John M. Hamlyn

Elevated concentrations of endogenous ouabain in patients with congestive heart failure.

BackgroundAn endogenous digitalis-like compound in mammals has long been postulated, but only recently has a substance indistinguishable from ouabain been identified in human plasma. Because of the potential significance of such a substance in patients with congestive heart failure, we sought to evaluate the pathophysiology of endogenous ouabain in these individuals. Methods and ResultsUsing an immunoassay, we determined plasma ouabain concentrations in 51 patients with heart failure and in 19 control subjects. Plasma ouabain concentrations in control subjects ranged from 0.16 to 0.77 nM (mean, 0.44±0.20 nM). In 19 matched heart failure patients receiving digoxin, the mean ouabain was significantly elevated at 1.59±2.2 nM (range, 0.17–8.76 nM, p < 0.05 versus control subjects). The ouabain concentration correlated inversely with both cardiac index (r = −0.62, p < 0.005) and mean arterial pressure (r = −0.51, p < 0.05). However, there was no correlation between ouabain and left ventricular filling (r = 0.19, NS) or right atrial pressures (r = 0.20, NS). In 16 heart failure patients not receiving digoxin, the mean ouabain was 1.52±2.58 nM. No relation between renal function and ouabain was detected. ConclusionsThe unanticipated lack of correlation of ouabain with atrial pressures indicates that volume is not the chief determinant of ouabain concentration in patients with congestive heart failure. However, the significant relations of plasma ouabain concentration with cardiac index and mean arterial pressure imply that endogenous ouabain may be an important homeostatic factor in humans.

How NaCl raises blood pressure: a new paradigm for the pathogenesis of salt-dependent hypertension

Excess dietary salt is a major cause of hypertension. Nevertheless, the specific mechanisms by which salt increases arterial constriction and peripheral vascular resistance, and thereby raises blood pressure (BP), are poorly understood. Here we summarize recent evidence that defines specific molecular links between Na(+) and the elevated vascular resistance that directly produces high BP. In this new paradigm, high dietary salt raises cerebrospinal fluid [Na(+)]. This leads, via the Na(+)-sensing circumventricular organs of the brain, to increased sympathetic nerve activity (SNA), a major trigger of vasoconstriction. Plasma levels of endogenous ouabain (EO), the Na(+) pump ligand, also become elevated. Remarkably, high cerebrospinal fluid [Na(+)]-evoked, locally secreted (hypothalamic) EO participates in a pathway that mediates the sustained increase in SNA. This hypothalamic signaling chain includes aldosterone, epithelial Na(+) channels, EO, ouabain-sensitive α(2) Na(+) pumps, and angiotensin II (ANG II). The EO increases (e.g.) hypothalamic ANG-II type-1 receptor and NADPH oxidase and decreases neuronal nitric oxide synthase protein expression. The aldosterone-epithelial Na(+) channel-EO-α(2) Na(+) pump-ANG-II pathway modulates the activity of brain cardiovascular control centers that regulate the BP set point and induce sustained changes in SNA. In the periphery, the EO secreted by the adrenal cortex directly enhances vasoconstriction via an EO-α(2) Na(+) pump-Na(+)/Ca(2+) exchanger-Ca(2+) signaling pathway. Circulating EO also activates an EO-α(2) Na(+) pump-Src kinase signaling cascade. This increases the expression of the Na(+)/Ca(2+) exchanger-transient receptor potential cation channel Ca(2+) signaling pathway in arterial smooth muscle but decreases the expression of endothelial vasodilator mechanisms. Additionally, EO is a growth factor and may directly participate in the arterial structural remodeling and lumen narrowing that is frequently observed in established hypertension. These several central and peripheral mechanisms are coordinated, in part by EO, to effect and maintain the salt-induced elevation of BP.

Sodium pump α2 subunits control myogenic tone and blood pressure in mice

A key question in hypertension is: How is long‐term blood pressure controlled? A clue is that chronic salt retention elevates an endogenous ouabain‐like compound (EOLC) and induces salt‐dependent hypertension mediated by Na+/Ca2+ exchange (NCX). The precise mechanism, however, is unresolved. Here we study blood pressure and isolated small arteries of mice with reduced expression of Na+ pump α1 (α1+/−) or α2 (α2+/−) catalytic subunits. Both low‐dose ouabain (1–100 nm; inhibits only α2) and high‐dose ouabain (≥1 μm; inhibits α1) elevate myocyte Ca2+ and constrict arteries from α1+/−, as well as α2+/− and wild‐type mice. Nevertheless, only mice with reduced α2 Na+ pump activity (α2+/−), and not α1 (α1+/−), have elevated blood pressure. Also, isolated, pressurized arteries from α2+/−, but not α1+/−, have increased myogenic tone. Ouabain antagonists (PST 2238 and canrenone) and NCX blockers (SEA0400 and KB‐R7943) normalize myogenic tone in ouabain‐treated arteries. Only the NCX blockers normalize the elevated myogenic tone in α2+/− arteries because this tone is ouabain independent. All four agents are known to lower blood pressure in salt‐dependent and ouabain‐induced hypertension. Thus, chronically reduced α2 activity (α2+/− or chronic ouabain) apparently regulates myogenic tone and long‐term blood pressure whereas reduced α1 activity (α1+/−) plays no persistent role: the in vivo changes in blood pressure reflect the in vitro changes in myogenic tone. Accordingly, in salt‐dependent hypertension, EOLC probably increases vascular resistance and blood pressure by reducing α2 Na+ pump activity and promoting Ca2+ entry via NCX in myocytes.

Ouabain-induced hypertension in the rat: relationships among plasma and tissue ouabain and blood pressure.

Objectives To determine the steady-state dose-dependence of blood pressure, plasma and tissue ouabain during continuous infusion of ouabain in the rat, and to evaluate the adrenal dependence and effect of a high salt intake on this form of hypertension. Design and methods Ouabain was administered, via subcutaneous osmotic pumps, to normal and adrenalectomized male Sprague-Dawley rats for 5 weeks. Blood pressure, plasma renin and aldosterone, and circulating and tissue levels of ouabain were determined. Results Following a latent period, blood pressures and circulating ouabain were significantly elevated dose-dependently in glycoside-infused rats at 5 weeks. Upon withdrawal of the ouabain infusion, blood pressure and plasma ouabain levels normalized within 1 week. In rats that received 30

Left Ventricular Mass, Stroke Volume, and Ouabain-Like Factor in Essential Hypertension

mUg ouabain/kg per day, the circulating, kidney, hypothalamic and anterior pituitary levels of ouabain were increased significantly (by 7-, 15-, 2.8− and 2.1-fold, respectively), whereas the content of other tissues tested was unchanged. Blood pressure and plasma levels of ouabain correlated with hypothalamic and kidney glycoside content in the infused rats. High-performance liquid chromatography of the adrenal, renal, hypothalamic and pituitary extracts showed one major peak of ouabain immunoreactivity, with a retention time equivalent to that of commercial ouabain. Plasma renin activity was normal, whereas aldosterone levels were increased significantly to 2.9− and sevenfold in rats that received 10 and 30

Sodium Transport Inhibition, Cell Calcium, and Hypertension: The Natriuretic Hormone/Na+-Ca2+ Exchange/Hypertension Hypothesis

mUg ouabain/kg per day, respectively. Dietary salt loading suppressed aldosterone and did not exacerbate hypertension. In bilaterally adrenalectomized rats the ambient circulating and kidney levels of ouabain were low and ouabain infusion raised glycoside levels and blood pressure significantly. Conclusions Prolonged infusion of ouabain in the normal rat raises the circulating, kidney, hypothalamic and anterior pituitary levels and induces a reversible hypertension with normal plasma renin activity. Although characterized by raised aldosterone levels, the hypertension does not require the adrenal glands and is not salt-sensitive. This model may be useful for exploring novel mechanisms of long-term regulation of blood pressure.

The Pump, the Exchanger and Endogenous Ouabain: Signaling Mechanisms that Link Salt Retention to Hypertension

Many patients with essential hypertension (EH) exhibit increased left ventricular mass. Similarly, elevated circulating levels of an endogenous ouabainlike factor (OLF) have been described in some but not all patients with EH. Moreover, ouabain has a hypertrophic influence on isolated cardiac myocytes. Accordingly, we investigated relationships among plasma OLF, left ventricular mass, and cardiac function in patients with EH. Plasma OLF was determined in 110 normotensive subjects and 128 patients with EH. Echocardiographic parameters and humoral determinants were measured in EH. Plasma OLF levels were increased (P<0.0001) in patients with EH (377+/-19 pmol/L) versus normotensive (253+/-53 pmol/L) subjects. The distribution of plasma OLF was unimodal in normotensives, whereas it was bimodal in EH. Twenty-four-hour diastolic ambulatory blood pressure was slighter higher in EH with high OLF compared with EH with normal OLF (93.2+/-1.14 versus 89.4+/-1.33 mm Hg, P=0.03). Left ventricular mass index and stroke volume in EH with high OLF were greater than in EH with normal OLF (101.9+/-3.3 versus 86.1+/-2.5 g/m(2), P=0.0003, and 57.10+/-1.48 versus 52.30+/-1.14 mL/m(2), P=0. 02, respectively), although heart rate was slower (74.2+/-1.3 versus 80.5+/-1.3 bpm, P=0.005). Multiple regression analysis that tested the influence of body mass index, age, gender, 24-hour blood pressure, and OLF on left ventricular mass revealed independent contributions of systolic (13.2%) and diastolic (12.4%) blood pressure and plasma OLF (11.6%) to left ventricular mass. We conclude that approximately 50% of patients with uncomplicated EH have elevated-high circulating OLF levels, higher diastolic blood pressure, greater left ventricular mass and stroke volume, and reduced heart rate. We propose that the OLF affects cardiovascular function and structure and should be considered as a factor that contributes to the risk of morbid events.

Ouabain-like Factor Quantification in Mammalian Tissues and Plasma Comparison of Two Independent Assays

Sodium plays a critical role in the etiology of essential hypertension, but the mechanism by which excess dietary sodium actually leads to the elevation of blood pressure is not understood. The hypothesis described shows how an excessive sodium load can lead to the development of hypertension. The underlying factor must be a genetic or acquired deficiency or limitation in renal sodium excretion that may be undetectable by standard renal function tests. The resultant tendency towards sodium, water, and extracellular fluid volume expansion is compensated by the secretion of a natriuretic hormone that promotes sodium excretion by inhibiting sodium pumps in the kidney tubule cells. The hormone also inhibits sodium pumps in other cells, including vascular smooth muscle cells, causing intracellular sodium to increase. Then, because the vascular smooth muscle cells contain a Na+-Ca2+ exchange transport system in their plasma membranes, more calcium than normal is delivered to these cells. This causes the increased contractility and reactivity that underlies the increased vascular tone and peripheral vascular resistance that elevates the blood pressure.

Endogenous ouabain and hemodynamic and left ventricular geometric patterns in essential hypertension

The central roles of salt (NaCl) and the kidneys in the pathogenesis of most forms of hypertension are well established.1,2 The linkage between NaCl retention and blood pressure (BP) elevation is often referred to as “whole body autoregulation.”3,4 Surprisingly, however, the precise mechanisms that underlie this linkage (ie, the signaling pathway) have escaped elucidation. Here, we examined the evidence that endogenous ouabain (EO), Na+ pumps (Na,K-ATPase), and the Na/Ca exchanger (NCX) are critical molecular mechanisms in this pathway. At constant cardiac output (CO), mean arterial BP=CO×TPR (where TPR is total peripheral vascular resistance).5 In most (chronic) hypertension, in humans and animals, the CO is relatively normal, and the high BP is maintained by an elevated TPR.1,4 TPR is controlled dynamically by vasoconstriction/dilation in small “resistance” arteries, which exhibit tonic constriction (“tone”). This can be studied in isolated, cannulated small arteries that develop spontaneous (myogenic) tone (MT),6 under constant or increasing intraluminal pressure. Indeed, the level of tone in isolated arteries “is often comparable to that observed in the same vessels in vivo,”6 and may even be used to predict BP changes7 (see below). MT is triggered by Ca2+ entry, primarily through voltage-gated Ca2+ channels in arterial smooth muscle (SM; ASM) cells,6 and contraction is activated by the rise in cytosolic Ca2+ concentration ([Ca2+]CYT).8 In NaCl-dependent hypertension, the enhanced vasoconstriction and increased tone and TPR are, at least in part, functional and reversible phenomena.9 Numerous mechanisms contribute to the regulation of myocyte [Ca2+]CYT and vasoconstriction, but the plasma membrane (PM) NCX provides an unique, direct link between Na+ and [Ca2+]CYT and, thus, Ca2+ signaling and contraction in ASM cells.10 NCX-mediated Ca2+ transport is …

Angiotensin II Stimulates Secretion of Endogenous Ouabain From Bovine Adrenocortical Cells via Angiotensin Type 2 Receptors

The resolution of controversies that concern the detectability of an endogenous ouabain-like factor (OLF) in mammalian tissues and plasma was approached by the application of a standardized method for its extraction and quantification. Two independent assays were used to quantify the OLF: (1) a radioimmunoassay, which used a polyclonal anti-ouabain antiserum, and (2) a radioenzymatic assay based on the inhibition of dog kidney Na+,K+-ATPase. Plasma and tissues were obtained from the Milan hypertensive strain (MHS) and the Milan normotensive strain (MNS) of rats and from healthy human volunteers. Results indicate that (1) a single high-performance liquid chromatography (HPLC) fraction identical to that of ouabain was identified by both assay methods in the rat hypothalamus and hypophysis and in both rat and human plasma; (2) dilution curves of OLF and standard ouabain were parallel and with a similar Kd, both in radioimmunoassay (3 nmol/L) and ATPase assay (14 nmol/L); (3) after HPLC, OLF was similarly quantified by the two methods in the hypothalamus, hypophysis, adrenals, and plasma of rats and in human plasma; (4) OLF was present in larger amounts in the hypothalamus, hypophysis, and plasma of MHS rats than that of MNS rats; (5) the HPLC fraction of human plasma was quantified similarly by both assays (range, 60 to 150 pmol/L); (6) recovery of standard ouabain in pre-HPLC plasma extracts was approximately 90%; and (7) pre-HPLC OLF concentrations in human plasma ranged between 0.05 and 0.75 nmol/L. Rat cerebral tissues and both rat and human plasma contained measurable amounts of OLF, which were quantified similarly by radioimmunoassay and ATPase assay, both before and after HPLC fractionation. The increased MHS tissue and plasma levels of OLF are in keeping with the pathogenetic role of this factor in MHS hypertension.