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

描述（由申请人提供）：膳食盐过量是高血压的常见原因，但盐升高血压（BP）的机制尚未明确。大量证据表明，盐潴留促进内源性瓦巴因（EO）的分泌，这是一种肾上腺皮质激素。EO激活动脉平滑肌细胞（ASMCs）和神经元中的Ca2+信号通路，增强血管张力并升高血压。该途径涉及a2/a3 Na+泵抑制，并通过Na/Ca交换器-1 （NCX1）和TRPC6通道增加Ca2+进入。这些蛋白的表达在几种形式的高血压中增加，并且通过用瓦巴因而不是地高辛慢性治疗培养的ASMCs来模拟。本项目的目的是关注急性和慢性瓦巴因-钠离子泵的相互作用以及钠离子泵在Ca2+信号通路中的具体作用。目的1：确定体内动脉肌细胞基底细胞质[Ca2+] （[Ca2+]CYT）、肌原性张力（MT）、激动剂诱发的ASMC和内皮反应是否在盐敏感性高血压小鼠和a2 Na+泵表达改变的小鼠中发生改变。在平滑肌中表达遗传编码的Ca2+生物传感器的麻醉小鼠体内，将同时测量血压、动脉[Ca2+]CYT和直径。这些数据将与平行的Ca2+成像和从这些小鼠身上分离的加压小动脉的收缩实验相关联。目的2：确定Na+泵浦高亲和力结合的功能效应。几种强心剂类固醇（CTS）的构效关系将通过测量其模拟或拮抗（如地高辛）纳摩尔瓦巴因在盐依赖性高血压中升高[Ca2+]CYT、增强肌原性张力和影响血压的作用的能力来研究。目的3：确定人类(h)ASMC a2 Na+泵是否像a2在啮齿类动物中一样介导钙素增强的Ca2+信号。我们将测试地高辛是否也增强了新解离的hASMCs中的Ca2+信号，和/或地高辛或其他CTS是否拮抗瓦巴因对Ca2+信号的作用。我们还将确定慢性乌巴因治疗对原代培养的hASMCs中a2、NCX1和TRPC6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.