Inhibition Of Na-K ATPase Research Articles

Silencing of ATP1A1 attenuates cell membrane disruption by nanosecond electric pulses

This study explored the role of the Na/K-ATPase (NKA) in membrane permeabilization induced by nanosecond electric pulses. Using CRISPR/Cas9 and shRNA, we silenced the ATP1A1 gene, which encodes α1 NKA subunit in U937 human monocytes. Silencing reduced the rate and the cumulative uptake of YoPro-1 dye after electroporation by 300-ns, 7–10 kV/cm pulses, while ouabain, a specific NKA inhibitor, enhanced YoPro-1 entry. We conclude that the α1 subunit supports the electropermeabilized membrane state, by forming or stabilizing electropores or by hindering repair mechanisms, and this role is independent of NKA's ion pump function.

Silencing of PKG1 Gene Mimics Effect of Aging and Sensitizes Rat Vascular Smooth Muscle Cells to Cardiotonic Steroids: Impact on Fibrosis and Salt Sensitivity.

Background Marinobufagenin, NKA (Na/K-ATPase) inhibitor, causes vasoconstriction and induces fibrosis via inhibition of Fli1 (Friend leukemia integration-1), a negative regulator of collagen synthesis. In vascular smooth muscle cells (VSMC), ANP (atrial natriuretic peptide), via a cGMP/PKG1 (protein kinase G1)-dependent mechanism, reduces NKA sensitivity to marinobufagenin. We hypothesized that VSMCfrom old rats, due to downregulation of ANP/cGMP/PKG-dependent signaling, would exhibit heightened sensitivity to the profibrotic effect of marinobufagenin. Methods and Results Cultured VSMCfrom the young (3-month-old) and old (24-month-old) male Sprague-Dawley rats and young VSMCwith silenced PKG1 gene were treated with 1 nmol/L ANP, or with 1 nmol/L marinobufagenin, or with a combination of ANP and marinobufagenin. Collagen-1, Fli1, and PKG1 levels were assessed by Western blotting analyses. Vascular PKG1 and Fli1 levels in the old rats were reduced compared with their young counterparts. ANP prevented inhibition of vascular NKA by marinobufagenin in young VSMC but not in old VSMC. In VSMC from the young rats, marinobufagenin induced downregulation of Fli1 and an increase in collagen-1 level, whereas ANP blocked this effect. Silencing of the PKG1 gene in young VSMC resulted in a reduction in levels of PKG1 and Fli1; marinobufagenin additionally reduced Fli1 and increased collagen-1 level, and ANP failed to oppose these marinobufagenin effects, similar to VSMC from the old rats with the age-associated reduction in PKG1. Conclusions Age-associated reduction in vascular PKG1 and the resultant decline in cGMP signaling lead to the loss of the ability of ANP to oppose marinobufagenin-induced inhibition of NKA and fibrosis development. Silencing of the PKG1 gene mimicked these effects of aging.

Shedding Light on the Role of Na,K-ATPase as a Phosphatase during Matrix-Vesicle-Mediated Mineralization

Matrix vesicles (MVs) contain the whole machinery necessary to initiate apatite formation in their lumen. We suspected that, in addition to tissue-nonspecific alkaline phosphatase (TNAP), Na,K,-ATPase (NKA) could be involved in supplying phopshate (Pi) in the early stages of MV-mediated mineralization. MVs were extracted from the growth plate cartilage of chicken embryos. Their average mean diameters were determined by Dynamic Light Scattering (DLS) (212 ± 19 nm) and by Atomic Force Microcopy (AFM) (180 ± 85 nm). The MVs had a specific activity for TNAP of 9.2 ± 4.6 U·mg-1 confirming that the MVs were mineralization competent. The ability to hydrolyze ATP was assayed by a colorimetric method and by 31P NMR with and without Levamisole and SBI-425 (two TNAP inhibitors), ouabain (an NKA inhibitor), and ARL-67156 (an NTPDase1, NTPDase3 and Ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) competitive inhibitor). The mineralization profile served to monitor the formation of precipitated calcium phosphate complexes, while IR spectroscopy allowed the identification of apatite. Proteoliposomes containing NKA with either dipalmitoylphosphatidylcholine (DPPC) or a mixture of 1:1 of DPPC and dipalmitoylphosphatidylethanolamine (DPPE) served to verify if the proteoliposomes were able to initiate mineral formation. Around 69-72% of the total ATP hydrolysis by MVs was inhibited by 5 mM Levamisole, which indicated that TNAP was the main enzyme hydrolyzing ATP. The addition of 0.1 mM of ARL-67156 inhibited 8-13.7% of the total ATP hydrolysis in MVs, suggesting that NTPDase1, NTPDase3, and/or NPP1 could also participate in ATP hydrolysis. Ouabain (3 mM) inhibited 3-8% of the total ATP hydrolysis by MVs, suggesting that NKA contributed only a small percentage of the total ATP hydrolysis. MVs induced mineralization via ATP hydrolysis that was significantly inhibited by Levamisole and also by cleaving TNAP from MVs, confirming that TNAP is the main enzyme hydrolyzing this substrate, while the addition of either ARL-6715 or ouabain had a lesser effect on mineralization. DPPC:DPPE (1:1)-NKA liposome in the presence of a nucleator (PS-CPLX) was more efficient in mineralizing compared with a DPPC-NKA liposome due to a better orientation of the NKA active site. Both types of proteoliposomes were able to induce apatite formation, as evidenced by the presence of the 1040 cm-1 band. Taken together, the findings indicated that the hydrolysis of ATP was dominated by TNAP and other phosphatases present in MVs, while only 3-8% of the total hydrolysis of ATP could be attributed to NKA. It was hypothesized that the loss of Na/K asymmetry in MVs could be caused by a complete depletion of ATP inside MVs, impairing the maintenance of symmetry by NKA. Our study carried out on NKA-liposomes confirmed that NKA could contribute to mineral formation inside MVs, which might complement the known action of PHOSPHO1 in the MV lumen.

Abstract 11566: Distribution and New Function of Na, K-ATPase and Its Phosphoprotein Regulator Phospholemman in Cardiac Myocytes

Introduction: Na, K-ATPase (NKA), regulated by phosphoprotein phospholemman (PLM), contributes to excitation-contraction via the Na,Ca-exchanger at cardiac T-tubules (TTs). It is known that the intercalated discs (IDs) contain concentrated specific Na channels and K channels, that rely on ion gradients set by NKA. Yet, the coordinated trafficking and localization of NKA and PLM in cardiomyocytes (CMs) remains incompletely understood, Hypothesis: NKA and PLM are concentrated in the ID and essential for cardiac conduction. Methods: We have made five antibodies (Abs) that specifically identify PLM (B8, PLM-N-term, and PLM-C-term) or NKA (NKA-α1 and Pan-α, recognizing both α1 and α2 subunits of NKA). Confocal immunofluorescence assays were used to determine the intracellular distribution of PLM and NKA. Optical mapping studies were performed in rabbit hearts. Results: These five Abs all displayed specific fluorescence signals in dog CMs that were more intense at the IDs than TTs. These signals were eliminated by competing peptides. B8 and NKA-α1 signals highly overlapped, with similar intensity ratios between ID and TT at ~3. Immunostaining of connexin 43, γ-catenin, or N-cadherin confirmed the co-localization of NKA and PLM to the ID. PLM and NKA co-enriched with ID protein markers in sarcolemma/ID membrane preparations. To corroborate the localization of PLM at the ID, we expressed dog PLM in rat CMs. By 30h post-infection, B8 detected newly synthesized dog PLM that paralleled the pattern of its steady-state distribution in tissue, with concentrated protein apparent at IDs (B8 fluorescence intensity ratio of ID/TT = 2.6 ± 0.5, 50CMs). Nocodazole prevented newly-made dog PLM trafficking to ID. Finally, optical mapping studies showed that the NKA inhibitor ouabain and digoxin significantly increased activation times in a dose-dependent manner (n=5 and 4 rabbits, respectively). Conclusions: The α1 subunit of NKA and PLM are each expressed at a ~3X higher concentration in ID compared to TT in CMs, contributing to cardiac conduction. Mechanisms of PLM and NKA protein trafficking to the two disparate subcellular sites, and associated effects of PLM regulation by adrenergic signaling, provide important new insights into the physiology of cardiac conduction.

Implications of Synthetic Modifications of the Cardiotonic Steroid Lactone Ring on Cytotoxicity.

Na,K-ATPase (NKA) and cardiotonic steroids (CTS) have shown potent cytotoxic and anticancer effects. Here, we have synthesized a series of CTS digoxin derivatives (γ-benzylidene) with substitutions in the lactone ring and evaluated the cytotoxicity caused by digoxin derivatives in tumor and non-tumor cells lines, as well as their effects on NKA. The cytotoxicity assay was determined in HeLa, A549, and WI-26 VA4 after they were treated for 48h with increased concentrations of CTS. The effects of CTS on NKA activity and immunoblotting of α1 and β1 isoforms were evaluated at IC50 concentrations in A549 cell membrane. NKA activity from mouse brain cortex was also measured. The majority of CTS exhibited low cytotoxicity in tumor and non-tumor cells, presenting IC50 values at micromolar concentrations, while digoxin showed cytotoxicity at nanomolar concentrations. BD-15 presented the lowest IC50 value (8µM) in A549 and reduced its NKA activity in 28%. In contrast, BD-7 was the compound that most inhibited NKA (56% inhibition) and presented high IC50 value for A549. In mouse cortex, only BD-15 modulated the enzyme activity in a concentration-dependent inhibition curve. These results demonstrate that the cytotoxicity of these compounds is not related to NKA inhibition. The substitutions in the lactone ring of digoxin led to an increase in the cytotoxic concentration in tumor cells, which may not be interesting for cancer, but it has the advantage of increasing the therapeutic margin of these molecules when compared to classic CTS, and can be used safely in research for other diseases.

The Na/K-ATPase α1/Src interaction regulates metabolic reserve and Western diet intolerance.

Highly prevalent diseases such as insulin resistance and heart failure are characterized by reduced metabolic flexibility and reserve. We tested whether Na/K-ATPase (NKA)-mediated regulation of Src kinase, which requires two NKA sequences specific to the α1 isoform, is a regulator of metabolic capacity that can be targeted pharmacologically. Metabolic capacity was challenged functionally by Seahorse metabolic flux analyses and glucose deprivation in LLC-PK1-derived cells expressing Src binding rat NKA α1, non-Src-binding rat NKA α2 (the most abundant NKA isoform in the skeletal muscle), and Src binding gain-of-function mutant rat NKA α2. Mice with skeletal muscle-specific ablation of NKA α1 (skα1-/-) were generated using a MyoD:Cre-Lox approach and were subjected to treadmill testing and Western diet. C57/Bl6 mice were subjected to Western diet with or without pharmacological inhibition of NKA α1/Src modulation by treatment with pNaKtide, a cell-permeable peptide designed by mapping one of the sites of NKA α1/Src interaction. Metabolic studies in mutant cell lines revealed that the Src binding regions of NKA α1 are required to maintain metabolic reserve and flexibility. Skα1-/- mice had decreased exercise endurance and mitochondrial Complex I dysfunction. However, skα1-/- mice were resistant to Western diet-induced insulin resistance and glucose intolerance, a protection phenocopied by pharmacological inhibition of NKA α1-mediated Src regulation with pNaKtide. These results suggest that NKA α1/Src regulatory function may be targeted in metabolic diseases. Because Src regulatory capability by NKA α1 is exclusive to endotherms, it may link the aerobic scope hypothesis of endothermy evolution to metabolic dysfunction.

Biased Effect of Cardiotonic Steroids on Na/K-ATPase-Mediated Signal Transduction.

Recent studies have revealed that Na/K-ATPase (NKA) can transmit signals through ion-pumping-independent activation of pathways relayed by distinct intracellular protein/lipid kinases, and endocytosis challenges the traditional definition that cardiotonic steroids (CTS) are NKA inhibitors. Although additional effects of CTS have long been suspected, revealing its agonist impact through the NKA receptor could be a novel mechanism in understanding the basic biology of NKA. In this study, we tested whether different structural CTS could trigger different sets of NKA/effector interactions, resulting in biased signaling responses without compromising ion-pumping capacity. Using purified NKA, we found that ouabain, digitoxigenin, and somalin cause comparable levels of NKA inhibition. However, although endogenous ouabain stimulates both protein kinases and NKA endocytosis, digitoxigenin and somalin bias to protein kinases and endocytosis, respectively, in LLC-PK1 cells. The positive inotropic effects of CTS are traditionally regarded as NKA inhibitors. However, CTS-induced signaling occurs at concentrations at least one order of magnitude lower than that of inotropy, which eliminates their well known toxic actions on the heart. The current study adds a novel mechanism that CTS could exert its biased signaling properties through the NKA signal transducer. SIGNIFICANCE STATEMENT: Although it is now well accepted that NKA has an ion-pumping-independent signaling function, it is still debated whether direct and conformation-dependent NKA/effector interaction is a key to this function. Therefore, this investigation is significant in advancing our understanding of the basic biology of NKA-mediated signal transduction and gaining molecular insight into the structural elements that are important for cardiotonic steroid's biased action.

Ouabain, a Na–K-ATPase inhibitor, enhances wakefulness in rats

Sleep disturbances are common findings in bipolar affective disorder (BD), and they may be related to the pathophysiology of the disease. REM sleep deprivation, which has an antidepressant effect, increases Na–K-ATPase (NKA) activity. NKA might be involved in the induction of rebound sleep. Decrements in NKA are thought to be implicated in the pathophysiology of BD. A single intracerebroventricular (ICV) microinjection of ouabain (OUA), an NKA inhibitor, results in long-lasting suppression of NKA activity. It has been proposed to be used as a model of BD. To elucidate the role of NKA, we studied the effects of ICV administered OUA on the sleep–wake activity of rats. Rats were ICV injected with saline on the baseline day and 2.5 μL 20 μM (n = 8) or 2 mM (n = 6) OUA on the subsequent OUA day. Saline was administered 1, 4, and 8 days after OUA injection. Sleep–wake activity was recorded for 12 h or 24 h post-injection. OUA enhanced wakefulness, but there was only little similarity between the changes in sleep and in BD after OUA treatment. These findings give only limited support for the NKA hypothesis of BD. Not the decrease in NKA activity itself that may be the important factor in the pathophysiology of BD, but rather the dysfunction of those regulatory processes that influence NKA.

Alphavirus Replication: The Role of Cardiac Glycosides and Ion Concentration in Host Cells.

Alphaviruses are arthropod-borne viruses that can cause fever, rash, arthralgias, and encephalitis. The mosquito species Aedes aegypti and Aedes albopictus are the most frequent transmitters of alphaviruses. There are no effective vaccines or specific antivirals available for the treatment of alphavirus-related infections. Interestingly, changes in ion concentration in host cells have been characterized as critical regulators of the alphavirus life cycle, including fusion with the host cell, glycoprotein trafficking, genome translation, and viral budding. Cardiac glycosides, which are classical inhibitors of the Na+ K+ ATPase (NKA), can inhibit alphavirus replication although their mechanisms of action are poorly understood. Nonetheless, results from multiple studies suggest that inhibition of NKA may be a suitable strategy for the development of alphavirus-specific antiviral treatments. This review is aimed at exploring the role of changes in ion concentration during alphavirus replication and at considering the possibility of NKA as a potential therapeutic target for antiviral drugs.

Istaroxime improves diabetic diastolic dysfunction through SERCA stimulation

Calcium handling is generally impaired in heart failure (HF). Mechanisms that can restore cardiac relaxation improving cellular Ca2 + cycling, represent a promising therapeutic approach for HF. Istaroxime is a Na-K ATPase (NKA) inhibitor with the property of accelerating Ca2 + re-uptake into sarcoplasmic reticulum (SR) through the SR Ca2 + pump (SERCA) stimulation by displacing the interaction between SERCA and its inhibitor, phospholamban (PLB). To characterize Istaroxime effects in a model of mild diabetes (type 1) with diastolic dysfunction and preserved global systolic function. Istaroxime was tested at a concentration mostly unaffecting NKA to isolate its effects dependent on SERCA only. Streptozotocin (STZ)-treated rats were evaluated at 9 weeks after STZ injection in comparison to control (CTR) ones. STZ-induced changes were evaluated in vivo (echocardiography), in isolated left ventricular myocytes and in SERCA2a-enriched microsomes. SERCA and PLB protein levels were measured by western blot and SERCA activity as 32P hydrolysis. Action potential rate-dependency and intracellular Ca2 + handling were evaluated in patch clamped or field-stimulated (2 Hz) cells. STZ-induced cardiomyopathy was characterized by impaired diastolic relaxation which was associated to reduced SERCA protein level and activity at the cellular level. Moreover, the monomeric PLB/SERCA ratio was increased. In STZ group, action potentials (AP) were significantly prolonged at each cycle length; Ca2 + transients were characterized by slower decay, delayed onset and increased diastolic Ca2 + . Istaroxime at a concentration of 100 nM significantly stimulated SERCA activity and SR Ca re-uptake after caffeine depletion in STZ group only. Moreover, Istaroxime reduced STZ-induced diastolic Ca2 + enhancement but not affected AP prolongation. SERCA stimulation can be considered a promising therapeutic approach for diastolic dysfunction treatment.

Acute salt loading and cardiotonic steroids in resistant hypertension.

The study addresses the association of marinobufagenin (MBG), a natriuretic and vasoconstrictor steroid, and Na/K-ATPase (NKA) activity with pressor response to salt-loading and arterial stiffness in resistant hypertension (RH). Thirty-four patients (18 males and 16 females; 56±8 years) with RH on a combined (lisnopril/amlodipine/hydrochlorothiazide) therapy and 11 healthy age-matched normotensive subjects (7 males and 4 females; 54±2 years) were enrolled in this study. Salt-loading was performed via intravenous infusion of 1000mL saline (0.9% NaCl) for 1h. Arterial stiffness was measured by Sphygmocor Px device with a calculation of pulse-wave velocity (PWV). Activity of NKA was measured in erythrocytes. We demonstrated that plasma levels of MBG and magnitude of NaCl-induced MBG-dependent NKA inhibition are associated with PWV, and that this association has gender- and age-specific fashion in RH patients.

Abstract 2328: ADCs that induce necrosis and thwart apoptotic resistance

Abstract Purpose: We have previously shown that targeting the Na,K-ATPase (NKA) with antibody drug conjugates is a powerful method for reducing tumors at levels found to be safe in cynomolgus monkeys. Currently no approved cancer drugs induce a potent necrotic type cell death even though experimental evidence shows that necrotic cell death has many notable and untapped benefits. Therefore, we wanted to better understand the driving mechanism by which ADCs targeting NKA inhibition induce necrosis. Experimental Design: Various cancer cell types were subjected to increasing and decreasing concentrations of Na+, K+ and Ca++, as well as various ADCs using bufalin as the warhead/payload/toxin. The cells were also subjected to siRNAs specific to the NKA and bufalin alone. The cells were then analyzed for the appearance and/or presence of necrosis, apoptosis, and markers indicating various stress pathways. Results: Conditions found to induce necrosis included: ADCs targeting specific NKA-protein complexes, cells subjected to media containing increased concentrations of K+, the specific Na,K-ATPase siRNAs, and bufalin alone. Conditions not found to induce necrosis included: media containing increased Na+, decreased Na+ or K+, control siRNAs, or ADCs that targeted proteins not complexed to the NKA or expressed on the cell surface. All cells undergoing necrosis lost their actin filaments, rounded up, and adherent cells detached from the surface and lost their motility. Markers indicated initial activation of the integrated stress responses (ISR). Minimal to no annexin staining indicated that apoptosis was not the dominate pathway in any experiment. Conclusion: Proper ion flux is important for maintaining cellular homeostasis. Cell swelling is a key process that is used to correct for changes in ion flux, induce inflammation and kill unwanted cells. The NKA is a key regulatory protein in this process and when blocked, leads to uncontrolled cells swelling and necrotic death. Precise inhibition of ion flux focused on cancer cells should be further investigated as a potential method for treating patients. Citation Format: James R. Prudent, David Marshall. ADCs that induce necrosis and thwart apoptotic resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2328.

Loss of NHERF-1 expression prevents dopamine-mediated Na-K-ATPase regulation in renal proximal tubule cells from rat models of hypertension: aged F344 rats and spontaneously hypertensive rats.

Dopamine decreases Na-K-ATPase (NKA) activity by PKC-dependent phosphorylation and endocytosis of the NKA α1. Dopamine-mediated regulation of NKA is impaired in aging and some forms of hypertension. Using opossum (OK) proximal tubule cells (PTCs), we demonstrated that sodium-hydrogen exchanger regulatory factor-1 (NHERF-1) associates with NKA α1 and dopamine-1 receptor (D1R). This association is required for the dopamine-mediated regulation of NKA. In OK cells, dopamine decreases NHERF-1 association with NKA α1 but increases its association with D1R. However, it is not known whether NHERF-1 plays a role in dopamine-mediated NKA regulation in animal models of hypertension. We hypothesized that defective dopamine-mediated regulation of NKA results from the decrease in NHERF-1 expression in rat renal PTCs isolated from animal models of hypertension [spontaneously hypertensive rats (SHRs) and aged F344 rats]. To test this hypothesis, we isolated and cultured renal PTCs from 22-mo-old F344 rats and their controls, normotensive 4-mo-old F344 rats, and SHRs and their controls, normotensive Wistar-Kyoto (WKY) rats. The results demonstrate that in both hypertensive models (SHR and aged F344), NHERF-1 expression, dopamine-mediated phosphorylation of NKA, and ouabain-inhibitable K+ transport are reduced. Transfection of NHERF-1 into PTCs from aged F344 and SHRs restored dopamine-mediated inhibition of NKA. These results suggest that decreased renal NHERF-1 expression contributes to the impaired dopamine-mediated inhibition of NKA in PTCs from animal models of hypertension.

L30A Mutation of Phospholemman Mimics Effects of Cardiac Glycosides in Isolated Cardiomyocytes.

To determine if mutations introduced into phospholemman (PLM) could increase the level of PLM-Na,K-ATPase (NKA) binding, we performed scanning mutagenesis of the transmembrane domain of PLM and measured Förster resonance energy transfer (FRET) between each mutant and NKA. We observed an increased level of binding to NKA for several PLM mutants compared to that of the wild type (WT), including L27A, L30A, and I32A. In isolated cardiomyocytes, overexpression of WT PLM increased the amplitude of the Ca2+ transient compared to the GFP control. The Ca2+ transient amplitude was further increased by L30A PLM overexpression. The L30A mutation also delayed Ca2+ extrusion and increased the duration of cardiomyocyte contraction. This mimics aspects of the effect of cardiac glycosides, which are known to increase contractility through inhibition of NKA. No significant differences between WT and L30A PLM-expressing myocytes were observed after treatment with isoproterenol, suggesting that the superinhibitory effects of L30A are reversible with β-adrenergic stimulation. We also observed a decrease in the extent of PLM tetramerization with L30A compared to WT using FRET, suggesting that L30 is an important residue for mediating PLM-PLM binding. Molecular dynamics simulations revealed that the potential energy of the L30A tetramer is greater than that of the WT, and that the transmembrane α helix is distorted by the mutation. The results identify PLM residue L30 as an important determinant of PLM tetramerization and of functional inhibition of NKA by PLM.

Abstract P244: Role of the Na-H Exchanger Regulatory Factor 1 (NHERF1) in Hypertension of Aging Animals

Aging animals develop hypertension when challenged with high salt diet due, in part, to desensitization of dopamine receptors (DR) in renal proximal tubules (RPT). We have demonstrated that NHERF1 associates with DR1 and Na-K ATPase (NKA) and is important for regulation of NKA in RPT. Preliminary data showed loss of NHERF1 expression in 22m old F344 rats. We hypothesized that loss of NHERF1 results in increased blood pressure (BP) and lack of natriuretic response to dopamine (DA) in aging animals. To address this hypothesis, Fischer Brown Norway (FBN) rats (1m, 4m, 12m, and 24m old) were fed diet containing 1% or 8% NaCl for one week and, BP was measured in anesthetized animals using an indwelling left femoral artery catheter. 8% NaCl did not increase BP in 1m or 4 month old rats. By contrast, 8% NaCl diet increased BP in 12m (84.3±3.5 vs 90.8±2.36) and 24m (73.5±7.58 vs 104±1.6) old animals. To determine if lack of NHERF1 is responsible for the increase in BP, we measured BP in 12 m old WT and NHERF1 KO mice. By contrast to WT mice, 8% NaCl diet did not increase BP in NHERF1 KO mice (84±4.9 vs 96.5±3.56 (WT) and 78.2±3.89 vs 81.8±9.2 (NHERF1 KO mice)). To confirm that NHERF1 is required for DA-mediated inhibition of NKA, NKA activity in primary proximal tubule cells (PTC) from young and old mice in culture was measured in the presence or absence of DA. DA decreased NKA activity in PTC from young animals (67.2±3.8 vs 32.7±5.3) but not in PTC from old animals. Transfection of NHERF1 restored NKA regulation by DA in PTC from old rats (58.4±4.2 vs 64.4±4.3 (in untransfected cells) 54.2±3.8 vs 31.1±3.4 (in NHERF1 transfected cells)). We conclude that NHERF1 regulates DA-mediated proximal tubule sodium handling; however, other factors modulate BP response to dietary sodium intake in young and old animals. The contribution of NHERF1 and dopamine signaling to sodium homeostasis requires further study.

Interaction between Na-K-ATPase and Bcl-2 proteins BclXL and Bak.

In silico analysis predicts interaction between Na-K-ATPase (NKA) and Bcl-2 protein canonical BH3- and BH1-like motifs, consistent with NKA inhibition by the benzo-phenanthridine alkaloid chelerythrine, a BH3 mimetic, in fetal human lens epithelial cells (FHLCs) (Lauf PK, Heiny J, Meller J, Lepera MA, Koikov L, Alter GM, Brown TL, Adragna NC. Cell Physiol Biochem 31: 257-276, 2013). This report establishes proof of concept: coimmunoprecipitation and immunocolocalization showed unequivocal and direct physical interaction between NKA and Bcl-2 proteins. Specifically, NKA antibodies (ABs) coimmunoprecipitated BclXL (B-cell lymphoma extra large) and BAK (Bcl-2 antagonist killer) proteins in FHLCs and A549 lung cancer cells. In contrast, both anti-Bcl-2 ABs failed to pull down NKA. Notably, the molecular mass of BAK1 proteins pulled down by NKA and BclXL ABs appeared to be some 4-kDa larger than found in input monomers. In silico analysis predicts these higher molecular mass BAK1 proteins as alternative splicing variants, encoding 42 amino acid (aa) larger proteins than the known 211-aa long canonical BAK1 protein. These BAK1 variants may constitute a pool separate from that forming mitochondrial pores by specifically interacting with NKA and BclXL proteins. We propose a NKA-Bcl-2 protein ternary complex supporting our hypothesis for a special sensor role of NKA in Bcl-2 protein control of cell survival and apoptosis.

Pathophysiologically-Relevant Levels of Endogenous Cardiotonic Steroids Inhibit the Cardiac Na/K ATPase and Activate ERK1/2 Hypertrophic Signaling In Vivo and In Vitro

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.

Istaroxime stimulates SERCA2a and accelerates calcium cycling in heart failure by relieving phospholamban inhibition

Calcium handling is known to be deranged in heart failure. Interventions aimed at improving cell Ca(2) (+) cycling may represent a promising approach to heart failure therapy. Istaroxime is a new luso-inotropic compound that stimulates cardiac contractility and relaxation in healthy and failing animal models and in patients with acute heart failure (AHF) syndrome. Istaroxime is a Na-K ATPase inhibitor with the unique property of increasing sarcoplasmic reticulum (SR) SERCA2a activity as shown in heart microsomes from humans and guinea pigs. The present study addressed the molecular mechanism by which istaroxime increases SERCA2a activity. To study the effect of istaroxime on SERCA2a-phospholamban (PLB) complex, we applied different methodologies in native dog healthy and failing heart preparations and heterologous canine SERCA2a/PLB co-expressed in Spodoptera frugiperda (Sf21) insect cells. We showed that istaroxime enhances SERCA2a activity, Ca(2) (+) uptake and the Ca(2) (+) -dependent charge movements into dog healthy and failing cardiac SR vesicles. Although not directly demonstrated, the most probable explanation of these activities is the displacement of PLB from SERCA2a.E2 conformation, independently from cAMP/PKA. We propose that this displacement may favour the SERCA2a conformational transition from E2 to E1, thus resulting in the acceleration of Ca(2) (+) cycling. Istaroxime represents the first example of a small molecule that exerts a luso-inotropic effect in the failing human heart through the stimulation of SERCA2a ATPase activity and the enhancement of Ca(2) (+) uptake into the SR by relieving the PLB inhibitory effect on SERCA2a in a cAMP/PKA independent way.

DigiFab Interacts With Endogenous Cardiotonic Steroids and Reverses Preeclampsia-Induced Na/K-ATPase Inhibition

Elevated levels of endogenous Na/K-ATPase (NKA) inhibitors, cardiotonic steroids (CTSs) including marinobufagenin (MBG), contribute to pathogenesis of preeclampsia (PE) and represent a target for immunoneutralization by Digibind (Ovine Digoxin Immune Antibody, Glaxo-Smith Kline). Because Digibind is no longer commercially available, we studied whether DigiFab (BTG International Ltd, UK) can substitute Digibind for immunoneutralization of CTS in patients with PE. We compared DigiFab, Digibind, and anti-MBG monoclonal antibody (mAb) with respect to their ability to interact with CTS in PE plasma and to restore NKA activity in erythrocytes from patients with PE. Using immunoassays based on DigiFab, Digibind, and anti-MBG mAb, we studied the elution profile of CTS following high-performance liquid chromatography (HPLC) fractionation of PE plasma. Totally, 7 patients with mild PE (28 ± 2 years; gestational age, 39 ± 0.5 weeks; blood pressure 156 ± 5/94 ± 2 mm Hg) and 6 normotensive pregnant participants (28 ± 1 years; gestational age, 39 ± 0.4 weeks; blood pressure 111 ± 2/73 ± 2 mm Hg) were enrolled. Preeclampsia was associated with a substantial inhibition of erythrocyte NKA (1.47 ± 0.17 vs 2.65 ± 0.16 µmol Pi/mL per h in control group, P < .001). Ex vivo, at 10 µg/mL concentration, which is consistent with the clinical dosing of Digibind administered previously in PE, DigiFab and Digibind as well as anti-MBG mAb (0.5 µg/mL) restored erythrocyte NKA activity. Following HPLC fractionation of pooled PE and control plasma, PE-associated increase in CTS material was detected by Digibind (176 vs 75 pmoles), DigiFab (221 vs 70 pmoles), and anti-MBG mAb (1056 vs 421 pmoles). Therefore, because DigiFab interacts with CTS from PE plasma and reverses PE-induced NKA inhibition, it can substitute Digibind for immunoneutralization of CTS in patients with PE.

Age-associated increase in salt sensitivity is accompanied by a shift in the atrial natriuretic peptide modulation of the effect of marinobufagenin on renal and vascular sodium pump

Marinobufagenin (MBG) promotes natriuresis via inhibition of renotubular Na/K-ATPase (NKA) and causes vasoconstriction via inhibition of vascular NKA. Atrial natriuretic peptide (ANP), via cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG)-dependent mechanism, sensitizes renal NKA to MBG but reduces MBG-induced inhibition of vascular NKA. As aging is associated with a downregulation of cGMP/PKG signaling, we hypothesized that in older rats, ANP would not potentiate renal effects of MBG and would not oppose vascular effects of MBG. In younger (3-month-old) and older (12-month-old) Sprague-Dawley rats, we compared SBP, natriuresis, activity of NKA in aorta and renal medulla, and levels of MBG and α-ANP at baseline and following acute NaCl loading (20%, 2.5 ml/kg, intraperitoneally), and studied modulation of MBG-induced NKA inhibition by α-ANP in vitro. As compared with younger rats, NaCl-loaded older rats exhibited a greater MBG response, greater SBP elevation (25 vs. 10 mmHg, P < 0.01) and greater inhibition of NKA in aorta (39 vs. 7%, P < 0.01), 30% less natriuresis, and less inhibition of renal NKA (25 vs. 42%, P < 0.05) in the presence of comparable responses of α-ANP and cGMP. In aorta and kidney of older rats, the levels of PKG were reduced, the levels of phosphodiesterase-5 were increased compared with that in young rats, and α-ANP failed to modulate MBG-induced NKA inhibition. Age-associated downregulation of cGMP/PKG-dependent signaling impairs the ability of ANP to modulate the effects of MBG on the sodium pump, which contributes to salt sensitivity.