Search

With an exception of few reports, the plasma concentration of ouabain and marinobufagenin, mostly studied cardiotonic steroids (CTS) assessed by immunoassay techniques, is less than 1 nM. During the last 3 decades, the implication of these endogenous CTS in the pathogenesis of hypertension and other volume-expanded disorders is widely disputed. The threshold for inhibition by CTS of human and rodent α1-Na,K-ATPase is ∼1 and 1000 nM, respectively, that rules out the functioning of endogenous CTS (ECTS) as natriuretic hormones and regulators of cell adhesion, cell-to-cell communication, gene transcription and translation, which are mediated by dissipation of the transmembrane gradients of monovalent cations. In several types of cells ouabain and marinobufagenin at concentrations corresponding to its plasma level activate Na,K-ATPase, decrease the [Na+]i/[K+]i-ratio and increase cell proliferation. Possible physiological significance and mechanism of non-canonical Na+i/K+i-dependent and Na+i/K+i-independent cell responses to CTS are discussed.

Marinobufagenin (MBG) is a bufadienolide cardiac inotrope implicated in volume expansion-mediated hypertensive states including essential hypertension and preeclampsia (PE). Endogenous MBG is an inhibitor of the α1-isoform of Na+,K+-ATPase with vasoconstrictive and cardiotonic properties, causing hypertension and natriuresis. Elevated endogenous MBG-like material levels have been described by immunoassays in salt-sensitive pregnant and preeclamptic rats as well as in preeclamptic human patients. The rise of endogenous MBG-like material appears prior the development of the main symptoms of PE, leading us to consider MBG as one of the potential biomarkers for PE. The weak specificity and the high variability of the published immunoassays gives no certification about endogenous MBG existence. This led us to set-up a highly specific and sensitive analytical method to detect MBG in plasma at low levels relying on liquid chromatography combined to mass spectrometry (UHPLC-MS/MS) with recording of 7 highly specific MRM transitions for MBG. Pure MBG standard used in the method development was obtained by purification from the Bufo marinus toad venom. d3-25-hydroxyvitamin D3 was used as internal standard. An increasing organic gradient with mobile phase A and B composed of 97:3 (v/v) H2O: MeOH and 50:45:5 (v/v/v) MeOH:IPA:H2O at pH 4.5 respectively was used on a Pursuit 3 PFP column (100 mm × 3 mm; 3 µm) to allow elution and separation of the plasmatic compounds. Chromatographic analyses of plasma samples were preceded by a precipitation of proteins pretreatment. The developed UHPLC-MS/MS assay has been applied to early-pregnant women plasma samples allowing us to investigate MBG plasma levels. Thanks to the high specificity of the assay we were able to authenticate and certify the presence of endogenous MBG in early-pregnant women plasma with the use of the 7 selected specific mass transitions. These pioneering preliminary results are giving a promising perspective for early preeclampsia risk assessment in pregnant women.

Sodium-potassium ATPase (NaKA) is a plasma membrane enzyme responsible for influencing membrane physiology by direct electrogenic activity. It determines cellular excitability and synaptic neurotransmission, thus affecting learning and memory processes. A principle catalytic α subunit of NaKA has development-specific expression pattern. There are two α isoforms, α1 and α3, in adult brain neurons. Although NaKA is a housekeeping enzyme, the physiological differences between these two α isoforms in different brain regions have not been well explored. Endogenous cardiotonic steroids, including Marinobufagenin and Ouabain, control the cell homeostasis and cell functions via inhibiting NaKA. Here we employed selective inhibition of α1 and α3 NaKA isoforms by Marinobufagenin and Ouabain respectively, to measure the contribution of α subunits in cellular physiology of three distinct mouse brain regions. The results of the whole cell recording demonstrated that α1 isoform predominated in layer-5 pyramidal cells at rostral motor cortex, while α3 isoform governed the pyramidal neurons at hippocampal CA1 region and to a lesser extent the layer-5 pyramidal neurons of parietal cortex. Furthermore, selective α isoform inhibition induced differential effects on distinct physiological properties even within the same brain region. In addition, our results supported the existence of synergism between two NaKA α isoforms. To conclude, this systematic study of NaKA α isoforms demonstrated their broader roles in neuronal functioning in a region-specific manner.

Ever since British Physician William Withering first described the use of foxglove extract for treatment of patients with congestive heart failure in 1785, cardiotonic steroids have been used clinically to treat heart failure and more recently atrial fibrillation. Due to their ability to bind and inhibit the ubiquitous transport enzyme sodium potassium pump, thus regulating intracellular Na+ concentration in every living cell, they are also an essential tool for research into the sodium potassium pump structure and function. Exogenous CTS have been clearly demonstrated to affect cardiovascular system through modulation of vagal tone, cardiac contraction (via ionic changes) and altered natriuresis. Reports of a number of endogenous CTS, since the 1980s, have intensified research into their physiologic and pathophysiologic roles and opened up novel therapeutic targets. Substantive evidence pointing to the role of endogenous ouabain and marinobufagenin, the two most prominent CTS, in development of cardiovascular disease has accumulated. Nevertheless, their presence, structure, biosynthesis pathways and even mechanism of action remain unclear or controversial. In this review the current state-of-the-art, the controversies and the remaining questions surrounding the role of endogenous cardiotonic steroids in health and disease are discussed.

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 chronic kidney disease, we suggest that sustained elevation of the slow CNS pathway, plasma EO and the cardiotonic steroid marinobufagenin (MBG), comprise a feed-forward system that raises BP and accelerates kidney and cardiac damage. Block of the slow CNS pathway and/or circulating EO and MBG may reduce BP and slow the progression to end stage renal disease.

Marinobufagenin (MBG), a steroid compound belonging to the bufadienolide cardiac inotropes, is a molecule enjoying a growing interest in the early diagnostic of volume expansion-mediated hypertensive states. This endogenous mammalian cardiotonic and natriuretic bufadienolide (characterized by vasoconstrictive activities) inhibits the α1 isoform of Na(+), K(+)-ATPase, implicating it in series of pathophysiological circumstances such as volume-expansion, essential hypertension and preeclampsia. Indeed, the enhanced production of MBG in preeclamptic patients has been confirmed in several studies, leading us to consider MBG as a biomarker for preeclampsia. The main source for MBG is located in the parotid and skin gland secretions of the toad Bufo marinus in which MBG is the major steroid cardiotonic component. This review emphasizes the key role of analytical development for dosage methods of MBG in biofluids, in the emergence of future perspectives in the diagnostic and therapeutic fields of preeclampsia (e.g. to investigate the biosynthetic origin of MBG and to better understand its implications).

Limited but provocative ecologic epidemiology suggests that dietary salt may play a central role in the genesis of not only of stroke, but also dementia, including Alzheimer’s disease. Impairment of nitric oxide bioactivity in the cerebral microvasculature is a likely mediator of this effect. Salted diets evoke increased adrenal secretion of the natriuretic steroid marinobufagenin (MBG), which promotes natriuresis via inhibition of renal tubular Na+/K+-ATPase; this effect is notably robust in salt-sensitive rodent strains in which other compensatory natriuretic mechanisms are subnormally efficient. MBG-mediated inhibition of sodium pumps in vascular smooth muscle likely plays a role in the hypertension induced by salty diets in these rodents. However, salt sensitivity in humans is associated with increased vascular mortality and ventricular hypertrophy independent of blood pressure; this suggests that MBG may be pathogenic via mechanisms unrelated to blood pressure control. Indeed, recent evidence indicates that MBG, via interaction with alpha1 isoforms of the sodium pump, can activate various intracellular signaling pathways at physiological concentrations too low to notably inhibit pump activity. An overview of current evidence suggests the hypothesis that MBG – as well as the cyclic strain induced by hypertension per se – may induce endothelial oxidative stress by activating NADPH oxidase. If so, this could rationalize the increase in vascular and systemic oxidative stress observed in salt-sensitive rodents fed salty diets, or in rodents infused with MBG; moreover, if this effect is a particularly prominent determinant of oxidative stress in cerebrovascular endothelium, it might help to explain the virtual absence of stroke and dementia in low-salt societies. As a corollary of this hypothesis, it can be predicted that spirulina-derived phycobilins, which appear to mimic the physiological role of bilirubin as an inhibitor of NAPDH oxidase complexes, may have potential for ameliorating the adverse health impacts of MBG and of salty diets. Potassium-rich diets are also likely to be protective in this regard, as they should suppress MBG production via their natriuretic impact, while their stimulatory effect on sodium pump activity may exert a hyperpolarizing effect on plasma membranes that suppresses NADPH oxidase activity.

Preeclampsia (PE) is a hypertensive disorder of pregnancy, in which marinobufagenin (MBG), a circulating cardiotonic steroid, is increased. The Gadd45a stress sensor protein is an upstream modulator of the pathophysiological changes observed in PE. However, the effects of MBG on Gadd45a stress signaling remain unknown. We examined the expression of Gadd45a, the sFlt-1 receptor, and p38, as well as caspase 3 and 8 activities in placental samples from four groups of rats. These were: normal pregnant (NP, n=8); pregnant rats which received weekly injections of desoxycorticosterone acetate and 0.9% saline as their drinking water (PDS, n=9); normal pregnant rats injected with MBG (NPM, n=8); and PDS rats injected with resibufogenin (RBG), an in vivo antagonist of MBG (PDSR, n=8). Utilizing human cytotrophoblast (CTB) cells, we examined the effect of MBG on these stress signaling proteins in vitro. Placental Gadd45a expression, caspase 3 and 8 activities, sFlt-1 concentrations, and sFlt-1 receptor expression were significantly higher in PDS and NPM compared to NP and PDSR rats. Gadd45a protein was significantly upregulated in the CTB cells when MBG was present in concentrations ≥1nM. Treatment with MBG (≥1nM) also significantly arrested cell cycle progression and activated the expression of the Gadd45a-mediated stress signaling proteins. Inhibition of Gadd45a through RNAi-mediation attenuated MBG-induced CTB cell stress signaling. In conclusion, MBG is involved in the alteration in Gadd45a stress signaling both in vivo and in vitro and RBG prevents these changes when administered in vivo.

The bufadienolides are a group of steroid hormones that circulate in blood and are excreted in urine. They have the ability to inhibit the adenosine triphosphatase sodium-potassium pump (Na(+)-K(+)-ATPase), with predilection for its alpha1 isoform. This capability enables them to share with other cardiac glycosides the facility to cause an increase in sodium excretion, produce vasoconstriction resulting in hypertension, and act as cardiac inotropes. Bufadienolides have been implicated in instances of volume expansion-mediated hypertension, syndromes in which they are considered capable of causing a vascular leak, interfering with cellular proliferation, and inhibiting cellular maturation. An antagonist to the most well-studied bufadienolide, marinobufagenin, is resibufogenin, a compound that provides promise for the treatment of disorders in which excessive levels of marinobufagenin are present and are etiopathogenetic.

The sodium–potassium ATPase (Na/K ATPase) is a major ionic transporter in the brain and is responsible for the maintenance of the Na + and K + gradients across the cell membrane. Cardiotonic steroids such as ouabain, digoxin and marinobufagenin are well-characterized inhibitors of the Na/K ATPase. Recently, cardiotonic steroids have been shown to have additional effects at concentrations below their IC 50 for pumping. The cardiotonic steroids ouabain, digoxin, and marinobufagenin all show an inverted U-shaped dose–response curve with inhibition of pumping at concentrations near their IC 50 , while increasing Na/K ATPase activity at doses below their IC 50 . This stimulatory effect of cardiotonic steroids was observed in vitro in hippocampal slice cultures as well as in the hippocampus in vivo . Increased Na/K ATPase activity has been shown to protect slice culture neurons from hypoxia–hypoglycemia. Ouabain protected slice culture neurons from experimental ischemia at concentrations that increased Na/K ATPase. This protective effect was observed when ouabain was dosed 30 min before, or 2 h following experimental ischemia. Ouabain no longer protected against experimental ischemia if the increase of Na/K ATPase was blocked. These data suggest that the protective effect of ouabain was due to increased Na/K ATPase activity. The demonstration of a neuroprotective effect of cardiotonic steroids could potentially assist in the treatment of stroke since digoxin, one of the cardiotonic steroids examined in this study, has approval by the Food and Drug Administration and can be safely administered at the concentrations that increase Na/K ATPase activity.

Digibind and DigiFab are commercial formulations of polyclonal, ovine, digoxin-specific Fabs in clinical use for treatment of digoxin intoxication. Of interest for extending its use to other clinical indications, Digibind has also been reported to neutralize the effect of endogenous digoxin-like molecules, including ouabain, that are linked to clinical disorders ranging from preeclampsia to congestive heart failure. Although Digibind and DigiFab are equivalent in their digoxin-binding activity, the antigens used to produce these Fabs are different. We therefore explored, using native (3)H-digoxin and (3)H-ouabain in four different types of solution-phase binding methods, whether they might exhibit different profiles with respect to ouabain and other digoxin-like factors. Consistent with previous results, both Fab preparations bound digoxin with the same affinities and capacities. However, (3)H-ouabain was found to bind with high affinity only to Fab sub-populations present in both products. Interestingly, this sub-population was twice as large for Digibind compared to DigiFab. Competition experiments also showed differences in specificity within Fab sub-populations. Therefore, the equivalence in digoxin-binding activity of the two Fab preparations does not extend to ouabain-binding capacity and Fab specificity, with implications for clinical differentiation between the preparations in treatment of disorders related to control of non-digoxin cardenolides. The existence of a small but perhaps clinically relevant sub-population of antibodies was detected using specific radioligands. This sub-population could not have been detected nor quantified using standard cross-reactivity in an ELISA assay.

An endogenous natriuretic and vasoconstrictor Na/K-ATPase inhibitor, marinobufagenin (MBG), is implicated in NaCl-induced hypertension and in ethanol addiction. In rats, MBG suppresses voluntary alcohol intake, while immunization against MBG induces alcohol-seeking behavior. Since alcohol withdrawal is associated with elevation of blood pressure (BP) and renal sodium retention, we hypothesized that MBG mediates pressor response to ethanol withdrawal. In male Sprague-Dawley rats, forced ethanol intake (20% v/v, 2.8+/-0.2 g/day for 7 days) did not affect BP and MBG excretion. Ethanol withdrawal was associated with a 21 mm Hg increase in BP, a 10% decrease in hematocrit, and a three-fold increase in renal MBG excretion. In vivo administration of anti-MBG antibody to rats prevented withdrawal-induced BP elevation. Therefore, MBG mediates pressor response to ethanol withdrawal, and may link mechanisms of ethanol dependence and hypertension.

Endogenous inhibitors of the Na/K-ATPase (NKA) and diabetes mellitus (DM) are both risk factors for preeclampsia and NaCl sensitive hypertension. Our goal was to test the hypothesis that NaCl supplementation, induces preeclampsia-like symptoms in pregnant rats with DM via stimulation of marinobufagenin (MBG), a natriuretic and vasoconstrictor inhibitor of the NKA. Type 2 DM in female Sprague-Dawley rats was induced by administration of 65 mg/kg streptozotocin at day 4 post partum. In intact rats, pregnancy was associated with a 2-fold increase in MBG levels and a mild impairment in glucose tolerance. Pregnant rats with DM exhibited fetal macrosomia, greater impairment of glucose tolerance, and higher levels of MBG as compared to that in normal pregnant rats. As compared to intact pregnant rats, NaCl supplementation of diabetic pregnant rats (drinking 1.8% NaCl during days 12-19 of pregnancy) was associated with an increase in systolic blood pressure, decreased fetal and placental weight, five-fold elevation of MBG excretion, and 42% inhibition of NKA in erythrocytes. In nonpregnant rats, in vivo pretreatment with anti-MBG antibody produced an exaggerated response of plasma levels of glucose and insulin in oral glucose tolerance test. These results suggest that MBG is a common factor in the pathogenesis of DM and preeclampsia, and that regulation of glucose tolerance may be one of the physiological functions of endogenous cardiotonic steroids.

Current evidence demonstrates that augmented production of endogenous cardiotonic steroids (CTS) such as ouabain and marinobufagenin is involved in the pathogenesis of hypertension and other cardiovascular diseases associated with volume expansion. It is also well documented that the development of hypertension and the cardiovascular complications of this disease are provoked by hypokalemia and suppressed by high-K + diet. We hypothesized that altered extracellular K + (K + )o handling contributes to the pathogenesis of hypertension via modulation of interaction of endogenous CTS with Na + /K + -ATPase. To examine this hypothesis, experiments were performed with C7-Madin-Darby canine kidney epithelial cells at [K + ]o detected in plasma under control conditions (4.5 mM), severe hypokalemia (2 mM), and hyperkalemia (7 mM). Elevation of [K + ]o from 2 to 7 mM increased the threshold of modulation of intracellular (Na + )i and (K + )i content by ouabain from 1 to 10 nM, which corresponds to the range of endogenous CTS detected in plasma from patients with volume-expanded disorders. In control medium, ∼30% activation of cell proliferation was observed with 3 nM ouabain, whereas the addition of 0.3 nM ouabain was sufficient to induce about the same increment of cell proliferation in K + -depleted medium. [K + ]o elevation up to 7 mM completely abolished the proliferative effect of ouabain. At [K + ]o = 2, 4.5 and 7 mM, the death of ouabain-treated cells was indicated in the presence of 10, 30 and 300 nM ouabain, respectively. In conclusion, our results showed that modulation of [K + ]o in a pathophysiologically reasonable range sharply affected efficacy of endogenous CTS in the elevation of the [Na + ]i/[K + ]i ratio and in triggering (Na + )i,(K + )i-independent signaling resulting in cell proliferation and death. We propose that Na + /K + -ATPase may be considered as a [K + ]o sensor involved in the crosstalk of (K + )o handling with the pathogenesis of cardiovascular diseases. © 2006 Elsevier Ireland Ltd. All rights reserved.

Preeclampsia is a pregnancy-specific syndrome that is the leading cause of maternal death during pregnancy in the developed world. In preeclampsia, a combination of immunological, genetic and environmental factors can lead to altered cytotrophoblast (CTB) invasion of the uterine wall, a process that is critical for normal placental development and pregnancy maintenance. Marinobufagenin (MBG) is an endogenous inhibitor of the sodium pump Na + /K + ATPase, and increased plasma MBG is associated with hypertension, chronic renal failure and preeclampsia. In the present study, the effects of MBG on CTB differentiation and invasion were investigated utilizing the first trimester extravillous CTB cell line SGHPL-4. MBG significantly inhibited SGHPL-4 proliferation in a dose-dependent manner. In addition, growth factor-induced migration and invasion were significantly inhibited by MBG treatment. These findings demonstrate that MBG impairs CTB differentiation along the invasive pathway. Elucidating the mechanisms by which MBG impairs placental development may increase our understanding of fetal and maternal pathologies associated with preeclampsia.

The cardiotonic steroid, ouabain, a specific inhibitor of Na(+),K(+)-ATPase, initiates protein-protein interactions that lead to an increase in growth and proliferation in different cell types. We explored the effects of ouabain on glucose metabolism in human skeletal muscle cells (HSMC) and clarified the mechanisms of ouabain signal transduction. In HSMC, ouabain increased glycogen synthesis in a concentration-dependent manner reaching the maximum at 100 nM. The effect of ouabain was additive to the effect of insulin and was independent of phosphatidylinositol 3-kinase inhibitor LY294002 but was abolished in the presence of a MEK1/2 inhibitor (PD98059) or a Src inhibitor (PP2). Ouabain increased Src-dependent tyrosine phosphorylation of alpha(1)- and alpha(2)-subunits of Na(+),K(+)-ATPase and promoted interaction of alpha(1)- and alpha(2)-subunits with Src, as assessed by co-immunoprecipitation with Src. Phosphorylation of ERK1/2 and GSK3alpha/beta, as well as p90rsk activity, was increased in response to ouabain in HSMC, and these responses were prevented in the presence of PD98059 and PP2. Incubation of HSMC with 100 nM ouabain increased phosphorylation of the alpha-subunits of the Na-pump at a MAPK-specific Thr-Pro motif. Ouabain treatment decreased the surface abundance of alpha(2)-subunit, whereas abundance of the alpha(1)-subunit was unchanged. Marinobufagenin, an endogenous vertebrate bufadienolide cardiotonic steroid, increased glycogen synthesis in HSMC at 10 nM concentration, similarly to 100 nM ouabain. In conclusion, ouabain and marinobufagenin stimulate glycogen synthesis in skeletal muscle. This effect is mediated by activation of a Src-, ERK1/2-, p90rsk-, and GSK3-dependent signaling pathway.

Preeclampsia is a disorder which is responsible for significant maternal morbidity and mortality as well as fetal wastage. Its pathogenesis remains obscure and its only treatment is the delivery of the placenta and the fetus. Over time it has become clear that this syndrome is not a single disease but a disorder with, most likely, multiple etiologic factors that have a common (or similar) phenotype(s). A leading hypothesis, first developed in the early 1970s, is that the hypertension, proteinuria and intrauterine growth restriction are the result of hypoperfusion of the maternal-fetal unit. However, the early events leading to this deranged circulatory event have not been extensively studied. We hypothesize that at least one of the early pathogenetic events is excessive expansion of the extracellular fluid volume. This leads to persistent elaboration of (a) circulating factor(s) that interfere(s) with remodeling of the decidual vasculature preventing normal placentation from occurring. Our experiments have dealt largely with the role that an endogenous bufadienolide, marinobufagenin (MBG), plays in this pathogenetic process. In this report, we provide evidence for this thesis and point to future studies aimed at testing this hypothesis. These will include evaluating large groups of preeclamptic patients to determine their blood and urinary levels of MBG. Efforts will also be made to determine if there are differences in sodium handling in those patients with elevated levels of MBG, compared to other preeclamptic patients and to normal pregnant subjects.

The search for an endogenous digitalis has led to the identification of the cardenolides ouabain and digoxin and the bufadienolide marinobufagenin in mammalian tissues and biological fluids. Ouabain's release from adrenal glands is under the control of epinephrine and angiotensin II; hence, its blood concentration changes rapidly on physical exercise. It also is controlled by brain areas sensing cerebrospinal Na+ concentration and apparently the body's K+ content because urinary K+ loss leads to an increase in its plasma concentration as well. Long-term treatment of rats with ouabain results in arterial hypertension, and 50% of Caucasians with low-renin hypertension have increased plasma concentrations of this cardenolide. Levels of digoxin, which is synthesized from acetate in adrenal glands, increase slightly in blood on prolonged exercise. It counteracts the hypertensinogenic action of ouabain in rats, as does the ouabain antagonist PST 2238. The plasma concentration of the bufadienolide marinobufagenin is increased after cardiac infarction. It may show natriuretic properties because it inhibits the alpha1 isoform of Na+/K+-adenosine triphosphatase (ATPase), the main sodium pump isoform of the kidney, much better than other sodium pump isoforms. These effects of endogenous cardiac glycosides are observed at concentrations that do not inhibit the sodium pump. Apparently, Na+/K+-ATPase is used by these steroids as a signal transducer to activate tissue proliferation, heart contractility, arterial hypertension, and natriuresis via various intracellular signaling pathways.

Backround and objectives: There are several potential endogenous digitalis-like factors (EDLF) in mammalian body fluids, and marinobufagenin (MBG) may be the most potent EDLF. Improved assays are needed to confirm the potency of these metabolites. In the present study, we have identified MBG and telocinobufagin (TCB) in human plasma by high-resolution mass spectrometry (MS) and nuclear magnetic resonance (NMR). Methods and results: The high-resolution MS analysis revealed the molecular masses of TCB and MBG to be the same as their respective theoretical values. Using a tandem mass spectrometer, the mass–charge ratio for TCB was determined to be 403.2 for the parent ion and 349.2 for the daughter ion. The mass–charge ratio for MBG was m/z 383.2 and m/z 401.2. The NMR study revealed that the signals for MBG and TCB were the same as those obtained by MS analysis. In human blood, MBG and TCB were also identified by liquid chromatography (LC) as well as MS. In the LC/MS assay, proscillaridin A was used as an internal standard. The plasma was pretreated with Sep-Pak C18, and then 50 μL was applied to the C8 high-performance liquid chromatography (HPLC) column. The mean plasma concentration of MBG in healthy volunteers (0.94 ± 0.28 ng/mL) was significantly lower than that in patients undergoing regular hemodialysis (3.81 ± 1.92 ng/mL). The concentration of TCB in the healthy volunteers (1.80 ± 0.55 ng/mL) was also significantly lower than that in patients with terminal renal failure (6.86 ± 4.30 ng/mL). Conclusion: These results indicate that the major EDLF is TCB because its plasma concentration is the highest among the reported endogenous digitalis candidates.

Individuals who eat salty diets and who are "salt-sensitive" tend to have increased left ventricular mass, independent of blood pressure; this phenomenon awaits an explanation. It is clear that local up-regulation of angiotensin II (AngII) production and activity play a key role in the induction of left ventricular hypertrophy (LVH). Recent evidence suggests that a healthy coronary microvascular endothelium opposes this effect by serving as a paracrine source of nitric oxide (NO), a natural antagonist of AngII activity, and that up-regulation of this mechanism can account for the protective role of bradykinin with respect to LVH. The coronary microvasculature also possesses NAD(P)H oxidase activity that can generate superoxide, inimical to the bioactivity of endothelial NO. There is now good reason to believe that the triterpenoid marinobufagenin (MBG), a selective inhibitor of the alpha-1 isoform of the sodium pump, mediates the impact of salty diets on blood pressure; production of MBG by the adrenal cortex is boosted when salt-sensitive animals are fed salty diets. It is hypothesized that coronary microvascular endothelium expresses the alpha-1 isoform of the sodium pump, and that MBG thus can target this endothelium. If that is the case, MBG would be expected to decrease membrane potential in these cells; as a consequence, superoxide production would be up-regulated, NO synthase activity would be down-regulated, and myocardial NO bioactivity would thus be suppressed. This would offer a satisfying explanation for the impact of salt and salt-sensitivity on risk for LVH. If expression of the alpha-1 isoform of the sodium pump is a more general property of vascular endothelium, MBG may suppress NO bioactivity in other regions of the vascular tree, thereby contributing to other adverse effects elicited by salty diets: reduced arterial compliance, medial hypertrophy, impaired endothelium-dependent vasodilation, hypertensive/diabetic glomerulopathy, increased risk for stroke, and hypertension.

In the present study the hypothesis was tested that sodium pump ligands (SPL) can modulate alcohol-seeking behavior and that this effect is related to changes in Na/K-ATPase activity in the central nervous system. Mice were tested for initiation of ethanol intravenous self-administration (IVSA) following i.p. pretreatment with vehicle or the endogenous SPL, marinobufagenin (MBG). Drug- and experimentally-naive mice acquired IVSA of 2% ethanol during a single 30-min session. MBG was found to dose-dependently attenuate (1.25–2.5 μg/kg) initiation of ethanol IVSA producing a decrease in the ratio and in the difference between operant responses of response-dependent and yoked animals as well as a decrease in percentage of mice demonstrating ethanol-seeking behavior. Attenuation of the reinforcing effect of ethanol resulting from MBG was associated with brain levels of this steroid capable of concurrently inhibiting Na/K-ATPase in the brain cortex. We hypothesize that endogenous digitalis-like factors could modulate the reinforcing effect of ethanol.

This study tested the hypothesis that endogenous digitalis-like factor (DLF) is involved in the development of alcohol dependence in rats. In 33 male Wistar rats in conditioned place preference (CPP) experiment, ethanol evoked increase in time spent in the ethanol-associated compartment (702+/-82 in ethanol-treated vs. 426+/-86 sec in the controls). Digoxin pretreatment (125 microg/kg, i/p) did not affect the time spent in the water-associated compartment (476+/-80 sec), but prevented the acquisition of ethanol CPP (385+/-112 sec in ethanol-paired side, P

Introduction Marinobufagenin (MBG) is an endogenous bufadienolide cardiac inotrope which is demonstrating growing interest in the early diagnosis of volume expansion-mediated hypertensive states such as preeclampsia (PE) and end-stage renal disease hypertension. Mammalian MBG is an inhibitor of the α 1 isoform of Na + , K + -ATPase with vasoconstrictive and cardiotonic properties, resulting in hypertension and natriuresis. Elevated endogenous MBG levels have been described in pregnant mammals and especially in preeclamptic patients [1,2,3]. The rise of endogenous MBG appears prior the development of the main symptoms of PE, leading us to consider MBG as one of the potential target in the biomarker panel for PE. A sensitive and accurate analytical method is needed to assess MBG in as lower level as possible in plasma. Currently, only marinobufagenin-like material has been found in humans using two published quantification methods based each on immunoassays [4,5]. These techniques suffer from a lack of specificity due to cross-reactivity and tend to exhibit high variability at low concentrations [6]. Objective Our aim is to develop a MBG assay using a more specific and easy to access technique, such as LC–MS/MS. An algorithm dealing with the MBG plasma levels might be established by clinicians in the future, in order to predict, in combination with other clinical and biological markers, the risk for preeclampsia in pregnant women. Methods As the major source for MBG is located in the parotid glands of the Bufo Marinus toad, we developed a purification method from toad venom in order to get pure MBG standard. A pre-extraction procedure was elaborated to concentrate and clean the plasma sample prior to its analysis. A LC–MS based assay designed to determine MBG in human plasma is being optimized, giving us the opportunity to investigate MBG in non-pregnant healthy volunteers plasma as well as in early pregnant women plasma. Results and discussion Pure MBG has been successfully extracted from the B. Marinus toad venom and the identity of the compound has been confirmed. An extraction procedure for MBG from plasma has been set up by use of solid phase extraction cartridges. Preliminary results allowed us to authenticate the presence of MBG by LC–MS/MS in non-pregnant women as well as in early pregnancy. Further optimization and validation of the LC–MS assay are needed to quantify MBG plasma levels. However, these pioneering observations, are giving the clinicians a promising perspective for early preeclampsia risk assessment in pregnant women.

Previously, we reported that the venom of Bufo marinus toad contains a Na+,K(+)-ATPase inhibitor with potent vasoconstrictor activity. In the present study, using thin-layer chromatography in Silicagel 60 F254 + 366, we separated a vasoactive substance from a mixture of steroids from Bufo marinus venom. Based on chromatographic mobility of this substance and typical color reaction after its vizualization with SbCl3, we identified it as a previously described steroid, marinobufagenin. Vasoconstrictor and Na+,K+ pump inhibitory properties of marinobufagenin were studied in isolated rat aortic rings and compared with those of ouabain. Ouabain (10-100 mumol.1-1) produced weak vasoconstriction, which was blocked by 2 mumol.1-1 phentolamine. 10 mumol.1-1 ouabain stimulated, and at higher concentrations inhibited, the Na+,K+ pump. 2 mumol.1-1 phentolamine abolished the activating effect of 10 mumol.1-1 ouabain on the Na+,K+ pump, but did not alter the inhibitory action of higher concentrations of ouabain. By contrast, marunibufagenin elicited rapid and strong vasoconstriction and inhibited ouabain-sensitive 86Rb uptake. Antidigoxin antibody antagonized the vasoconstrictor responses to marinobufagenin, but not to ouabain. 2 mumol.1-1 phentolamine did not alter the constrictor effect of marinobufagenin. In solid-phase digoxin immunoassay, marinobufagenin demonstrated higher digoxin-like immunoreactivity than ouabain.

Background— Plasma levels of cardiotonic steroids are elevated in volume-expanded states, such as chronic kidney disease, but the role of these natriuretic hormones in subjects with heart failure (HF) is unclear. We sought to determine the prognostic role of the cardiotonic steroids marinobufagenin (MBG) in HF, particularly in relation to long-term outcomes. Methods and Results— We first measured plasma MBG levels and performed comprehensive clinical, laboratory, and echocardiographic assessment in 245 patients with HF. All-cause mortality, cardiac transplantation, and HF hospitalization were tracked for 5 years. In our study cohort, median (interquartile range) MBG was 583 (383–812) pM. Higher MBG was associated with higher myeloperoxidase ( r =0.42, P r =0.25, P =0.001), and asymmetrical dimethylarginine ( r =0.32, P s ′, r =−0.39, P P =0.014) even after adjustment for age, sex, diabetes mellitus, and ischemic pathogenesis. In mice, a left anterior descending coronary artery ligation model of HF lead to increases in MBG, whereas infusion of MBG into mice for 4 weeks lead to significant increases in myeloperoxidase, asymmetrical dimethylarginine, and cardiac fibrosis. Conclusions— In the setting of HF, elevated plasma levels of MBG are associated with right ventricular dysfunction and predict worse long-term clinical outcomes in multivariable models adjusting for established clinical and biochemical risk factors. Infusion of MBG seems to directly contribute to increased nitrative stress and cardiac fibrosis.

Type-4 cardiorenal syndrome patients develop heart failure as result of kidney failure. While the mechanism of cardiac dysfunction is unknown, elevated levels of circulating endogenous Na/K ATPase inhibitors (NKAi) are detected in patients and animals with kidney failure. We investigated the pathophysiological consequences of NKAi elevation in rat myocytes and hearts of animals with chronic kidney disease (CKD).Methods: Intracellular Na concentration ([Nai]) was measured using fluorescent ratio-imaging microscopy and ERK1/2 phosphorylation by western blotting, in adult rat ventricular myocytes. Cardiac Na/K ATPase (NKA) activity was measured using biochemical NKA assays. CKD was induced in rats via 5/6 partial-nephrectomy (PN) or in mice via folic-acid administration (FA). Creatinine serum levels were increased in both models, with kidney dysfunction (and cardiac hypertrophy) being more severe in PN model.Results: Treatment of field-stimulated rat cardiac myocytes with bufalin (10nM) or ouabain (100nM) significantly increased [Nai] by 1.2±0.3 and 1.9±0.2mM, respectively, whereas, concentrations of marinobuganenin below 1μM did not. Furthermore, NKAi activated hypertrophic signaling cascades (assessed by ERK1/2 phosphorylation) with a potency order of bufalin (EC50=70nM)>ouabain (EC50=93nM)> marinobuganenin (EC50=6.4μM). Despite small but significant increases in NKA alpha-1/2 expression, NKA activity in hearts of animals with kidney dysfunction was reduced by 45±19% in PN (6 weeks post-PN) and 34±9% in FA animals (12 weeks post FA-injection). NKA inhibition was reversed by incubation with a polyclonal anti-Digoxin antibody (binds most structurally-related NKAi), confirming involvement of NKAi.Conclusions: Pathophysiologically-relevant concentrations of bufalin and ouabain, but not marinobufagenin, raise [Nai] and activate hypertrophic signaling pathways in the heart, thus contributing to Na and diastolic Ca overload and hypertrophy, during CKD.

syndrome in preeclampsia: in vivo and in vitro study Mohammad Uddin, Steven Allen, Richard Jones, David Zawieja, Thomas Kuehl Texas A&M Health Science Center College of Medicine/Scott & White Memorial Hospital, Department of Obstetrics and Gynecology, Temple, TX, Texas A&M Health Science Center College of Medicine, Dept. of Systems Biology and Translational Medicine, Temple, TX OBJECTIVE: Preeclampsia (PreE) has multiple pathophysiologic triggers leading to a common cascade of symptoms. In an animal model of this syndrome, we have shown that the urinary excretion of a bufadienolide, marinobufagenin (MBG), is elevated prior to the development of symptoms. Vasogenic cerebral edema represents a breach of the blood-brain barrier (BBB) and is a potential preE complication. We investigated alterations in the endothelial cells of the cerebral circulation in rats rendered “preE”, the effects of MBG on these alterations, and the underlying molecular mechanisms. STUDY DESIGN: (1) BBB permeability in preE rats was assessed by Evans blue (EB) dye extravasation into brain parenchyma by a fluorescent assay. (2) Human brain microvascular endothelial cells (HBMEC) were used to test for effects of MBG in vitro on monolayer permeability. In HBMEC, phosphorylation of ERK1/2, Jnk, p38, and Src was evaluated after MBG treatment. Apoptosis was evaluated by examining alterations in caspase 3/7 and annexin-V staining with and without pretreatment with p38 inhibitor before MBG treatment. Effects of MBG on endothelial tight junction proteins were assessed by immunofluorescence microscopy. RESULTS: (1) In the striatum, the extent of dye extravasation was greater (p 0.05) in preE rats (9.1 g EB/mg dry tissue) than in controls (6.2 g EB/mg dry tissue). (2) Concentrations of MBG 1 nM significantly inhibited the proliferation of HBMEC by 46-50%. MBG also significantly increased monolayer permeability within 6 hours (1.5 / 0.18 fold), caused a significant decrease in the phosphorylation of ERK1/2 (80%), and activated the phosphorylation of Jnk (56%), p38 (71%), and Src (75%). The expression of caspases 3/7 was increased indicating activation of apoptosis (1.5 / 0.12 fold). The activation of apoptosis was prevented by pretreatment with a p38 inhibitor. Additionally, MBG caused the disruption of endothelial adherens tight junction proteins. CONCLUSION: These data provide evidence for the view that MBG may play an important role in cerebral edema and neurologic abnormalities seen in preE.

The cardiotonic receptor on the Na,K-ATPase and an endogenous ligand play a physiological role in regulating its enzyme activity in mammalian skeletal muscle (Radzyukevich et al. 2009. PNAS 106(8):2565-70). The goal of this study was to develop an approach to identify the endogenous ligand(s). The regulation is preserved in vitro and crude muscle extracts retain active factor(s). Therefore, we used muscle as a source of concentrated bound ligand, in combination with a batch affinity purification method which uses the receptor itself to capture its ligands. We applied affinity extraction with a purified Na,K-ATPase to a MeOH extract from skeletal muscle. Ligands were bound to the receptor in Mg2+-containing buffer and released in EDTA. The efficiency of ligand binding was confirmed and optimized using 3H-ouabain. Binding of Mg2+ to a single Mg binding site enhances binding at the cardiotonic receptor site by a factor of 215, and thereby favors specific over potentially non-specific ligand binding. The resulting compounds were desalted on hydrophobic SPE cartridge and separated on HPLC with a C18 column. One of the fractions from muscle showed a single peak in MS (m/z 381 M + Na+). This compound is distinct from ouabain (m/z 606), ouabagenin (m/z 460), marinobufagenin (m/z 423) and other known cardiotonic steroids. We obtained the same factor from pig kidney, plus 3 additional compounds. These results demonstrate that at least one novel cardiotonic hormone is common to different mammalian species and tissues. Given the broad scientific and therapeutic implications of the cardiotonic hormones, our current goal is to accumulate a sufficient amount of this compound for structural identification by cryo NMR. This goal is feasible using a scaled up procedure which yields nanogram amounts (in ouabain equivalent units) per run.