挥类似的功能。 公共卫生相关性：该项目的目标是确定将过量盐与啮齿动物血压升高联系起来的特定机制是否也存在于人类中。我计划通过测试人类动脉细胞中啮齿动物机制的已识别成分来实现这一目标。我预计，这些结果将提供第一个直接证据，证明啮齿动物盐依赖性高血压的潜在关键分子机制在人类中的功能相似。

DESCRIPTION (provided by applicant): Salt retention is generally regarded as the most common cause of high blood pressure (BP) in humans. One in three US adults has high BP (hypertension), which can lead to more serious complications such as heart failure and stroke. Therefore, it is important to understand the mechanisms that link salt retention to hypertension in humans in an effort to better control, or even prevent this disease. Recent studies have identified several key steps in the pathway linking salt to hypertension in rodents. First, salt retention promotes secretion of the adrenocortical hormone, endogenous ouabain (EO). EO then specifically binds and inhibits arterial smooth muscle cell (ASMC) ouabain-sensitive a2 Na+ pumps, while the more abundant ouabain-resistant a1 pumps are unaffected. The a2 Na+ pumps localize specifically to plasma membrane (PM) microdomains overlying the sarcoplasmic reticulum (SR); thus ouabain increases only the local Na+ concentration in the tiny "junctional spaces" between the PM and SR. The exchange of cell Na+ for extracellular Ca2+ by the Na+/Ca2+ exchanger-1 (NCX1), which is also located at PM-SR junctions, increases Ca2+ retention. This in turn raises vascular tone and peripheral vascular resistance, and thereby elevates BP. Parallels of the rodent mechanism can be seen in humans. One major issue, however, with translating the rodent mechanism to humans is that the predominant ("housekeeping") a1 Na+ pumps in humans are ouabain-sensitive. Therefore, it is not clear whether the a1 and/or a2 Na+ pumps are involved in ouabain-dependent BP increases in humans. My goal here is to test the hypothesis that in human ASMCs (hASMCs), inhibition of a2 Na+ pumps, and not a1 Na+ pumps, by nanomolar ouabain augments Ca2+ signaling, as in rodent ASMCs. I will test this hypothesis by first determining what Na+ pump a (catalytic) subunit isoforms are expressed in hASMCs and what is their relative abundance (Aim 1). Next, I will determine how the Na+ pump a isoforms are localized/distributed in hASMC PM by performing high-resolution confocal immunocytochemistry on primary hASMCs (Aim 2). Finally, I will determine whether low dose (1-10 nM) ouabain augments Ca2+ signaling in response to vasoconstrictors (serotonin, ATP, and high K+) using fura-2 (Aim 3). If augmentation is seen, I will determine whether this effect is due to a1 or a2 Na+ pumps by lipofectamine transfection of hASMCs with either an ouabain-resistant (OR) or dominant negative (DN) rat a2 Na+ pump construct. Verification of my hypothesis via these experiments will provide the first direct evidence that key molecular mechanisms that underlie salt-dependent hypertension in rodents are present and function similarly in humans. PUBLIC HEALTH RELEVANCE: The goal of this project is to determine whether the specific mechanisms that link excess salt to elevated blood pressure in rodents also exist in humans. I plan to achieve this goal by testing for the identified components of the rodent mechanism in human arterial cells. I anticipate that the results will provide the first direct evidence that the underlying key molecular mechanisms in salt-dependent hypertension in rodents function similarly in humans.