Alpha-2 Na+ Pumps, [Ca2+], Arterial Contraction & Hypertension

描述（由申请人提供）：过量的膳食盐是高血压的常见原因，但盐升高血压（BP）的机制尚未解决。大量证据表明，盐潴留促进内源性哇巴因（EO），肾上腺皮质激素的分泌。EO激活动脉平滑肌细胞（ASMC）和神经元中的Ca 2+信号通路，增强血管张力并升高BP。该途径涉及α 2/α 3 Na+泵抑制，以及经由Na/Ca交换器-1（NCX 1）和TRPC 6通道增加的Ca 2+进入。这些蛋白质的表达在几种形式的高血压中增加，并且通过用哇巴因而不是地高辛长期处理培养的ASMCs来模拟。本项目的目的是关注急性和慢性哇巴因-Na+泵相互作用以及Na+泵在Ca 2+信号通路中的特定作用。 目标1：确定盐敏感性高血压小鼠和α 2 Na+泵表达改变的小鼠体内动脉肌细胞基础胞浆[Ca 2 +]（[Ca 2 +]CYT）、肌源性张力（MT）和激动剂诱发的ASMC和内皮反应是否发生改变。将在表达平滑肌中遗传编码的Ca 2+生物传感器的麻醉小鼠中同时体内测量BP和动脉[Ca 2 +]CYT和直径。这些数据将与来自这些小鼠的分离的加压小动脉的平行Ca 2+成像和收缩实验相关。目的2：研究哇巴因高亲和结合Na+泵的功能效应。几种强心类固醇（CTS）的结构-活性关系将通过测量其模拟或拮抗（如地高辛）纳摩尔哇巴因在盐依赖性高血压中升高[Ca 2 +]CYT、增强肌源性张力和影响BP的作用的能力来研究。目的3：确定人（h）ASMC α 2 Na+泵是否像α 2在啮齿动物中那样介导哇巴因增强的Ca 2+信号传导。我们将测试地高辛是否也增强新鲜解离的hASMCs中的Ca 2+信号传导，和/或地高辛或其他CTS是否拮抗哇巴因对Ca 2+信号传导的作用。我们还将确定长期哇巴因处理对原代培养的hASMCs中α 2、NCX 1和TRPC 6蛋白表达的影响，以及这种影响是否被地高辛和某些其他CTS拮抗。

DESCRIPTION (provided by applicant): Excess dietary salt is a common cause of hypertension, but the mechanism(s) by which salt elevates blood pressure (BP) are unresolved. Extensive evidence indicates that salt retention promotes the secretion of endogenous ouabain (EO), an adrenocortical hormone. EO activates a Ca2+ signaling pathway in arterial smooth muscle cells (ASMCs) and neurons that augments vascular tone and elevates BP. This pathway involves a2/a3 Na+ pump inhibition, and increased Ca2+ entry via Na/Ca exchanger-1 (NCX1) and TRPC6 channels. Expression of these proteins is increased in several forms of hypertension, and is mimicked by chronic treatment of cultured ASMCs with ouabain but not digoxin. The aims of this project focus on acute and chronic ouabain-Na+ pump interactions and the specific role of Na+ pumps in the Ca2+ signaling pathway. Aim 1: To determine if in vivo arterial myocyte basal cytosolic [Ca2+] ([Ca2+]CYT), myogenic tone (MT), and agonist-evoked ASMC and endothelial responses are altered in mice with salt-sensitive hypertension and in mice with altered a2 Na+ pump expression. BP, and arterial [Ca2+]CYT and diameter will be measured simultaneously in vivo in anesthetized mice expressing a genetically-encoded Ca2+ biosensor in smooth muscle. The data will be correlated with parallel Ca2+ imaging and contraction experiments on isolated, pressurized small arteries from these mice. Aim 2: To determine the functional effects of Na+ pump ouabain high-affinity binding. The structure-activity relationship of several cardiotonic steroids (CTS) will be investigated by measuring their ability to mimic, or antagonize (as digoxin does), the action of nanomolar ouabain in raising [Ca2+]CYT, augmenting myogenic tone, and influencing BP in salt-dependent hypertension. Aim 3: To determine whether human (h)ASMC a2 Na+ pumps mediate ouabain-augmented Ca2+ signaling, as a2 does in rodents. We will test whether digoxin also augments Ca2+ signaling in freshly-dissociated hASMCs, and/or whether digoxin or other CTS antagonize the effect of ouabain on Ca2+ signaling. We also will determine the effects of chronic ouabain treatment on a2, NCX1 and TRPC6 protein expression in primary cultured hASMCs, and whether this effect is antagonized by digoxin and certain other CTS.