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

描述(申请人提供)：盐滞留通常被认为是人类高血压(BP)的最常见原因。每三个美国成年人中就有一个患有高血压，这会导致更严重的并发症，如心力衰竭和中风。因此，为了更好地控制甚至预防这种疾病，了解盐沉积与人类高血压之间的联系机制是很重要的。最近的研究已经确定了将盐与啮齿类动物的高血压联系起来的几个关键步骤。首先，盐滞留促进了肾上腺皮质激素内源性哇巴因(EO)的分泌。然后，EO特异性地结合和抑制动脉平滑肌细胞(ASMC)对哇巴因敏感的A2 Na+泵，而更丰富的对哇巴因敏感的A1泵不受影响。A2 Na+泵仅定位于肌浆网(SR)上的质膜(PM)微区；因此哇巴因只增加PM和SR之间微小“连接空间”的局部Na+浓度。同样位于PM-SR连接处的Na+/Ca~(2+)交换器-1(NCX1)将细胞内的Na+交换为细胞外的Ca~(2+)，增加了细胞内的Ca~(2+)滞留。这反过来会增加血管张力和外周血管阻力，从而升高血压。在人类身上也可以看到啮齿动物机制的相似之处。然而，将啮齿动物的机制转化为人类的一个主要问题是，人类中占主导地位的A1 Na+泵是哇巴因敏感的。因此，目前尚不清楚A1和/或A2钠泵是否与哇巴因依赖性血压升高有关。我在这里的目标是检验这样一个假设，即在人ASMCs(HASMCs)中，纳米分子哇巴因抑制a2 Na+泵，而不是A1 Na+泵，会增强钙信号，就像在啮齿动物ASMCs中一样。我将通过首先确定在hASMC中表达哪些Na+泵A(催化)亚单位亚基以及它们的相对丰度来检验这一假设(目标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亚型在hASMC PM中的定位/分布(目标2)。最后，我将使用Fura-2(目标3)确定低剂量(1-10 NM)哇巴因是否在血管收缩药(5-羟色胺、三磷酸腺苷和高K+)的反应中增强钙信号。如果观察到增强，我将确定这种作用是由于通过脂质体转导具有哇巴因抗性(OR)或显性负性(DN)的hASMCs的a1或a2Na+泵所致。通过这些实验验证我的假设将提供第一个直接证据，证明啮齿动物盐依赖型高血压背后的关键分子机制在人类中存在，并具有类似的功能。 公共卫生相关性：该项目的目标是确定将过量盐分与啮齿动物血压升高联系起来的具体机制是否也存在于人类身上。我计划通过测试人类动脉细胞中已识别的啮齿动物机制组件来实现这一目标。我预计，这些结果将提供第一个直接证据，证明啮齿类动物盐依赖高血压的潜在关键分子机制在人类中具有类似的功能。

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.