digoxin and bufalin

Topics: Animals; Antiviral Agents; Betacoronavirus; Biological Products; Bufanolides; Cardiac Glycosides; Cell Survival; Chlorocebus aethiops; Chloroquine; Coronavirus Infections; COVID-19; Digoxin; Gene Expression Regulation; High-Throughput Screening Assays; Host-Pathogen Interactions; Humans; Janus Kinases; Mitogen-Activated Protein Kinases; NF-E2-Related Factor 2; NF-kappa B; Pandemics; Phenanthrenes; Pneumonia, Viral; SARS-CoV-2; Signal Transduction; Sodium-Potassium-Exchanging ATPase; Vero Cells; Virus Replication

Binding to Na+,K+-ATPase, cardiotonic steroids (CTS) activate intracellular signaling cascades that affect gene expression and regulation of proliferation and apoptosis in cells. Ouabain is the main CTS used for studying these processes. The effects of other CTS on nervous tissue are practically uncharacterized. Previously, we have shown that ouabain affects the activation of mitogen-activated protein kinases (MAP kinases) ERK1/2, p38, and JNK. In this study, we compared the effects of digoxin and bufalin, which belong to different subclasses of CTS, on primary culture of rat cortical cells. We found that CTS toxicity is not directly related to the degree of Na+,K+-ATPase inhibition, and that bufalin and digoxin, like ouabain, are capable of activating ERK1/2 and p38, but with different concentration and time profiles. Unlike bufalin and ouabain, digoxin did not decrease JNK activation after long-term incubation. We concluded that the toxic effect of CTS in concentrations that inhibit less than 80% of Na+,K+-ATPase activity is related to ERK1/2 activation as well as the complex profile of MAP kinase activation. A direct correlation between Na+,K+-ATPase inhibition and the degree of MAP kinase activation is only observed for ERK1/2. The different action of the three CTS on JNK and p38 activation may indicate that it is associated with intracellular signaling cascades triggered by protein-protein interactions between Na+,K+-ATPase and various partner proteins. Activation of MAP kinase pathways by these CTS occurs at concentrations that inhibit Na+,K+-ATPase containing the α1 subunit, suggesting that these signaling cascades are realized via α1. The results show that the signaling processes in neurons caused by CTS can differ not only because of different inhibitory constants for Na+,K+-ATPase.

Topics: Animals; Bufanolides; Cell Survival; Cells, Cultured; Cerebrum; Digoxin; Enzyme Activation; JNK Mitogen-Activated Protein Kinases; Microsomes; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neurons; Ouabain; p38 Mitogen-Activated Protein Kinases; Rats; Rats, Wistar; Sodium-Potassium-Exchanging ATPase

Cardiotonic steroids (CTSs) are specific and potent inhibitors of the Na(+),K(+)-ATPase, with highest affinity to the phosphoenzyme (E2P) forms. CTSs are comprised of a steroid core, which can be glycosylated, and a varying number of substituents, including a five- or six-membered lactone. These functionalities have specific influence on the binding properties. We report crystal structures of the Na(+),K(+)-ATPase in the E2P form in complex with bufalin (a nonglycosylated CTS with a six-membered lactone) and digoxin (a trisaccharide-conjugated CTS with a five-membered lactone) and compare their characteristics and binding kinetics with the previously described E2P-ouabain complex to derive specific details and the general mechanism of CTS binding and inhibition. CTSs block the extracellular cation exchange pathway, and cation-binding sites I and II are differently occupied: A single Mg(2+) is bound in site II of the digoxin and ouabain complexes, whereas both sites are occupied by K(+) in the E2P-bufalin complex. In all complexes, αM4 adopts a wound form, characteristic for the E2P state and favorable for high-affinity CTS binding. We conclude that the occupants of the cation-binding site and the type of the lactone substituent determine the arrangement of αM4 and hypothesize that winding/unwinding of αM4 represents a trigger for high-affinity CTS binding. We find that the level of glycosylation affects the depth of CTS binding and that the steroid core substituents fine tune the configuration of transmembrane helices αM1-2.

Topics: Animals; Bufanolides; Crystallography, X-Ray; Digoxin; Fluorescence; Glycosylation; Kinetics; Models, Molecular; Ouabain; Protein Binding; Protein Conformation; Sodium-Potassium-Exchanging ATPase; Structure-Activity Relationship; Swine; X-Ray Diffraction

Cardiotonic steroids have long been in clinical use for treatment of heart failure and are now emerging as promising agents in various diseases, especially cancer. Their main target is Na(+)/K(+)-ATPase, a membrane protein involved in cellular ion homeostasis. Na(+)/K(+)-ATPase has been implicated in cancer biology by affecting several cellular events and signaling pathways in both sensitive and drug-resistant cancer cells. Hence, we investigated the cytotoxic activities of 66 cardiotonic steroids and cardiotonic steroid derivatives in sensitive CCRF-CEM and multidrug-resistant CEM/ADR5000 leukemia cells. Data were then subjected to quantitative structure-activity relationship analysis (QSAR) and molecular docking into Na(+)/K(+)-ATPase, which both indicated a possible differential expression of the pump in the mentioned cell lines. This finding was confirmed by western blotting, intracellular potassium labeling and next generation sequencing which showed that Na(+)/K(+)-ATPase was less expressed in multidrug-resistant than in sensitive cells.

Topics: Antineoplastic Agents; Bufanolides; Cardiac Glycosides; Cell Line, Tumor; Cell Survival; Digoxin; Doxorubicin; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Gene Expression; Humans; Leukocytes, Mononuclear; Molecular Docking Simulation; Primary Cell Culture; Quantitative Structure-Activity Relationship; Signal Transduction; Sodium-Potassium-Exchanging ATPase; Verapamil

Sodium taurocholate cotransporting polypeptide (NTCP) has been reported as a functional receptor for hepatitis B virus (HBV) infection. However, HBV could not efficiently infect HepG2 cells expressing NTCP (NTCP-HepG2 cells) under adherent monolayer-cell conditions. In this study, NTCP was mainly detected in the basolateral membrane region, but not the apical site, of monolayer NTCP-HepG2 cells. We hypothesized that non-adherent cell conditions of infection would enhance HBV infectivity. Non-adherent NTCP-HepG2 cells were prepared by treatment with trypsin and EDTA, which did not degrade NTCP in the membrane fraction. HBV successfully infected NTCP-HepG2 cells at a viral dose 10 times lower in non-adherent phase than in adherent phase. Efficient infection of non-adherent NTCP-HepG2 cells with blood-borne or cell-culture-derived HBV was observed and was remarkably impaired in the presence of the myristoylated preS1 peptide. HBV could also efficiently infect HepaRG cells under non-adherent cell conditions. We screened several compounds using our culture system and identified proscillaridin A as a potent anti-HBV agent with an IC50 value of 7.2 nM. In conclusion, non-adherent host cell conditions of infection augmented HBV infectivity in an NTCP-dependent manner, thus providing a novel strategy to identify anti-HBV drugs and investigate the mechanism of HBV infection.

Topics: Antiviral Agents; Bufanolides; Cell Adhesion; Digitoxin; Digoxin; Gene Expression; Hep G2 Cells; Hepatitis B virus; High-Throughput Screening Assays; Humans; Organic Anion Transporters, Sodium-Dependent; Phthalazines; Proscillaridin; Receptors, Virus; Simvastatin; Strophanthins; Symporters; Transgenes; Viral Envelope Proteins; Virus Internalization

Virtually all transcription factors partner with coactivators that recruit chromatin remodeling factors and interact with the basal transcription machinery. Coactivators have been implicated in cancer cell proliferation, invasion, and metastasis, including the p160 steroid receptor coactivator (SRC) family composed of SRC-1 (NCOA1), SRC-2 (TIF2/GRIP1/NCOA2), and SRC-3 (AIB1/ACTR/NCOA3). Given their broad involvement in many cancers, they represent candidate molecular targets for new chemotherapeutics. Here, we report on the results of a high-throughput screening effort that identified the cardiac glycoside bufalin as a potent small-molecule inhibitor for SRC-3 and SRC-1. Bufalin strongly promoted SRC-3 protein degradation and was able to block cancer cell growth at nanomolar concentrations. When incorporated into a nanoparticle delivery system, bufalin was able to reduce tumor growth in a mouse xenograft model of breast cancer. Our work identifies bufalin as a potentially broad-spectrum small-molecule inhibitor for cancer.

Topics: Animals; Bufanolides; Cell Line; Cell Line, Tumor; Cell Proliferation; Digoxin; HeLa Cells; Humans; MCF-7 Cells; Mice; Mice, SCID; Nuclear Receptor Coactivator 1; Nuclear Receptor Coactivator 3; Transcription Factors

All cardiac steroids have a similar structure, bind to and inhibit the ubiquitous transmembrane protein Na(+), K(+)-ATPase and increase the force of contraction of heart muscle. However, there are diverse biological responses to different cardiac steroids both at the cellular and at the molecular level. Moreover, we have recently shown that ouabain inhibits digoxin- and bufalin-induced changes in membrane traffic. The present study was designed to test the hypothesis that ouabain also has an inhibitory effect on cardiotoxicity induced by other cardiac steroids.. The hypothesis was tested in isolated heart muscle preparations and in an in vivo model of cardiotoxicity in guinea pigs.. Ouabain at a low dose attenuated the toxicity induced by bufalin and digoxin in heart muscle preparations. In addition, ouabain at the low dose (91 ng.kg(-1).h(-1)), but not at a higher dose (182 ng.kg(-1).h(-1)), delayed the development of digoxin-induced (500 microg.kg(-1).h(-1)) cardiotoxicity in anaesthetized guinea pigs, as manifested by delayed arrhythmia and terminal ventricular fibrillation, as well as a reduced heart rate. In addition, as observed with ouabain, the phosphoinositide 3-kinase inhibitor wortmannin (100 microg.kg(-1).h(-1)) delayed the digoxin-induced arrhythmia in anaesthetized guinea pigs.. The present study demonstrates the inhibitory effect, probably through signal transduction pathways, of ouabain on digoxin- and bufalin-induced cardiotoxicity in guinea pigs. Further understanding of this phenomenon could be beneficial for increasing the therapeutic window for cardiac steroids in the treatment of chronic heart failure.

Topics: Androstadienes; Animals; Arrhythmias, Cardiac; Bufanolides; Cardiotonic Agents; Digoxin; Dose-Response Relationship, Drug; Guinea Pigs; Heart Rate; Male; Myocardium; Ouabain; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Signal Transduction; Sodium-Potassium-Exchanging ATPase; Ventricular Fibrillation; Wortmannin

Chan Su and Lu-Shen-Wan are Chinese medicines that crossreact with digoxin immunoassays. Recently, Abbott Laboratories released a new digoxin immunoassay, Digoxin III. We studied potential interference of Chan Su and Lu-Shen-Wan with the Digoxin III assay by comparing results obtained by using Digoxin II and fluorescence polarization immunoassay, also manufactured by Abbott Laboratories. Aliquots of a drug-free serum pool were supplemented with aqueous extract of Chan Su or Lu-Shen-Wan and apparent digoxin concentrations were measured using all three digoxin assays. Significant crossreactivity of Chan Su and Lu-Shen-Wan was observed with the new Digoxin III assay. Moreover, when mice were fed with Chan Su or Lu-Shen-Wan, significant apparent digoxin concentrations were also observed in the sera of mice using the Digoxin III assay indicating that such interferences are also present in vivo. When serum pools prepared from patients receiving digoxin were further supplemented with Chan Su or Lu-Shen-Wan extract, falsely elevated digoxin values were observed with both Digoxin III and fluorescence polarization immunoassay, but digoxin values were falsely lowered using the Digoxin II assay. For example, when one aliquot of Digoxin Serum Pool 1 containing 0.94 ng/mL of digoxin was supplemented with 5.0 microg/mL of Chan Su extract, the digoxin concentration was falsely elevated to 6.60 ng/mL as measured by the Digoxin III assay and 6.99 as measured by the fluorescence polarization immunoassay assay. In contrast, the observed digoxin value was falsely lowered to 0.72 ng/mL using the Digoxin II assay. Interference of Chan Su and Lu-Shen-Wan in the Digoxin III assay cannot be eliminated by monitoring free digoxin concentrations. Digibind neutralizes digoxin-like immunoreactive components of Chan Su and such effect can be monitored by measuring apparent free digoxin concentrations using the Digoxin III assay. We conclude the both Chan Su and Lu-Shen-Wan significantly interfere with serum digoxin measurements by the new Digoxin III assay.

Topics: Animals; Bufanolides; Cross Reactions; Digoxin; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; False Negative Reactions; False Positive Reactions; Fluorescence Polarization; Herb-Drug Interactions; Humans; Immunoassay; Mice; Sensitivity and Specificity

Plasma membrane Na(+)-K(+)-ATPase, which drives potassium into and sodium out of the cell, has important roles in numerous physiological processes. Cardiac steroids (CS), such as ouabain and bufalin, specifically interact with the pump and affect ionic homeostasis, signal transduction, and endocytosed membrane traffic. CS-like compounds are present in mammalian tissues, synthesized in the adrenal gland, and considered to be new family of steroid hormones. In this study, the mechanism of Na(+)-K(+)-ATPase involvement in the regulation of endocytosis is explored. We show that the effects of various CS on changes in endosomal pH are mediated by the pump and correspond to their effects on endosomal membrane traffic. In addition, it was found that CS-induced changes in endocytosed membrane traffic were dependent on alterations in [Na(+)] and [H(+)] in the endosome. Furthermore, we show that various CS differentially regulate endosomal pH and membrane traffic. The results suggest that these differences are due to specific binding characteristics. Based on our observations, we propose that Na(+)-K(+)-ATPase is a key player in the regulation of endosomal pH and endocytosed membrane traffic. Furthermore, our results raise the possibility that CS-like hormones regulate differentially intracellular membrane traffic.

Topics: Acids; Bufanolides; Cardiotonic Agents; Cell Line; Cell Membrane; Digoxin; Endocytosis; Endosomes; Enzyme Inhibitors; Humans; Hydrogen-Ion Concentration; Neurons; Ouabain; Potassium; Protein Transport; Sodium; Sodium-Potassium-Exchanging ATPase; Stem Cells; Transferrin; Tritium

Cardiac steroids (CS) are specific inhibitors of Na+, K+-ATPase activity. Although the presence of CS-like compounds in animal tissues has been established, their physiological role is not clear. In a previous study we showed that in pulse-chase membrane-labeling experiments, long term (hours) interaction of CS at physiological concentrations (nM) with Na+, K+-ATPase, caused changes in endocytosed membrane traffic in human NT2 cells. This was associated with the accumulation of large vesicles adjacent to the nucleus. For this sequence of events to function in the physiological setting, however, CS would be expected to modify membrane traffic upon short term (min) exposure and membrane labeling. We now demonstrate that CS affects membrane traffic also following a short exposure. This was reflected by the CS-induced accumulation of FM1-43 and transferrin in the cells, as well as by changes in their colocalization with Na+, K+-ATPase. We also show that the CS-induced changes in membrane traffic following up to 2 hrs exposure are reversible, whereas longer treatment induces irreversible effects. Based on these observations, we propose that endogenous CS-like compounds are physiological regulators of the recycling of endocytosed membrane proteins and cargo in neuronal cells, and may affect basic mechanisms such as neurotransmitter release and reuptake.

Topics: Apoptosis; Biological Transport, Active; Bufanolides; Cardiac Glycosides; Cell Line; Cell Membrane; Cells, Cultured; Digoxin; Enzyme Activation; Humans; Neurons; Ouabain; Signal Transduction; Sodium-Potassium-Exchanging ATPase

In 1992, apparent digoxin concentrations determined by the Abbott TDx II assay 5 hours after the ingestion of 10 pills of traditional Chinese medicine containing toad secretions (chan su) by 7 volunteers, yielded results that were equimolar to bufalin measured by 2 in-house bufalin radioimmunoassays (RIAs). Recently, a 17-year-old Chinese female unintentionally took 100 (instead of 10) of these pills for a sore throat but suffered no ill effects. The blood bufalin concentration at 3 hours by 1 of the 2 RIAs was 10.93 nmol/L, which was commensurate with the dose. However, the apparent digoxin measured by a TDx II assay produced in 2004 was only 3.08 nmol/L, which probably reflects the change in the specificity of the polyclonal digoxin antisera used in the assay over the years. In 1989, the TDx assay was commended for its ability to detect poisoning from plant and animal cardenolides, a property that seems to be waning and, thus, bad news for those wishing to use the assay to detect alternative cardenolides. But, on the other hand, it possibly eliminates the "specter" of digoxin-like immunoreactive substance (DLIS) that has afflicted some digoxin assays, which can only be good news.

Topics: Adolescent; Bufanolides; Cross Reactions; Digoxin; Female; Fluorescence Polarization Immunoassay; Humans; Medicine, Chinese Traditional

Chan Su, a traditional Chinese medicine, is used for treating the heart diseases and other systemic illnesses. Our studies with animal model have revealed its role in increasing intracellular calcium concentration in cardiomyocytes. Nitric oxide (NO), a second messenger molecule, and its metabolites have been demonstrated to maintain and modulate multiple physiologic functions including the cardiovascular and reproductive systems. In order to explore the mechanism of action of Chan Su, we studied the ability of Chan Su to stimulate NO production in cultured trophoblastic BeWo cells.. BeWo cell is a cloned established cell line purified from human choriocarcinoma. These cells have some similarities in biological behavior with endothelial cells. Therefore, BeWo cell line may act as a model system for production of nitric oxide by Chan Su both in placenta and in cardiovascular tissue, and the results can easily be extrapolated to cardiomyocytes. Very small amount of ethanol extract of Chan Su was added to the cultured cells in KBM buffer and a chemiluminescence system was used for the measurement of nitric oxide. The amounts of Chan Su extract added to cultured cells were comparable to expected level of Chan Su in human serum after ingestion. We also repeated these experiments with bufalin, the active component of Chan Su.. The ethanol extract of Chan Su (5 and 10 microg/ml) significantly increased NO production up to 110% of basal control value, but higher concentrations (40 and 80 microg/ml) of Chan Su (as expected in an overdose) resulted in decreased NO production below the control level. This biphasic effect on nitric oxide production was also observed with bufalin, the active component of Chan Su responsible for its digoxin-like immunoreactivity. The presence of bufalin in Chan Su preparation was confirmed by thin layer chromatography (TLC) analysis.. Chan Su as well as bufalin is able to modulate the production of NO in BeWo cell line.

Topics: Amphibian Venoms; Bufanolides; Cell Line; Chromatography, Thin Layer; Digoxin; Fluorescence Polarization Immunoassay; Humans; Medicine, Chinese Traditional; Nitric Oxide; Trophoblasts

Asian medicines are widely used as alternative medicine. However, interactions between drugs and Asian medicines have been poorly studied. Chan Su and Lu-Shen-Wan are Asian medicines that contain the cardiaoactive compound bufalin. Bufalin is structurally similar to digitoxin and is also strongly bound to serum albumin. The authors studied possible displacement of digitoxin from the protein binding site by bufalin. The authors prepared three serum pools from patients taking digitoxin and supplemented aliquots of each serum pool with no bufalin (control) and 25 ng/mL, 50 ng/mL, 100 ng/mL, 250 ng/mL, 500 ng/mL, and 1000 ng/mL bufalin. The authors observed significant displacement of digitoxin by bufalin as evidenced by increased free digitoxin concentrations. For example, the concentration of free digitoxin increased from a control value of 1.6 ng/mL to 2.5 ng/mL in the presence of 1000 ng/mL bufalin (total digitoxin: 36.3 ng/mL) in the serum pool 1. The authors observed similar increases in free digitoxin concentrations in other serum pools in the presence of various concentrations of bufalin. The authors used a chemiluminescent assay and ACS:180 analyzer to measure both total (in the original serum) and free (in the protein-free ultrafiltrate) digitoxin concentrations because the chemiluminescent assay does not cross-react with bufalin. When an acetone/water (1:1 by volume) extract of Chan Su was added to a serum pool containing digitoxin, the authors observed a significantly increased free digitoxin concentration, indicating that Chan Su can displace digitoxin from the protein binding site in vitro. The authors conclude that bufalin and acetone/water extract of Chan Su can cause significant displacement of digitoxin from the protein binding site.

Topics: Binding Sites; Bufanolides; Cardiotonic Agents; Digoxin; Drug Interactions; Humans; Materia Medica; Protein Binding

Na+,K+-ATPase activity in the epithelial layer is fundamental to the maintenance of ionic concentration gradients and transparency of the lens. Recently we have identified endogenous digitalislike compounds (DLC), 19-norbufalin and its peptide derivatives, in human cataractous lenses (Lichtstein et al. Eur J Biochem 216: 261-268, 1993). Lenses were treated with 10 nM ouabain, bufalin or 19-norbufalin derivative for 24 h and were compared to control lenses. Differential display analysis revealed that one of the down-regulated genes was 14-3-3 theta. Down-regulation was confirmed by Northern blot and by RT-PCR analysis. RT-PCR of additional 14-3-3 isoforms revealed that the eta and gamma isoforms of 14-3-3 are also down-regulated by ouabain, bufalin and 19-norbufalin derivative, whereas the zeta isoform is down-regulated only by bufalin. These results demonstrate that one of the consequences of Na+,K+-ATPase inhibition by exogenous or endogenous inhibitors is the down-regulation of mRNA transcripts encoding several isoforms of 14-3-3. Since the 14-3-3 proteins are multifunctional regulatory proteins, the reduction in the abundance of various isoforms will have profound effects on cell function. Furthermore, These results, together with the demonstration of digitalislike compounds in the normal lens, and their increased level in human cataractous lenses, strongly suggests their involvement in the molecular mechanisms responsible for cataract formation.

Topics: 14-3-3 Proteins; Animals; Blotting, Northern; Bufanolides; Cardenolides; Cardiotonic Agents; Crystallins; Digoxin; Gene Expression; Lens, Crystalline; Ouabain; Rats; Rats, Sprague-Dawley; RNA, Messenger; Saponins; Signal Transduction; Sodium-Potassium-Exchanging ATPase; Tyrosine 3-Monooxygenase

Intoxication caused by digitalis-like substances after ingestion of cooked toad soup has been reported. Bufalin, a cardioactive compound, is found in toad. Bufalin is also found in many Chinese medicines. Earlier reports demonstrated cross reactivity of bufalin with fluorescence polarization immunoassay for digoxin. In this report, the authors demonstrated a significantly higher cross reactivity of bufalin with the fluorescence polarization assay for digitoxin. They supplemented aliquots of normal plasma that had various concentrations of bufalin (1 to 50 micrograms/ml) from a local blood bank and measured apparent digitoxin concentrations using fluorescence polarization immunoassay and chemiluminescent assays (ACS digitoxin) for digitoxin. They measured apparent digoxin and digitoxin concentrations using fluorescence polarization, microparticle enzyme immunoassay, and chemiluminescent assays for digitoxin. They observed apparent digitoxin or digoxin concentrations in sera supplemented with bufalin only with the fluorescence polarization assays. For example, the apparent digitoxin concentration observed in a serum supplemented with 25 ng/ml of bufalin was 24.3 ng/ml of digitoxin equivalent. The apparent digoxin concentration observed in the same specimen was 1.33 ng/ml digoxin equivalent. Bufalin caused positive interference in serum digoxin or digitoxin measurements in specimens containing digoxin or digitoxin when concentrations were measured by fluorescence polarization assays. In contrast, bufalin lowered the measured digoxin concentrations in serum pools containing digoxin when digoxin concentrations were measured by the microparticle enzyme immunoassay. The authors conclude that bufalin toxicity can be rapidly detected by the fluorescence polarization assay for digitoxin.

Topics: Animals; Anura; Bufanolides; Cardiotonic Agents; Digitoxin; Digoxin; Drug Interactions; Fluorescence Polarization Immunoassay; Humans

The microparticle enzyme immunoassay (MEIA for digoxin (Abbott Laboratories, Abbott Park, Ill) requires no sample pretreatment and is widely used in clinical toxicology laboratories for monitoring serum digoxin concentrations. One advantage of the new MEIA is the lower cross-reactivities with such cross-reactants as digitoxin, oleandrin, and bufalin compared with the fluorescence polarization immunoassay (FPIA)for digoxin. Digitoxin, oleandrin, and bufalin showed positive cross-reactivity with MEIA and FPIAs for digoxin in the absence of the primary analyte, digoxin. A surprising finding was that digoxin concentrations were falsely decreased by these cross-reactants when serum pools containing digoxin were supplemented with various concentrations of these cross-reactants and when digoxin concentrations were measured by the MEIA. In contrast, digoxin concentrations were falsely elevated when measured by the FPIA. For example, when a serum pool containing 2.15 nmol/L of digoxin was supplemented with 129.5 nmol/L of bufalin, the apparent digoxin concentrations were 1.45 nmol/L with the MEIA and 3.00 nmol/L with the FPIA. Taking the advantage of only 25% protein binding of digoxin and more than 95% protein binding of digitoxin and bufalin, we demonstrated that monitoring free digoxin instead of total digoxin eliminated negative interference of digoxin by these cross-reactants in the MEIA and positive interference in the FPIA. Although oleandrin is also strongly bound to serum protein, high concentrations of oleandrin still modestly affect the free digoxin assay for both MEIA and FPIAs.

Topics: Bufanolides; Cardenolides; Cardiac Glycosides; Cardiotonic Agents; Cross Reactions; Digitoxin; Digoxin; False Positive Reactions; Fluorescence Polarization Immunoassay; Humans; Immunoenzyme Techniques; Microspheres

Oleandrin plant poisoning is common in children and the plant extract is used in Chinese medicines. The toxicity is due to oleandrin and the deglycosylated metabolite oleandrigenin. Bufalin and cinobufotalin (toad cardiac toxins) are also widely used in Chinese medicines like Chan SU, and Lu-Shen -WU. Severe toxicity from bufalin after consumption of toad soup has been reported. Taking advantage of structural similarities of these toxins with digitoxin, we demonstrated that these compounds can be rapidly detected in blood by the fluorescence polarization immunoassay for digitoxin. The cross reactivities of these compounds with digoxin assay were much lower. For example, when a drug free serum was supplemented with 10 microg/ml of oleandrin, we observed 127.7 ng/ml of digitoxin equivalent but only 2.4 ng/ml of digoxin equivalent concentration. Digibind neutralized all cardiac toxins studied as evidenced by significant fall of free concentrations. When aliquots of serum pool containing 50.0 microg/ml of oleandrin were supplemented with 0, 10.0, 25.0, 50.0, 100, and 200 microg/ml of digibind, the mean free concentrations were 30.6, 23.3, 16.0, 10.7, 7.8 and 5.5 microg/ml respectively. Similarly, with 50.0 microg/ml of oleandrigenin (total concentration: 36.2 ng/ml), the free concentration was 14.5 ng/ml digitoxin equivalent in the absence of digibind and 5.4 ng/ml in the presence of 200 microg/ml of digibind. In another specimen containing 500 ng/ml bufalin (total concentration: 156.9 ng/ml), the free concentration was 8.6 ng/ml in the absence of digibind and none detected in the presence of 100.0 microg/ml digibind. Because such neutralization may also occur in vivo, digibind may be useful in treating patients exposed to these toxins.

Topics: Bufanolides; Cardenolides; Cardiotonic Agents; Chromatography, High Pressure Liquid; Cross Reactions; Digitoxin; Digoxin; Humans; Immunoassay; Immunoglobulin Fab Fragments; Mass Spectrometry; Neutralization Tests

Compromised renal function predisposes to volume-dependent hypertension. Increased plasma levels of a sodium pump inhibitor as a possible pathogenetic factor have been demonstrated by many investigators in such patients, but efforts to identify the responsible agent have led to many, diverse candidates. Our premise in this study is that candidacy must depend on the satisfaction of rigorous criteria, including a specific action of the agent on the sodium pump. These criteria included reversibility, concentration dependence, receptor mediation, and an action at the appropriate step in the enzyme cycle. These criteria were applied to a potent [Na,K]ATPase inhibitor we have identified in the peritoneal dialysate of patients with chronic renal failure, present only during extracellular fluid volume expansion, the levels of which are correlated with the blood pressure rise that results from excessive NaCl and water intake. In microsomes that contained both [Na,K]ATPase and other ATPases, this candidate inhibited only the Na and K dependent, ouabain-sensitive ATPase. It displaced ouabain from the cardioglycoside binding site and its binding was linked to inhibition. Inhibition was produced by slowing the pump's dephosphorylation step, the exact action of all cardioglycosides. Finally, the candidate cross reacted with a digoxin Fab fragment and this Fab reversed its inhibition of [Na,K]ATPase. Together, these experiments demonstrate that the PD candidate specifically, and reversibly, inhibits the sodium pump via the cardioglycoside binding site, and hence, meets this crucial criterion for candidacy.

Topics: Antineoplastic Agents; Binding Sites; Binding, Competitive; Bufanolides; Cross Reactions; Dialysis Solutions; Digoxin; Enzyme Inhibitors; Humans; Immunoglobulin Fab Fragments; Iodine Radioisotopes; Kidney Failure, Chronic; Lysophosphatidylcholines; Oleic Acid; Ouabain; Peritoneal Dialysis; Peritoneum; Sensitivity and Specificity; Sodium-Potassium-Exchanging ATPase; Strophanthidin

Toxicity from toad venom poisoning is similar to digoxin toxicity and carries a high mortality rate. We report on six previously healthy men who developed vomiting and bradycardia after ingesting a purported topical aphrodisiac. Each patient had positive apparent digoxin levels and the first four patients died of cardiac dysrhythmias. The last two patients recovered following treatment with digoxin Fab fragments. We analyzed samples of the purported aphrodisiac and found that it was identical to Chan Su, a Chinese medication made from toad venom. To our knowledge, this is the first reported use of digoxin Fab fragments to treat toad venom poisoning.

Topics: Adolescent; Adult; Amphibian Venoms; Animals; Aphrodisiacs; Bradycardia; Bufanolides; Bufonidae; Bufotenin; Digoxin; Fatal Outcome; Humans; Immunoglobulin Fab Fragments; Male; Materia Medica; Ventricular Fibrillation; Vomiting

Efforts to study the endogenous sodium pump inhibitor (ESPI) have been complicated by the limited specificity of available assays. We recently developed an assay of [Na,K]ATPase inhibition more sensitive than conventional assays. This enhancement reflects a prereaction step that increases binding affinity of digitalislike molecules to the digitalis receptor. We tested the possibility that this enhanced inhibition is limited to inhibitors acting specifically through the digitalis-binding site. Using both the standard and sensitive [Na,K]ATPase methods, known specific inhibitors of the sodium pump (ouabain, digoxin, bufalin) showed significant increases in the inhibition in the sensitive as compared with the standard [Na,K]ATPase assay (ouabain 34.4 +/- 7.3 vs. 8.3 +/- 0.5%, digoxin 43.2 +/- 9.1 vs. 7.2 +/- 3.1%, bufalin 46.9 +/- 5.0 vs. 22.6 +/- 1.6%). Some proposed candidates for the ESPI, acknowledged to be nonspecific inhibitors, showed no enhancement (oleic acid 36.0 +/- 4.5 vs. 34.8 +/- 5.6%, lysophosphatidyl choline 10.8 +/- 3.5 vs. 12.8 +/- 5.2%, and vanadate 34.3 +/- 8.8 vs. 33.8 +/- 1.4%). Other candidates, whose inhibitory specificity is unknown, including an ESPI from the peritoneal dialysate of patients with renal failure were also studied. The ESPI showed enhanced inhibition (24.1 +/- 4.9 vs. 5.3 +/- 2.0%). These studies suggest that the sensitive assay in conjunction with a standard [Na,K]ATPase assay may allow the early determination of candidates interacting specifically with the digitalis receptor to inhibit the sodium pump.

Topics: Animals; Antineoplastic Agents; Binding, Competitive; Bufanolides; Cattle; Digitalis; Digoxin; Dose-Response Relationship, Drug; Enzyme Inhibitors; Kidney; Lysophosphatidylcholines; Oleic Acid; Oleic Acids; Ouabain; Plants, Medicinal; Plants, Toxic; Regression Analysis; Silver Nitrate; Sodium-Potassium-Exchanging ATPase; Steroids; Tamoxifen; Vanadates

The existence of a mammalian natriuretic substance similar to plant digitalis, which inhibits Na,K-ATPase, has been speculated about, but as yet no definite substance has been found. Digoxin-like immunoreactive substance (DLIS) has been reported in various clinical states including new born infants. Using bufalin (a cardioactive substance of animal origin) as antigen, four polyclonal antisera have been produced from 2 separate rabbits and characterised for cross-reactivity with 32 compounds. One antiserum showed a marked change in its cross-reactivity after resting the animal for a year. Of the endogenous substances tested, progesterone was found to be the most cross-reactive. Radioimmunoassay of foetal cord sera with different antisera, gave different levels of bufalin-like immunoactivity. However, after a novel "affinity-immunoassay" procedure, this apparent bufalin-like immunoactivity disappeared. It is concluded that bufalin-like immunoactivity in the cord blood is caused by the cross-reaction of endogenous steroids with bufalin antiserum, and the same may be true for DLIS.

Topics: Animals; Blood Proteins; Bufanolides; Cardenolides; Cattle; Chromatography, Affinity; Cross Reactions; Digoxin; Fetal Blood; Humans; Immune Sera; Infant, Newborn; Rabbits; Radioimmunoassay; Saponins

A study was conducted on the pharmacological actions of the toad venom-containing drug "Kyushin" (KY-2 and KY-R) on urinary volume and electrolytes excretion, regional blood flow, renal artery blood flow and carrageenin-induced hind-paw edema. In rabbits, KY-2 and KY-R significantly increased urinary volume after intravenous administration of 8 mg/kg. In guinea pigs, KY-2 and KY-R produced a significant increase in urinary volume after intraduodenal administration (i.d.) of 80 mg/kg. In guinea pigs treated with propranolol, KY-2 at 20 and 40 mg/kg p.o. and KY-R at 40 mg/kg p.o. increased urinary volume. At 40 mg/kg i.d. both KY-2 and KY-R produced an increase in regional blood flow, as determined by the hydrogen gas clearance method, of the brain areas including the amygdaloid nucleus, but did not affect regional blood flow in liver, kidney and skeletal muscle, or renal artery blood flow. In rats, carrageenin-induced hind-paw edema was inhibited by KY-2 or KY-R at 600 mg/kg p.o.

Topics: Animals; Bufanolides; Carrageenan; Digoxin; Drug Evaluation, Preclinical; Duodenum; Edema; Electrolytes; Guinea Pigs; Hemodynamics; Injections, Intravenous; Intubation, Gastrointestinal; Liver; Male; Materia Medica; Muscles; Propranolol; Rabbits; Rats; Rats, Wistar; Renal Artery; Renal Circulation; Urine

1. The presence of an endogenous digitalis-like factor (EDLF) in the plasma of both normal and volume expanded animals is well documented. In this study we have used ouabain and bufalin as pharmacological analogues to mimic the renal effects of EDLF and to investigate whether any interaction occurs between atrial natriuretic factor (ANF) and EDLF. 2. Conscious Na replete sheep with chronically indwelling catheters in the renal artery received renal arterial infusion of ouabain (1000 micrograms h-1) or bufalin (500 micrograms h-1) for 60 min. Renal arterial infusion of bufalin increased sodium excretion (UNaV) from 120 +/- 13 to 596 +/- 161 mumol min-1 after 45 min. Bufalin infusion did not alter glomerular filtration rate (GFR), effective renal plasma flow (ERPF), or lithium clearance. Ouabain infusion increased UNaV from 124 +/- 57 to 764 +/- 123 mumol min-1 in the first hour after infusion. 3. ANF infusion increased UNaV from 159 +/- 34 to 583 +/- 134 mumol min-1. When renal arterial bufalin infusion was followed by renal arterial ANF infusion (50 micrograms h-1) UNaV was increased from 155 +/- 31 to 795 +/- 96 mumol min-1. This increase in UNaV is approximately equal to the sum of the separate effects of bufalin and ANF. 4. The natriuretic effects of ouabain at pharmacological doses in sheep are confirmed by this study. The data presented here do not support the hypothesis that EDLF sensitizes the kidney to the natriuretic effects of ANF.

Topics: Animals; Atrial Natriuretic Factor; Blood Pressure; Blood Proteins; Bufanolides; Cardenolides; Digoxin; Female; Heart Rate; Infusions, Intra-Arterial; Natriuresis; Ouabain; Potassium; Renal Artery; Renal Circulation; Saponins; Sheep; Sodium; Sodium-Potassium-Exchanging ATPase

Digitalis glycoside-like properties of the Bufo marinus toad crude venom and one of its constituents, bufalin, were studied in various assay systems. In concentrations 0.3-30 micrograms/ml crude venom increased the contractility of isolated electrically driven rat atria, constricted rat aortic rings, inhibited ouabain-sensitive Na+,K(+)-ATPase in rat erythrocytes and the Na+,K(+)-pump in rat aorta, and cross-reacted with antidigoxin antibody from the dissociation enhanced lanthanide fluoroimmunoassay (DELFIA). These effects were unaffected by adrenoceptor blockers and the 5-HT antagonist, deseril, but were blocked by antidigoxin antibody. Bufalin (10-30 microM) increased myocardial contractility and inhibited Na+,K(+)-ATPase in rat erythrocytes similarly to crude Bufo marinus venom. In rat aorta bufalin showed weak and delayed vasoconstrictor activity which was antagonized by 2 microM phentolamine, and had a biphasic effect on the Na+,K(+)-pump; 0.5-1.0 microM bufalin stimulated the pump, while higher concentrations inhibited its activity. Although the effects of bufalin were blocked by antidigoxin antibody, bufalin showed very low digoxin-like immunoreactivity in the DELFIA. These observations suggest that, in addition to bufalin, Bufo marinus venom contains at least one more digitalis-like steroid with significant intrinsic vasoconstrictor activity which, unlike bufalin, constricts the blood vessels acting directly via inhibition of the sodium pump in the vascular smooth muscle membrane.

Topics: Amphibian Venoms; Animals; Aorta, Abdominal; Blood Proteins; Bufanolides; Bufo marinus; Cardenolides; Digitalis Glycosides; Digoxin; Erythrocytes; Female; Fluorescent Antibody Technique; Immunoglobulin G; In Vitro Techniques; Male; Muscle, Smooth, Vascular; Ouabain; Parotid Gland; Rats; Rats, Wistar; Saponins; Sodium Channels; Sodium-Potassium-Exchanging ATPase; Vasoconstriction

Topics: Bufanolides; Digoxin; Humans; Medicine, Chinese Traditional; Radioimmunoassay; Reagent Kits, Diagnostic

Increased levels of a circulating digoxin-like factor (DLF) occur in a number of physiologic states in which sodium homeostasis is altered, and may contribute to the pathogenesis of hypertension. We exploited the different affinities for DLF of seven antisera directed at digoxin to develop an immunochemical profile, and then employed this index to address two questions: does the same DLF species exist in several conditions associated with increased DLF levels, including pregnancy, renal failure, hepatic failure, and neonatal cord blood? Will this approach prove useful in assessing candidates proposed to be DLF? An identical profile was identified in serum from pregnant women and patients with renal or hepatic failure, and a highly significant correlation existed between DLF levels measured with antisera of high and intermediate affinity in 42 subjects with increased levels (r = 0.93; P less than .001). In patients with renal failure, when endogenous DLF levels were too low to assess the profile, concentration of the serum resulted in measurable DLF levels that had an identical profile. The profile was somewhat altered in umbilical cord blood, perhaps reflecting an influence of increased steroid hormone levels. Among agents suggested as candidates for DLF, neither lysophosphatidylcholine nor ouabain showed a profile resembling DLF. Progesterone, 17-OH-progesterone, and bufalin, on the other hand, did show substantial similarity, perhaps providing a clue to the structure of DLF. The normal plasma levels of progesterone and 17-OH-progesterone are 100- to 1000-fold too low to be candidates for DLF and bufalin was sufficiently dissimilar not to be a candidate. DLF in at least three different patient populations probably represents identical chemical species.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Antibodies; Antibody Affinity; Antibody Specificity; Blood Proteins; Bufanolides; Cardenolides; Digoxin; Female; Fetal Blood; Humans; Kidney Diseases; Liver Diseases; Pregnancy; Progesterone; Radioimmunoassay; Saponins

Highly specific antisera produced against cardenolide (digoxin) and bufodienolide (bufalin) may bind the endogenous ouabain-displacing compounds from human urine. However, it should be borne in mind that the degree of recognition by the antisera significantly differ between two ouabain-displacing compounds.

Topics: Antigen-Antibody Reactions; Binding, Competitive; Blood Proteins; Bufanolides; Cardenolides; Cross Reactions; Digoxin; Humans; In Vitro Techniques; Ouabain; Saponins

Substances structurally and functionally similar to digitalis glycosides are produced by several vertebrate species. There also is evidence for a digitalis-like substance of human origin. Standard microelectrode techniques were used to study the direct effects on the cellular electrophysiology of canine Purkinje fibers of 1) bufalin, an unconjugated cardiotonic steroid molecule that is produced by the toad Bufo marinus, and 2) an extract of human bile that showed digitalis-like immunoreactivity on radioimmunoassay. The goal of this study was to determine whether these substances have arrhythmogenic effects comparable with those seen with toxic doses of digitalis glycosides. Bufalin, 2 x 10(-8) M, significantly (p less than 0.05) reduced maximal diastolic potential, action potential amplitude and duration and maximal rate of rise of phase 0 (Vmax) within 40 min of onset of exposure. All six fibers developed delayed afterdepolarizations and two developed triggered rhythms. Ouabain was less potent, in that a 2 x 10(-7) M concentration was required to comparably reduce maximal diastolic potential, action potential amplitude and duration and Vmax within 30 min. These Purkinje fibers also developed delayed afterdepolarizations and triggered rhythms. A sample of an extract of human bile that showed digitalis-like immunoreactivity with an antibufalin serum also reduced maximal diastolic potential, action potential amplitude and duration and Vmax, and produced delayed afterdepolarizations and triggered activity. In contrast, immunologically unreactive bile extracts had no appreciable effect on the action potential. In summary, the cardiac toxicity of digitalis substances produced by lower vertebrates is comparable with that induced by the glycosides. Moreover, it appears that humans may produce digitalis-like substances that may be cardiotoxic.

Topics: Action Potentials; Animals; Bile; Blood Proteins; Bufanolides; Cardenolides; Digoxin; Dogs; Electrophysiology; Female; Heart Conduction System; Humans; Male; Membrane Potentials; Ouabain; Purkinje Fibers; Radioimmunoassay; Saponins; Sodium-Potassium-Exchanging ATPase