Calcium and Sodium Transport in Hypertension

说明(申请人提供)：盐滞留和随之而来的血浆容量膨胀不可避免地会导致血压(BP)升高，在慢性状态(高血压)下，血压升高是通过增加总外周血管阻力(TPR)来维持的。令人惊讶的是，盐分滞留增加TPR和BP的“信号通路”尚未解决。我们的目标是利用小鼠模型阐明盐沉积与高血压之间的特定分子机制，因为盐依赖型高血压在人类和啮齿动物中是相似的。我们将利用药理学工具和基因工程小鼠来检验这一假说：i)内源性哇巴因(EO)，ii)通过哇巴因敏感的a2催化亚基抑制动脉Na+泵的活性，以及iii)通过动脉Na-Ca交换器-1(NCX1)增加Ca~(2+)内流是该信号通路的关键步骤。具体目标涉及三个亚假说：1)转基因G蛋白偶联受体激酶-4[GRK4？(A486V)]小鼠的盐依赖性高血压是由eO、A2 Na+泵和NCX1介导的。非转基因GRK4？日粮盐对血浆EO和BP的影响。和盐敏感的GRK4？(A486V)小鼠。将确定DigiBind(哇巴因结合抗体)、PST-2238(哇巴因拮抗剂)和SEA0400(NCX1阻滞剂)以及下调2 Na+泵或降低NCX1表达对盐依赖的BP升高的影响。2)a2Na+泵的表达减少增加了对盐的敏感性，而盐依赖的？BP是通过作用于a2Na+泵的EO和NCX1介导的。饮食盐对WT(A2S/S)小鼠和具有一个突变A2等位基因(A2S/-)的小鼠血浆EO和BP的影响将被测量。DigiBind、PST-2238、SEA0400和降低NCX1表达的作用将在盐依赖的BP上进行测试。3)平滑肌(Sm)特异性NCX1.3过度表达引起的盐敏感性与哇巴因的高敏感性相关，并随着a2 Na+泵丰度的降低而增强。将在WT小鼠和过表达smNCX1.3(NCX1.3smTg/TG)的小鼠中测量饮食盐对血浆EO和BP的影响；DigiBind和PST-2238以及下调sm-A2泵表达的效果将在盐依赖的BP上进行测试。哇巴因和血管紧张素II输注对WT、NCX1.3smTg/Tg和sm特异性NCX1基因敲除小鼠血压的影响将进行比较。这些结果将阐明一些将盐与高血压联系起来的关键机制，将为人类研究提供路线图，并将确定新的治疗靶点。 与公共卫生相关：该项目的目标是确定摄入过量盐会引起血压升高的具体机制。我们将使用转基因小鼠来研究将盐与高血压联系起来的途径中的拟议步骤，在这些步骤中，这些步骤被增强或抑制。这些结果将阐明在人类和啮齿动物中参与这一盐-高血压途径的关键机制，将为人类研究提供路线图，并将确定药物治疗的新靶点。

DESCRIPTION (provided by applicant): Salt retention and consequent plasma volume expansion inevitably cause elevation of blood pressure (BP) which, in the chronic state (hypertension), is sustained by increased total peripheral vascular resistance (TPR). Surprisingly, the "signaling pathway" by which salt retention increases TPR and BP is unresolved. Our goal is to elucidate the specific molecular mechanisms that link salt retention to hypertension using mouse models because salt-dependent hypertension is similar in humans and rodents. We will employ pharmacological tools and genetically-engineered mice to test the hypothesis that: i) Endogenous ouabain (EO), ii) Inhibited activity of arterial Na+ pumps with a (ouabain-sensitive, S) a2 catalytic subunit, and iii) Increased Ca2+ entry via arterial Na-Ca exchanger-1 (NCX1) are key steps in the signaling pathway. The Specific Aims address three sub-hypotheses: 1) That salt-dependent hypertension in mice with transgenic G-protein coupled receptor kinase-4 [GRK4?(A486V)] is mediated by EO, a2 Na+ pumps, and NCX1. The effects of dietary salt on plasma EO and BP will be measured in non-transgenic GRK4? and salt-sensitive GRK4?(A486V) mice. The effects of Digibind (antibodies that bind ouabain), PST-2238 (ouabain antagonist) and SEA0400 (NCX1 blocker), and of knock-down of a2 Na+ pump or reduced NCX1 expression, on the salt-dependent rise in BP (?BP) will be determined. 2) That reduced a2 Na+ pump expression increases salt-sensitivity, and that salt-dependent ?BP is mediated by EO acting on a2 Na+ pumps, and by NCX1. The effects of dietary salt on plasma EO and BP will be measured in WT (a2S/S) mice and mice with one null mutant a2 allele (a2S/-). The effects of Digibind, PST-2238, SEA0400 and reduced NCX1 expression will be tested on the salt-dependent ?BP. 3) That salt sensitivity due to smooth muscle (sm)-specific NCX1.3 overexpression correlates with hypersensitivity to ouabain and is augmented by reduced a2 Na+ pump abundance. The effects of dietary salt on plasma EO and BP will be measured in WT mice and mice that overexpress smNCX1.3 (NCX1.3smTg/Tg); the effects of Digibind and PST-2238, and of knock-down of sm-a2 pump expression, will be tested on salt-dependent ?BP. The effects of ouabain and angiotensin II infusion on BP will be compared in WT, NCX1.3smTg/Tg, and sm-specific NCX1 knockout mice. The results will elucidate some key mechanisms that link salt to hypertension, will provide a roadmap for human studies, and will pinpoint new therapeutic targets. PUBLIC HEALTH RELEVANCE: The goal of this project is to determine the specific mechanisms by which ingestion of excess salt raises blood pressure. We will study proposed steps in the pathway that links salt to hypertension by using genetically-modified mice in which these steps are augmented or suppressed. The results will elucidate key mechanisms that participate in this salt-hypertension pathway in humans as well as in rodents, will provide a roadmap for human studies, and will identify new targets for drug therapy.