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Immunoneutralization of elevated circulating levels of endogenous digitalis-like Na/K-ATPase inhibitors (i.e. cardiotonic steroids (CTS)) represents a novel approach in the treatment of preeclampsia (PE). Recently we demonstrated that DigiFab (Fab fragments of affinity-purified ovine digoxin antibody) restores PE-induced inhibition of Na/K-ATPase in erythrocytes ex vivo. Previously magnesium ions were shown to antagonize digitalis-induced toxicity, which is mediated by Na/K-ATPase inhibition. We hypothesized that magnesium sulfate would potentiate the effect of DigiFab in the reversal of CTS-induced Na/K-ATPase inhibition.To test this hypothesis, we studied the ex vivo effect of DigiFab on Na/K-ATPase activity in erythrocytes from patients with PE in the absence and in the presence of 3 mmol/L magnesium sulfate.Compared with 11 normotensive pregnant subjects (29 ± 1 years; gestational age = 39.0 ± 0.2 weeks; blood pressure = 111 ± 2/73 ± 2 mm Hg), the 12 patients with PE (30 ± 1 years; gestational age = 37.9 ± 0.3 weeks; blood pressure = 159 ± 5/99 ± 3 mm Hg) had plasma levels of marino-bufagenin increased 3-fold (1.38 ± 0.40 vs. 0.38 ± 0.10 nmol/L; P0.01) and activity of Na/K-ATPase in erythrocytes was inhibited (1.16 ± 0.11 vs. 2.80 ± 0.20 μmol Pi/ml/h; P0.01). Ex vivo, DigiFab (1 µg/ml) restored erythrocyte Na/K-ATPase activity (1.72 ± 0.13 µmol Pi/ml/h; P0.01), and 3 mmol magnesium sulfate potentiated the effect of DigiFab (2.30 ± 0.20 µmol Pi/ml/h; P0.01).Magnesium is capable of increasing the efficacy of immunoneutralization of marinobufagenin-induced Na/K-ATPase inhibition.

Cardiotonic steroids (CTS) are implicated in pathophysiology of uremic cardiomyopathy. In the present study, we tested whether a monoclonal antibody (mAb) against the bufadienolide CTS, marinobufagenin (MBG), alleviates cardiac hypertrophy and fibrosis in partially nephrectomized (PNx) rats.In PNx rats, we compared the effects of 3E9 anti-MBG mAb and of Digibind, an affinity-purified digoxin antibody, on blood pressure and cardiac hypertrophy and fibrosis following 4 weeks after the surgery.In PNx rats, a fourfold elevation in plasma MBG levels was associated with hypertension, increased cardiac levels of carbonylated protein, cardiac hypertrophy, a reduction in cardiac expression of a nuclear transcription factor which is a negative regulator of collagen synthesis, Friend leukemia integration-1 (Fli-1), and an increase in the levels of collagen-1. A single intraperitoneal administration of 3E9 mAb to PNx rats reduced blood pressure by 59 mm Hg for 7 days and produced a significant reduction in cardiac weight and cardiac levels of oxidative stress, an increase in the expression of Fli-1, and a reduction in cardiac fibrosis. The effects of Digibind were similar to those of 3E9 mAb, but were less pronounced.In experimental chronic renal failure, elevated levels of MBG contribute to hypertension and induce cardiac fibrosis via suppression of Fli-1, representing a potential target for therapy.

The development of hypertension is firmly associated with high salt intake, and reducing daily salt intake results in a decrease in both systolic blood pressure (SBP) and diastolic blood pressure of mildly hypertensive patients.1–3 It is, therefore, vital to understand the detailed mechanisms relating salt intake and blood pressure regulation. In this regard, isolation of endogenous cardiotonic steroids such as ouabain and marinobufagenin from human and experimental animals, and findings of their involvement in cardiovascular and renal diseases, have a significant impact on the understanding of complex mechanisms of hypertension.4 Although there are inconsistent findings about the levels of plasma endogenous ouabain after salt administration, acute NaCI loading, deoxycorticosterone acetate (DOCA)-salt treatment, and high-salt diet have been shown to be associated with an increase in plasma ouabain in rats5,6 and humans.7 Augmented levels of plasma marinobufagenin have been detected in acute plasma volume expansion,8 high-salt diet,9 pregnancy-related hypertension,10 essential hypertension, primary aldosteronism, renal failure,11 and adrenocorticotropic hormone (ACTH)-induced hypertension.12 Furthermore, Huang et al. and Krep et al. reported that central and peripheral administration of Digibind, an antibody Fab fragment to digoxin, significantly lowered the blood pressure of hypertensive high-salt and DOCA-salt treated rats.13,14 Na,K-ATPase consists of three subunits, α, β and FXYD protein family.15 Four α isoforms of Na,K-ATPase, α1–α4 have been identified and show variable tissue distribution: the αl isoform is expressed abundantly in most tissues; the α2 isoform is detected in brain, heart, skeletal and vascular smooth muscle, and adipocytes; the α3 isoform is predominant in neurons and ovaries; the α4 isoform is exclusively expressed in sperm. In most mammals, including humans, all four α isoforms are sensitive to ouabain, but in mice and rats, the αl Na,K-ATPase is remarkably resistant to ouabain,16,17 leading investigators to postulate that the sensitive α2 isoform plays an important regulatory role in the cardiovascular system, despite its more restricted expression pattern. In an attempt to explore the specific role of these various isoforms, we have generated genetically modified mice with specifically altered sensitivities to ouabain: a ouabain-resistant α2 Na,K-ATPase (α1R/Rα2R/R) and a ouabain-sensitive (“humanized”) α1 Na,K-ATPase (αS/Sα2R/R).18,19 We have shown that mice expressing either a ouabain-sensitive α1 or α2 isoform (α1R/Rα2S/S or α1S/Sα2R/R) can develop ACTH-dependent hypertension, whereas the mice with two resistant isoforms (α1R/Rα2R/R) remain normotensive during ACTH treatment. α1R/Rα2R/R mice also show lower blood pressure during pregnancy.20 By contrast, we found that 2-kidney, 1-clip renovascular hypertension is equivalent in all three genotypes, and therefore does not depend on a ouabain-sensitive α1 or α2 subunit.21 Since previous studies have specifically reported a link between DOCA-salt hypertension and endogenous cardiotonic steroids, the present study was performed to further investigate the role of the cardiotonic steroid-binding site of Na,K-ATPase in DOCA-salt hypertension, as another model of high blood pressure. Our findings indicate that DOCA salt hypertension does not require a sensitive α2 subunit.

BACKGROUND Marinobufagenin (MBG) is an endogenous Na/K-ATPase inhibitor, a natriuretic and a vasoconstrictor. MBG is implicated in salt-sensitive hypertension, cardiac hypertrophy, and initiate the pro-fibrotic signaling. Previously it was demonstrated that immunoneutralization of an endogenous MBG by 3E9 anti-MBG-antibody (mAb) in vivo lowered blood pressure (BP) and reversed cardiac fibrosis in salt-sensitive, and in partially nephrectomized rats. In the present study, we investigated whether mAb alleviates vascular remodeling induced in normotensive rats on high salt intake. METHODS Wistar rats (5 months old) received normal (CTRL; n = 8) or high salt intake (2% NaCl in drinking water) for 4 weeks ( n = 16). Rats from the group on a high salt intake were administered vehicle (SALT; n = 8) or mAb (50 µg/kg) (SALT-AB; n = 8) during the last week of high salt diet. BP, erythrocyte Na/K-ATPase activity, levels of MBG in plasma and 24-hour urine, and sensitivity of aortic explants to the vasorelaxant effect of sodium nitroprusside (SNP) were measured. Aortic collagen abundance was determined immunohistochemically. RESULTS In SALT vs. CTRL, heightened levels of MBG were associated with inhibition of erythrocyte Na/K-ATPase in the absence of BP changes. High salt intake was accompanied by a 2.5-fold increase in aortic collagen abundance and by a reduction of sensitivity of aortic explants to the vasorelaxant effect of SNP following endothelin-1-induced constriction. In the SALT-AB group, all NaCl-mediated effects were reversed by immunoneutralization of MBG. CONCLUSIONS High salt intake in young normotensive rats can induce vascular fibrosis via pressure-independent/MBG-dependent mechanisms, and this remodeling is reduced by immunoneutralization of MBG.

Recently, an endogenous digitalis-like factor (EDLF) was shown to be stimulated in corticotropin (ACTH) hypertension in the rat. We have shown that mammalian plasma contains a vasoconstrictor Na,K-ATPase inhibitor, which cross-reacts with an antibody to amphibian EDLF, marinobufagenin. In the present experiment, the effect of 8 days of intramuscular ACTH treatment (0.5 mg/kg/day) of male Fisher 344 x NB rats on blood pressure, plasma ouabain-like and marinobufagenin-like immunoreactivity, and on the activity of Na,K-ATPase in aortic sarcolemma were studied. The ACTH treatment for 8 days resulted in increased systolic blood pressure (151 +/- 12.4 v 121 +/- 4.0 mm Hg, P.01), inhibition of Na,K-ATPase in aortic sarcolemma (2.99 +/- 0.35 v 5.43 +/- 0.17 micromol ADP/mg(prot)/h), and increases in plasma concentration of marinobufagenin-like (0.44 +/- 0.06 v 0.21 +/- 0.05 nmol/L), but not ouabain-like (0.09 +/- 0.01 v 0.10 +/- 0.04 nmol/L) immunoreactivity. In dissociation enhanced lanthanide fluoroimmunoassay (DELFIA), serial dilutions of plasma from ACTH-treated rats extracted with 25% and 80% acetonitrile, respectively, demonstrated parallelism to the calibration curves of ouabain and marinobufagenin. These findings suggest that an endogenous bufodienolide Na,K-ATPase inhibitor, rather than an endogenous ouabain-like compound, is increased after 8 days of treatment of rats with ACTH.

Recently, we have shown that mammalian plasma cross-reacts with an antibody to a bufodienolide Na/K ATPase inhibitor, marinobufagenin. In rat aorta, marinobufagenin induced vasoconstriction via inhibition of the vascular smooth muscle Na/K pump, whereas ouabain had its predominant effect on pumps localized to nerve endings. Na/K ATPase inhibitory effects of ouabain and marinobufagenin were studied in two membrane fractions isolated from Fisher 344xBN rat thoracic aorta by sucrose density gradient centrifugation. One fraction contained predominantly the alpha-3 isoform of Na/K ATPase and represented membranes from the perivascular nerve endings (neuronal plasmalemma). The other membrane fraction, containing predominantly the alpha-1 isoform, was derived from the vascular smooth muscle sarcolemma. The IC50 for inhibition of the Na/K ATPase by ouabain and marinobufagenin were 2.6 nmol/L and 0.14 micromol/L in the neuronal plasmalemma, and 50 nmol/L and 2.1 nmol/L in sarcolemma, respectively. These results confirm the view that differential responsiveness to endogenous digitalis-like factors is a functional feature of alpha isoforms of Na/K ATPase.

Vasoconstrictor and Na/K pump inhibitory properties of a bufodienolide Na/K-ATPase inhibitor, marinobufagenin, were studied in isolated rings of 2 to 3 order branches of human pulmonary arteries respectively. Marinobufagenin displayed concentration-dependent vasoconstrictor activity (0.01 to 10 mmol/L). In sarcolemma membranes prepared from pulmonary artery marinobufagenin inhibited Na/K-ATPase (IC50 = 50 nmol/L). In eight healthy male Caucasians, concentrations of marinobufagenin-like immunoreactive material in C-18 extracted plasma were 1.38 +/- 0.60 nmol/L. Twenty-four-hour urinary release of marinobufagenin-like immunoreactive material in eight healthy males was 1.20 +/- 0.95 nmol/day. Chloroform extract of human urine was fractionated using reverse-phase high-performance liquid chromatography (32% acetonitrile, Deltapak). The HPLC fraction coeluting with marinobufagenin in 7 min, cross reacted with antimarinobufagenin and antidigoxin, but not antiouabain antibody. These results demonstrate that human plasma and urine contains a bufodienolide vasoconstrictor EDLF, marinobufagenin-like immunoreactive Na,K pump inhibitor.