Evaluation of Anti-Inflammatory Activity of the New Cardiotonic Steroid γ-Benzylidene Digoxin 8 (BD-8) in Mice.

Cardiotonic steroids are known to bind to Na+/K+-ATPase and regulate several biological processes, including the immune response. The synthetic cardiotonic steroid γ-Benzylidene Digoxin 8 (BD-8) is emerging as a promising immunomodulatory molecule, although it has remained largely unexplored. Therefore, we tested the immunomodulatory potential of BD-8 both in vitro and in vivo. Hence, primary mouse macrophages were incubated with combinations of BD-8 and the pro-inflammatory fungal protein zymosan (ZYM). Nitric oxide (NO) production was determined by Griess reagent and cytokines production was assessed by enzyme-linked immunosorbent assay. Inducible nitric oxide synthase (iNOS), reactive oxygen species (ROS), p-nuclear factor kappa B p65 (NF-κB p65), p-extracellular signal-regulated kinase (p-ERK), and p-p38 were evaluated by flow cytometry. Macrophages exposed to BD-8 displayed reduced phagocytic activity, NO levels, and production of the proinflammatory cytokine IL-1ß induced by ZYM. Furthermore, BD-8 diminished the expression of iNOS and phosphorylation of NF-κB p65, ERK, and p38. Additionally, BD-8 exhibited anti-inflammatory capacity in vivo in a carrageenan-induced mouse paw edema model. Taken together, these findings demonstrate the anti-inflammatory activity of BD-8 and further reinforce the potential of cardiotonic steroids and their derivatives as immunomodulatory molecules.

Comparative Therapeutic Potential of Cardioactive Glycosides in Doxorubicin Model of Heart Failure.

In the present study, we investigated the cardioactive glycosides oleandrin and ouabain, and compared them to digoxin in a model of cardiotoxicity induced by doxorubicin. Adult rats were distributed into four experimental groups. Each group was challenged with a single intraperitoneal application of doxorubicin at a dose of 12 mg/kg. Then, they were treated with saline solution and the glycosides oleandrin, ouabain, and digoxin at a dose of 50 µg/kg, for 7 days. They underwent echocardiography, electrocardiography, hematologic, biochemical tests, and microscopic evaluation of the heart. All animals presented congestive heart failure, which was verified by a reduction in the ejection fraction. Oleandrin and digoxin were able to significantly reduce (p < 0.05) the eccentric remodeling caused by doxorubicin. Oleandrin and digoxin were significantly lower (p < 0.05) than the control group in maintaining systolic volume and left ventricular volume in diastole. Other parameters evaluated did not show significant statistical differences. All animals showed an increase in erythrocyte count, and an increase in the duration of the QRS complex on the ECG and myocardial necrosis at the histopathological analysis. It is concluded that the glycosides oleandrin, ouabain, and digoxin in the used dosage do not present therapeutic potential for the treatment of congestive heart failure caused by doxorubicin.

The γ-Benzylidene Digoxin Derivative BD-15 Increases the α3-Na, K-ATPase Activity in Rat Hippocampus and Prefrontal Cortex and no Change on Heart.

Our study aimed to investigate the effects of the new cardiotonic steroid BD-15 (γ-benzylidene derivatives) in the behavioral parameters, oxidative stress and the Na, K-ATPase activity in the hippocampus, prefrontal cortex and heart from rats to verify the safety and possible utilization in brain disorders. For this study, groups of male Wistar rats were used after intraperitoneal injection of 20, 100 and 200 µg/Kg with BD-15. The groups were treated for three consecutive days and the control group received 0.9% saline. BD-15 did not alter behavior of rats treated with different doses. An increase in the specific α2,3-Na, K-ATPase activity was observed for all doses of BD-15 tested in the hippocampus. However, in the prefrontal cortex, only the dose of 100 µg/Kg increased the activity of all Na, K-ATPase isoforms. BD-15 did not cause alteration in the lipid peroxidation levels in the hippocampus, but in the prefrontal cortex, a decrease of lipid peroxidation (~ 25%) was observed. In the hippocampus, GSH levels increased with all doses tested, while in the prefrontal cortex no changes were found. Subsequently, when the effect of BD-15 on cardiac tissue was analyzed, no changes were observed in the tested parameters. BD-15 at a dosage of 100 µg/Kg proved to be promising because it is considered therapeutic for brain disorders, since it increases the activity of the α3-Na, K-ATPase in the hippocampus and prefrontal cortex, as well as decreasing the oxidative stress in these brain regions. In addition, this drug did not cause changes in the tissues of the heart and kidneys, preferentially demonstrating specificity for the brain.

Digoxin Combined with Aerobic Interval Training Improved Cardiomyocyte Contractility.

Digoxin is a cardiotonic that increases the cardiac output without causing deleterious effects on heart, as well as improves the left ventricular performance during physical exercise. We tested whether the association between chronic digoxin administration and aerobic interval training (AIT) promotes beneficial cardiovascular adaptations by improving the myocardial contractility and calcium (Ca2+) handling. Male Wistar rats were randomly assigned to sedentary control (C), interval training (T), sedentary digoxin (DIGO) and T associated to digoxin (TDIGO). AIT was performed on a treadmill (1h/day, 5 days/week) for 60 days, consisting of successive 8-min periods at 80% and 20% of VO2máx for 2 min. Digoxin was administered by orogastric gavage for 60 days. Left ventricle samples were collected to analysis of Ca2+ handling proteins; contractility and Ca2+ handling were performed on isolated cardiomyocytes. TDIGO group had a greater elevation in fractional shortening (44%) than DIGO, suggesting a cardiomyocyte contractile improvement. In addition, T or TDIGO groups showed no change in cardiomyocytes properties after Fura2-acetoxymethyl ester, as well as in sarcoplasmic reticulum Ca2+-ATPase (SERCA2a), phospholamban and calcineurin expressions. The main findings indicate that association of digoxin and aerobic interval training improved the cardiomyocyte contractile function, but these effects seem to be unrelated to Ca2+ handling.

Digoxin in Atrial Fibrillation: An Old Topic Revisited.

Digoxin has been used for more than 50 years in patients with Atrial Fibrillation (AF), with the goal of Controlling Heart Rate (HR) and restoring sinus rhythm. In the last two decades, several studies have correlated therapeutic use of digoxin with increased mortality. However, such studies have potential biases that cannot be disregarded, mainly because they are cross-sectional experiments or post-hoc analyses of Randomized Controlled Trials (RCTs). Despite uncertainties regarding the safety of digoxin in this setting, it remains one of the most prescribed drugs for AF worldwide. On the other hand, the absence of any RCTs designed to evaluate mortality makes a definitive conclusion more difficult to reach; therefore, this medication must be used with care. In this review, we explored the therapeutic use of digoxin in the context of AF, discussed mortality data by means of critical analysis in the light of the best available evidence, and position ourselves in relation to more rigorous control of serum levels of this drug in daily practice.

21-Benzylidene digoxin decreases proliferation by inhibiting the EGFR/ERK signaling pathway and induces apoptosis in HeLa cells.

Cardiotonic steroids (CTS) are agents traditionally known for their capacity to bind to the Na,K-ATPase (NKA), affecting the ion transport and the contraction of the heart. Natural CTS have been shown to also have effects on cell signaling pathways. With the goal of developing a new CTS derivative, we synthesized a new digoxin derivative, 21-benzylidene digoxin (21-BD). Previously, we have shown that this compound binds to NKA and has cytotoxic actions on cancer, but not on normal cells. Here, we further studied the mechanisms of actions of 21-BD. Working with HeLa cells, we found that 21-BD decreases the basal, as well as the insulin stimulated proliferation. 21-BD reduces phosphorylation of the epidermal growth factor receptor (EGFR) and extracellular-regulated kinase (ERK), which are involved in pathways that stimulate cell proliferation. In addition, 21-BD promotes apoptosis, which is mediated by the translocation of Bax from the cytosol to mitochondria and the release of mitochondrial cytochrome c to the cytosol. 21-BD also activated caspases-8, -9 and -3, and induced the cleavage of poly (ADP-ribose) polymerase-1 (PARP-1). Altogether, these results show that the new compound that we have synthesized exerts cytotoxic actions on HeLa cells by inhibition of cell proliferation and the activation of both the extrinsic and intrinsic apoptotic pathways. These results support the relevance of the cardiotonic steroid scaffold as modulators of cell signaling pathways and potential agents for their use in cancer.

Conformational states of the pig kidney Na+/K+-ATPase differently affect bufadienolides and cardenolides: A directed structure-activity and structure-kinetics study.

There is a renewed interest in the Na+/K+-ATPase (NKA, EC 3.6.3.9) either as a target for new therapeutic uses or for understanding the putative pathophysiological role of its mammalian endogenous ligands. Recent data indicate that bufalin binds to the pig kidney NKA in a way different from ouabain and digoxin, raising the question of a putative class difference between bufadienolides and cardenolides. The purpose of this work was to perform a study of the relationship between structure and both activity and kinetics, focusing mainly on the influence of the lactone ring in C17 (5 vs. 6 membered), the effect of C14-15 cyclization and the carbohydrate moiety in C3. We compared the potency of fourteen related cardiotonic steroids (CTS) for inhibition of the cycling pig kidney NKA in two different concentrations of K+, as well as the affinity for binding to the E2P conformation of the enzyme (Mg-Pi medium) and the potency for inhibiting the E2[2K] conformation of the NKA (K+-pNPPase activity). Cardenolides were clearly sensitive to the antagonistic effect of high K+ concentrations whereas bufadienolides were not or less sensitive. The C14-15 cyclization observed in some bufadienolides, such as resibufogenin and marinobufagin, caused a drastic fall in the affinity for binding to the NKA in the E2P conformation and increased the velocity of K+-pNPPase inhibition. The absence of a carbohydrate moiety in C3 increased the velocity of inhibition. Cardenolides were much more dependent on the E2P conformation for binding than bufadienolides since their ratios of E2[2K] IC50 to E2P Ki were higher than for bufadienolides. Therefore, the present data established the remarkable influence of C14-15 cyclization and of the carbohydrate moiety in C3 on both affinity and kinetics of CTS and indicate that, as a class, bufadienolides would harbor qualitative differences from cardenolides with respect to the NKA conformations to which they can bind.

Evaluation of neuroprotective activity of digoxin and semisynthetic derivatives against partial chemical ischemia.

Recently, cardiotonic steroids (CTS) have been shown to lead to the activation of Na,K-ATPase at low concentrations in brain, promoting neuroprotection against ischemia. We report here the results of the use of digoxin and its semisynthetic derivatives BD-14, BD-15, and BD-16 against partial chemical ischemic induction followed by reperfusion in murine neuroblastoma cells neuro-2a (N2a). For chemical ischemic induction, sodium azide (5 mM) was used for 5 hours, and then reperfusion was induced for 24 hours. Na,K-ATPase activity and protein levels were analyzed in membrane preparation of N2a cells pretreated with the compounds (150 nM), in the controls and in induced chemical ischemia. In the Na,K-ATPase activity and protein levels assays, the steroids digoxin and BD-15 demonstrated a capacity to modulate the activity of the enzyme directly, increasing its levels of expression and activity. Oxidative parameters, such as superoxide dismutase (SOD) activity, lipid peroxidation (thiobarbituric acid reactive substance), glutathione peroxidase (GPx), glutathione (GSH) levels, hydrogen peroxide content, and the amount of free radicals (reactive oxygen species) during induced chemical ischemia were also evaluated. Regarding the redox state, lipid peroxidation, hydrogen peroxide content, and GPx activity, we have observed an increase in the chemical ischemic group, and a reduction in the groups treated with CTS. SOD activity increased in all treated groups when compared to control and GSH levels decreased when treated with sodium azide and did not change with CTS treatments. Regarding the lipid profile, we saw a decrease in the content of phospholipids and cholesterol in the chemical ischemic group, and an increase in the groups treated with CTS. In conclusion, the compounds used in this study demonstrate promising results, since they appear to promote neuroprotection in cells exposed to chemical ischemia.

Antagonistic anticancer effect of paclitaxel and digoxin combination.

Despite the growing interest in the antitumor effect of cardiotonic steroids, combination treatments with well-established chemotherapy drugs like paclitaxel have been rarely investigated. Moreover, paclitaxel has been suggested as a Na+ /K+ -ATPase inhibitor. Here we investigated the effect of paclitaxel and digoxin alone or in combination on the viability of human lung (A549) and cervical cancer (HeLa) cell lines and the inhibitory effect of paclitaxel on several mammalian Na+ /K+ -ATPases. Although the viability of both tumor cell lines was concentration-dependently affected by digoxin treatment after 48 hours (A549 IC50 = 31 nM and HeLa IC50 = 151 nM), a partial effect was observed for paclitaxel, with a maximal inhibitory effect of 45% at 1000 nM with A549 and around 70% with HeLa cells (IC50 = 1 nM). Although the two drugs were cytotoxic, their combined effect in HeLa cells was revealed to be antagonistic, as estimated by the combination index. No direct inhibitory effect of paclitaxel was detected in human, pig, rat, and mouse Na+ /K+ -ATPase enzymes, but high concentrations of paclitaxel decreased the Na+ /K+ -ATPase activity in HeLa cells after 48 hours without affecting protein expression. Our findings demonstrate that, under our conditions, paclitaxel and digoxin cotreatment produce antagonistic cytotoxic effects in HeLa cells, and the mechanism of action of paclitaxel does not involve a direct inhibition of Na+ /K+ -ATPase. More studies shall be designed to evaluate the consequences of the interaction of cardiotonic steroids and chemotherapy drugs.

21­Benzylidene digoxin, a novel digoxin hemi-synthetic derivative, presents an anti-inflammatory activity through inhibition of edema, tumour necrosis factor alpha production, inducible nitric oxide synthase expression and leucocyte migration.

Recent findings have demonstrated new therapeutic functions of cardiotonic steroids, a process that is termed drug repositioning. Despite the confirmed anti-inflammatory effects of cardiotonic steroids, their clinical use has been discouraged due to toxicity related to inhibition of the Na+/K+ ATPase. A novel synthetic compound derived from digoxin, 21­benzylidene digoxin (21­BD), does not inhibit this enzyme. Herein, we evaluated the anti-inflammatory and antinociceptive effects and acute toxicity of 21­BD. Murine (Swiss mice) models of paw oedema induced by carrageenan, acetic acid-induced abdominal writhing, and formalin and acute toxicity tests were used. Oral administration of 21­BD (0.3 mg/kg) showed a significant and prolonged inhibition of paw oedema. Histological analysis demonstrated a reduction in inflammatory cells and expression of inducible nitric oxide synthase (iNOS) in footpads 6 h after administration of carrageenan. 21­BD (0.3 mg/kg) also reduced the levels of tumour necrosis factor (TNF)-α 2 and 4 h after carrageenan. 21­BD demonstrated antinociceptive activity, inhibiting abdominal writhes at all tested doses. However, in the formalin test, 21­BD did not present antinociceptive activity. In the acute toxicity test, 21­BD did not cause symptoms of toxicity or mortality. The present study demonstrated, for the first time, that 21­BD is safe and exhibits a marked anti-inflammatory activity in acute local inflammation. This effect might be a consequence of its ability to inhibit the release of the PMN leucocyte-derived mediators, including TNF-α, and iNOS expression as well as its inhibitory effect on oedema and PMN leucocyte infiltration.

Mechanism of Rhinella icterica (Spix, 1824) toad poisoning using in vitro neurobiological preparations.

The biological activity of Rhinella icterica toxic secretion (RITS) was evaluated on chick neuromuscular junctions, rat heart́s tissue and mice hippocampal slices. At chick biventer cervicis preparation, RITS (5, 10 and 20µg/mL) produced a concentration-independent irreversible neuromuscular blockade, which was preceded by a transitory increase of muscle twitch tension with the lowest concentration, in 120min recordings. In this set of experiments, RITS incubation partially prevented the curare neuromuscular blockade. The assessment of chick biventer cervicis muscle acetylcholinesterase (AChE) in the presence of RITS showed a significant inhibition of the enzyme, similarly to neostigmine. The incubation of muscles with digoxin or ouabain mimicked the poison activity by increasing the amplitude of the twitches followed by a progressive depression of the muscle strength. In addition, RITS demonstrated a digitalic-like activity, by inhibiting significantly the cardiac Na+, K+-ATPase. When the central nervous system was accessed, RITS induced an increase in the cell viability, in the lowest concentration. In addition, the poison protected slices subject to oxygen/glucose deprivation. Altogether, these data indicate that the poisonous extract of R. icterica is able to interfere with peripheral and central neurotransmission, probably due to a direct interaction with AChE, calcium channels and Na+, K+-ATPase. A further investigation upon the poison toxic components will unveil the components involved in such a pharmacological activity and the potential biotechnological application of this poison.

Selectivity analyses of γ-benzylidene digoxin derivatives to different Na,K-ATPase α isoforms: a molecular docking approach.

Digoxin and other cardiotonic steroids (CTS) exert their effect by inhibiting Na,K-ATPase (NKA) activity. CTS bind to the various NKA isoforms that are expressed in different cell types, which gives CTS their narrow therapeutic index. We have synthesised a series of digoxin derivatives (γ-Benzylidene digoxin derivatives) with substitutions in the lactone ring (including non-oxygen and ether groups), to obtain CTS with better NKA isoform specificity. Some of these derivatives show some NKA isoform selective effects, with BD-3, BD-8, and BD-13 increasing NKA α2 activity, BD-5 inhibiting NKA α1 and NKA α3, BD-10 reducing NKA α1, but stimulating NKA α2 and α3; and BD-14, BD-15, and BD-16 enhancing NKA α3 activity. A molecular-docking approach favoured NKA isoform specific interactions for the compounds that supported their observed activity. These results show that BD compounds are a new type of CTS with the capacity to target NKA activity in an isoform-specific manner.

Involvement of Src signaling in the synergistic effect between cisplatin and digoxin on cancer cell viability.

Cisplatin and other platinum-containing drugs have played a crucial role in anticancer treatments for over 30 years. However, treatment with cisplatin may cause serious side effects, such as myelosuppression, nausea, ototoxicity, nephrotoxicity, and cell resistance processes. In addition, cardiotonic steroids, particularly digoxin, have recently been suggested to exert potent anticancer effects. Therefore, it is possible that the combined treatment of HeLa cells with cisplatin and digoxin can ameliorate the cytotoxic effects and decrease the side effects of cisplatin. In this study, we demonstrated that the interaction between cisplatin and digoxin had a synergistic effect on cervical cancer cells and a significantly positive cytotoxic and antiproliferative effect on this cell line compared to the control and single cisplatin treatments. Although a decrease in the Na,K-ATPase α1 subunit expression was observed in total extracts, its expression remains unchanged in the membrane, as does the Na,K-ATPase activity. The antiproliferative effect of the synergistic treatment appears to depend on Src kinase activation, indicating the possible involvement of the Scr-EGFR-ERK1/2 pathway in the antitumor effect. The inhibition of ERK1/2 provoked the same synergism with 1 µM cisplatin as that observed with 1 nM digoxin plus 1 µM cisplatin but not with 1 nM digoxin. Pretreatment with PP2 during combined treatment abolished the synergistic effect on the antiproliferative activity. Cisplatin and digoxin are already used in the clinical setting; therefore, this study opens possibilities for future clinical trials of combined treatments to improve treatment outcomes with a lower incidence of toxicity and side effects.

Differences of lipid membrane modulation and oxidative stress by digoxin and 21-benzylidene digoxin.

Cardiotonic steroids (CTS) are compounds which bind to the Na,K-ATPase, leading to its inhibition and in some cases initiating signaling cascades. Long utilized as a treatment for congestive heart disease, CTS have more recently been observed to inhibit proliferation and cause apoptosis in several cancer cell lines. A synthetic derivative of the CTS digoxin, called 21-benzylidene digoxin (21-BD), activates the Na,K-ATPase rather than cause its inhibition, as its parent compound does. Here, the mechanism behind the unique effects of 21-BD are further explored. In HeLa cancer cells, low (5µM) and high (50µM) doses of 21-BD activated and inhibited the Na,K-ATPase, respectively, without altering the membrane expression of the Na,K-ATPase. While digoxin did not affect HeLa membrane cholesterol or phospholipid content, 50µM 21-BD increased both lipids via a mechanism reliant on an intact cell. Afterwards, the direct action of 21-BD was evaluated on erythrocyte membranes; however, no effect was observed. As CTS may generate reactive oxygen species (ROS) which can affect plasma membrane fluidity and therefore Na,K-ATPase activity, several markers involved in ROS generation were analyzed such as, lipid peroxidation (TBARS), reduced glutathione (GSH), catalase (CAT) and superoxide dismutase (SOD). GSH content and catalase activity were unaffected by digoxin or 21-BD. Surprisingly, TBARS and SOD activity was decreased with digoxin and with 50µM 21-BD. Thus, 21-BD and digoxin altered components involved in ROS generation and inhibition in a similar fashion. This study suggests alterations to the Na,K-ATPase and membrane lipids by 21-BD is not reliant on ROS generation.

[Analysis of the mitochondrial function and ultrastructure in healthy albino mice treated with heart failure medications] / Análisis de la función y ultraestructura mitocondrial en ratones albinos sanos tratados con medicamentos para insuficiencia cardíaca.

Background: Mitochondrial activity is essential for cardiac and skeletal muscle. The relationship between mitochondrial dysfunction and different cardiovascular conditions has been well described. Pharmacological treatment for heart failure involves different drugs as: angiotensin-converting enzyme inhibitors, B-adrenergic blockers, digitalis glycosides and diuretics. The clinical benefit from medication is clear, however, the role of this drugs in mitochondrial metabolisms is not well understood. Aim of the study: The objective of our study was to analyze structural and functional characteristics of cardiac and skeletal muscle mitochondria in mice treated with drugs normally used for heart failure and compare it to a control group. Methods: Twenty-five Albino Mice divided in five groups were treated with heart failure medication during 30 days (group I to IV). 30 days after treatment they were sacrificed, heart and skeletal muscle were analyzed and compared with a control group (V). Results: Enzymatic activity was slightly increased in groups treated with heart failure medication compared to control group (p>0.05). Mitochondrial morphology was significantly altered in groups treated compared to control group, in addition, mitochondrial area was significantly increased in the treated groups, in both cardiac and skeletal muscle. Conclusions: We concluded that heart failure medication could produce modifications in mitochondrial function; we believe that mitochondria maintains the enzymatic activity by increasing size and modifying morphology. Methods: Twenty-five Albino Mice divided in five groups were treated with heart failure medication during 30 days (group I to IV). 30 days after treatment they were sacrificed, heart and skeletal muscle were analyzed and compared with a control group (V). Results: Enzymatic activity was slightly increased in groups treated with heart failure medication compared to control group (p>0.05). Mitochondrial morphology was significantly altered in groups treated compared to control group, in addition, mitochondrial area was significantly increased in the treated groups, in both cardiac and skeletal muscle. Conclusions: We concluded that heart failure medication could produce modifications in mitochondrial function; we believe that mitochondria maintains the enzymatic activity by increasing size and modifying morphology. Results: Enzymatic activity was slightly increased in groups treated with heart failure medication compared to control group (p>0.05). Mitochondrial morphology was significantly altered in groups treated compared to control group, in addition, mitochondrial area was significantly increased in the treated groups, in both cardiac and skeletal muscle. Conclusions: We concluded that heart failure medication could produce modifications in mitochondrial function; we believe that mitochondria maintains the enzymatic activity by increasing size and modifying morphology. Conclusions: We concluded that heart failure medication could produce modifications in mitochondrial function; we believe that mitochondria maintains the enzymatic activity by increasing size and modifying morphology.

Introducción: la actividad mitocondrial es esencial para el músculo cardíaco y esquelético. La relación entre la disfunción mitocondrial y diferentes condiciones cardiovasculares ha sido bien descrita. El tratamiento farmacológico de la insuficiencia cardíaca implica diferentes medicamentos como: inhibidores de la enzima convertidora de la angiotensina, bloqueadores B-adrenérgicos, glucósidos digitálicos y diuréticos. Los beneficios clínicos del tratamiento son claros, sin embargo, el papel de estos fármacos en el metabolismo mitocondrial no esta bien establecido.Objetivo del estudio: El objetivo de nuestro estudio fue analizar las características estructurales y funcionales de las mitocondrias del músculo cardíaco y esquelético en ratones tratados con fármacos habitualmente utilizados para la insuficiencia cardíaca y compararlo con un grupo control.Métodos: Veinticinco ratones albinos divididos en cinco grupos fueron tratados con la medicación para insuficiencia cardíaca durante 30 días (grupo I a IV). 30 días después del tratamiento se sacrificaron, el corazón y el músculo esquelético se analizaron y se compararon con un grupo control (V).Resultados: La actividad enzimática se incrementó ligeramente en los grupos tratados con medicamentos insuficiencia cardiaca en comparación con el grupo control (p> 0,05). morfología mitocondrial se modificó significativamente en los grupos tratados en comparación con el grupo control, además, el área mitocondrial fue significativamente mayor en los grupos tratados, tanto en el músculo cardíaco y estriado.Conclusiones: Concluimos que la medicación insuficiencia cardíaca podría producir modificaciones en la función mitocondrial; creemos que las mitocondrias pueden mantener la actividad enzimática mediante el aumento de tamaño y modificación de la morfología. Objetivo: El objetivo de nuestro estudio fue analizar las características estructurales y funcionales de las mitocondrias del músculo cardíaco y esquelético en ratones tratados con fármacos habitualmente utilizados para la insuficiencia cardíaca y compararlo con un grupo control. Métodos: Veinticinco ratones albinos divididos en cinco grupos fueron tratados con la medicación para insuficiencia cardíaca durante 30 días (grupo I a IV). 30 días después del tratamiento se sacrificaron, el corazón y el músculo esquelético se analizaron y se compararon con un grupo control (V).Resultados: La actividad enzimática se incrementó ligeramente en los grupos tratados con medicamentos insuficiencia cardiaca en comparación con el grupo control (p> 0,05). morfología mitocondrial se modificó significativamente en los grupos tratados en comparación con el grupo control, además, el área mitocondrial fue significativamente mayor en los grupos tratados, tanto en el músculo cardíaco y estriado.Conclusiones: Concluimos que la medicación insuficiencia cardíaca podría producir modificaciones en la función mitocondrial; creemos que las mitocondrias pueden mantener la actividad enzimática mediante el aumento de tamaño y modificación de la morfología. Resultados: La actividad enzimática se incrementó ligeramente en los grupos tratados con medicamentos insuficiencia cardiaca en comparación con el grupo control (p> 0,05). morfología mitocondrial se modificó significativamente en los grupos tratados en comparación con el grupo control, además, el área mitocondrial fue significativamente mayor en los grupos tratados, tanto en el músculo cardíaco y estriado.Conclusiones: Concluimos que la medicación insuficiencia cardíaca podría producir modificaciones en la función mitocondrial; creemos que las mitocondrias pueden mantener la actividad enzimática mediante el aumento de tamaño y modificación de la morfología. Conclusiones: Concluimos que la medicación insuficiencia cardíaca podría producir modificaciones en la función mitocondrial; creemos que las mitocondrias pueden mantener la actividad enzimática mediante el aumento de tamaño y modificación de la morfología.

21-Benzylidene digoxin: a proapoptotic cardenolide of cancer cells that up-regulates Na,K-ATPase and epithelial tight junctions.

Cardiotonic steroids are used to treat heart failure and arrhythmia and have promising anticancer effects. The prototypic cardiotonic steroid ouabain may also be a hormone that modulates epithelial cell adhesion. Cardiotonic steroids consist of a steroid nucleus and a lactone ring, and their biological effects depend on the binding to their receptor, Na,K-ATPase, through which, they inhibit Na+ and K+ ion transport and activate of several intracellular signaling pathways. In this study, we added a styrene group to the lactone ring of the cardiotonic steroid digoxin, to obtain 21-benzylidene digoxin (21-BD), and investigated the effects of this synthetic cardiotonic steroid in different cell models. Molecular modeling indicates that 21-BD binds to its target Na,K-ATPase with low affinity, adopting a different pharmacophoric conformation when bound to its receptor than digoxin. Accordingly, 21-DB, at relatively high µM amounts inhibits the activity of Na,K-ATPase α1, but not α2 and α3 isoforms. In addition, 21-BD targets other proteins outside the Na,K-ATPase, inhibiting the multidrug exporter Pdr5p. When used on whole cells at low µM concentrations, 21-BD produces several effects, including: 1) up-regulation of Na,K-ATPase expression and activity in HeLa and RKO cancer cells, which is not found for digoxin, 2) cell specific changes in cell viability, reducing it in HeLa and RKO cancer cells, but increasing it in normal epithelial MDCK cells, which is different from the response to digoxin, and 3) changes in cell-cell interaction, altering the molecular composition of tight junctions and elevating transepithelial electrical resistance of MDCK monolayers, an effect previously found for ouabain. These results indicate that modification of the lactone ring of digoxin provides new properties to the compound, and shows that the structural change introduced could be used for the design of cardiotonic steroid with novel functions.

Effects of digoxin and Na, K-ATPase immunoexpression on human oral squamous carcinomas.

AIM: The present study evaluated the expression of α1 and ß1 Na,K-ATPase, as well as the effects of digoxin (DGX) on oral squamous cell carcinomas (OSCCs). PATIENTS AND METHODS: Immunohistochemical expression of α1 and ß1 Na,K-ATPase were evaluated in 60 patients who underwent treatment at the São João de Deus Hospital. SCC-25 viability was assessed by the colorimetric assay. Chi-square or Fisher's exact tests were used to analyze the association of α1 and ß1 Na,K-ATPase expression with the variables. RESULTS: Immunoexpression of α1 and ß1 Na,K-ATPase were observed in 28% and 55% of the tumors, however these proteins were not significant prognostic factors. Tobacco was significantly associated with α1 expression. SCC-25 viability decreased significantly after treatment with 1 µM DGX at 24 h. CONCLUSION: The smoking status of OSCC patients was significantly associated with α1 expression and DGX affected the SCC-25 viability in a dose- and duration-dependent manner.

Acetaminophen inhibits intestinal p-glycoprotein transport activity.

Repeated acetaminophen (AP) administration modulates intestinal P-glycoprotein (P-gp) expression. Whether AP can modulate P-gp activity in a short-term fashion is unknown. We investigated the acute effect of AP on rat intestinal P-gp activity in vivo and in vitro. In everted intestinal sacs, AP inhibited serosal-mucosal transport of rhodamine 123 (R123), a prototypical P-gp substrate. R123 efflux plotted against R123 concentration adjusted well to a sigmoidal curve. Vmax decreased 50% in the presence of AP, with no modification in EC50, or slope, ruling out the possibility of inhibition to be competitive. Inhibition by AP was absent at 0°C, consistent with interference of the active transport of R123 by AP. Additionally, AP showed no effect on normal localization of P-gp at the apical membrane of the enterocyte and neither affected paracellular permeability. Consistent with absence of a competitive inhibition, two further strategies strongly suggested that AP is not a P-gp substrate. First, serosal-mucosal transport of AP was not affected by the classical P-gp inhibitors verapamil or Psc 833. Second, AP accumulation was not different between P-gp knock-down and wild-type HepG2 cells. In vivo intestinal absorption of digoxin, another substrate of P-gp, was assessed in the presence or absence of AP (100 µM). Portal digoxin concentration was increased by 214%, in average, by AP, as compared with digoxin alone. In conclusion, AP inhibited P-gp activity, increasing intestinal absorption of digoxin, a prototypical substrate. These results suggest that therapeutic efficacy of P-gp substrates can be altered if coadministered with AP.

Inhibitory effect of combinations of digoxin and endogenous cardiotonic steroids on Na+/K+-ATPase activity in human kidney membrane preparation.

AIMS: Cardiac glycosides have been extensively used in the treatment of congestive heart failure for more than 200 years. Recently, cardenolides and bufadienolides were isolated from mammalian tissue and are considered as a new class of steroidal hormones. The aim of the present work was to characterize the interaction between the most clinical used cardiac glycoside digoxin and the cardiac glycosides known to exist endogenously, i.e., ouabain, marinobufagin and telocinobufagin, on human kidney Na(+)/K(+)-ATPase. MAIN METHODS: Inhibition of Na(+)/K(+)-ATPase activity from crude membrane preparations of human kidney was performed using increasing concentrations of the drugs alone or mixtures of ouabain:digoxin, telocinobufagin:digoxin and marinobufagin:digoxin in a fixed ratio 1:4, 2:3 and 3:2, respectively. The colorimetric method of Fiske and Subbarow was used to measure the inorganic phosphate released. KEY FINDINGS: Analyses of inhibition curves showed that the experimental curves for all combinations were superimposed on the theoretical additive curves indicating that an additive effect occurs among distinct cardenolides and bufadienolides combinations on the human α1ß1 Na(+)/K(+)-ATPase protomer. SIGNIFICANCE: Considering the extensive use of digoxin in the treatment of heart failure and the recent findings that endogenous cardiac glycosides may have altered levels in many diseases, including heart failure, the demonstration of additive effect between cardiac glycosides can help in the understanding of recent clinical observations, including that lower than usual doses of cardiac glycosides are necessary for decreasing mortality in these patients.

Cardiovascular drugs in human mechanical nociception: digoxin, amlodipine, propranolol, pindolol and atenolol.

Calcium channel blockers, beta adrenergic receptor blockers and Na/K ATPase inhibitors are widely used drugs, mainly for cardiovascular diseases. Their pharmacological targets are not restricted to the cardivascular tissue, nociceptive system structures also express similar targets, which strongly suggests a direct effect on pain sensation. To evaluate the pain intensity changes in outpatient groups, who receive these drugs as a therapy, a cross-sectional sampled, randomized patient groups receiving the calcium channel blocker amlodipine for blood hypertension (n=45), beta adrenergic receptor blockers (propranolol, atenolol or pindolol; n=40) for blood hypertension, or digoxin (n=40) for heart failure, were compared to an aparently healthy volunteers control group (n=60). A calibrated noxious pressure of 890 g/mm2 was applied for 5 seconds on the patient's sternum. Subjective pain intensity was reported by the visual analog scale (VAS, 0 to 10). Pain modulation system was evaluated by the application of a second stimulus with a 5 minutes delay. The analgesic effect of the beta blockers group (propanolol, atenolol, pindolol) was dosage-dependant (-36.8%; P = 0.0000003), without differences among them. The calcium channel blocker amlodipine showed lower pain scores (-50.6%; P = 0.0000003) than beta-receptor blockers (P = 0.0000003). Digoxin presented the highest pain scores (+56.5%; P = 0.0000003). All pain scores for the second stimulus were lower than the first stimulus and were differentially affected by beta-blockers (atenolol, pindolol and propanolol) and calcium channel blocker (amlodipine), but not by digoxin. These results suggest the influence of widely clinically used cardiovascular drugs on nociception.