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Background: Endogenous ouabain (EO) and atrial natriuretic peptide (ANP) are important in regulation of sodium and fluid balance. There is indirect evidence that ANP may be involved in the regulation of endogenous cardenolides., Methods: H295R are human adrenocortical cells known to release EO. Cells were treated with ANP at physiologic concentrations or vehicle (0.1% DMSO), with or without guanylyl cyclase inhibitor 1,2,4 oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). Cyclic guanosine monophosphate (cGMP), the intracellular second messenger of ANP, was measured by a chemiluminescent immunoassay and EO was measured by radioimmunoassay of C18 extracted samples., Results: EO secretion is inhibited by ANP treatment, with the most prolonged inhibition (90 min vs ≤ 60 min) occurring at physiologic ANP concentrations (50 pg/mL). Inhibition of guanylyl cyclase with ODQ, also reduces EO secretion. The inhibitory effects on EO release in response to cotreatment with ANP and ODQ appeared to be additive., Conclusions: ANP inhibits basal EO secretion, and it is unlikely that this is mediated through ANP-A or ANP-B receptors (the most common natriuretic peptide receptors) or their cGMP second messenger; the underlying mechanisms involved are not revealed in the current studies. The role of ANP in the control of EO synthesis and secretion in vivo requires further investigation., Competing Interests: I have read the journal’s policy and Dr. El-Mallakh is on the speakers’ bureaus of Eisai, Indivior, Intra-Cellular Therapies, Janssen, Lundbeck, Noven, Otsuka, Sunovion, and Teva. The other authors of this manuscript do not have any competing interest.

Accumulating evidence obtained over the last three decades has revealed a neuroendocrine system in the brain that mediates long term increases in blood pressure. The system involves distinct ion transport pathways including the alpha-2 isoform of the Na,K pump and epithelial sodium channels, as well as critical hormone elements such as angiotensin II, aldosterone, mineralocorticoid receptors and endogenous ouabain. Activation of this system either by circulating or central sodium ions and/or angiotensin II leads to a cascading sequence of events that begins in the hypothalamus and involves the participation of several brain nuclei including the subfornical organ, supraoptic and paraventricular nuclei and the rostral ventral medulla. Key events include heightened aldosterone synthesis and mineralocorticoid receptor activation, upregulation of epithelial sodium channels, augmented synthesis and secretion of endogenous ouabain from hypothalamic magnocellular neurons, and sustained increases in sympathetic outflow. The latter step depends upon increased production of angiotensin II and the primary amplification of angiotensin II type I receptor signaling from the paraventricular nucleus to the rostral ventral lateral medulla. The transmission of sympathetic traffic is secondarily amplified in the periphery by increased short- and long-term potentiation in sympathetic ganglia and by sustained actions of endogenous ouabain in the vascular wall that augment expression of sodium calcium exchange, increase cytosolic Ca 2+ and heighten myogenic tone and contractility. Upregulation of this multi-amplifier system participates in forms of hypertension where salt, angiotensin and/or aldosterone are elevated and contributes to adverse outcomes in heart failure., Competing Interests: Declaration of Competing Interest None., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In this brief review we discuss some aspects of the Na + pump and its roles in mediating the effects of ouabain and endogenous ouabain (EO): i) in regulating the cytosolic Ca 2+ concentration ([Ca 2+ ] CYT ) via Na/Ca exchange (NCX), and ii) in activating a number of protein kinase (PK) signaling cascades that control a myriad of cell functions. Importantly, [Ca 2+ ] CYT and the other signaling pathways intersect at numerous points because of the influence of Ca 2+ and calmodulin in modulating some steps in those other pathways. While both mechanisms operate in virtually all cells and tissues, this article focuses primarily on their functions in the cardiovascular system, the central nervous system (CNS) and the kidneys., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Rationale & Objective: Studies of humans and animals have suggested that endogenous ouabain (EO) and related genes are mediators of acute (AKI) and chronic kidney injury. We sought to examine the relationship among EO levels, genetic variants in lanosterol synthase (LSS; an enzyme that catalyzes synthesis of cholesterol, a precursor of EO), and both AKI and chronic kidney injury., Study Design: 2 prospective observational cohort studies and a cross-sectional study of kidney tissue., Setting & Participants: (1) A prospective cohort study of patients undergoing cardiovascular surgery, (2) measurement of EO concentration in kidney tissue removed because of an adjacent tumor, and (3) a prospective cohort study of patients with newly diagnosed essential hypertension., Exposure: Missense variant in LSS (A instead of C allele at rs2254524), which leads to a valine to leucine substitution at amino acid 642., Outcomes: Development of postoperative AKI in the cardiovascular surgery cohort, EO concentration in kidney tissue, and estimated glomerular filtration rate (eGFR) reductions in the essential hypertension cohort., Analytical Approach: Logistic regression for analysis of postoperative AKI, analysis of variance for EO concentration in kidney tissue, and generalized linear models for changes in eGFR over time., Results: AKI incidence following cardiovascular surgery was greater among those with the LSS rs2254524 AA genotype (30.7%) than in those with the CC genotype (17.4%; P=0.001). LSS rs2254524 AA kidneys had higher EO concentrations than CC kidneys (2.14±0.29 vs 1.25±0.08ng/g; P<0.001). In the longitudinal study of patients with essential hypertension (median follow-up, 4 years; range, 1-15 years), eGFR decline was greater among the LSS rs2254524 AA genotype group (-4.39±1.18mL/min/1.73m 2 per year) than in the AC or CC genotype groups (-1.07±0.55 and -2.00±0.45mL/min/1.73m 2 per year respectively; P = 0.03)., Limitations: These associations do not necessarily represent causal relationships; LSS rs2254524 variants may have effects on other steroid hormones., Conclusions: These findings support the potential value of LSS rs2254524 genotype-based risk stratification to identify patients at high risk for AKI before cardiovascular surgery, as well as predict accelerated eGFR in the setting of hypertension. These findings also suggest that LSS may in part drive EO-mediated kidney damage. EO may represent a new potential therapeutic target for the prevention of AKI and slowing of kidney damage in the setting of hypertension., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Reduced smooth muscle (SM)-specific α2 Na + pump expression elevates basal blood pressure (BP) and increases BP sensitivity to angiotensin II (Ang II) and dietary NaCl, whilst SM-α2 overexpression lowers basal BP and decreases Ang II/salt sensitivity. Prolonged ouabain infusion induces hypertension in rodents, and ouabain-resistant mutation of the α2 ouabain binding site (α2 R/R mice) confers resistance to several forms of hypertension. Pressure overload-induced heart hypertrophy and failure are attenuated in cardio-specific α2 knockout, cardio-specific α2 overexpression and α2 R/R mice. We propose a unifying hypothesis that reconciles these apparently disparate findings: brain mechanisms, activated by Ang II and high NaCl, regulate sympathetic drive and a novel neurohumoral pathway mediated by both brain and circulating endogenous ouabain (EO). Circulating EO modulates ouabain-sensitive α2 Na + pump activity and Ca 2+ transporter expression and, via Na + /Ca 2+ exchange, Ca 2+ homeostasis. This regulates sensitivity to sympathetic activity, Ca 2+ signalling and arterial and cardiac contraction., (© 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.)

Objective: In the setting of normal sodium (Na) intake, many patients with hypertension have inappropriately elevated plasma aldosterone (Aldo) levels and may be at increased risk for tissue damage. Moreover, other adrenocortical steroids, including endogenous ouabain can stimulate tissue damage. As endogenous ouabain is often elevated in chronically Na-loaded states, is a vasoconstrictor, raises blood pressure (BP), and also promotes tissue fibrosis, we investigated the extent to which plasma Aldo and endogenous ouabain were coelevated among naïve hypertensive patients (NHP). We also investigated the impact of an acute salt load on these steroids, BP, and renal function., Methods: NHP (590) were grouped in tertiles based on their baseline plasma Aldo (mean ± SEM first 7.59 ± 0.18, versus third 24.15 ± 0.31 ng/dl). Baseline plasma renin activity (2.4 ± 0.1 versus 1.2 ± 0.1 ng/ml per h, P < 0.001), endogenous ouabain (268 ± 14.9 pmol/l versus 239.0 ± 13.6 pmol, P < 0.01) and DBP (91.9 ± 0.76 versus 89.6 ± 0.71 mmHg, P = 0.017) were higher in NHP in the third versus the first Aldo tertile, respectively., Results: Acute Na loading showed that the BP of the third Aldo tertile NHP was especially salt-sensitive (slope of pressure-natriuresis relationship 0.015 ± 0.002 versus 0.003 ± 0.001 μEq/mmHg per min, P = 0.00024 after adjustment for sex, BMI, and age). Regression analyses showed that plasma Aldo and endogenous ouabain were linearly related (β = 0.181, P = 0.0003)., Conclusion: Among patients with essential hypertension, circulating endogenous ouabain and Aldo are typically coelevated and their BP is salt-sensitive. In conditions where Aldo is inappropriately elevated, both Aldo and endogenous ouabain may contribute to adverse cardiovascular and renal outcomes.

Circulating levels of endogenous ouabain (EO), a vasopressor hormone of adrenocortical origin, are increased by sodium depletion. Furthermore, lanosterol synthase, an enzyme involved in cholesterol biosynthesis, has a missense polymorphism (rs2254524 V642L) that affects EO biosynthesis in adrenocortical cells. Here, we investigated the hypothesis that lanosterol synthase rs2254524 alleles in vivo impact the blood pressure (BP) and EO responses evoked by a low dietary Na intake (<100 mEq/d, 2 weeks) among patients with mild essential hypertension. During the low salt diet, the declines in both systolic BP (SBP: -8.7±1.7 versus -3.0±1.5; P=0.013) and diastolic BP (DBP: -5.1±0.98 versus -1.4±0.94 mm Hg; P<0.05), and the slope of the long-term pressure-natriuresis relationship affected significantly the presence of the lanosterol synthase rs2254524 A variant (AA: 0.71±0.22, AC 0.09±0.13, and CC 0.04±0.11 mEq/mm Hg/24 h; P=0.028). In addition, BP rose in ≈25% of the patients in response to the low salt diet and this was associated with increased circulating EO. Lanosterol synthase gene polymorphisms influence both the salt sensitivity of BP and changes in circulating EO in response to a low salt diet. The response of BP and EO to the low salt diet is markedly heterogeneous. Approximately 25% of patients experienced adverse effects, that is, increased BP and EO when salt intake was reduced and may be at increased long-term risk. The augmented response of EO to the low salt diet further supports the view that adrenocortical function is abnormal in some essential hypertensives., (© 2015 American Heart Association, Inc.)

In response to progressive nephron loss, volume and humoral signals in the circulation have increasing relevance. These signals, including plasma sodium, angiotensin II, and those related to volume status, activate a slow neuromodulatory pathway within the central nervous system (CNS). The slow CNS pathway includes specific receptors for angiotensin II, mineralocorticoids, and endogenous ouabain (EO). Stimulation of the pathway leads to elevated sympathetic nervous system activity (SNA) and increased circulating EO. The sustained elevation of circulating EO (or ouabain) stimulates central and peripheral mechanisms that amplify the impact of SNA on vascular tone. These include changes in synaptic plasticity in the brain and sympathetic ganglia that increase preganglionic tone and amplify ganglionic transmission, amplification of the impact of SNA on arterial tone in the vascular wall, and the reprogramming of calcium signaling proteins in arterial myocytes. These increase SNA, raise basal and evoked arterial tone, and elevate blood pressure (BP). In the setting of CKD, we suggest that sustained activation/elevation of the slow CNS pathway, plasma EO, and the cardiotonic steroid marinobufagenin, comprises a feed-forward system that raises BP and accelerates kidney and cardiac damage. Block of the slow CNS pathway and/or circulating EO and marinobufagenin may reduce BP and slow the progression to ESRD., (Copyright © 2015 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.)

'Classic' cardiotonic steroids (CTSs) such as digoxin and ouabain selectively inhibit Na+, K+ -ATPase (the Na+ pump) and, via Na+ / Ca2+ exchange (NCX), exert cardiotonic and vasotonic effects. CTS action is more complex than previously thought: prolonged subcutaneous administration of ouabain, but not digoxin, induces hypertension, and digoxin antagonizes ouabain's hypertensinogenic effect. We studied the acute interactions between CTSs in two indirect assays of Na+ pump function: myogenic tone (MT) in isolated, pressurized rat mesenteric small arteries, and Ca2+ signalling in primary cultured rat hippocampal neurones. The 'classic' CTSs (0.3-10 nm) behaved as 'agonists': all increased MT70 (MT at 70 mmHg) and augmented glutamate-evoked Ca2+ (Fura-2) signals. We then tested one CTS in the presence of another. Most CTSs could be divided into ouabain-like (ouabagenin, dihydroouabain (DHO), strophanthidin) or digoxin-like CTS (digoxigenin, digitoxin, bufalin). Within each group, the CTSs were synergistic, but ouabain-like and digoxin-like CTSs antagonized one another in both assays: For example, the ouabain-evoked (3 nm) increases in MT70 and neuronal Ca2+ signals were both greatly attenuated by the addition of 10 nm digoxin or 10 nm bufalin, and vice versa. Rostafuroxin (PST2238), a digoxigenin derivative that displaces 3H-ouabain from Na+, K+ -ATPase, and attenuates some forms of hypertension, antagonized the effects of ouabain, but not digoxin. SEA0400, a Na+ / Ca2+ exchanger (NCX) blocker, antagonized the effects of both ouabain and digoxin. CTSs bind to the α subunit of pump αβ protomers. Analysis of potential models suggests that, in vivo, Na+ pumps function as tetraprotomers ((αβ)4) in which the binding of a single CTS to one protomer blocks all pumping activity. The paradoxical ability of digoxin-like CTSs to reactivate the ouabain-inhibited complex can be explained by de-oligomerization of the tetrameric state. The interactions between these common CTSs may be of considerable therapeutic relevance.

Objectives: Acute kidney injury is a frequent complication of cardiac surgery and increases morbidity and mortality. As preoperative biomarkers predicting the development of acute kidney injury are not available, we have tested the hypothesis that preoperative plasma levels of endogenous ouabain may function as this type of biomarker., Rationale and Design: Endogenous ouabain is an adrenal stress hormone associated with adverse cardiovascular outcomes. Its involvement in acute kidney injury is unknown. With studies in patients and animal settings, including isolated podocytes, we tested the above mentioned hypothesis., Patients: Preoperative endogenous ouabain was measured in 407 patients admitted for elective cardiac surgery and in a validation population of 219 other patients. We also studied the effect of prolonged elevations of circulating exogenous ouabain on renal parameters in rats and the influence of ouabain on podocyte proteins both "in vivo" and "in vitro.", Main Results: In the first group of patients, acute kidney injury (2.8%, 8.3%, 20.3%, p < 0.001) and ICU stay (1.4±0.38, 1.7±0.41, 2.4±0.59 days, p = 0.014) increased with each incremental preoperative endogenous ouabain tertile. In a linear regression analysis, the circulating endogenous ouabain value before surgery was the strongest predictor of acute kidney injury. In the validation cohort, acute kidney injury (0%, 5.9%, 8.2%, p < 0.0001) and ICU stay (1.2±0.09, 1.4±0.23, 2.2±0.77 days, p = 0.003) increased with the preoperative endogenous ouabain tertile. Values for preoperative endogenous ouabain significantly improved (area under curve: 0.85) risk prediction over the clinical score alone as measured by integrate discrimination improvement and net reclassification improvement. Finally, in the rat model, elevated circulating ouabain reduced creatinine clearance (-18%, p < 0.05), increased urinary protein excretion (+ 54%, p < 0.05), and reduced expression of podocyte nephrin (-29%, p < 0.01). This last finding was replicated ex vivo by incubating podocyte primary cell cultures with low-dose ouabain., Conclusions: Preoperative plasma endogenous ouabain levels are powerful biomarkers of acute kidney injury and postoperative complications and may be a direct cause of podocyte damage.

Cardiotonic steroids (CTS) of the strophanthus and digitalis families have opposing effects on long-term blood pressure (BP). This implies hitherto unrecognized divergent signaling pathways for these CTS. Prolonged ouabain treatment upregulates Ca(2+) entry via Na(+)/Ca(2+) exchanger-1 (NCX1) and TRPC6 gene-encoded receptor-operated channels in mesenteric artery smooth muscle cells (ASMCs) in vivo and in vitro. Here, we test the effects of digoxin on Ca(2+) entry and signaling in ASMC. In contrast to ouabain treatment, the in vivo administration of digoxin (30 μg·kg(-1)·day(-1) for 3 wk) did not raise BP and had no effect on resting cytolic free Ca(2+) concentration ([Ca(2+)](cyt)) or phenylephrine-induced Ca(2+) signals in isolated ASMCs. Expression of transporters in the α2 Na(+) pump-NCX1-TRPC6 Ca(2+) signaling pathway was not altered in arteries from digoxin-treated rats. Upregulated α2 Na(+) pumps and a phosphorylated form of the c-SRC protein kinase (pY419-Src, ~4.5-fold) were observed in ASMCs from rats treated with ouabain but not digoxin. Moreover, in primary cultured ASMCs from normal rats, treatment with digoxin (100 nM, 72 h) did not upregulate NCX1 and TRPC6 but blocked the ouabain-induced upregulation of these transporters. Pretreatment of ASMCs with the c-Src inhibitor PP2 (1 μM; 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) but not its inactive analog eliminated the effect of ouabain on NCX1 and TRPC6 expression and ATP-induced Ca(2+) entry. Thus, in contrast to ouabain, the interaction of digoxin with α2 Na(+) pumps is unable to activate c-Src phosphorylation and upregulate the downstream NCX1-TRPC6 Ca(2+) signaling pathway in ASMCs. The inability of digoxin to upregulate c-Src may underlie its inability to raise long-term BP.

Purpose of Review: Endogenous cardiotonic steroids (CTS) exert long-term effects on salt and blood pressure homeostasis. Here we discuss recent observations on mechanisms of salt sensitivity that involve endogenous ouabain and novel pathways in the brain and discuss their possible relationship to arterial and renal function in hypertension., Recent Findings: Chronic elevation of brain sodium promotes sustained hypertension mediated by central endogenous ouabain and the Na(+) pump α-2 catalytic subunit. The intermediary pressor mechanism in the brain involves aldosterone biosynthesis, activation of mineralocorticoid receptors and increased epithelial sodium channel activity. In the periphery, elevated plasma CTS raise contractility and blood pressure by augmentation of sympathetic nerve responses, increasing arterial Ca(2+) signaling and blunting nitric oxide production in the renal medulla and collecting ducts., Summary: Endogenous ouabain in the brain appears to play a critical role in salt sensitivity and hypertension. In the periphery, the J-shaped relationship of plasma endogenous ouabain in response to short-term changes in salt balance in humans raises the possibility that endogenous ouabain contributes to the increased risk of adverse cardiovascular events associated with both low and high salt intakes.

Endogenous cardiotonic steroids (CTS) raise blood pressure (BP) via vascular sodium calcium exchange (NCX1.3) and transient receptor-operated channels (TRPCs). Circulating CTS are superelevated in pregnancy-induced hypertension and preeclampsia. However, their significance in normal pregnancy, where BP is low, is paradoxical. Here we test the hypothesis that vascular resistance to endogenous ouabain (EO) develops in normal pregnancy and is mediated by reduced expression of NCX1.3 and TRPCs. We determined plasma and adrenal levels of EO and the impact of exogenous ouabain in pregnancy on arterial expression of Na(+) pumps, NCX1.3, TRPC3, and TRPC6 and BP. Pregnant (embryonic day 4) and nonpregnant rats received infusions of ouabain or vehicle. At 14-16 days, tissues and plasma were collected for blotting and EO assay by radioimmunoassay (RIA), liquid chromatography (LC)-RIA, and LC-multidimensional mass spectrometry (MS3). BP (-8 mmHg; P < 0.05) and NCX1.3 expression fell (aorta -60% and mesenteric artery -30%; P < 0.001) in pregnancy while TRPC expression was unchanged. Circulating EO increased (1.14 ± 0.13 nM) vs. nonpregnant (0.6 ± 0.08 nM; P < 0.05) and was confirmed by LC-MS3 and LC-RIA. LC-MS3 revealed two previously unknown isomers of EO; one increased ∼90-fold in pregnancy. Adrenal EO but not isomers were increased in pregnancy. In nonpregnant rats, similar infusions of ouabain raised BP (+24 ± 3 mmHg; P < 0.001). In ouabain-infused rats, impaired fetal and placental growth occurred with no BP increase. In summary, normal pregnancy is an ouabain-resistant state associated with low BP, elevated circulating levels of EO, two novel steroidal EO isomers, and increased adrenal mass and EO content. Ouabain raises BP only in nonpregnant animals. Vascular resistance to the chronic pressor activity of endogenous and exogenous ouabain is mediated by suppressed NCX1.3 and reduced sensitivity of events downstream of Ca(2+) entry. The mechanisms of EO resistance and the impaired fetal and placental growth due to elevated ouabain may be important in pregnancy-induced hypertension (PIH) and preeclampsia (PE).

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.

Objective: To evaluate whether the renin-angiotensin-aldosterone system (RAAS) and endogenous ouabain system differently affect renal Na handling and blood pressure., Methods: 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., Results: 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., Conclusion: 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.

The Na(+) pump and its Endogenous modulator Ouabain (EO) can be considered as an ancestral enzymatic system, conserved among species ranging from Drosophila to humans, related to Na handling. In this review, we examine how EO is linked with vascular function in hypertension and if it impacts the pathogenesis of heart and renal failure. Moreover, the molecular mechanism of endogenous ouabain-linked hypertension involves the sodium pump/sodium-calcium exchanger duet. Biosynthesis of EO occurs in adrenal glands and is under the control of angiotensin II, ACTH and epinephrine. Elevated concentrations of EO and in the sub-nanomolar concentration range were found to stimulate proliferation and differentiation of cardiac and smooth muscle cells. They may have a primary role in the development of cardiac dysfunction and failure. Experimental data suggest that the Na/K-ATPase α(2)-catalytic subunit causes EO-induced vasoconstriction. Finally, maneuvers that promote Na depletion, as diuretic therapy or reduced Na intake, raise the EO levels. Taken together, these findings suggest a key role for EO in body Na homeostasis., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Salt retention as a result of chronic, excessive dietary salt intake, is widely accepted as one of the most common causes of hypertension. In a small minority of cases, enhanced Na(+) reabsorption by the kidney can be traced to specific genetic defects of salt transport, or pathological conditions of the kidney, adrenal cortex, or pituitary. Far more frequently, however, salt retention may be the result of minor renal injury or small genetic variation in renal salt transport mechanisms. How salt retention actually leads to the increase in peripheral vascular resistance (the hallmark of hypertension) and the elevation of blood pressure remains an enigma. Here we review the evidence that endogenous ouabain (an adrenocortical hormone), arterial smooth muscle α2 Na(+) pumps, type-1 Na/Ca exchangers, and receptor- and store-operated Ca(2+) channels play key roles in the pathway that links salt to hypertension. We discuss cardenolide structure-function relationships in an effort to understand why prolonged administration of ouabain, but not digoxin, induces hypertension, and why digoxin is actually anti-hypertensive. Finally, we summarize recent observations which indicate that ouabain upregulates arterial myocyte Ca(2+) signaling mechanisms that promote vasoconstriction, while simultaneously downregulating endothelial vasodilator mechanisms. In sum, the reports reviewed here provide novel insight into the molecular mechanisms by which salt retention leads to hypertension., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Prolonged ouabain administration (25 microg kg(-1) day(-1) for 5 wk) induces "ouabain hypertension" (OH) in rats, but the molecular mechanisms by which ouabain elevates blood pressure are unknown. Here, we compared Ca(2+) signaling in mesenteric artery smooth muscle cells (ASMCs) from normotensive (NT) and OH rats. Resting cytosolic free Ca(2+) concentration ([Ca(2+)](cyt); measured with fura-2) and phenylephrine-induced Ca(2+) transients were augmented in freshly dissociated OH ASMCs. Immunoblots revealed that the expression of the ouabain-sensitive alpha(2)-subunit of Na(+) pumps, but not the predominant, ouabain-resistant alpha(1)-subunit, was increased (2.5-fold vs. NT ASMCs) as was Na(+)/Ca(2+) exchanger-1 (NCX1; 6-fold vs. NT) in OH arteries. Ca(2+) entry, activated by sarcoplasmic reticulum (SR) Ca(2+) store depletion with cyclopiazonic acid (SR Ca(2+)-ATPase inhibitor) or caffeine, was augmented in OH ASMCs. This reflected an augmented expression of 2.5-fold in OH ASMCs of C-type transient receptor potential TRPC1, an essential component of store-operated channels (SOCs); two other components of some SOCs were not expressed (TRPC4) or were not upregulated (TRPC5). Ba(2+) entry activated by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol [a measure of receptor-operated channel (ROC) activity] was much greater in OH than NT ASMCs. This correlated with a sixfold upregulation of TRPC6 protein, a ROC family member. Importantly, in primary cultured mesenteric ASMCs from normal rats, 72-h treatment with 100 nM ouabain significantly augmented NCX1 and TRPC6 protein expression and increased resting [Ca(2+)](cyt) and ROC activity. SOC activity was also increased. Silencer RNA knockdown of NCX1 markedly downregulated TRPC6 and eliminated the ouabain-induced augmentation; silencer RNA knockdown of TRPC6 did not affect NCX1 expression but greatly attenuated its upregulation by ouabain. Clearly, NCX1 and TRPC6 expression are interrelated. Thus, prolonged ouabain treatment upregulates the Na(+) pump alpha(2)-subunit-NCX1-TRPC6 (ROC) Ca(2+) signaling pathway in arterial myocytes in vitro as well as in vivo. This may explain the augmented myogenic responses and enhanced phenylephrine-induced vasoconstriction in OH arteries (83) as well as the high blood pressure in OH rats.

Prolonged ouabain administration to normal rats causes sustained blood pressure (BP) elevation. This ouabain-induced hypertension (OH) has been attributed, in part, to the narrowing of third-order resistance arteries (approximately 320 microm internal diameter) as a result of collagen deposition in the artery media. Here we describe the structural and functional properties of fourth-order mesenteric small arteries from control and OH rats, including the effect of low-dose ouabain on myogenic tone in these arteries. Systolic BP in OH rats was 138 +/- 3 versus 124 +/- 4 mmHg in controls (P < 0.01). Pressurized (70 mmHg) control and OH arteries, with only a single layer of myocytes, both had approximately 165-microm internal diameters and approximately 20-microm wall thicknesses. Even after fixation, despite vasoconstriction, the diameters and wall thicknesses did not differ between control and OH fourth-order arteries, whereas in third-order arteries, both parameters were significantly smaller in OH than in controls. Myogenic reactivity was significantly augmented in OH fourth-order arteries. Nevertheless, phenylephrine- (1 microM) and high K(+)-induced vasoconstrictions and acetylcholine-induced vasodilation were comparable in control and OH arteries. Vasoconstrictions induced by 5 microM phenylephrine and by 10 mM caffeine in Ca(2+)-free media indicated that releasable sarcoplasmic reticulum Ca(2+) stores were normal in OH arteries. Importantly, 100 nM ouabain constricted both control and OH arteries by approximately 26 microm, indicating that this response was not downregulated in OH rats. This maximal ouabain-induced constriction corresponds to a approximately 90% increase in resistance to flow in these small arteries; thus ouabain at EC(50) of approximately 0.66 nM should raise resistance by approximately 35%. We conclude that dynamic constriction in response to circulating nanomolar ouabain in small arteries likely makes a major contribution to the increased vascular tone and BP in OH rats.

Background: 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., Methods: 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., Results: 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., Conclusions: 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.

An endogenous ouabain has been isolated and conclusively identified from several mammalian tissues, including human plasma, by a number of independent laboratories. Substantial evidence from independent laboratories in several continents is consistent with an adrenal source for most if not all of the circulating endogenous ouabain. Accumulating evidence suggests that circulating levels of endogenous ouabain in humans are modulated by dietary salt and chronic volume status. Endogenous ouabain is linked significantly with vascular function in hypertension and likely impacts the pathogenesis of heart and renal failure. Moreover, the molecular mechanism of endogenous ouabain-linked hypertension involves the sodium pump/sodium-calcium exchange duet. The outstanding analytical issues include the elucidation of distal events in the biosynthetic pathway for endogenous ouabain and identification of molecular mechanisms that regulate its secretion and clearance.

Objective: 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., Methods: 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)., Results: 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., Conclusion: 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.

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.

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.

Objective: 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., Methods: 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., Results: 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-., Conclusions: 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.

Accumulating evidence indicates that mammals use steroidal glycosides with "digitalis-like" activity. An endogenous ouabain (EO) has been described and is linked with long-term changes in sodium balance and cardiovascular structure and function. In the adrenal gland, the biosynthesis of EO and similar compounds appears to involve cholesterol side-chain cleavage with sequential metabolism of pregnenolone and progesterone. The more distal events in the biosynthesis have not been elucidated. Preliminary work using primary cell cultures from the bovine adrenal cortex suggests that the biosynthesis of EO is affected by inhibitors of 11beta-hydroxylase. Direct participation of 11-hydoxylase in EO synthesis would lead to an 11beta isomer of ouabain in mammals and, in vivo, an 11beta-oriented hydroxyl group would spontaneously form a mixture of two 11-19 hemiketal isomers. The latter isomers would likely be converted back to a single 11beta isomer of ouabain during isolation. The existence of an additional ring in the hemiketals, along with reduced flexion of the steroidal A, B, and C rings, raises the possibility that their in vivo physiological targets and actions differ from the isolated form of EO.

Sodium-potassium pumps (Na pumps) are the only known plasma membrane receptors for cardiac glycosides. However, adrenocortical cells secrete an endogenous ouabain via an unknown mechanism that is subject to feedback inhibition via the cell surface. In addition, recent studies suggest that the induction of sustained hypertension by ouabain analogs in rats may be independent of Na pump inhibition. Accordingly, we used bovine adrenocortical cells and membranes to search for novel binding sites for ouabain. In high extracellular potassium solutions, the binding of ouabain to the Na pumps of cultured cells was suppressed, yet residual specific binding of (3)H-ouabain was observed. In high extracellular potassium, Scatchard analyses revealed a novel class of ouabain binding sites with high affinity (<50 nmol/L, < 2.5 x 10(5) sites/cell) that was distinct from the low-affinity Na pump sites (>1 micromol/L, 4.5 x 10(6) sites/cell). Analysis of the kinetics for the dissociation of (3)H-ouabain from intact cells revealed components whose t(0.5) values were 6.5 minutes, 3.3 hours, and 33 hours and associated with novel sites, Na pumps, and lysosomal recycling, respectively. Studies with isolated membranes under ligand conditions where the participation of Na pumps was minimized revealed specific ouabain binding to novel sites that was saturable, time-dependent, of high affinity (K(d) approximately 15 nmol/L), and of low density (apparent B(max)=0.23 pmol/mg, c.f., Na pumps=10.2 pmol/mg). Ouabain binding to the novel sites was stimulated by high concentrations of KCl but was not affected by aldosterone or cortisol up to 30 micromol/L. Novel sites were not detected in skeletal muscle or liver membranes. Photoaffinity studies followed by SDS-PAGE showed ouabain-protectable labeling of membrane polypeptides with apparent molecular weights of 143, 113, and 65 kDa. We conclude that adrenocortical cells express ouabain receptors that are distinct from Na pumps. These novel receptors may be involved in the regulation and/or secretion of endogenous ouabain.

Cardiotonic steroids (CTS), used by certain insects, toads, and rats for protection from predators, became, thanks to Withering's trailblazing 1785 monograph, the mainstay of heart failure (HF) therapy. In the 1950s and 1960s, we learned that the CTS receptor was part of the sodium pump (NKA) and that the Na+/Ca2+ exchanger was critical for the acute cardiotonic effect of digoxin- and ouabain-related CTS. This "settled" view was upended by seven revolutionary observations. First, subnanomolar ouabain sometimes stimulates NKA while higher concentrations are invariably inhibitory. Second, endogenous ouabain (EO) was discovered in the human circulation. Third, in the DIG clinical trial, digoxin only marginally improved outcomes in patients with HF. Fourth, cloning of NKA in 1985 revealed multiple NKA α and β subunit isoforms that, in the rodent, differ in their sensitivities to CTS. Fifth, the NKA is a cation pump and a hormone receptor/signal transducer. EO binding to NKA activates, in a ligand- and cell-specific manner, several protein kinase and Ca2+-dependent signaling cascades that have widespread physiological effects and can contribute to hypertension and HF pathogenesis. Sixth, all CTS are not equivalent, e.g., ouabain induces hypertension in rodents while digoxin is antihypertensinogenic ("biased signaling"). Seventh, most common rodent hypertension models require a highly ouabain-sensitive α2 NKA and the elevated blood pressure is alleviated by EO immunoneutralization. These numerous phenomena are enabled by NKA's intricate structure. We have just begun to understand the endocrine role of the endogenous ligands and the broad impact of the ouabain-binding site on physiology and pathophysiology. [ABSTRACT FROM AUTHOR]

Cloning of the “Naþ pump” (Naþ,Kþ -ATPase or NKA) and identification of a circulating ligand, endogenous ouabain (EO), a cardiotonic steroid (CTS), triggered seminal discoveries regarding EO and its NKA receptor in cardiovascular function and the pathophysiology of heart failure (HF) and hypertension. Cardiotonic digitalis preparations were a preferred treatment for HF for two centuries, but digoxin was only marginally effective in a large clinical trial (1997). This led to diminished digoxin use. Missing from the trial, however, was any consideration that endogenous CTS might influence digitalis’ efficacy. Digoxin, at therapeutic concentrations, acutely inhibits NKA but, remarkably, antagonizes ouabain’s action. Prolonged treatment with ouabain, but not digoxin, causes hypertension in rodents; in this model, digoxin lowers blood pressure (BP). Furthermore, NKA-bound ouabain and digoxin modulate different protein kinase signaling pathways and have disparate long-term cardiovascular effects. Reports of “brain ouabain” led to the elucidation of a new, slow neuromodulatory pathway in the brain; locally generated EO and the α2 NKA isoform help regulate sympathetic drive to the heart and vasculature. The roles of EO and α2 NKA have been studied by EO assay, ouabain-resistant mutation of α2 NKA, and immunoneutralization of EO with ouabain-binding Fab fragments. The NKA α2 CTS binding site and its endogenous ligand are required for BP elevation in many common hypertension models and full expression of cardiac remodeling and dysfunction following pressure overload or myocardial infarction. Understanding how endogenous CTS impact hypertension and HF pathophysiology and therapy should foster reconsideration of digoxin’s therapeutic utility. [ABSTRACT FROM AUTHOR]

Reduced smooth muscle (SM)-specific α2 Na+ pump expression elevates basal blood pressure (BP) and increases BP sensitivity to angiotensin II (Ang II) and dietary NaCl, whilst SM-α2 overexpression lowers basal BP and decreases Ang II/salt sensitivity. Prolonged ouabain infusion induces hypertension in rodents, and ouabain-resistant mutation of the α2 ouabain binding site (α2R/R mice) confers resistance to several forms of hypertension. Pressure overload-induced heart hypertrophy and failure are attenuated in cardio-specific α2 knockout, cardio-specific α2 overexpression and α2R/R mice. We propose a unifying hypothesis that reconciles these apparently disparate findings: brain mechanisms, activated by Ang II and high NaCl, regulate sympathetic drive and a novel neurohumoral pathway mediated by both brain and circulating endogenous ouabain (EO). Circulating EO modulates ouabain-sensitive α2 Na+ pump activity and Ca2+ transporter expression and, via Na+/Ca2+ exchange, Ca2+ homeostasis. This regulates sensitivity to sympathetic activity, Ca2+ signalling and arterial and cardiac contraction. [ABSTRACT FROM AUTHOR]

The work of de Wardener and colleagues stimulated longstanding interest in natriuretic hormones (NHs). In addition to the atrial peptides (APs), the circulation contains unidentified physiologically relevant NHs. One NH is controlled by the central nervous system (CNS) and likely secreted by the pituitary. Its circulating activity is modulated by salt intake and the prevailing sodium concentration of the blood and intracerebroventricular fluid, and contributes to postprandial and dehydration natriuresis. The other NH, mobilized by a trial stretch, promotes natriuresis by increasing the production of intrarenal dopamine and/or nitric oxide (NO). Both NHs have short (<35 min) circulating half lives, depress renotubular sodium transport, and neither requires the renal nerves. The search for NHs led to endogenous cardiotonic steroids (CTS) including ouabain-, digoxin-, and bufadienolide-like materials. These CTS, given acutely in high nanomole to micromole amounts into the general or renal circulations, inhibit sodium pumps and are natriuretic. Among these CTS, only bufalin is cleared sufficiently rapidly to qualify for an NH-like role. Ouabain-like CTS are cleared slowly, and when given chronically in low daily nanomole amounts, promote sodium retention, augment arterial myogenic tone, reduce renal blood flow and glomerular filtration, suppress NO in the renal vasa recta, and increase sympathetic nerve activity and blood pressure. Moreover, lowering total body sodium raises circulating endogenous ouabain. Thus, ouabain-like CTS have physiological actions that, like aldosterone, support renal sodium retention and blood pressure. In conclusion, the mammalian circulation contains two non-AP NHs. Identification of the CNS NH should be a priority. [ABSTRACT FROM AUTHOR]

The Milan hypertensive strain (MHS) rats are a genetic model of hypertension with adducin gene polymorphisms linked to enhanced renal tubular Na+ reabsorption. Recently we demonstrated that Ca2+ signaling is augmented in freshly isolated mesenteric artery myocytes from MHS rats. This is associated with greatly enhanced expression of Na+/Ca2+ exchanger-1 (NCX1), C-type transient receptor potential (TRPC6) protein, and sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2) compared with arteries from Milan normotensive strain (MNS) rats. Here, we test the hypothesis that the enhanced Ca2+ signaling in MHS arterial smooth muscle is directly reflected in augmented vasoconstriction [myogenic and phenylephrine (PE)-evoked responses] in isolated mesenteric small arteries. Systolic blood pressure was higher in MHS (145 ± 1 mmHg) than in MNS (112 ± 1 mmHg; P < 0.001; n = 16 each) rats. Pressurized mesenteric resistance arteries from MHS rats had significantly augmented myogenic tone and reactivity and enhanced constriction to low-dose (1-100 nM) PE. Isolated MHS arterial myocytes exhibited approximately twofold increased peak Ca2+ signals in response to 5 μM PE or ATP in the absence and presence of extracellular Ca2+. These augmented responses are consistent with increased vasoconstrictor-evoked sarcoplasmic reticulum (SR) Ca2+ release and increased Ca2+ entry, respectively. The increased SR Ca2+ release correlates with a doubling of inositol 1,4,5-trisphosphate receptor type 1 and tripling of SERCA2 expression. Pressurized MHS arteries also exhibited a ~70% increase in 100 nM ouabain-induced vasoconstriction compared with MNS arteries. These functional alterations reveal that, in a genetic model of hypertension linked to renal dysfunction, multiple mechanisms within the arterial myocytes contribute to enhanced Ca2+ signaling and myogenic and vasoconstrictor-induced arterial constriction. MHS rats have elevated plasma levels of endogenous ouabain, which may initiate the protein upregulation and enhanced Ca2+ signaling. These molecular and functional changes provide a mechanism for the increased peripheral vascular resistance (whole body autoregulation) that underlies the sustained hypertension. [ABSTRACT FROM AUTHOR]

• Endogenous ouabain (EO), a novel hormone, helps regulate the cytosolic Ca2+ concentration ([Ca2+] CYT) and Ca2+ signaling via Na/Ca exchange. • EO also activates a number of protein kinase (PK) signaling cascades that control a myriad of cell functions. • [Ca2+] CYT and the PK pathways intersect at numerous points because Ca2+ and calmodulin also modulate some steps in those other pathways. • Both mechanisms operate in virtually all cells and tissues. In this brief review we discuss some aspects of the Na+ pump and its roles in mediating the effects of ouabain and endogenous ouabain (EO): i) in regulating the cytosolic Ca2+ concentration ([Ca2+] CYT) via Na/Ca exchange (NCX), and ii) in activating a number of protein kinase (PK) signaling cascades that control a myriad of cell functions. Importantly, [Ca2+] CYT and the other signaling pathways intersect at numerous points because of the influence of Ca2+ and calmodulin in modulating some steps in those other pathways. While both mechanisms operate in virtually all cells and tissues, this article focuses primarily on their functions in the cardiovascular system, the central nervous system (CNS) and the kidneys. [ABSTRACT FROM AUTHOR]