Evaluation of Ouabain's Tissue Distribution in C57/Black Mice Following Intraperitoneal Injection, Using Chromatography and Mass Spectrometry.

Cardiotonic steroids (CTSs), such as digoxin, are used for heart failure treatment. However, digoxin permeates the brain-blood barrier (BBB), affecting central nervous system (CNS) functions. Finding a CTS that does not pass through the BBB would increase CTSs' applicability in the clinic and decrease the risk of side effects on the CNS. This study aimed to investigate the tissue distribution of the CTS ouabain following intraperitoneal injection and whether ouabain passes through the BBB. After intraperitoneal injection (1.25 mg/kg), ouabain concentrations were measured at 5 min, 15 min, 30 min, 1 h, 3 h, 6 h, and 24 h using HPLC-MS in brain, heart, liver, and kidney tissues and blood plasma in C57/black mice. Ouabain was undetectable in the brain tissue. Plasma: Cmax = 882.88 ± 21.82 ng/g; Tmax = 0.08 ± 0.01 h; T1/2 = 0.15 ± 0.02 h; MRT = 0.26 ± 0.01. Cardiac tissue: Cmax = 145.24 ± 44.03 ng/g (undetectable at 60 min); Tmax = 0.08 ± 0.02 h; T1/2 = 0.23 ± 0.09 h; MRT = 0.38 ± 0.14 h. Kidney tissue: Cmax = 1072.3 ± 260.8 ng/g; Tmax = 0.35 ± 0.19 h; T1/2 = 1.32 ± 0.76 h; MRT = 1.41 ± 0.71 h. Liver tissue: Cmax = 2558.0 ± 382.4 ng/g; Tmax = 0.35 ± 0.13 h; T1/2 = 1.24 ± 0.7 h; MRT = 0.98 ± 0.33 h. Unlike digoxin, ouabain does not cross the BBB and is eliminated quicker from all the analyzed tissues, giving it a potential advantage over digoxin in systemic administration. However, the inability of ouabain to pass though the BBB necessitates intracerebral administration when used to investigate its effects on the CNS.

Sensational site: the sodium pump ouabain-binding site and its ligands.

Cardiotonic steroids (CTS), used by certain insects, toads, and rats for protection from predators, became, thanks to Withering's trailblazing 1785 monograph, the mainstay of heart failure (HF) therapy. In the 1950s and 1960s, we learned that the CTS receptor was part of the sodium pump (NKA) and that the Na+/Ca2+ exchanger was critical for the acute cardiotonic effect of digoxin- and ouabain-related CTS. This "settled" view was upended by seven revolutionary observations. First, subnanomolar ouabain sometimes stimulates NKA while higher concentrations are invariably inhibitory. Second, endogenous ouabain (EO) was discovered in the human circulation. Third, in the DIG clinical trial, digoxin only marginally improved outcomes in patients with HF. Fourth, cloning of NKA in 1985 revealed multiple NKA α and ß subunit isoforms that, in the rodent, differ in their sensitivities to CTS. Fifth, the NKA is a cation pump and a hormone receptor/signal transducer. EO binding to NKA activates, in a ligand- and cell-specific manner, several protein kinase and Ca2+-dependent signaling cascades that have widespread physiological effects and can contribute to hypertension and HF pathogenesis. Sixth, all CTS are not equivalent, e.g., ouabain induces hypertension in rodents while digoxin is antihypertensinogenic ("biased signaling"). Seventh, most common rodent hypertension models require a highly ouabain-sensitive α2 NKA and the elevated blood pressure is alleviated by EO immunoneutralization. These numerous phenomena are enabled by NKA's intricate structure. We have just begun to understand the endocrine role of the endogenous ligands and the broad impact of the ouabain-binding site on physiology and pathophysiology.

Broad spectrum post-entry inhibitors of coronavirus replication: Cardiotonic steroids and monensin.

A small molecule screen identified several cardiotonic steroids (digitoxin and ouabain) and the ionophore monensin as potent inhibitors of HCoV-229E, HCoV-OC43, and SARS-CoV-2 replication with EC50s in the low nM range. Subsequent tests confirmed antiviral activity in primary cell models including human nasal epithelial cells and lung organoids. Addition of digitoxin, ouabain, or monensin strongly reduced viral gene expression as measured by both viral protein and RNA accumulation. Furthermore, the compounds acted post virus entry. While the antiviral activity of digitoxin was dependent upon activation of the MEK and JNK signaling pathways but not signaling through GPCRs, the antiviral effect of monensin was reversed upon inhibition of several signaling pathways. Together, the data demonstrates the potent anti-coronavirus properties of two classes of FDA approved drugs that function by altering the properties of the infected cell, rendering it unable to support virus replication.

Endogenous Digitalis-like Factors as a Key Molecule in the Pathophysiology of Pregnancy-Induced Hypertension and a Potential Therapeutic Target in Preeclampsia.

Preeclampsia (PE), the most severe presentation of hypertensive disorders of pregnancy, is the major cause of morbidity and mortality linked to pregnancy, affecting both mother and fetus. Despite advances in prophylaxis and managing PE, delivery of the fetus remains the only causative treatment available. Focus on complex pathophysiology brought the potential for new treatment options, and more conservative options allowing reduction of feto-maternal complications and sequelae are being investigated. Endogenous digitalis-like factors, which have been linked to the pathogenesis of preeclampsia since the mid-1980s, have been shown to play a role in the pathogenesis of various cardiovascular diseases, including congestive heart failure and chronic renal disease. Elevated levels of EDLF have been described in pregnancy complicated by hypertensive disorders and are currently being investigated as a therapeutic target in the context of a possible breakthrough in managing preeclampsia. This review summarizes mechanisms implicating EDLFs in the pathogenesis of preeclampsia and evidence for their potential role in treating this doubly life-threatening disease.

The Effects of Cinobufagin on Hepatocellular Carcinoma Cells Enhanced by MRT68921, an Autophagy Inhibitor.

Cinobufagin, a cardiotonic steroid derived from toad venom extracts, exhibits significant anticancer properties by inhibiting Na[Formula: see text]/K[Formula: see text]-ATPase in cancer cells. It is frequently used in clinical settings to treat advanced-stage cancer patients, improving their quality of life and survival time. However, its long-term use can result in multidrug resistance to other chemotherapy drugs, and the exact mechanism underlying this effect remains unknown. Therefore, this study explores the molecular mechanism underlying the anticancer effects of cinobufagin in hepatocellular carcinomas (HCCs), specifically in HepG2 and Huh-7 cells. As determined using transcriptome analysis, cinobufagin-triggered protective autophagy suppressed cell apoptosis in liver cancer HepG2 and Huh-7 cells by inhibiting the phosphoinositide-3-Kinase (PI3K)-AKT serine/threonine kinase (AKT)-mammalian target of rapamycin (mTOR) pathway. Cinobufagin-inhibited cell proliferation, induced apoptosis, and generated cell autophagy by upregulating the expression of MAP1 light chain 3 protein II, Beclin1, and autophagy-related protein 12-5. In addition, the autophagy inhibitor MRT68921 improved the antiproliferative and proapoptotic effects of cinobufagin in the studied cell lines. Overall, this study suggests that combining cinobufagin with an autophagy inhibitor can effectively treat HCC, providing a potential strategy for cancer therapy.

Na+,K+-ATPase and Cardiotonic Steroids in Models of Dopaminergic System Pathologies.

In recent years, enough evidence has accumulated to assert that cardiotonic steroids, Na+,K+-ATPase ligands, play an integral role in the physiological and pathophysiological processes in the body. However, little is known about the function of these compounds in the central nervous system. Endogenous cardiotonic steroids are involved in the pathogenesis of affective disorders, including depression and bipolar disorder, which are linked to dopaminergic system dysfunction. Animal models have shown that the cardiotonic steroid ouabain induces mania-like behavior through dopamine-dependent intracellular signaling pathways. In addition, mutations in the alpha subunit of Na+,K+-ATPase lead to the development of neurological pathologies. Evidence from animal models confirms the neurological consequences of mutations in the Na+,K+-ATPase alpha subunit. This review is dedicated to discussing the role of cardiotonic steroids and Na+,K+-ATPase in dopaminergic system pathologies-both the evidence supporting their involvement and potential pathways along which they may exert their effects are evaluated. Since there is an association between affective disorders accompanied by functional alterations in the dopaminergic system and neurological disorders such as Parkinson's disease, we extend our discussion to the role of Na+,K+-ATPase and cardiotonic steroids in neurodegenerative diseases as well.

Hypoxic Stress-Dependent Regulation of Na,K-ATPase in Ischemic Heart Disease.

In cardiomyocytes, regular activity of the Na,K-ATPase (NKA) and its Na/K pump activity is essential for maintaining ion gradients, excitability, propagation of action potentials, electro-mechanical coupling, trans-membrane Na+ and Ca2+ gradients and, thus, contractility. The activity of NKA is impaired in ischemic heart disease and heart failure, which has been attributed to decreased expression of the NKA subunits. Decreased NKA activity leads to intracellular Na+ and Ca2+ overload, diastolic dysfunction and arrhythmias. One signal likely related to these events is hypoxia, where hypoxia-inducible factors (HIF) play a critical role in the adaptation of cells to low oxygen tension. HIF activity increases in ischemic heart, hypertension, heart failure and cardiac fibrosis; thus, it might contribute to the impaired function of NKA. This review will mainly focus on the regulation of NKA in ischemic heart disease in the context of stressed myocardium and the hypoxia-HIF axis and argue on possible consequences of treatment.

Ouabain Effects on Human Anaplastic Thyroid Carcinoma 8505C Cells.

Anaplastic thyroid carcinoma (ATC) is a rare, but aggressive, carcinoma derived from follicular cells. While conventional treatments may improve patients' survival, the lethality remains high. Therefore, there is an urgent need for more effective ATC treatments. Cardiotonic steroids, such as ouabain, have been shown to have therapeutic potential in cancer treatment. Thus, we aimed to evaluate ouabain's effects in human anaplastic thyroid cells. For this, 8505C cells were cultured in the presence or absence of ouabain. Viability, cell death, cell cycle, colony formation and migratory ability were evaluated in ouabain-treated and control 8505C cells. The expression of differentiation and epithelial-to-mesenchymal transition (EMT) markers, as well as IL-6, TGFb1 and their respective receptors were also quantified in these same cells. Our results showed that ouabain in vitro decreased the number of viable 8505C cells, possibly due to an inhibition of proliferation. A reduction in migration was also observed in ouabain-treated 8505C cells. In contrast, decreased mRNA levels of PAX8 and TTF1 differentiation markers and increased levels of the N-cadherin EMT marker, as well as IL-6 and TGFb1, were found in ouabain-treated 8505C cells. In short, ouabain may have anti-proliferative and anti-migratory effect on 8505C cells, but maintains an aggressive and undifferentiated profile.

Epistatic Effects Between Amino Acid Insertions and Substitutions Mediate Toxin resistance of Vertebrate Na+,K+-ATPases.

The recurrent evolution of resistance to cardiotonic steroids (CTS) across diverse animals most frequently involves convergent amino acid substitutions in the H1-H2 extracellular loop of Na+,K+-ATPase (NKA). Previous work revealed that hystricognath rodents (e.g., chinchilla) and pterocliform birds (sandgrouse) have convergently evolved amino acid insertions in the H1-H2 loop, but their functional significance was not known. Using protein engineering, we show that these insertions have distinct effects on CTS resistance in homologs of each of the two species that strongly depend on intramolecular interactions with other residues. Removing the insertion in the chinchilla NKA unexpectedly increases CTS resistance and decreases NKA activity. In the sandgrouse NKA, the amino acid insertion and substitution Q111R both contribute to an augmented CTS resistance without compromising ATPase activity levels. Molecular docking simulations provide additional insight into the biophysical mechanisms responsible for the context-specific mutational effects on CTS insensitivity of the enzyme. Our results highlight the diversity of genetic substrates that underlie CTS insensitivity in vertebrate NKA and reveal how amino acid insertions can alter the phenotypic effects of point mutations at key sites in the same protein domain.

Quantification of Cardiotonic Steroids Potentially Regulated by Paraoxonase 3 in a Rat Model of Chronic Kidney Disease Using UHPLC-Orbitrap-MS.

Endogenous cardiotonic steroids (CTSs), such as telocinobufagin (TCB) and marinobufagin (MBG) contain a lactone moiety critical to their binding and signaling through the Na+/K+-ATPase. Their concentrations elevate in response to sodium intake and under volume-expanded conditions. Paraoxonase 3 (PON3) is an enzyme that can hydrolyze lactone substrates. Here, we examine the role of PON3 in regulating CTS levels in a rat model of chronic kidney diseases (CKD). TCB and MBG were extracted from rat urine samples, and the analyses were carried out using ultra-high pressure liquid chromatography−Orbitrap-mass spectrometry (UHPLC-Orbitrap-MS). Ten-week-old Dahl salt-sensitive wild type (SS-WT) and Dahl salt-sensitive PON3 knockout (SS-PON3 KO) rats were maintained on a high-salt diet (8% NaCl) for 8 weeks to initiate salt-sensitive hypertensive renal disease characteristic of this model. CTS extraction recovery from urine >80% was achieved. For animals maintained on a normal chow diet, the baseline amount of TCB excreted in 24 h urine of SS-PON3 KO rats (6.08 ± 1.47 ng/24 h; or 15.09 ± 3.25 pmol) was significantly higher than for SS-WT rats (1.48 ± 0.69 ng/24 h; or 3.67 ± 1.54 pmol, p < 0.05). Similarly, for the same animals, the amount of excreted MBG was higher in the urine of SS-PON3 KO rats (4.74 ± 1.30 ng/24 h versus 1.03 ± 0.25 ng/24 h in SS-WT; or 11.83 ± 2.91 pmol versus 2.57 ± 0.56 pmol in SS-WT, p < 0.05). For animals on a high-salt diet, the SS-PON3 KO rats had significantly increased levels of TCB (714.52 ± 79.46 ng/24 h; or 1774.85 ± 175.55 pmol) compared to SS-WT control (343.84 ± 157.54 ng/24 h; or 854.09 ± 350.02 pmol, p < 0.05), and comparatively higher levels of MBG were measured for SS-PON3 KO (225.55 ± 82.61 ng/24 h; or 563.19 ± 184.5 pmol) versus SS-WT (157.56 ± 85.53 ng/24 h; or 393.43 ± 191.01 pmol, p > 0.05) rats. These findings suggest that the presence and absence of PON3 dramatically affect the level of endogenous CTSs, indicating its potential role in CTS regulation.

Chemistry and the Potential Antiviral, Anticancer, and Anti-Inflammatory Activities of Cardiotonic Steroids Derived from Toads.

Cardiotonic steroids (CTS) were first documented by ancient Egyptians more than 3000 years ago. Cardiotonic steroids are a group of steroid hormones that circulate in the blood of amphibians and toads and can also be extracted from natural products such as plants, herbs, and marines. It is well known that cardiotonic steroids reveal effects against congestive heart failure and atrial fibrillation; therefore, the term "cardiotonic" has been coined. Cardiotonic steroids are divided into two distinct groups: cardenolides (plant-derived) and bufadienolides (mainly of animal origin). Cardenolides have an unsaturated five-membered lactone ring attached to the steroid nucleus at position 17; bufadienolides have a doubly unsaturated six-membered lactone ring. Cancer is a leading cause of mortality in humans all over the world. In 2040, the global cancer load is expected to be 28.4 million cases, which would be a 47% increase from 2020. Moreover, viruses and inflammations also have a very nebative impact on human health and lead to mortality. In the current review, we focus on the chemistry, antiviral and anti-cancer activities of cardiotonic steroids from the naturally derived (toads) venom to combat these chronic devastating health problems. The databases of different research engines (Google Scholar, PubMed, Science Direct, and Sci-Finder) were screened using different combinations of the following terms: "cardiotonic steroids", "anti-inflammatory", "antiviral", "anticancer", "toad venom", "bufadienolides", and "poison chemical composition". Various cardiotonic steroids were isolated from diverse toad species and exhibited superior anti-inflammatory, anticancer, and antiviral activities in in vivo and in vitro models such as marinobufagenin, gammabufotalin, resibufogenin, and bufalin. These steroids are especially difficult to identify. However, several compounds and their bioactivities were identified by using different molecular and biotechnological techniques. Biotechnology is a new tool to fully or partially generate upscaled quantities of natural products, which are otherwise only available at trace amounts in organisms.

Research Progress in Pharmacological Activities and Applications of Cardiotonic Steroids.

Cardiotonic steroids (CTS) are a group of compounds existing in animals and plants. CTS are commonly referred to cardiac glycosides (CGs) which are composed of sugar residues, unsaturated lactone rings and steroid cores. Their traditional mechanism of action is to inhibit sodium-potassium ATPase to strengthen the heart and regulate heart rate, so it is currently widely used in the treatment of cardiovascular diseases such as heart failure and tachyarrhythmia. It is worth noticing that recent studies have found an avalanche of inestimable values of CTS applications in many fields such as anti-tumor, anti-virus, neuroprotection, and immune regulation through multi-molecular mechanisms. Thus, the pharmacological activities and applications of CTS have extensive prospects, which would provide a direction for new drug research and development. Here, we review the potential applications of CTS in cardiovascular system and other systems. We also provide suggestions for new clinical practical strategies of CTS, for many diseases. Four main themes will be discussed, in relation to the impact of CTS, on 1) tumors, 2) viral infections, 3) nervous system diseases and 4) immune-inflammation-related diseases.

Cryoelectron microscopy of Na+,K+-ATPase in the two E2P states with and without cardiotonic steroids.

Cryoelectron microscopy (cryo-EM) was applied to Na+,K+-ATPase (NKA) to determine the structures of two E2P states, one (E2PATP) formed by ATP and Mg2+ in the forward reaction, and the other (E2PPi) formed by inorganic phosphate (Pi) and Mg2+ in the backward reaction, with and without ouabain or istaroxime, representatives of classical and new-generation cardiotonic steroids (CTSs). These two E2P states exhibit different biochemical properties. In particular, K+-sensitive acceleration of the dephosphorylation reaction is not observed with E2PPi, attributed to the presence of a Mg2+ ion in the transmembrane cation binding sites. The cryo-EM structures of NKA demonstrate that the two E2P structures are nearly identical but Mg2+ in the transmembrane binding cavity is identified only in E2PPi, corroborating the idea that it should be denoted as E2PPi·Mg2+. We can now explain why the absence of transmembrane Mg2+ in E2PATP confers the K+ sensitivity in dephosphorylation. In addition, we show that ATP bridges the actuator (A) and nucleotide binding (N) domains, stabilizing the E2PATP state; CTS binding causes hardly any changes in the structure of NKA, both in E2PATP and E2PPi·Mg2+, indicating that the binding mechanism is conformational selection; and istaroxime binds to NKA, extending its aminoalkyloxime group deep into the cation binding site. This orientation is upside down compared to that of classical CTSs with respect to the steroid ring. Notably, mobile parts of NKA are resolved substantially better in the electron microscopy (EM) maps than in previous X-ray structures, including sugars sticking out from the ß-subunit and many phospholipid molecules.

Canrenone Restores Vasorelaxation Impaired by Marinobufagenin in Human Preeclampsia.

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

The Janus face of ouabain in Na+ /K+ -ATPase and calcium signalling in neurons.

Na+ /K+ -ATPase, a transmembrane protein essential for maintaining the electrochemical gradient across the plasma membrane, acts as a receptor for cardiotonic steroids such as ouabain. Cardiotonic steroids binding to Na+ /K+ -ATPase triggers signalling pathways or inhibits Na+ /K+ -ATPas activity in a concentration-dependent manner, resulting in a modulation of Ca2+ levels, which are essential for homeostasis in neurons. However, most of the pharmacological strategies for avoiding neuronal death do not target Na+ /K+ -ATPase activity due to its complexity and the poor understanding of the mechanisms involved in Na+ /K+ -ATPase modulation. The present review aims to discuss two points regarding the interplay between Na+ /K+ -ATPase and Ca2+ signalling in the brain. One, Na+ /K+ -ATPase impairment causing illness and neuronal death due to Ca2+ signalling and two, benefits to the brain by modulating Na+ /K+ -ATPase activity. These interactions play an essential role in neuronal cell fate determination and are relevant to find new targets for the treatment of neurodegenerative diseases. LINKED ARTICLES: This article is part of a themed issue on Building Bridges in Neuropharmacology. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.8/issuetoc.

Depth of the Steroid Core Location Determines the Mode of Na,K-ATPase Inhibition by Cardiotonic Steroids.

Cardiotonic steroids (CTSs) are specific inhibitors of Na,K-ATPase (NKA). They induce diverse physiological effects and were investigated as potential drugs in heart diseases, hypertension, neuroinflammation, antiviral and cancer therapy. Here, we compared the inhibition mode and binding of CTSs, such as ouabain, digoxin and marinobufagenin to NKA from pig and rat kidneys, containing CTSs-sensitive (α1S) and -resistant (α1R) α1-subunit, respectively. Marinobufagenin in contrast to ouabain and digoxin interacted with α1S-NKA reversibly, and its binding constant was reduced due to the decrease in the deepening in the CTSs-binding site and a lower number of contacts between the site and the inhibitor. The formation of a hydrogen bond between Arg111 and Asp122 in α1R-NKA induced the reduction in CTSs' steroid core deepening that led to the reversible inhibition of α1R-NKA by ouabain and digoxin and the absence of marinobufagenin's effect on α1R-NKA activity. Our results elucidate that the difference in signaling, and cytotoxic effects of CTSs may be due to the distinction in the deepening of CTSs into the binding side that, in turn, is a result of a bent-in inhibitor steroid core (marinobufagenin in α1S-NKA) or the change of the width of CTSs-binding cavity (all CTSs in α1R-NKA).

Evaluation of Cardiotonic Steroid Modulation of Cellular Cholesterol and Phospholipid.

We have previously shown that 21-benzylidene digoxin (21-BD) increases the total cholesterol and phospholipid content on the membrane of HeLa cells. Lipid modulation caused by cardiotonic steroids (CTS) is still unexplored. Therefore, the aim of the present study was to evaluate the cholesterol and phospholipid modulation of the cell membrane caused by ouabain and 21-BD and the possible involvement of the caveolae on this modulation. For this, one cell line containing caveolae (HeLa) and other not containing (Caco-2) were used. The modulation of the lipid profile was evaluated by total cholesterol and phospholipids measurements, and identification of membrane phospholipids by HPTLC. The cholesterol distribution was evaluated by filipin staining. The caveolin-1 expression was evaluated by Western Blotting. Ouabain had no effect on the total membrane lipid content in both cell lines. However, 21-BD increased total membrane phospholipid content and had no effect on the membrane cholesterol content in Caco-2 cells. CTS were not able to alter the specific phospholipids content. In the filipin experiments, 21-BD provoked a remarkable redistribution of cholesterol to the perinuclear region of HeLa cells. In Caco-2 cells, it was observed only a slight increase in cholesterol, especially as intracellular vesicles. The caveolin-1 expression was not altered by any of the compounds. Our data mainly show different effects of two cardiotonic steroids. Ouabain had no effect on the lipid profile of cells, whereas 21-BD causes important changes in cholesterol and phospholipid content. Therefore, the modulation of cholesterol content in the plasma membrane of HeLa cells is not correlated with the expression of caveolin-1.

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 48 h with increased concentrations of CTS. The effects of CTS on NKA activity and immunoblotting of α1 and ß1 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 (8 µM) 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.

Na,K-ATPase as a target for endogenous cardiotonic steroids: What's the evidence?

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

Cartiotonic steroids affect monolayer permeability in lymphatic endothelial cells.

Edema is common in preeclampsia (preE), a hypertensive disorder of pregnancy. Cardiotonic steroids (CTSs) such as marinobufagenin (MBG) are involved in the pathogenesis of preE. To assess whether CTSs are involved in the leakage of lymphatic endothelial cell (LEC), we evaluated their effect on monolayer permeability of LECs (MPLEC) in culture. A rat mesenteric LECs were treated with DMSO (vehicle), and CTSs (MBG, CINO, OUB) at concentrations of 1, 10, and 100 nM. Some LECs were pretreated with 1 µM L-NAME (N-Nitro-L-Arginine Methyl Ester) before adding 100 nM MBG or cinobufotalin (CINO). Expression of ß-catenin and vascular endothelial (VE)-cadherin in CTS-treated LECs was measured by immunofluorescence and MPLEC was quantified using a fluorescence plate reader. Western blot was performed to measure ß-catenin and VE-cadherin protein levels and myosin light chain 20 (MLC20) phosphorylation. MBG (≥ 1 nM) and CINO (≥ 10 nM) caused an increase (p < 0.05) in the MPLEC compared to DMSO while ouabain (OUB) had no effect. Pretreatment of LECs with 1 µM L-NAME attenuated (p < 0.05) the MPLEC. The ß-catenin expression in LECs was downregulated (p < 0.05) by MBG and CINO. However, there was no effect on the LECs tight junctions for the CINO group. VE-cadherin expression was downregulated (p < 0.05) by CINO, and MLC20 phosphorylation was upregulated (p < 0.05) by MBG. We demonstrated that MBG and CINO caused an increase in the MPLEC, which were attenuated by L-NAME pretreatment. The data suggest that CTSs exert their effect via nitric-oxide-dependent signaling pathway and may be involved in vascular leak syndrome of LEC lining in preE.