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) 2 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, 2 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 2 Na+ pump or reduced NCX1 expression, on the salt-dependent rise in BP (BP) will be determined. 2) That reduced 2 Na+ pump expression increases salt-sensitivity, and that salt- dependent BP is mediated by EO acting on 2 Na+ pumps, and by NCX1. The effects of dietary salt on plasma EO and BP will be measured in WT (2S/S) mice and mice with one null mutant 2 allele (2S/-). 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 2 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-2 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.