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

描述(申请人提供)：过量的食盐是高血压的常见原因，但盐升高血压的机制(S)尚未解决。大量证据表明，盐滞留促进内源性哇巴因(EO)的分泌，哇巴因是一种肾上腺皮质激素。EO激活动脉平滑肌细胞(ASMC)和神经元中的钙信号通路，从而增加血管张力和升高血压。这一途径包括抑制A2/A3Na+泵，并通过Na/Ca交换器-1(NCX1)和TRPC6通道增加钙内流。这些蛋白的表达在几种形式的高血压中都会增加，并被哇巴因而不是地高辛慢性处理培养的ASMC所模拟。本项目的目标是研究急性和慢性哇巴因-Na+泵的相互作用以及Na+泵在钙信号通路中的特殊作用。目的：研究盐敏感型高血压小鼠和a2Na+泵表达改变的小鼠在体动脉心肌细胞基础胞浆[Ca~(2+)]_(Cyt)、肌张力(MT)、激动剂诱导的ASMC和内皮反应是否发生改变。在表达基因编码的钙离子生物传感器的麻醉小鼠中，将在体内同时测量血压、动脉[钙]细胞周期和直径。这些数据将与这些小鼠分离的加压小动脉上的平行钙成像和收缩实验相关联。目的：测定Na+泵哇巴因高亲和力结合的功能效应。几种强心类固醇(CTS)的构效关系将通过测量它们在盐依赖型高血压中模拟或拮抗纳米分子哇巴因(如地高辛所做的)升高[Ca+]细胞色素T、增强肌源性张力和影响血压的作用来研究。目的3：确定人(H)ASMC a~(2+)Na+泵是否像啮齿类动物的a~(2+)一样，介导哇巴因增强的钙信号。我们将测试地高辛是否也增强新鲜分离的hASMCs的钙信号，和/或地高辛或其他CTS是否拮抗哇巴因对钙信号的影响。我们还将确定慢性哇巴因处理对原代培养的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其他某些CTS所拮抗。

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.