Plasma Endogenous Ouabain Research Articles

Abstract 419: Central Angiotensin II Increases Circulating Endogenous Ouabain, Arterial Myocyte Na/Ca Exchanger Expression and Blood Pressure via Brain Aldosterone and Mineralocorticoid Receptors

The brain, together with altered arterial function, plays a crucial role in the pathogenesis of hypertension. We have suggested that endogenous ouabain (EO), and its actions on arterial Na + and Ca 2+ transporters, are key in some forms of hypertension; these entities may be controlled by brain mechanisms (review: Blaustein et al., Am J Physiol Heart Circ Physiol 302: H1031-49, 2012). One commonly-used hypertension model involves infusion of angiotensin II (Ang II) in rodents. When Ang II was infused (subcutaneous minipump, 400 ng/kg/min x 15 days) into 8 normal ∼35 g C57Bl/6 mice, 24 hr mean blood pressure (MBP in mm Hg, telemetry), was 119±4, vs 97±2 in 8 vehicle-infused controls, C ( P <0.001). Expression of Na/Ca exchanger-1 (NCX1, immunoblots with R3F1 antibody) was increased by 171±21% ( P <0.001 vs C ) in the aortas of the Ang II-treated mice. To test the role of the brain in Ang II-induced hypertension, vehicle ( C ; n = 7) or Ang II ( A ; n = 8) was administered by intra-cerebroventricular infusion (Ang II, 3.6 μg/day x 12 days) in 200-250 g Wistar rats. In some rats, the Ang II infusion contained the mineralocorticoid receptor (MR) blocker, eplerenone ( E , 5 μg/day x 12 days; n = 7), or the aldosterone synthase inhibitor, FAD-286 ( F , 25 μg/day x 12 days; n = 8). At the end of the treatment, the following MBPs were recorded (mm Hg, telemetry): C : 101±2; A : 123±4 ( P <0.05 vs C, E & F ); E : 108±2; F : 112±2 ( P <0.05 vs C ). Upon euthanasia, blood, pituitary and adrenal samples were taken for EO radioimmunoassay (RIA), and aortas were harvested for NCX1 immunoblotting. The following results were obtained: 1) Plasma EO (nM): C , 0.34±0.07; A , 0.58±0.08 ( P <0.05 vs C ; P <0.01 vs F ); E , 0.37±0.08; F , 0.30±0.05. The plasma EO increase in A was confirmed by off-line liquid chromatography-RIA on pooled samples. 2) NCX1 expression (normalized to C = 1.0±0.2): A , 2.6±0.3 ( P <0.01 vs C ; P <0.05 vs E ); E , 0.9±0.3; F , 1.3±0.2. 3) Adrenal and pituitary EO were not changed. These results provide the first evidence that circulating EO levels and arterial NCX expression are regulated by the brain Ang II-aldosterone-MR system. Thus, in addition to regulating BP by direct, neurally-mediated vasoconstriction, the brain also influences BP by modulating ion transport pathways involved in arterial Na + and Ca 2+ homeostasis.

Left Ventricular Structure and Function in Relation to Steroid Biosynthesis Genes in a White Population

Both endogenous ouabain (EO) and aldosterone are steroid hormones which might play a role in the pathogenesis of left ventricular (LV) hypertrophy and cardiac remodeling. Cholesterol side-chain cleavage enzyme (CYP11A1) and 3β-hydroxysteroid dehydrogenase (HSD3B1) are two key enzymes in the pathway of steroid biosynthesis. We investigated in 532 individuals (mean age, 50.3 years; 51.5% women) randomly recruited from a white European population whether LV structure and function were related to genetic variations in CYP11A1 and HSD3B1. We measured LV structure by conventional echocardiography and LV diastolic function by Doppler imaging of the transmitral blood flow and the mitral annular movement. We genotyped tag single nucleotide polymorphisms (SNPs) rs2279357, rs11638442 and rs2073475 in CYP11A1, and rs2236780, rs3765945, and rs6203 in HSD3B1. While adjusting for covariables and accounting for family clusters, LV mass index decreased (P ≤ 0.049) across the CYP11A1 genotypes in rs2279357 (CC vs. CT vs. TT), rs11638442 (GG vs. GC vs. CC), and rs2073475 (GG vs. GA+AA). Carriers of the CYP11A1 TCG haplotype had lower (P ≤ 0.017) LV mass and LV mass index than noncarriers. Carriers of HSD3B1 GCC haplotype had lower peak early (Ea; P = 0.004) and higher peak late (Aa; P = 0.066) diastolic mitral annular velocities and therefore a lower Ea/Aa ratio (P = 0.041) as compared with noncarriers. Neither plasma endogenous ouabain nor 24-h urinary aldosterone were related to any of the SNPs or haplotypes (P ≥ 0.07). Pending confirmation in other studies, LV mass and LV diastolic function seem to be related to genetic variation in the steroid biosynthesis.

Endogenous ouabain and the renin–angiotensin–aldosterone system: distinct effects on Na handling and blood pressure in human hypertension

To evaluate whether the renin-angiotensin-aldosterone system (RAAS) and endogenous ouabain system differently affect renal Na handling and blood pressure. Three hundred and one patients in whom we compared blood pressure, and renal Na tubular reabsorption in the basal condition and 2 h (T120) after saline infusion. Following multivariate-adjusted linear and quartiles analysis, baseline mean blood pressure (MBP) was significantly higher (113.7 ± 1.33 mmHg) in the fourth versus the first endogenous ouabain quartile (103.8 ± 1.04 mmHg) and the trend across the quartiles was highly significant (β = 0.23, P = 3.53e-04). In contrast, an inverse relationship was present in the renin activity (PRA) quartiles with MBP highest in the first (112.5 ± 1.26) and lowest in the fourth PRA quartile (107.6 ± 1.48, P = 0.039). Following an acute saline load, changes in MBP and the slope of the pressure-natriuresis relationship were inversely related across the PRA quartiles. The fractional excretion of sodium (FENa) showed a negative linear trend going from the first to the third endogenous ouabain quartiles (2.35 ± 0.17 and 1.90 ± 0.14%, P = 0.05). Patients in the fourth endogenous ouabain quartile (>323 pmol/l) showed increased FENa T120 (2.78 ± 0.18%, P < 0.01) and increased Na tubular rejection fraction (P = 0.007) after Na load. After the saline load, there was a biphasic relationship between plasma endogenous ouabain and FENa favoring Na retention at low endogenous ouabain and Na excretion at high endogenous ouabain levels. The RAAS and endogenous ouabain system are two independent and complementary systems having an inverse (RAAS) or a direct (endogenous ouabain system) relationship with hemodynamic parameters.

Endogenous Ouabain: A Link Between Sodium Intake and Hypertension

The sodium pump, an ancestral enzyme with conserved ability to bind ouabain, plays a key role in salt conservation and is regulated by aldosterone and endogenous ouabain (EO). Plasma EO is elevated in about 45% of patients with essential hypertension and correlates with blood pressure. The relationship of EO with Na(+) balance is complex. Na(+) depletion raises circulating EO, whereas acute saline loads have no effect on EO in essential hypertension, and ambient levels of EO are unrelated to the saline sensitivity of blood pressure. Short-term periods of high dietary salt elevate EO and the relationship with salt balance in normal individuals is V-shaped, whereas the long-term relationship is likely to be L-shaped. Normal individuals suppress the high EO transient triggered by high-salt diets and avoid hypertension. In contrast, patients with elevated EO on normal Na(+) intakes have hypertension related to poor modulation of EO biosynthesis, clearance, or both.

The Ouabain-Binding Site of the 2 Isoform of Na,K-ATPase Plays a Role in Blood Pressure Regulation During Pregnancy

The cardiotonic steroid/ouabain-binding site of the α subunit of Na,K-ATPase is thought to play an important role in cardiovascular homeostasis. Previously, we demonstrated the cardiotonic steroid-binding site of the α2 Na,K-ATPase is involved in adrenocorticotropic hormone (ACTH)-induced hypertension by using gene-modified α2(R/R) mice in which the cardiotonic steroid-binding site is relatively resistant to ouabain compared to the ouabain-sensitive wild-type α2(S/S) mice. To further explore the importance of this site in the cardiovascular system, we investigated blood pressure regulation during pregnancy in mice with the α2(R/R) isoform. The systolic blood pressure (SBP) of the α2(S/S) and α2(R/R) mice was measured before and during pregnancy by tail-cuff. The expression of the α isoforms of Na, K-ATPase in various tissues and plasma endogenous ouabain contents were assessed prior to pregnancy as well as days 7 and 17 of gestation. The α2(S/S) mice showed a gradual decrease in the SBP during the first two trimesters, followed by an increase above the preconceptional level in the third trimester. However, the α2(R/R) mice exhibited a lower blood pressure in the third trimester. The cardiac expression of the α2 Na,K-ATPase in the α2(S/S) mice was significantly less than that of the α2(R/R) mice throughout the pregnancy. The plasma endogenous ouabain concentration significantly increased by twofold at day 17 of pregnancy in the α2(R/R) mice but not in the α2(S/S) mice. The cardiotonic steroid-binding site of the α2 Na,K-ATPase plays a role in maintaining normal SBP during pregnancy.

Pregnancy and Pseudopregnancy: Physiological States with High Endogenous Ouabain, Coexistent Suppression of Maternal Arterial Myocyte NCX and α2 Na+ Pumps, and Resistance to Ouabain‐Induced Hypertension

Endogenous ouabain (EO) is elevated and correlates with blood pressure (BP) in ~50% of patients with essential hypertension. “Ouabain‐like” inhibitors are increased in pre‐eclampsia (PE) and correlate with BP. EO and ouabain inhibit the α2‐Na+ pump isoform in arterial myocytes, enhance Ca2+ entry via NCX1.3, and raise BP. We explored whether EO per se is elevated in pregnancy and pseudopregnancy (PSP) and determined whether exogenous ouabain induces PE in otherwise normal pregnant rats. We implanted mini‐osmotic pumps containing ouabain in pregnant (E10), non‐pregnant, and PSP rats and measured BP by tail cuff. At E20 or equivalent, blood, adrenals, and aortas were collected to determine ouabain (RIA), ouabain‐like materials (RRA), NCX, and α2‐Na+ pump expression (Western blot). Plasma EO increased in pregnant (4.7 ± 1.9 nM plasma equiv) vs non‐pregnant (0.3 ± 0.02 nM plasma equiv, p&lt;0.001) rats and was confirmed by HPLC‐RIA and mass spectrometry. Adrenal EO content was lower in pregnant (12.3 ± 5.0 nmoles/kg) vs non‐pregnant rats (32.5 ± 15.3 nmoles/kg, p&lt;0.01). NCX and α2‐Na+ pump expression decreased in pregnancy. Ouabain infusion raised BP in non‐pregnant rats (+21.3 mmHg, p&lt;0.01), but had no effect on BP in their pregnant or PSP counterparts. These data show: 1) that the pregnant and PSP conditions are physiologically high EO states; 2) both conditions are associated with the development of a physiological resistance to ouabain that prevents BP from increasing. 3) The mechanism of the ouabain resistance is exclusively maternal, and likely related to decreased expression of, and reduced Ca2+ entry by, vascular NCX. Failure of the maternal circulation to develop resistance to EO may underlie hypertension in PE.

Selective association of endogenous ouabain with subclinical organ damage in treated hypertensive patients

According to previous studies endogenous ouabain (EO) closely correlates with high blood pressure, congestive heart failure and kidney disease in humans. Our aims were to analyse associations between plasma, urinary EO level and various markers of cardiovascular damage in treated hypertensive patients. Forty-one adult patients with hypertension and/or diabetes mellitus (DM) and/or chronic kidney disease (CKD) were studied. We assessed plasma and urinary EO, pro-brain natriuretic peptide and catecholamines, profile of ambulatory blood pressure monitor and cardiovascular status by echocardiography and echo-tracking. The highest level of plasma EO (19.7±9.5 pmol l⁻¹) was measured in hypertensive patients with DM and CKD. The nighttime mean arterial blood pressure independently correlated with the level of plasma EO (P=0.004), while independent predictor of the β-stiffness of carotid artery was the urinary EO (P=0.011). Elevated level of EO was associated with nighttime blood pressure and subclinical organ damage in treated hypertensive patients, suggesting possible role of EO in the pathogenesis of impaired diurnal blood pressure rhythm and arterial stiffness.

Highlights From The Literature

Highlights From The Literature

Endogenous Ouabain in Ménière's Disease

Endogenous Ouabain (EO) has been demonstrated to modulate the activity of Na+, K+ -ATPase. Our purpose was to measure plasma levels of EO in Ménière's Disease (MD) subjects as a possible predisposing factor to developing and maintaining hydrops. Case-control study. University hospital. Thirty-nine MD subjects and 29 controls with a lifetime negative history for vertigo and dizziness. Plasma levels of EO. Plasma EO in MD subjects was in the range between 33 and 504 pmol/L (median, 135.5 pmol/L), whereas in the control group, plasma EO varied between 70 and 724 pmol/L (median, 205 pmol/L). The Mann-Whitney U test detected a statistically significant difference (p = 0.0001). Low plasma levels of EO have been proposed to augment Na-K pump activity, whereas high EO levels show an inhibitory effect on the pump activity. A proper pump activity may be necessary to keep the right ionic amount and osmolarity in endolymph. Although other possibilities may be considered, we suggest that altered control mechanisms of pump activity may be related to the pathogenesis and maintenance of MD.

Physiological roles of endogenous ouabain in normal rats

Endogenous ouabain (EO)-like compounds are synthesized in and released from the adrenal gland. Although EO has been implicated in several pathological states such as hypertension and heart and kidney failure, its physiological roles in normal animal have not been elucidated. To address this issue, we studied the effects of reduction in plasma EO resulting from antiouabain antibody administration. Normal rats were treated for 28 days with antiouabain antibodies or rabbit IgG as control. Infusions were delivered through a jugular vein cannula by osmotic pumps, and blood pressure was monitored by tail-cuff plethysmography. The animals were housed in metabolic cages to measure water and food consumption and urine excretion. After 28 days, the thoracic aorta was isolated and used to study phenylephrine-induced contraction and atrial natriuretic peptide (ANP)-induced vasorelaxation. The adrenal gland cortex was enlarged in the antiouabain antibody-treated rats. Moreover, on the second day of treatment, there was a significant transient reduction in natriuresis in the antiouabain antibody-treated rats, suggesting that EO is a natriuretic hormone. Reduction in natriuresis was also observed when EO levels were reduced by active immunization resulting from sequential injection of ouabain-albumin. Furthermore, following 28 days of treatment, the response to phenylephrine was significantly lowered and that to ANP was significantly increased in aortic rings from antiouabain antibody-treated rats. These findings show for the first time that circulatory ouabain plausibly originating in the adrenal has physiological roles controlling vasculature tone and sodium homeostasis in normal rats.

Left ventricular geometry and endogenous ouabain in a Flemish population

Endogenous ouabain, an endogenous digitalis-like steroid, is synthesized in the adrenal glands and possibly in the hypothalamus. In vitro and in vivo, endogenous ouabain triggers growth and proliferation of cardiomyocytes. We therefore investigated the association between left ventricular structure and function and plasma endogenous ouabain in a general population. We randomly recruited 536 individuals from a general population (50.7% women, mean age 53.1 years). Measurements included echocardiographic left ventricular structure and function, blood pressure, plasma endogenous ouabain, and the 24-h urinary excretion of sodium. The geometric mean plasma endogenous ouabain was 95.5 pmol/l (interquartile range 79.4-120.2 pmol/l). We expressed effect sizes for a 1-SD increase in plasma endogenous ouabain (0.21 on the logarithmic scale), while accounting for important covariables. For a 1-SD increment in plasma endogenous ouabain, SBP, left ventricular posterior wall, the interventricular septum, and relative wall thickness increased by 1.59 mmHg (P = 0.009), 0.138 mm (P = 0.003), 0.152 mm (P = 0.013), and 0.71 x 10 (P = 0.008), respectively. In a sensitivity analysis, involving 431 individuals aged 29-71 years (10-90th percentile interval), a 1-SD increase in plasma endogenous ouabain was associated with opposite trends in ejection fraction (+0.90%, P = 0.005) and left ventricular systolic longitudinal strain (-0.48%, P = 0.011). Moreover, in individuals whose sodium excretion was above median (160 mmol/24 h), the aforementioned associations reached a higher level of statistical significance. Our population-based study suggested that endogenous ouabain might have a trophic effect on the myocardium, independent of blood pressure and other covariables. The clinical implication of these findings remains to be elucidated.

Steroid Biosynthesis and Renal Excretion in Human Essential Hypertension: Association With Blood Pressure and Endogenous Ouabain

Endogenous ouabain (EO) has been linked with long-term changes in sodium balance and cardiovascular structure and function. The biosynthesis of EO involves, cholesterol side-chain cleavage (CYP11A1), 3-beta-hydroxysteroid dehydrogenase (HSD3B) with sequential metabolism of pregnenolone and progesterone. Furthermore, the renal excretion of cardiac glycosides is mediated by the organic anion transporter (SLCO4C1) at the basolateral membrane and the P-glycoprotein (PGP) (encoded by MDR1) at the apical membrane of the nephron. Average 24-h ambulatory blood pressures were recorded in 729 untreated essential hypertensives. Aldosterone (Aldo), EO, urinary Na+, and K+ excretions were determined. Single-nucleotide polymorphism (SNP) and haplotype-based association study was performed with a total of 26 informative SNPs. Plasma EO was significantly directly related to both day (r = 0.131, P < 0.01) and nighttime diastolic blood pressure (DBP) (r = 0.143, P < 0.01), and remained significantly related after correction for confounders (sex, body mass index, age). Genotype analysis for EO levels and daytime DBP gave significant results for CYP11A1 rs11638442 and MDR1 rs1045642 (T/C Ile1145) in which the minor allele tracked with higher EO levels (T/T 210.3 (147-272) vs. C/C 270.7 (193-366) pmol/l, P < 0.001). Association was found between HSD3B1 polymorphisms and/or haplotypes with blood pressure (systolic blood pressure (SBP) 140.3 (11.7) vs. 143.8 (11.2) mm Hg, P < 0.01) and plasma Aldo (P < 0.05). Haplotype-based analyses support the data of SNP analysis. Among patients with essential hypertension, cholesterol side-chain cleavage and MDR1 loci are related to circulating EO and DBP, most likely by influencing EO synthesis and transmembrane transport, respectively. In contrast, variants in HSD3B1 are related with SBP probably via Aldo.

Relationships among endogenous ouabain, α-adducin polymorphisms and renal sodium handling in primary hypertension

The basolateral Na pump drives renotubular reabsorption. In cultured renal cells, mutant adducins, as well as sub-nanomolar ouabain concentrations, stimulate the Na-K pump. To determine whether these factors interact and affect Na handling and blood pressure (BP) in vivo, we studied 155 untreated hypertensive patients subdivided on the basis of their plasma endogenous ouabain or alpha-adducin genotype (ADD1 Gly460Trp-rs4961). Under basal conditions, proximal tubular reabsorption and plasma Na were higher in patients with mutated Trp ADD1 or increased endogenous ouabain (P = 0.002 and 0.05, respectively). BPs were higher in the high plasma endogenous ouabain group (P = 0.001). Following volume loading, the increment in BP (7.73 vs. 4.81 mmHg) and the slopes of the relationship between BP and Na excretion were greater [0.017 +/- 0.002 vs. 0.009 +/- 0.003 mmHg/(muEq min)] in ADD1 Trp vs. ADD1 Gly carriers (P < 0.05). BP changes were similar, whereas the slopes of the relationship between BP and Na excretion were lower [0.016 +/- 0.003 vs. 0.008 +/- 0.002 mmHg/(muEq min)] in patients with low vs. high endogenous ouabain (P < 0.05). In patients with high endogenous ouabain, volume loading increased the BP in the ADD1 Trp group but not in the Gly group (P < 0.05). Thus, patients with ADD1 Trp alleles are sensitive to salt and tubular Na reabsorption remains elevated after volume expansion. With saline loading, BP changes are similar in high and low endogenous ouabain patients, whereas tubular Na reabsorption increases in the high endogenous ouabain group. Saline loading unmasks differences in renal Na handling in patients with mutant adducin or high endogenous ouabain and exposes an interaction of endogenous ouabain and Trp alleles on BP.

Endogenous ouabain and cardiomyopathy in dialysis patients

Endogenous ouabain (EO) is markedly raised in patients with chronic renal failure. As high EO induces myocardial cell hypertrophy in vitro and it is associated with left ventricular hypertrophy (LVH) in essential hypertensives and in patients with heart failure we investigated the relationship between plasma EO and LV mass and geometry in 156 end-stage renal disease (ESRD) patients. EO was measured by a specific radioimmunoassay and by mass spectrometry. On univariate analysis, plasma EO was directly related to LV mass (r = 0.26, P = 0.001) and LV end diastolic volume (r = 0.25, P = 0.002) and these relationships held true in multiple linear regression models including a series of potential confounders. Patients with eccentric LVH (n = 41, i.e. 26%) had the highest plasma levels of EO when compared to patients with other patterns of LV geometry (P = 0.001). Furthermore, plasma EO had diagnostic value for eccentric LVH because the area under the corresponding ROC curve (68%) was significantly greater (P = 0.002) than the threshold of diagnostic indifference. In this analysis, the sensitivity was 91% and the specificity was 36%. The positive predictive value was 33% but EO had a remarkably high negative predictive value (92%) for the exclusion of eccentric hypertrophy. In ESRD patients, plasma EO is independently associated with LV mass, LV volume and eccentric LVH. The results of this study are compatible with the hypothesis that EO is involved in alterations of LV mass in ESRD.

Salt intake and depletion increase circulating levels of endogenous ouabain in normal men

High-salt diets elevate circulating Na+ pump inhibitors, vascular resistance, and blood pressure. Ouabain induces a form of hypertension mediated via the alpha2-Na+ pump isoform and the calcium influx mode of the vascular sodium calcium exchanger (NCX). Whereas elevated levels of an endogenous ouabain (EO) and NCX have been implicated in salt-sensitive hypertension, acute changes in sodium balance do not affect plasma EO. This study investigated the impact of longer-term alterations in sodium balance on the circulating levels and renal clearance of EO in normal humans. Thirteen normal men consumed a normal diet, high-salt diet, and hydrochlorothiazide (HCTZ), each for 5-day periods to alter sodium balance. EO and other humoral and urinary variables were determined daily. On a normal diet, urinary sodium excretion (140 +/- 16 meq/day), plasma EO (0.43 +/- 0.08 nmol/l) and urinary EO excretion (1.04 +/- 0.13 nmol/day) were at steady state. On the 3rd day of a high-salt diet, urine sodium excretion (315 +/- 28 meq/day), plasma EO (5.8 +/- 2.2 nmol/l), and the urinary EO excretion (1.69 +/- 0.27 nmol/day) were significantly increased, while plasma renin activity and aldosterone levels were suppressed. The salt-evoked increase in plasma EO was greater in older individuals, in subjects whose baseline circulating EO was higher, and in those with low renal clearance. During HCTZ, body weight decreased and plasma renin activity, aldosterone, and EO (1.71 +/- 0.77 nmol/l) rose, while urinary EO excretion remained within the normal range (1.44 +/- 0.31 nmol/day). Blood pressure fell in one subject during HCTZ. HPLC of the plasma extracts showed one primary peak of EO immunoreactivity with a retention time equivalent to ouabain. High-salt diets and HCTZ raise plasma EO by stimulating EO secretion, and a J-shaped curve relates sodium balance and EO in healthy men. Under normal dietary conditions, approximately 98% of the filtered load of EO is reabsorbed by the kidney, and differences in the circulating levels of EO are strongly influenced by secretion and urinary excretion of EO. The dramatic impact of high-salt diets on plasma EO is consistent with its proposed role as a humoral vasoconstrictor that links salt intake with vascular function in hypertension.

How does salt retention raise blood pressure?

A critical question in hypertension research is: How is long-term blood pressure controlled? Excessive NaCl ingestion or NaCl retention by the kidneys and the consequent tendency toward plasma volume expansion lead to hypertension. Nevertheless, the precise mechanisms linking salt to high blood pressure are unresolved. The discovery of endogenous ouabain, an adrenocortical hormone, provided an important clue. Ouabain, a selective Na+ pump inhibitor, has cardiotonic and vasotonic effects. Plasma endogenous ouabain levels are significantly elevated in approximately 40% of patients with essential hypertension and in animals with several forms of salt-dependent hypertension. Also, prolonged ouabain administration induces hypertension in rodents. Mice with mutant Na+ pumps or Na/Ca exchangers (NCX) and studies with a ouabain antagonist and an NCX blocker are revealing the missing molecular mechanisms. These data demonstrate that alpha2 Na+ pumps and NCX1 participate in long-term regulation of vascular tone and blood pressure. Pharmacological agents or mutations in the alpha2 Na+ pump that interfere with the action of ouabain on the pump, and reduced NCX1 expression or agents that block NCX all impede the development of salt-dependent or ouabain-induced hypertension. Conversely, nanomolar ouabain, reduced alpha2 Na+ pump expression, and smooth muscle-specific overexpression of NCX1 all induce hypertension. Furthermore, ouabain and reduced alpha2 Na+ pump expression increase myogenic tone in isolated mesenteric small arteries in vitro, thereby tying these effects directly to the elevation of blood pressure. Thus, endogenous ouabain, and vascular alpha2 Na+ pumps and NCX1, are critical links between salt and hypertension. New pharmacological agents that act on these molecular links have potential in the clinical management of hypertension.

Independent and incremental prognostic value of endogenous ouabain in idiopathic dilated cardiomyopathy

Increased circulating levels of endogenous ouabain (EO) have been observed in some heart failure patients, but their long term clinical significance is unknown. This study investigated the prognostic value of EO for worsening heart failure among 140 optimally treated patients (age 50+/-14 years; 104 male; NYHA class 1.9+/-0.7) with idiopathic dilated cardiomyopathy. Plasma EO was determined by RIA and by liquid chromatography mass spectrometry, values were linearly correlated (r = 0.89) in regression analysis. During follow-up (13+/-5 months), heart failure progression was defined as worsening clinical condition leading to one or more of the following: sustained increase in conventional therapies, hospitalization, cardiac transplant, or death. NYHA functional class, age, LVEF, peak VO2 and plasma levels of EO were predictive for heart failure progression. Heart failure worsened 1.5 fold (HR: 1.005; 95% CI: 1.001-1.007; p<0.01) for each 100 pmol/L increase in plasma EO. Moreover, those patients with higher plasma EO values had an odds ratio of 5.417 (95% CI: 2.044-14.355; p<0.001) for heart failure progression. Following multivariate analysis, LVEF, NYHA class and plasma EO remained significantly linked with clinical events. This study provides the first evidence that circulating EO is a novel, independent and incremental marker that predicts the progression of heart failure.

A New Antihypertensive Agent that Antagonizes the Prohypertensive Effect of Endogenous Ouabain and Adducin

Endogenous Ouabain (EO) and Adducin enhance the Na-K pump function and play an important role in sodium homeostasis and blood pressure (BP) regulation. In the general population, plasma EO modulates BP either by inhibiting the prohypertensive effect of an excessive salt intake or counteracting the depressor action of normal-moderate salt intake. Almost 50% of hypertensive patients have increased circulating plasma levels of EO. EO has been associated both to left ventricular dysfunction and hypertrophy. A new antihypertensive agent, PST2238, (17beta-(3-furyl)-5beta-androstan-3beta, 14beta, 17alpha-triol a digitoxigenin derivative) able to selectively antagonize both the EO and adducin prohypertensive and molecular effects, has been developed. In hypertensive rats (MHS strain) carrying both adducin mutations and increased plasma EO and in ouabain-infused rats (OS), PST2238 lowers BP by normalizing the renal Na-K pump function. In OS rats, PST antagonized the cardiac and renal pro-hypertrophic ouabain effect associated to the activation of the Src-EGFr-ERK(1/2) signaling cascade. Phase 1 clinical studies demonstrated a high tolerability of PST2238. In a preliminary phase 2 study on 42 mild never-treated hypertensive patients, PST2238 given for 3 months at 0.5 mg/day, significantly reduced BP in subjects with moderate salt intake, implying that it may be selectively effective in conditions where EO plays a prohypertensive role. In conclusion, PST2238, because of its peculiar action mechanism, represents a new tool to disentangle the complex relationship between salt intake, genetic control of renal sodium handling and EO effect.

High circulating levels of endogenous ouabain in the offspring of hypertensive and normotensive individuals

Impaired diastolic function and left ventricular hypertrophy can occur early in the natural history of essential hypertension. High circulating levels of endogenous ouabain (EO) have been described in essential hypertension and have also been associated with left ventricular hypertrophy. The aim of this study was to investigate whether these cardiac modifications are related to plasma EO levels in the offspring of hypertensive families. The study involved 41 subjects with (FAM+) and 45 subjects without (FAM-) a family history of hypertension. Arterial blood pressure, left ventricular geometry and function, and plasma EO levels were measured in each subject. Plasma EO levels were higher in the FAM+ subjects (221.5 +/- 10.95 versus 179.6 +/- 9.58 pmol/l, P = 0.004), and directly correlated with both systolic (r = 0.417, P < 0.0001) and diastolic blood pressure (r = 0.333, P = 0.002). Plasma EO was inversely related to an index of cardiac diastolic function determined as the ratio between the early and late peak flow velocity (r = -0.286, P = 0.012) and isovolumetric relaxation time (IVRT) (r = 0.32, P = 0.003). The IVRT was also significantly higher in FAM+, correlated with the IVRT (r = 0.32, P = 0.003). The IVRT was also significantly higher in FAM+, whereas the other echocardiographic parameters were similar to FAM-. Among the offspring of families with a positive history of hypertension, circulating EO levels and blood pressure are increased. Plasma EO levels are related to alterations of some indexes of diastolic heart function in these individuals.

Endogenous Ouabain and Acute Salt Loading in Low-Renin Hypertension

Endogenous ouabain (EO), which is structurally identical to the cardiac glycoside ouabain, has been isolated in human plasma. The substance EO has been implicated in states of volume expansion and in some types of arterial hypertension, especially as a factor of salt sensitivity of blood pressure. On the other hand, salt sensitivity has been described in low-renin hypertension. The aim of this study was to determine the response of plasma EO to acute sodium expansion in low-renin hypertension. We performed an acute intravenous sodium load (2 L of saline in 4 h) in 13 hypertensive subjects with low renin values (<0.65 ng/mL/h). We measured EO in plasma extracts by a radioimmunoassay and compared it with other endocrine parameters including atrial natriuretic peptide (ANP), aldosterone (ALDO), cortisol (CORT), adrenocorticotropic hormone (ACTH), and plasma renin activity (PRA). We found that EO showed only a mild nonsignificant decrease after saline infusion (from 938.8+/-218.8 pmol/L to 781.4+/-181.4 pmol/L ouabain equivalents), whereas ALDO and PRA significantly decreased (P<.0001; P<.05 respectively). The ANP significantly increased, as a marker of effective volume expansion (P<.01). At the end of the infusion, we found that EO was positively related to ACTH levels and to ALDO changes from baseline. Our results do not support the hypothesis that EO is stimulated in low-renin hypertension by acute salt-volume expansion. ACTH could be a factor modulating EO secretion in this condition.