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.

Public Health Relevance

Hypertension is prevalent in adults in industrialized societies, and is a major risk factor for serious cardiac and vascular pathologies. Excessive dietary salt and the tendency to salt retention is a common cause of hypertension. This project is designed to elucidate some specific mechanisms in the pathway linking salt to the elevation of blood pressure.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Hypertension and Microcirculation Study Section (HM)
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OH, Youngsuk
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University of Maryland Baltimore
Schools of Medicine
United States
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Hamlyn, John M; Blaustein, Mordecai P (2016) Endogenous Ouabain: Recent Advances and Controversies. Hypertension 68:526-32
Lanzani, Chiara; Gatti, Guido; Citterio, Lorena et al. (2016) Lanosterol Synthase Gene Polymorphisms and Changes in Endogenous Ouabain in the Response to Low Sodium Intake. Hypertension 67:342-8
Blaustein, Mordecai P; Chen, Ling; Hamlyn, John M et al. (2016) Pivotal role of ?2 Na+ pumps and their high affinity ouabain binding site in cardiovascular health and disease. J Physiol 594:6079-6103
Chen, Ling; Song, Hong; Wang, Youhua et al. (2015) Arterial ?2-Na+ pump expression influences blood pressure: lessons from novel, genetically engineered smooth muscle-specific ?2 mice. Am J Physiol Heart Circ Physiol 309:H958-68
Hamlyn, John M; Manunta, Paolo (2015) Endogenous cardiotonic steroids in kidney failure: a review and an hypothesis. Adv Chronic Kidney Dis 22:232-44
Song, Hong; Karashima, Eiji; Hamlyn, John M et al. (2014) Ouabain-digoxin antagonism in rat arteries and neurones. J Physiol 592:941-69
Blaustein, Mordecai P (2014) Why isn't endogenous ouabain more widely accepted? Am J Physiol Heart Circ Physiol 307:H635-9
Hamlyn, John M; Linde, Cristina I; Gao, Junjie et al. (2014) Neuroendocrine humoral and vascular components in the pressor pathway for brain angiotensin II: a new axis in long term blood pressure control. PLoS One 9:e108916
Song, Hong; Thompson, Scott M; Blaustein, Mordecai P (2013) Nanomolar ouabain augments Ca2+ signalling in rat hippocampal neurones and glia. J Physiol 591:1671-89
Hamlyn, John M; Blaustein, Mordecai P (2013) Salt sensitivity, endogenous ouabain and hypertension. Curr Opin Nephrol Hypertens 22:51-8

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