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Marinobufagenin (MBG) is implicated in chronic kidney disease, where it removes Fli1-induced inhibition of the collagen-1. We hypothesized that (i) in nephrectomized rats, aortic fibrosis develops due to elevated plasma MBG and inhibited Fli1, and (ii) that the antibody to MBG reduces collagen-1 and improves vasodilatation. A partial nephrectomy was performed in male Sprague-Dawley rats. Sham-operated animals comprised the control group. At 5 weeks following nephrectomy, rats were administered the vehicle ( n = 8), or the anti-MBG antibody ( n = 8). Isolated aortic rings were tested for their responsiveness to sodium nitroprusside following endothelin-1-induced constriction. In nephrectomized rats, there was an increase in the intensity of collagen staining in the aortic wall vs. the controls. In antibody-treated rats, the structure of bundles of collagen fibers had ordered organization. Western blots of the aorta had lower levels of Fli1 (arbitrary units, 1 ± 0.05 vs. 0.2 ± 0.01; p < 0.001) and greater collagen-1 (arbitrary units, 1 ± 0.01 vs. 9 ± 0.4; p < 0.001) vs. the control group. Administration of the MBG antibody to rats reversed the effect of the nephrectomy on Fli1 and collagen-1 proteins. Aortic rings pretreated with endothelin-1 exhibited 50% relaxation following the addition of sodium nitroprusside (EC 50 = 0.28 μmol/L). The responsiveness of the aortic rings obtained from nephrectomized rats was markedly reduced (EC 50 = 3.5 mol/L) compared to the control rings. Treatment of rats with the antibody restored vasorelaxation. Thus, the anti-MBG antibody counteracts the Fli1-collagen-1 system and reduces aortic fibrosis.

Background: Previously we demonstrated that in patients with preeclampsia elevated levels of endogenous Na/K-ATPase inhibitor, marinobufagenin, cause inhibition of Friend leukemia virus integration 1 (Fli1), a negative regulator of collagen-1 synthesis. We hypothesized that in vitro silencing of Fli1 in healthy human umbilical arteries would be associated with an increase in collagen-1 output, similar to the effect of preeclampsia in rat and human tissues., Methods: The isolated segments of healthy human umbilical arteries were tested for sensitivity to MBG and Fli1 silencing with Fli1 siRNA or control siRNA., Results: Following 24-hour incubation of arteries with nanomolar concentrations of marinobufagenin, Fli1 expression was inhibited 5-fold (P < 0.001), and synthesis of collagen-1 increased 3 times (P < 0.01). Twenty-four-hour incubation of umbilical artery fragments with Fli1 siRNA caused a dramatic decrease of Fli1 (7-fold; P < 0.001) and cytoplasmic PKC δ (4-fold; P < 0.001) expression in comparison to control siRNA or untreated control, followed by elevation in procollagen (3-fold; P < 0.001) and collagen-1 (3-fold; P < 0.001) levels in vascular tissue., Conclusions: Our results show that after silencing the Fli1 gene in healthy human umbilical arteries a new phenotype emerges which is typical for preeclampsia and is associated with vascular fibrosis., (© The Author(s) 2022. Published by Oxford University Press on behalf of American Journal of Hypertension, Ltd. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Previous studies implicated cardiotonic steroids, including Na/K-ATPase inhibitor marinobufagenin (MBG), in the pathogenesis of preeclampsia (PE). Recently, we demonstrated that (i) MBG induces fibrosis in rat tissues via a mechanism involving Fli1, a negative regulator of collagen-1 synthesis, and (ii) MBG sensitive Na/K-ATPase inhibition is reversed by mineralocorticoid antagonists. We hypothesized that in human PE elevated MBG level is associated with the development of fibrosis of the umbilical arteries and that this fibrosis can be attenuated by canrenone. Fifteen patients with PE (mean BP = 118 ± 4 mmHg; 34 ± 2 years; 38 ± 0.3 weeks gest. age) and twelve gestational age-matched normal pregnant subjects (mean BP = 92 ± 2 mmHg; 34 ± 1 years; 39 ± 0.2 weeks gest. age) were enrolled in the study. PE was associated with a higher plasma MBG level, with a four-fold decrease in Fli1 level and a three-fold increase in collagen-1 level in the PE umbilical arteries vs. those from the normal subjects (p < 0.01). Isolated rings of umbilical arteries from the subjects with PE exhibited impaired responses to the relaxant effect of sodium nitroprusside vs. control vessels (EC50 = 141 nmol/L vs. EC50 = 0.9 nmol/L; p < 0.001). The effects of PE on Fli1 and collagen-1 were blocked by the in vitro treatment of umbilical arteries by 10 μmol/L canrenone. Similar results were obtained for umbilical arteries pretreated with MBG. These data demonstrate that elevated MBG level is implicated in the development of the fibrosis of umbilical arteries in PE, and that this could be blocked by mineralocorticoid antagonists.

Background: Previous studies implicated cardiotonic steroids, including Na/K-ATPase inhibitor marinobufagenin (MBG), in the pathogenesis of preeclampsia (PE). We demonstrated that MBG induces fibrosis via mechanism involving inhibition of Fli1, a nuclear transcription factor and a negative regulator of collagen-1 synthesis. We hypothesized that PE blockade of increased MBG with antibody would lessen the fibrosis of umbilical arteries and lower the blood pressure in rats with PE., Methods: We tested 36 pregnant Sprague-Dawley rats in which 12 were made hypertensive by 1.8% Na supplementation (days 6-19 of gestation), 12 pregnant rats served controls. At day 19, PE rats received one intraperitoneal injection of polyclonal anti-MBG-4 antibody (0.5 ug/ml) for 4 hours., Results: PE was associated with higher blood pressure (117 ± 2 vs. 107 ± 2 mm Hg; P < 0.01), plasma MBG levels (1.54 ± 0.34 vs. 0.49 ± 0.11 nmol/L; P < 0.01), protein excretion (26 vs. 12 mg/24 hours), sFlt-1 (3-fold), decrease in Fli1 (7-fold) and increase in collagen-1 in aorta (4-fold) vs. control rats (all P < 0.01). In 12 rats treated with polyclonal anti-MBG-4 antibody blood pressure dropped (93 ± 3 mm Hg) and Fli1 was decreased much less (2-fold; P < 0.01 vs. nontreated rats)., Conclusions: These results demonstrate that in experimental PE elevated MBG level is implicated in umbilical fibrosis via suppression of Fli1., (© American Journal of Hypertension, Ltd 2019. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Endogenous cardiotonic steroid, marinobufagenin (MBG), induces Fli1-dependent tissue fibrosis. We hypothesized that an increase in MBG initiates the development of aortic fibrosis in salt-loaded rats with type 2 diabetes mellitus (DM2) via pressure-independent mechanism. DM2 was induced by a single intraperitoneal administration of 65 mg/kg streptozotocin to neonatal (4-5 days) male Wistar rats. Eight-week-old DM2 rats received water or 1.8% NaCl (DM-NaCl) solution for 4 weeks (n = 16); half of DM-NaCl rats were treated with anti-MBG monoclonal antibody (mAb) (DM-NaCl-AB) during week 4 of salt loading; control intact rats received water (n = 8/group). Blood pressure, MBG, erythrocyte Na/K-ATPase activity, aortic weights, levels of fibrosis markers (Fli1, protein kinase Cδ, transforming growth factor-β1, receptors of the transforming growth factor beta5, fibronectin, collagen-1), and sensitivity of the aortic explants to the vasorelaxant effect of sodium nitroprusside were assessed. No changes in systolic blood pressure were observed while erythrocyte Na/K-ATPase was inhibited by 30%, plasma MBG was doubled, and aortic markers of fibrosis became elevated in DM-NaCl rats versus control. Treatment of DM-NaCl rats with anti-MBG mAb activated Na/K-ATPase, prevented increases in aortic weights, and the levels of fibrosis markers returned to the control levels. The responsiveness of the aortic rings from DM-NaCl rats to the relaxant effect of sodium nitroprusside was reduced (half maximal effective concentration (EC50) = 29 nmol/L) versus control rings (EC50 = 7 nmol/L) and was restored by anti-MBG mAb (EC50 = 9 nmol/L). Our results suggest that in salt-loaded diabetic rats, MBG stimulates aortic collagen synthesis in a pressure-independent fashion and that 2 profibrotic mechanisms, Fli1 dependent and transforming growth factor-β dependent, underlie its effects.

Background: Previous studies implicated cardiotonic steroids, including Na/K-ATPase inhibitor marinobufagenin (MBG), in the pathogenesis of preeclampsia (PE). Immunoneutralization of heightened MBG by Digibind, a digoxin antibody, reduces blood pressure (BP) in patients with PE, and anti-MBG monoclonal antibody lessens BP in a rat model of PE. Recently, we demonstrated that MBG induces fibrosis in cardiovascular tissues via a mechanism involving inhibition of Fli-1, a nuclear transcription factor and a negative regulator of collagen-1 synthesis., Objectives and Methods: We hypothesized that in PE, elevated placental MBG levels are associated with development of fibrosis in umbilical arteries. Eleven patients with PE (mean BP 124 ± 4 mmHg; age 29 ± 2 years; 39 weeks gest. age) and 10 gestational age-matched normal pregnant subjects (mean BP 92 ± 2 mmHg; controls) were enrolled in the clinical study., Results: PE was associated with a higher placental (0.04 ± 0.01 vs. 0.49 ± 0.11 pmol/g; p < 0.01) and plasma MBG (0.5 ± 0.1 vs. 1.6 ± 0.5 nmol/L; p < 0.01), lower Na/K-ATPase activity in erythrocytes (2.7 ± 0.2 vs. 1.5 ± 0.2 µmol Pi/mL/hr; p < 0.01), 9-fold decrease of Fli-1 level and 2.5-fold increase of collagen-1 in placentae ( p < 0.01) vs. control. Incubation of umbilical arteries from control patients with 1 nmol/L MBG was associated with four-fold decrease in Fli-1 level and two-fold increase in collagen-1 level vs. those incubated with placebo ( p < 0.01), i.e., physiological concentration of MBG mimicked effect of PE in vitro. Collagen-1 abundance in umbilical arteries from PE patients was 4-fold higher than in control arteries, and this PE-associated fibrosis was reversed by monoclonal anti-MBG antibody ex vivo., Conclusion: These results demonstrate that elevated placental MBG level is implicated in the development of fibrosis of the placenta and umbilical arteries in PE.

Background: Endogenous sodium pump inhibitors promote sodium excretion in normotensives and contribute to vasoconstriction in NaCl-sensitive hypertension. Marinobufagenin (MBG), an endogenous bufadienolide inhibitor of alpha-1 sodium pump, contributes to hypertension in Dahl salt-sensitive rats (DS). We hypothesized that in NaCl-loaded DS and normotensive Sprague-Dawley rats (S-D), MBG would elicit different patterns of sodium pump inhibition., Methods: We compared systolic blood pressure (SBP), renal sodium excretion, activity of the sodium pump in aorta and renal medulla, and levels of MBG, atrial natriuretic peptide (ANP), and cyclic guanosine monophosphate (cGMP) in salt-loaded DS and S-D (20% NaCl, 2.5 ml/kg, intraperitoneally)., Results: NaCl loading produced sustained elevations in renal MBG excretion in both DS (2.41 +/- 0.24 vs. 0.79 +/- 0.08 pmol/h/kg, P < 0.01) and S-D (1.97 +/- 0.37 vs. 0.60 +/- 0.07 pmol/h/kg, P < 0.01) vs. that at baseline (n = 10 for each group). In NaCl-loaded DS, SBP rose by 18 mm Hg (P < 0.01) and aortic sodium pump was inhibited by 22% (P < 0.05 vs. control), while in S-D, SBP and activity of aortic sodium pump did not change. NaCl-loaded S-D excreted twice as much sodium as DS; in S-D, renal sodium pump was inhibited by 24% vs. 14% inhibition in DS (P < 0.05). NaCl loading elicited increases in plasma ANP and in renal cGMP excretion in S-D but not in DS., Conclusions: Our present observations demonstrate that in NaCl-loaded S-D and DS, a comparable MBG response is associated with preferential inhibition of the sodium pump in the kidney and in vascular smooth muscle, respectively, resulting in an adaptive natriuresis in S-D but sodium retention and pressor response in DS.

Background: Levels of marinobufagenin (MBG), an endogenous bufadienolide Na/K-ATPase (NKA) inhibitor, increase in preeclampsia and in NaCl-sensitive hypertension., Methods: We tested a 3E9 monoclonal anti-MBG antibody (mAb) for the ability to lower blood pressure (BP) in NaCl-sensitive hypertension and to reverse the preeclampsia-induced inhibition of erythrocyte NKA. Measurements of MBG were performed via immunoassay based on 4G4 anti-MBG mAb., Results: In hypertensive Dahl-S rats, intraperitoneal administration of 50 microg/kg 3E9 mAb lowered BP by 32 mmHg and activated the Na/K-pump in the thoracic aorta by 51%. NaCl supplementation of pregnant rats (n = 16) produced a 37 mmHg increase in BP, a 3.5-fold rise in MBG excretion, and a 25% inhibition of the Na/K-pump in the thoracic aorta, compared with pregnant rats on a normal NaCl intake. In eight pregnant hypertensive rats, 3E9 mAb reduced the BP (21 mmHg) and restored the vascular Na/K-pump. In 14 patients with preeclampsia (mean BP, 126 +/- 3 mmHg; 26.9 +/- 1.4 years; gestational age, 37 +/- 0.8 weeks), plasma MBG was increased three-fold and erythrocyte NKA was inhibited compared with that of 12 normotensive pregnant women (mean BP, 71 +/- 3 mmHg) (1.5 +/- 0.1 vs. 3.1 +/- 0.2 micromol Pi/ml/h, respectively; P < 0.01). Ex-vivo 3E9 mAb restored NKA activity in erythrocytes from patients with preeclampsia. As compared with 3E9 mAb, Digibind, an affinity-purified antidigoxin antibody, was less active with respect to lowering BP in both hypertensive models and to restoration of NKA from erythrocytes from patients with preeclampsia., Conclusion: Anti-MBG mAbs may be a useful tool in studies of MBG in vitro and in vivo and may offer treatment of preeclampsia.

Background: Brain endogenous ouabain (EOU) raises blood pressure (BP) via an angiotensin II (ATII)-sensitive pathway in NaCl-loaded Dahl salt-sensitive rats (DSS). Brain EOU activates central and adrenocortical renin-angiotensin systems, and stimulates marinobufagenin, a vasoconstrictor and natriuretic inhibitor of sodium pump., Methods: We studied effects of acute NaCl loading (17 mmol/kg NaCl, intraperitoneally) on levels of EOU and marinobufagenin in several brain areas in DSS. We then studied effects of intrahippocampal administration of very-low-dose ouabain (60 pg) on EOU, marinobufagenin, BP, sodium excretion, and sodium-pump activity in the aorta and renal medulla in the absence and presence of anti-marinobufagenin and anti-ouabain antibodies, and losartan., Results: NaCl loading of DSS induced transient increases of EOU in the hippocampus and amygdala (15 min; 300%), supraoptical nucleus of hypothalamus (SON) (30 min, 230%) and pituitary (30 min; 85%), and ATII elevation in the SON (30 min). Intrahippocampal administration of ouabain (60 pg) stimulated ATII in the SON, produced natriuresis, 40 mmHg rise in BP, inhibition of sodium-pump in the renal medulla (19.6%) and aorta (25%), and a two-fold increase in renal marinobufagenin excretion. Pretreatment of rats with anti-marinobufagenin antibody prevented ouabain-induced pressor and natriuretic responses and sodium-pump inhibition. Pressor responses to ouabain were also prevented by losartan (intravenously) and by administration of anti-ouabain antibody into the SON., Conclusions: NaCl loading of DSS induces a cascade of events, triggered by brain EOU and ATII. Intrahippocampal administration of a low-dose ouabain mimics effects of NaCl loading and stimulates marinobufagenin, which produces natriuresis, and inhibits the vascular sodium-pump, inducing an increase in BP.

NaCl loading and plasma volume expansion stimulate 2 natriuretic systems, vasoconstrictor, digitalis-like Na/K-ATPase inhibitors and vasorelaxant ANP peptides. Several hormones, including ANP, regulate activity of the Na/K-ATPase by modulation of its phosphorylation state. We studied effects of ANP on Na/K-ATPase phosphorylation and inhibition by an endogenous sodium pump ligand, marinobufagenin, in the aorta and renal medulla from male Sprague-Dawley rats. Marinobufagenin dose-dependently inhibited the Na/K-ATPase in renal and vascular membranes at the level of higher (nanomolar) and lower affinity (micromolar) binding sites. Marinobufagenin (1 nmol/L) inhibited Na/K-ATPase in aortic sarcolemma (18%) and in renal medulla (19%). prepro-ANP 104 to 123 (ppANP) and alpha-human ANP ([alpha-hANP] both 1 nmol/L) potentiated marinobufagenin-induced Na/K-ATPase inhibition in the kidney, but reversed the effect of marinobufagenin in the aorta. Similarly, ppANP and alpha-hANP modulated the sodium pump (ouabain-sensitive (86)Rb uptake) inhibitory effects of marinobufagenin in the aorta and renal medulla. In renal medulla, ppANP and alpha-hANP induced alpha-1 Na/K-ATPase phosphorylation, whereas in aorta, both peptides dephosphorylated Na/K-ATPase. The effect of ppANP on Na/K-ATPase phosphorylation and inhibition was mimicked by a protein kinase G activator, 8-Br-PET-cGMP (10 micromol/L), and prevented by a protein kinase G inhibitor, KT5823 (60 nmol/L). Our results suggest that alpha-1 Na/K-ATPase inhibition by marinobufagenin in the kidney is enhanced via Na/K-ATPase phosphorylation by ANP, whereas in the aorta, ANP exerts an opposite effect. The concurrent production of a vasorelaxant, ANP, and a vasoconstrictor, marinobufagenin, potentiate each other's natriuretic effects, but ANP peptides may offset the deleterious vasoconstrictor effect of marinobufagenin.

Objective: In NaCl-loaded Dahl salt-sensitive (DS) rats the transient stimulation of brain endogenous ouabain (EO) precedes the increase in renal excretion of marinobufagenin (MBG), a vasoconstrictor and natriuretic. In hypertensive DS rats, EO raises blood pressure (BP) via an ATII-sensitive pathway. We hypothesized that an NaCl-induced increase in MBG is linked to the EO-stimulated ATII pathway., Methods: We studied the effects of 3 h of NaCl loading (17 mmol/kg, intraperitoneally) in male DS rats treated with antibodies to MBG or ouabain, or with losartan (25 mg/kg)., Results: NaCl loading alone induced a transient stimulation of pituitary EO (22.4 +/- 1.8 versus 12.2 +/- 1.3 pmol/g) and ATII (39.4 +/- 2.8 versus 18.4 +/- 3.2 ng/g), a sustained increase in MBG excretion (5.2 +/- 0.6 versus 1.1 +/- 0.2 pmol/h), a 40% inhibition of the renal sodium pump, a natriuretic response, a 35 mmHg increase in systolic BP, and an increase in adrenocortical ATII and MBG levels and in plasma norepinephrine. The anti-MBG antibody reduced the natriuresis (36%) and BP (40 mmHg), and restored renal sodium pump activity. The anti-ouabain antibody prevented the increase in pituitary ATII, reduced MBG excretion, natriuresis and BP, increased sodium pump activity, and prevented increases in plasma norepinephrine, pituitary and adrenocortical ATII, and adrenocortical MBG. Losartan mimicked the effects of the anti-ouabain antibody, but did not affect the excretion of EO. In adrenocortical cells of DS rats, ATII stimulated MBG secretion, and losartan blocked this effect., Conclusions: In response to NaCl loading, brain EO, via an AT1 receptor pathway and probably via sympathetic activation, stimulates adrenocortical MBG, which inhibits the renal sodium pump and elevates BP.

Objective: The pathogenesis of pre-eclampsia (PE), a major cause of maternal and fetal mortality, is not fully understood. Digitalis-like sodium pump ligands (SPLs) are believed to be implicated in PE, as illustrated by clinical observations that DIGIBIND, a digoxin antibody that binds SPLs, lowers blood pressure (BP) in PE. We recently reported that plasma levels of marinobufagenin (MBG), a vasoconstrictor SPL, are increased four-fold in patients with PE. In the present study, we tested whether a polyclonal antibody to MBG can lower BP in rats with pregnancy-associated hypertension., Methods: Systolic BP (SBP), 24-h renal excretion of MBG and endogenous ouabain (EO), and sodium pump activity in the thoracic aortae were measured in virgin and pregnant Sprague-Dawley rats without and with NaCl supplementation (drinking 1.8% NaCl solution)., Results: NaCl supplementation of virgin rats stimulated renal excretion of MBG by 60%, but not that of EO, and did not change the BP. Compared with virgin rats, the last week of pregnancy in non-NaCl-loaded rats was associated with a decrease in SBP (106 +/- 2 versus 117 +/- 2 mmHg); a moderate increase in renal excretion of MBG (97.6 +/- 4.9 versus 57.4 +/- 7.0 pmoles/24 h) and EO (36.2 +/- 4.3 versus 24.1 +/- 3.2 pmoles/24 h). NaCl-loaded pregnant rats exhibited elevation in SBP (139 +/- 3 mmHg; P < 0.01 versus non-NaCl-loaded pregnant rats), in renal excretion of MBG (160.0 +/- 17.5 pmoles/24 h; P < 0.01 versus non-NaCl-loaded pregnant rats), but not in EO, and showed fetal growth retardation. Administration of the anti-MBG antibody to NaCl-loaded pregnant rats lowered SBP (111 +/- 2 mmHg; P < 0.01) and increased aortic sodium pump activity (144 +/- 3 versus 113 +/- 5 nmol Rb/g per min; P < 0.01 versus non-NaCl-loaded pregnant rats)., Conclusions: These observations provide evidence that MBG contributes to BP elevation in pregnant rats rendered hypertensive by NaCl supplementation.

Marinobufagenin (MBG), an endogenous ligand of alpha-1 Na/K-ATPase, becomes elevated and contributes to hypertension in NaCl-loaded Dahl-S rats (DS). Protein kinase C (PKC) phosphorylates alpha-1 Na/K-ATPase and increases its MBG sensitivity. Cicletanine, an antihypertensive compound with PKC-inhibitory activity, reverses MBG-induced Na/K-ATPase inhibition and vasoconstriction. We hypothesized that increased PKC levels in sodium-loaded hypertensive DS would sensitize alpha-1 Na/K-ATPase to MBG and that PKC inhibition by cicletanine would produce an opposite effect. We studied the effects of cicletanine on systolic blood pressure, left ventricular PKC isoforms, cardiac alpha-1 Na/K-ATPase levels, and sensitivity to MBG in hypertensive DS. Seven DS received 50 mg x kg(-1) x d(-1) cicletanine, and 7 DS received vehicle during 4 weeks of an 8% NaCl diet. Vehicle-treated rats exhibited an increase in blood pressure, left ventricular mass, MBG excretion (74+/-11 vs 9+/-1 pmol/24 h, P<0.01), myocardial alpha-1 Na/K-ATPase protein, and PKC beta2 and delta. The sensitivity of Na/K-ATPase to MBG was enhanced at the level of high-affinity binding sites (IC50, 0.8 vs 4.4 nmol/L, P<0.01). Cicletanine-treated rats exhibited a 56-mm Hg reduction in blood pressure (P<0.01) and a 30% reduction in left ventricular weight, whereas cardiac alpha-1 Na/K-ATPase protein and MBG levels were unchanged. In cicletanine-treated rats, PKC beta2 was not increased, the sensitivity of Na/K-ATPase to MBG was decreased (IC50=20 micromol/L), and phorbol diacetate-induced alpha-1 Na/K-ATPase phosphorylation was reduced versus vehicle-treated rats. In vitro cicletanine treatment of sarcolemma from vehicle-treated rats also desensitized Na/K-ATPase to MBG, indicating that this effect was not solely attributable to a reduction in blood pressure. Thus, PKC-induced phosphorylation of cardiac alpha-1 Na/K-ATPase is a likely target for cicletanine treatment.

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 microg/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.

Background: A reduced cardiac output in chronic heart failure (CHF) evokes renal NaCl and water retention, and, therefore, activates mechanisms promoting natriuresis. Atrial natriuretic peptide (ANP) is one such factor. We hypothesized that another NaCl sensitive endogenous natriuretic factor, i.e., marinobufagenin (MBG), a specific ligand of the alpha-1 subunit of Na/K ATPase (the main kidney isoform) and also a vasoconstrictor and cardiotonic substance, would be elevated in CHF patients in a graded manner with the severity of CHF., Methods and Results: We measured the plasma levels of MBG, alpha-hANP, ouabain-like compound (OLC) and left ventricular (LV) volumes and ejection fraction in 23 consecutive hypertensive male patients with CHF. Plasma MBG levels exhibited progressive increases (0.59 +/- 0.15, 1.08 +/- 0.20, 1.35 +/- 0.17 and 1.88 +/- 0.05 nmol/l NYHA 1-4, respectively) and paralleled the changes of alpha-hANP. Conversely, plasma OLC did not exhibit such increases. Plasma MBG correlated with alpha-hANP (r = 0.82; P < 0.0001). Both MBG and alpha-hANP correlated with LV systolic (r = 0.55 and r = 0.47; P < 0.01) diameter and inversely with ejection fraction (r = -0.73 and r = -0.60; P < 0.01). OLC did not exhibit correlations with alpha-hANP or LV volumes, but positively correlated with systolic brachial blood pressure and with pulse pressure., Conclusions: In CHF, MBG exhibits progressive increases similar to ANP, varies with CHF severity and correlates with LV systolic function. We hypothesize, that, in CHF, the concurrent production of these two natriuretic hormones, a vasorelaxant, ANP, and a vasoconstrictor, MBG, potentiate each other's natriuretic effects, but may offset their vasoactive actions.

Background: Digitalis-like sodium pump ligands (SPLs) effect natriuresis via inhibition of renal tubular Na(+),K(+)-ATPase but may induce vasoconstriction. The present study investigated the potential roles of 2 putative endogenous SPLs, an ouabain-like compound (OLC) and an alpha(1) Na(+),K(+)-ATPase inhibitor, marinobufagenin (MBG), in regulating natriuresis and blood pressure (BP) responses to sustained and acute NaCl loading in Dahl salt-sensitive rats (DS)., Methods and Results: During 4 weeks of an 8% NaCl diet, DS exhibited a progressive increase in MBG renal excretion (66 +/-13 pmol/24 hours at week 4 versus 11 +/- 1 pmol/24 hours at baseline, n=48), which paralleled an increase in systolic BP (174 +/- 10 mm Hg at week 4 versus 110 +/- 2 mm Hg at baseline). By contrast, OLC excretion peaked at week 1 and returned to baseline levels. Administration of an anti-MBG, but not anti-ouabain antibody, to DS after 3 weeks of a high NaCl diet lowered BP (139 +/- 7 versus 175 +/- 5 mm Hg, P<0.001, n=5). Acute NaCl loading (2 hours) of DS (n=5) increased MBG and OLC excretion and natriuresis. Pretreatment of acutely NaCl-loaded DS with an anti-MBG antibody (n=5) reduced the excretion of sodium and MBG but not that of OLC. An anti-ouabain antibody (n=5) reduced sodium excretion and both OLC and MBG., Conclusions: An initial transient stimulation of OLC induced by NaCl loading of DS precedes an MBG response. A sustained increase in MBG production in DS contributes to the chronic BP elevation induced by a sustained high NaCl intake.

Dahl salt-sensitive rats (DS), which have a mutation in the alpha-1 subunit of Na(+)/K(+)-ATPase, exhibit impaired pressure natriuresis and on a high-salt diet, retain Na(+) and exhibit increased blood pressure. Recently, we have shown that mammalian tissues contain a bufadienolide Na(+)/K(+)-ATPase inhibitory factor, marinobufagenin (MBG), that exhibits greater affinity for the alpha-1 than alpha-3 sodium pump isoform. The present study investigated the possible role of MBG in hypertension in DS on a high NaCl intake. Eight DS and 8 Dahl salt-resistant rats (DR) were placed on an 8% NaCl diet. Within 2 weeks, systolic blood pressure increased in DS (162+/-9 mm Hg at week 2 versus 110+/-2 mm Hg in baseline, P<0.01), and increased less in DR (124+/-3 mm Hg at week 2 versus 112+/-2 mm Hg in baseline). Renal excretion of MBG increased 4-fold (38.9+/-7.6 pmol versus 9.1+/-1.3 pmol in baseline, P<0.01) in DS, but by only 25% in DR (13.2+/-0.9 pmol versus 10.3+/-0.7 pmol in baseline). Excretion of endogenous ouabain did not change in either strain. MBG-immunoreactive material was purified from the urine of hypertensive DS by means of 2 steps of reverse-phase high performance liquid chromatography (HPLC) and compared with plant ouabain and amphibian MBG for its ability to inhibit the Na(+)/K(+)-ATPase from rat kidney (which expresses only alpha-1 Na(+)/K(+)-ATPase isoform). Unlike ouabain (IC(50)=248 micromol/L), serially diluted, HPLC-purified MBG immunoreactivity from DS and authentic MBG potently inhibited rat kidney Na(+)/K(+)-ATPase (IC(50)=70 and 78 nmol/L, respectively). Our results suggest that an alpha-1 Na(+)/K(+)-ATPase ligand, MBG, is elaborated to promote natriuresis in hypertensive DS. MBG acts as a selective inhibitor of the ouabain-resistant alpha-1 Na(+)/K(+)-ATPase subunit, ie, the major sodium pump isoform of the kidneys, as would be expected of a putative natriuretic hormone.