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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]

The Milan hypertensive strain (MHS) of rats is a model for hypertension in humans. Inherited defects in renal function have been well studied in MHS rats, but the mechanisms that underlie the elevated vascular resistance are unclear. Altered [Ca.sup.2+] signaling plays a key role in the vascular dysfunction associated with arterial hypertension. Here we compared [Ca.sup.2+] signaling in mesenteric artery smooth muscle cells from MHS rats and its normotensive counterpart (MNS). Systolic blood pressure was higher in MHS than in MNS rats (144 [+ or -] 2 vs. 113 [+ or -] 1 mmHg, P < 0.05). Resting cytosolic free [Ca.sup.2+] concentration (measured with fura-2) and ATP-induced [Ca.sup.2+] transients were augmented in freshly dissociated arterial myocytes from MHS rats. [Ba.sup.2+] entry activated by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol (a measure of receptor-operated channel activity) was much greater in MHS than MNS arterial myocytes. This correlated with a threefold upregulation of transient receptor potential canonical 6 (TRPC6) protein. TRPC3, the other component of receptor-operated channels, was marginally, but not significantly, upregulated. The expression of TRPC1/5, components of store-operated channels, was not altered in MHS mesenteric artery smooth muscle. Immunoblots also revealed that the [Na.sup.+]/[Ca.sup.2+] exchanger-1 (NCX1) was greatly upregulated in MHS mesenteric artery (by ~13-fold), whereas the expression of plasma membrane [Ca.sup.2+]-ATPase was not altered. [Ca.sup.2+] entry via the reverse mode of NCX1 evoked by the removal of extracellular [Na.sup.+] induced a rapid increase in cytosolic free [Ca.sup.2+] concentration that was significantly larger in MHS arterial myocytes. The expression of [[alpha].sub.1]/[[alpha].sub.2] [Na.sup.+] pumps in MHS mesenteric arteries was not changed. Immunocytochemical observations showed that NCX1 and TRPC6 are clustered in plasma membrane microdomains adjacent to the underlying sarcoplasmic reticulum. In summary, MHS arteries exhibit upregulated TRPC6 and NCX1 and augmented [Ca.sup.2+] signaling. We suggest that the increased [Ca.sup.2+] signaling contributes to the enhanced vasoconstriction and elevated blood pressure in MHS rats. adducin; hypertension; Milan normotensive rats; C-type transient receptor potential channels; receptor-operated calcium entry doi: 10.1152/ajpheart.00356.2010.

Am J Physiol Heart Circ Physiol 298: H263-H274, 2010. First published November 6, 2009; doi: 10.1152/ajpheart.00784.2009.--Prolonged ouabain administration (25 [micro]g x [kg.sup.-1] x [day.sup.-1] for 5 wk) induces 'ouabain hypertension' (OH) in rats, but the molecular mechanisms by which ouahain elevates blood pressure are unknown. Here, we compared [Ca.sup.2+] signaling in mesenteric artery smooth muscle cells (ASMCs) from normotensive (NT) and OH rats. Resting cytosolic free [Ca.sup.2+] concentration ([[Ca.sup.2+].sub.cyt]; measured with fura-2) and phenylephrine-induced [Ca.sup.2+] transients were augmented in freshly dissociated OH ASMCs. Immunoblots revealed that the expression of the ouabain-sensitive [[alpha].sub.2]-subunit of [Na.sup.+] pumps, but not the predominant, ouabain-resistant [[alpha].sub.1]-subunit, was increased (2.5-fold vs. NT ASMCs) as was [Na.sup.+]/ [Ca.sup.2+] exchanger-1 (NCX1; 6-fold vs. NT) in OH arteries. [Ca.sup.2+] entry, activated by sarcoplasmic reticulum (SR) [Ca.sup.2+] store depletion with cyclopiazonic acid (SR [Ca.sup.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 TRPC 1, an essential component of store-operated channels (SOCs); two other components of some SOCs were not expressed (TRPC4) or were not upregulated (TRPC5). [Ba.sup.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.sup.2+]].sub.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, NCXI and TRPC6 expression are interrelated. Thus, prolonged ouabain treatment upregulates the [Na.sup.+] pump [[alpha].sub.2]-subunit-NCX1-TRPC6 (ROC) [Ca.sup.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. [Na.sup.+] pumps; [Na.sup.+]/[Ca.sup.2+] exchange; store-operated [Ca.sup.2+] entry; receptor-operated [Ca.sup.2+] entry; arterial myocytes

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 (~320 [micro]m internal diameter) as a result of collagen deposition in the artery media (see Ref. 6). 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 lone in these arteries. Systolic BP in OH rats was 138 [+ or -] 3 versus 124 [+ or -] 4 mmHg in controls (P myogenic reactivity: resistance artery

High-salt diets elevate circulating [Na.sup.+] pump inhibitors, vascular resistance, and blood pressure. Ouabain induces a form of hypertension mediated via the [[alpha].sub.2]-[Na.sup.+] 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 [+ or -] 16 meq/day), plasma EO (0.43 [+ or -] 0.08 nmol/1) and urinary EO excretion (1.04 [+ or -] 0.13 nmol/day) were at steady state. On the 3rd day of a high-salt diet, urine sodium excretion (315 [+ or -] 28 meq/day), plasma EO (5.8 [+ or -] 2.2 nmol/1), and the urinary EO excretion (1.69 [+ or -] 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 [+ or -] 0.77 nmol/l) rose, while urinary EO excretion remained within the normal range (1.44 [+ or -] 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, ~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. cardiac glycosides; sodium; sodium pump; inhibitor; circulation; hypertension

Results demonstrate that the binding site of the potent sodium pump steroid inhibitor, ouabain, exhibits structurally mobile elelments and this conformational flexibility slows its initial binding to the sodium pump. Data indicate that the biologcal activity of the system depends upon synchronization between receptor and ligand dynamics.

Humans and various mammals have plasma and adrenal glands which reveal the presence of ouabain. Splenectomy and unilateral adrenal venous (AV) cannulation are used to prepare trained adult dogs to examine the potential adrenal release of ouabain in vivo and in reaction to modification in the quantity of blood. Hemorrhage or expansion of blood volume is seen in the dogs two days after an overnight fast. Ouabain-specific immounoassay is used to calculate ouabain in AV and arterial plasma (ART), while radioimmunoassay is used to calculate cortisol and aldosterone.

Altered sympathetic nervous system activity has been implicated often in hypertension. We examined short-term potentiation [posttetanic potentiation (PTP)] and long-term potentiation (LTP) in the isolated superior cervical ganglia (SCG) from Sprague-Dawley (SD) rats given vehicle, digoxin, or ouabain by subcutaneous implants as well as in animals with ouabain-induced hypertension (OHR), and inbred Baltimore ouabain-resistant (BOR) and Baltimore ouabain-sensitive (BOS) strains of rats. Postganglionic compound action potentials (CAP) were used to determine PTP and LTP following a tetanic stimulus (20 Hz, 20 s). Baseline CAP magnitude was greater in ganglia from OHR than in vehicle-treated SD rats before tetanus, but the decay time constant of PTP was significantly decreased in OHR and in rats infused with digoxin that were normotensive. In hypertensive BOS and OHR, the time constants for the decay of both PTP and LTP ([t.sub.L]) were increased and correlated with blood pressure (slope = 0.15 min/mmHg, r = 0.52, P [is less than] 0.047 and 6.7 min/mmHg, r = 0.906, P [is less than] 0.0001, respectively). In BOS and OHR, [t.sub.L] (minutes) was 492 [+ or -] 40 (n = 7) and 539 [+ or -] 41 (n = 5), respectively, and differed (P [is less than] 0.05) from BOR (257 [+ or -] 48, n = 4), SD vehicle rats (240 [+ or -] 18, n = 4), and captopril-treated OHR (370 [+ or -] 52, n = 5). After the tetanus, the CAP at 90 min in BOS and OHR SCG declined less rapidly vs. SD vehicle rats or BOR. Captopril normalized blood pressure and [t.sub.L] in OHR. We conclude that the duration of ganglionic LTP and blood pressure are tightly linked in ouabain-dependent hypertension. Our results favor the possibility that enhanced duration of LTP in sympathetic neurons contributes to the increase in sympathetic nerve activity in ouabain-dependent hypertension and suggest that a captopril-sensitive step mediates the link of ouabain with LTP. neurons; angiotensin; breeding; sodium pump; captopril; long-term potentiation

Arnon, Assaf, John M. Hamlyn, and Mordecai P. Blaustein. Ouabain augments [Ca.sup.2+] transients in arterial smooth muscle without raising cytosolic [Na.sup.+]. Am J Physiol Heart Circ Physiol 279: H679-H691, 2000.--Ouabain and other cardiotonic steroids (CTS) inhibit [Na.sup.+] pumps and are widely believed to exert their cardiovascular effects by raising the cytosolic [Na.sup.+] concentration ([[[Na.sup.+]].sub.cyt]) and [Ca.sup.2+]. This view has not been rigorously reexamined despite evidence that low-dose CTS may act without elevating [[[Na.sup.+]].sub.cyt]; also, it does not explain the presence of multiple, functionally distinct isoforms of the [Na.sup.+] pump in many cells. We investigated the effects of [Na.sup.+] pump inhibition on [[[Na.sup.+]].sub.cyt] (with [Na.sup.+] binding benzofuran isophthalate) and [Ca.sup.2+] transients (with fura 2) in primary cultured arterial myocytes. Low concentrations of ouabain (3-100 nM) or human ouabain-like compound or reduced extracellular [K.sup.+] augmented hormone-evoked mobilization of stored [Ca.sup.2+] but did not increase bulk [[[Na.sup.+]].sub.cyt]. Augmentation depended directly on external [Na.sup.+], but not external [Ca.sup.2+], and was inhibited by 10 mM [Mg.sup.2+] or 10 [micro]M [La.sup.3+]. Evoked [Ca.sup.2+] transients in pressurized small resistance arteries were also augmented by nanomolar ouabain and inhibited by [Mg.sup.2+]. These results suggest that [Na.sup.+] enters a tiny cytosolic space between the plasmalemma (PL) and the adjacent sarcoplasmic reticulum (SR) via an [Mg.sup.2+]- and [La.sup.3+]-blockable mechanism that is activated by SR store depletion. The [Na.sup.+] and [Ca.sup.2+] concentrations within this space may be controlled by clusters of high ouabain affinity ([Alpha]3) [Na.sup.+] pumps and Na/Ca exchangers located in PL microdomains overlying the SR. Inhibition of the [Alpha]3 pumps by low-dose ouabain should raise the local concentrations of [Na.sup.+] and [Ca.sup.2+] and augment hormone-evoked release of [Ca.sup.2+] from SR stores. Thus the clustering of small numbers of specific PL ion transporters adjacent to the SR can regulate global [Ca.sup.2+] signaling. This mechanism may affect vascular tone and blood flow and may also influence [Ca.sup.2+] signaling in many other types of cells. vasoconstrictors; sodium pump; sodium/calcium exchange; junctional sarcoplasmic reticulum

Arnon, Assaf, John M. Hamlyn, and Mordecai P. Blaustein. [Na.sup.+] entry via store-operated channels modulates [Ca.sup.2+] signaling in arterial myocytes. Am J. Physiol. Cell Physiol. 278: C163-C173, 2000.--In many nonexcitable cells, hormones and neutransmitters activate [Na.sup.+] influx and mobilize [Ca.sup.2+] from intracellular stores. The stores are replenished by [Ca.sup.2+] influx via 'store-operated' [Ca.sup.2+] channels (SOC). The main routes of [Na.sup.+] entry in these cells are unresolved, and no role for [Na.sup.+] in signaling has been recognized. We demonstrate that the SOC are a major [Na.sup.+] entry route in arterial myocytes. Unloading of the [Ca.sup.2+] stores with cyclopiazonic acid (a sarcoplasmic reticulum [Ca.sup.2+] pump inhibitor) and caffeine induces a large external [Na.sup.+]-dependent rise in the cytosolic [Na.sup.+] concentration. One component of this rise in cytosolic [Na.sup.+] concentration is likely due to [Na.sup.+]/[Ca.sup.2+] exchange; it depends on elevation of cytosolic [Ca.sup.2+] and is insensitive to 10 mM [Mg.sup.2+] and 10 [micro]M [La.sup.3+]. Another component is inhibited by [Mg.sup.2+] and [La.sup.3+], blockers of SOC; this component persists in cells preloaded with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N',-tetraacetic acid to buffer [Ca.sup.2+] transients and prevent [Na.sup.+]/[Ca.sup.2+] exchange-mediated [Na.sup.+] entry. This [Na.sup.+] entry apparently is mediated by SOC. The [Na.sup.+] entry influences [Na.sup.+] pump activity and [Na.sup.+]/ [Ca.sup.2+] exchange and has unexpectedly large effects on cell-wide [Ca.sup.2+] signaling. The SOC pathway may be a general mechanism by which [Na.sup.+] participates in signaling in many types of cells. sodium-calcium exchange; magnesium; sodium pump; cytosolic sodium concentration

Aims High salt intake markedly enhances hypertension induced by angiotensin II (Ang II). We explored central and peripheral slow-pressor mechanisms which may be activated by Ang II and salt. Methods and results In protocol I, Wistar rats were infused subcutaneously with low-dose Ang II (150 ng/kg/min) and fed regular (0.4%) or high salt (2%) diet for 14 days. In protocol II, Ang II-high salt was combined with intracerebroventricular infusion of mineralocorticoid receptor (MR) blockers (eplerenone, spironolactone), epithelial sodium channel (ENaC) blocker (benzamil), angiotensin II type 1 receptor (AT1R) blocker (losartan) or vehicles. Ang II alone raised mean arterial pressure (MAP) ∼10 mmHg, but Ang II-high salt increased MAP ∼50 mmHg. Ang II-high salt elevated plasma corticosterone, aldosterone and endogenous ouabain but not Ang II alone. Both Ang II alone and Ang II-high salt increased mRNA and protein expression of CYP11B2 (aldosterone synthase gene) in the adrenal cortex but not of CYP11B1 (11-β-hydroxylase gene). In the aorta, Ang II-high salt increased sodium-calcium exchanger-1 (NCX1) protein. The Ang II-high salt induced increase in MAP was largely prevented by central infusion of MR blockers, benzamil or losartan. Central blockades significantly lowered plasma aldosterone and endogenous ouabain and markedly decreased Ang II-high salt induced CYP11B2 mRNA expression in the adrenal cortex and NCX1 protein in the aorta. Conclusion These results suggest that in Ang II-high salt hypertension, MR-ENaC-AT1R signalling in the brain increases circulating aldosterone and endogenous ouabain, and arterial NCX1. These factors can amplify blood pressure responses to centrally-induced sympatho-excitation and thereby contribute to severe hypertension. [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 article presents a summary of reports about endogenous ouabain (EO), a cardiotonic steroid hormone. Topics include the role of EO in regulating intracellular signaling, the discovery of EO isomers, the link between EO and the development of chronic kidney diseases, and its role as a biomarker of heart failure.