Ouabain, Local, Ca2+ Control & Myogenic Tone
Most rat models of salt-dependent hypertension are associated with elevated plasma levels of endogenous ouabain (EO, an adrenocortical hormone), and prolonged administration of a high-salt diet to humans raises plasma EO levels. Moreover, chronic ouabain administration induces hypertension in rodents. These and related observations indicate that ouabain-induced hypertension (OH) is a good model for salt-dependent hypertension.
The steps leading from ouabain (or EO) to high blood pressure are unknown. This project addresses the hypothesis that specific arterial smooth muscle Na+ pumps with high ouabain affinity (α2 Na+ pumps in vascular smooth muscle) and the Na/Ca exchanger (NCX) play key roles in the pathogenesis of salt-dependent hypertension. The NCX translates ouabain’s inhibitory action on the α2 Na+ pumps into modulation of cytosolic Ca2+ and control of arterial contractility and myogenic tone. The proposed sequence of steps is illustrated in Figure 1.
Proposed sequence of steps that link renal salt retention to BP elevation. Interventions discussed in this proposal that test the hypothesis are shown at the left. ACTH, adrenocorticotropic hormone; Na+ pump α2R/R, ouabain-resistant α2 Na+ pump; Na+ pump α+/-, α2 subunit heterozygous null mutant; NCX, Na/Ca exchanger (type 1/1.3 is expressed in vascular smooth muscle); +/-, heterozygous null mutant; SM-/-, smooth muscle-specific knockout; Tg/Tg, smooth muscle-specific overexpressor (see Blaustein et al., 2006; Iwamoto et al., 2004; Zhang et al., 2005).
Project 1 has four Specific Aims
- For Aim 1, we are characterizing the Ca2+ (and Na+) entry mechanisms, especially those mediated by store-operated channels (SOCs) and by NCX, in small resistance arteries. We are also determining how these transporters participate in Ca2+ homeostasis and the control of myogenic tone (see video 1 below). Early results indicate that a small, but critical, component of myogenic tone depends upon NCX-mediated Ca2+ entry (Iwamoto et al., 2004; Zhang et al., 2005; Zhang et al., 2006).
- For Aim 2, we are testing the hypothesis that genetically or pharmacologically reduced activity of Na+ pumps with α2 (but not α1) subunits (the two isoforms expressed in mesenteric artery myocytes) modulates intracellular Ca2+ and myogenic tone in small arteries (see Video 2 below). The idea that certain agents with anti-hypertensive activity may interfere with ouabain’s action on these Na+ pump α2 subunits also is being explored. The initial evidence that supports these ideas has recently been published (Zhang et al., 2005; Blaustein et al., 2006).
- For Aim 3, we are testing the hypothesis that smooth muscle NCX mediates the effects of reduced activity of Na+ pumps with α2 subunits on cytosolic Ca2+ and myogenic tone. We are exploring the idea that these effects of ouabain can be abrogated by agents with antihypertensive activity that block NCX. The initial data supporting these ideas was recently published (Iwamoto et al., 2004; Zhang et al., 2005).
- For Aim 4, we are testing the hypothesis that chronic in vivo ouabain administration (manifested as OH) and acute in vitro ouabain administration have similar effects on the mechanisms that control arterial contractility and myogenic tone in small arteries. Rat and mouse pressurized small mesenteric arteries loaded with Ca2+ and Na+ indicators are being used to study ion concentration changes (confocal and widefield microscopy), membrane potential and myogenic tone simultaneously. Transgenic mice with altered Na+ pump and NCX genes, and novel anti-hypertensive agents that directly block ouabain’s action or that selectively block NCX, are being used to identify specific steps in the sequence from ouabain to altered arterial contractility. The results of these studies are enhancing our understanding of the pathogenesis of salt-dependent hypertension and are pinpointing new targets for innovative anti-hypertensive therapy.
The results of these studies are enhancing our understanding of the pathogenesis of salt-dependent hypertension and are pinpointing new targets for innovative anti-hypertensive therapy.
Blaustein,M.P., J. Zhang, L. Chen, and B.P. Hamilton. How does salt retention raise blood pressure? Am. J. Physiol., Regul., Integr., Comp. Physiol. 290:R514-R523 (2006).
Iwamoto, T., S. Kita, J. Zhang, M.P. Blaustein, Y. Arai, S. Yoshida, K. Wakimoto, I. Komuro & T. Katsuragi. Salt-sensitive hypertension is triggered by Ca2+ entry via Na+/Ca2+ exchanger type-1 in vascular smooth muscle. Nature Med. 10:1193-1999 (2004).
Zhang, J., M-Y. Lee, M. Cavalli, L. Chen, R. Berra-Romani, C.W. Balke, G. Bianchi, P. Ferrari, J.M. Hamlyn, T. Iwamoto, J.B. Lingrel, D.R. Matteson, W.G. Wier, and M.P. Blaustein. Sodium pump alpha-2 subunits control myogenic tone and blood pressure in mice. J. Physiol. 569:243-256 (2005).
Zhang J., R. Berra-Romani , M.J. Sinnegger-Brauns, J. Striessnig , M.P. Blaustein, D.R. Matteson. Role of Cav1.2 L-type Ca2+ channels in vascular tone: effects of nifedipine and Mg2+. Am. J. Physiol. Heart Circ. Physiol. 292:H415-H425 (2006).
Iwamoto, T., Y. Watanabe, and M.P. Blaustein. Na+/Ca2+ exchange inhibitors: A new class of calcium regulators. Cardiovasc. Hematol. Disorders-Drug Targets, 7:188-198 (2007).
Blaustein, M.P., and W.G. Wier. Editorial. Local Sodium, Global Reach. Filling the gap between salt and hypertesnion. Circ. Res. 101:959-961 (2007).
Blaustein, M.P., D.J. Weber, and T. Charpentier. Commentary. Getting a grip on calcium regulation. PNAS 104:18349-18350 (2007).
Blaustein, M.P., J. Zhang, L. Chen, H. Song, H. Raina, S.P. Kinsey, M. Izuka, T. Iwamoto, M.I. Kotlikoff, J.B. Lingrel, K.D. Philipson, W.G. Wier, J.M. Hamlyn. The pump, the exchanger, and endogenous ouabain: signaling mechanisms that link salt retention to hypertension. Hypertension 53:291-298 (2009).
Cytosolic Ca2+ fluctuations in individual arterial smooth muscle cells during development of myogenic tone. Fluorescent images are tangential sections through the wall of an intact, pressurized mesenteric small artery. Myocytes were loaded with the Ca2+-sensitive fluorochrome, fluo-4.
Nanomolar ouabain-induced increase in cytosolic Ca2+ concentrations [Ca2+]CYT in individual arterial smooth muscle cells of an intact artery with myogenic tone. Fluorescent (Fluo-4) images are longitudinal sections through the middle of an intact, pressurized mesenteric small artery. The ouabain-induced rise in [Ca2+]CYT (increased fluorescence) is followed by vasoconstriction (movement of the artery wall to the right) (see Iwamoto et al., 2004; Zhang et al., 2005).