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There is an intrinsic relationship between psychiatric disorders and neuroinflammation, including bipolar disorder. Ouabain, an inhibitor of Na + /K + -ATPase, has been implicated in the mouse model with manic-like behavior. However, the molecular mechanisms linking neuroinflammation and manic-like behavior require further investigation. CCAAT/Enhancer-Binding Protein Delta (CEBPD) is an inflammatory transcription factor that contributes to neurological disease progression. In this study, we demonstrated that the expression of CEBPD in astrocytes was increased in ouabain-treated mice. Furthermore, we observed an increase in the expression and transcript levels of CEBPD in human primary astrocytes following ouabain treatment. Transcriptome analysis revealed high MMP8 expression in human primary astrocytes following CEBPD overexpression and ouabain treatment. We confirmed that MMP8 is a CEBPD-regulated gene that mediates ouabain-induced neuroinflammation. In our animal model, treatment of ouabain-injected mice with M8I (an inhibitor of MMP8) resulted in the inhibition of manic-like behavior compared to ouabain-injected mice that were not treated with M8I. Additionally, the reduction in the activation of astrocytes and microglia was observed, particularly in the hippocampal CA1 region. Excessive reactive oxygen species formation was observed in ouabain-injected mice, and treating these mice with M8I resulted in the reduction of oxidative stress, as indicated by nitrotyrosine staining. These findings suggest that MMP8 inhibitors may serve as therapeutic agents in mitigating manic symptoms in bipolar disorder., (© 2024. The Author(s).)

Background: Bipolar disorder (BD) is a psychiatric disorder with complex therapy, besides the treatment with antidepressants induce a mania switch., Objective: Investigate the effect of the administration of imipramine (IMI) in rats submitted to intracerebroventricular (ICV) administrations of ouabain (OUA)., Methods: Adult Wistar rats (n = 28) were submitted to only one ICV administration of OUA or artificial cerebrospinal fluid. On the 7th and 9th days following the ICV administration, animals were submitted to a behavioral analysis comprising open field task and forced swimming test. Between the 9th and 14th days, the rats received one daily intraperitoneal administration of IMI or saline (Sal). On the 15th day rats were submitted to the last session of behavioral analysis, followed by euthanasia. The frontal cortex and hippocampus were dissected for the subsequent biochemical assessments: oxidative parameters, and Na+/K+-ATPase activity., Results: OUA administration induced a manic-like effect on the 7th day and a depressive-like behavior on the 14th day. In contrast, IMI administration elicited significant mania switch-like effect on this same stage in animals who received OUA. OUA increased oxidative damage and activity of antioxidant enzymes in the brain of rats. IMI potentialized the oxidative damage of OUA. No significant differences between groups were observed in the Na+/K+-ATPase activity., Conclusion: The present study suggests that residual effects from inhibition of the Na+K+ATPase could be involved in the manic-switch observed in bipolar patients. Besides, the OUA model of bipolar disorder could be used to study bipolar disorder in the context of mania switch., (Copyright © 2021. Published by Elsevier B.V.)

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., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Diastolic dysfunction is a major feature of hypertrophic cardiomyopathy (HCM). Data from patient tissue and animal models associate increased Ca 2+ sensitivity of myofilaments with altered Na + and Ca 2+ ion homeostasis in cardiomyocytes with diastolic dysfunction. In this study, we tested the acute effects of ouabain on ventricular myocytes of an HCM mouse model. The effects of ouabain on contractility and Ca 2+ transients were tested in intact adult mouse ventricular myocytes (AMVMs) of Mybpc3-targeted knock-in (KI) and wild-type (WT) mice. Concentration-response assessment of contractile function revealed low sensitivity of AMVMs to ouabain (10 μM) compared to literature data on human cardiomyocytes (100 nM). Three hundred μM ouabain increased contraction amplitude (WT ~1.8-fold; KI ~1.5-fold) and diastolic intracellular Ca 2+ in both WT and KI (+12-18%), but further decreased diastolic sarcomere length in KI cardiomyocytes (-5%). Western Blot analysis of whole heart protein extracts revealed 50% lower amounts of Na + /K + ATPase (NKA) in KI than in WT. Ouabain worsened the diastolic phenotype of KI cardiomyocytes at concentrations which did not impair WT diastolic function. Ouabain led to an elevation of intracellular Ca 2+ , which was poorly tolerated in KI showing already high cytosolic Ca 2+ at baseline due to increased myofilament Ca 2+ sensitivity. Lower amounts of NKA in KI could amplify the need to exchange excessive intracellular Na + for Ca 2+ and thereby explain the general tendency to higher diastolic Ca 2+ in KI., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Cardiac glycosides (CGs), toxins well-known for numerous human and cattle poisoning, are natural compounds, the biosynthesis of which occurs in various plants and animals as a self-protective mechanism to prevent grazing and predation. Interestingly, some insect species can take advantage of the CG's toxicity and by absorbing them, they are also protected from predation. The mechanism of action of CG's toxicity is inhibition of Na + /K + -ATPase (the sodium-potassium pump, NKA), which disrupts the ionic homeostasis leading to elevated Ca 2+ concentration resulting in cell death. Thus, NKA serves as a molecular target for CGs (although it is not the only one) and even though CGs are toxic for humans and some animals, they can also be used as remedies for various diseases, such as cardiovascular ones, and possibly cancer. Although the anticancer mechanism of CGs has not been fully elucidated, yet, it is thought to be connected with the second role of NKA being a receptor that can induce several cell signaling cascades and even serve as a growth factor and, thus, inhibit cancer cell proliferation at low nontoxic concentrations. These growth inhibitory effects are often observed only in cancer cells, thereby, offering a possibility for CGs to be repositioned for cancer treatment serving not only as chemotherapeutic agents but also as immunogenic cell death triggers. Therefore, here, we report on CG's chemical structures, production optimization, and biological activity with possible use in cancer therapy, as well as, discuss their antiviral potential which was discovered quite recently. Special attention has been devoted to digitoxin, digoxin, and ouabain.

Background: The present study aims to evaluate the effects of ouabain on memory and neurotrophic parameters in the brains of rats., Methods: Wistar rats received an intracerebroventricular (ICV) injection of ouabain or artificial cerebrospinal fluid (aCSF). Seven and 14 days after ICV administration, the animals were subjected to the open-field and splash tests. Furthermore, the pro-BDNF, BDNF, TrkB, and CREB were assessed in the frontal cortex and hippocampus of the rats, in both seven and 14 days after ICV injection. The memory of the animals was tested by novel object recognition test (NOR) and inhibitory avoidance task (IA), only 14 days after ICV administration., Results: Ouabain increased locomotion and exploration in the animals seven days after its administration; however, 14 days after ICV, these behavioral parameters return to the basal level. Seven days after ouabain administration increased grooming behavior in the splash test; on the other hand, seven days after ouabain injection decreased the grooming behavior, which is considered an anhedonic response. Besides, ouabain decreased recognition index in the NOR and decreased aversive memory in the IA, when compared to the control group. The levels of pro-BDNF and BDNF decreased in the frontal cortex seven days after ouabain; but its receptor (TrkB) and CREB decreased seven and 14 days after ouabain, in both cerebral structures evaluated., Conclusion: Ouabain-induced animal model of BD is an excellent model to assess memory alteration, observed in bipolar patients. Besides, the memory impairment induced by ouabain seems to be related to BDNF signaling pathway alterations., (Copyright © 2021. Published by Elsevier B.V.)

The aim of this study was to evaluate the comparative effects of CGs on heart physiology. Twenty-eight Wistar rats were distributed into four groups (n = 7), control group received NaCl 0.9% every 24 h for 21 days; treated groups received respectively 50 μg/kg of digoxin (DIG), ouabain (OUA) and oleandrin (OLE) every 24 h for 21 days. Serial ECGs were performed, as well as serum levels of creatinine kinase (CK), its MB fraction, troponin I (cTnI), calcium (Ca 2+ ) and lactic dehydrogenase (LDH). Heart tissue was processed for histology, scanning electron microscopy and Western blot analysis for cTnI, brain natriuretic peptide (BNP), sodium potassium pump alpha-1 and alpha-2. Ventricle samples were also analyzed for thiobarbituric acid reactive substances and antioxidant enzymes (SOD, GPX, and CAT). ECGs showed decrease in QT and progressive shortening of QRS. No arrhythmias were observed. No significant differences were associated with CGs treatment and serum levels of CK, CK-MB, and cTnI. Only oleandrin increased LDH levels. Histological analysis showed degenerative changes and only oleandrin promoted moderate focal necrosis of cardiomyocytes. Scanning microscopy also confirmed the greatest effect of oleandrin, with rupture and shortening of cardiac fibers. The expression of troponin I and alpha-1 isoform were not altered, however, the protein levels of BNP and alpha-2 were higher in the groups that received oleandrin and ouabain in relation to the digoxin group. All GCs affected the production of ROS, without causing lipid peroxidation, through the activation of different antioxidant pathways. It is concluded that the administration of digoxin, ouabain, and oleandrin at 50 µg/kg for 21 days caused cardiovascular damage that represent an important limitation into its future use in heart failure and antineoplastic therapy.

A post hoc analysis of the PALLAS trial suggested life-threatening interactions of digitalis and dronedarone. Thus, there is concern about an interplay between digitalis and other drugs that influence cardiac electrophysiology. We therefore investigated the interaction between digitalis and flecainide or ranolazine. Twenty-five rabbit hearts were Langendorff-perfused and treated with flecainide (2 µM, 12 hearts) or ranolazine (10 µM, 13 hearts). Infusion of flecainide prolonged mean action potential duration [APD 90 , from 153 ms (interquartile range (IQR): 29.7 ms) to 159 ms (IQR: 24.9 ms, p = 0.04)] and effective refractory period [ERP, 170 ms (IQR: 40 ms) vs. 200 ms (IQR: 32.5 ms, p < 0.01)]. Administration of ranolazine prolonged APD 90 [144 ms (IQR: 34.3 ms)) vs. 157 ms (IQR: 31.2 ms, p < 0.01)] and ERP [180 ms (IQR: 40 ms) vs. 200 ms (IQR: 30 ms, p < 0.01)]. Additional infusion of the digitalis glycoside ouabain (0.2 µM) abbreviated APD 90 and ERP in both groups (flecainide: APD 90 : to 128 ms (IQR: 19 ms), ERP: to 170 ms (IQR: 20 ms), p < 0.01 each; ranolazine: APD 90 : to 141 ms (IQR: 40 ms), ERP: to 160 ms (IQR: 30 ms), p < 0.01 each). Ventricular vulnerability was assessed by a pacing protocol employing premature extra stimuli and burst stimulation. No proarrhythmic effect was observed with flecainide (1 vs. 3 episodes at baseline) or ranolazine (3 vs. 11 episodes at baseline). However, further infusion of ouabain had a proarrhythmic effect for both drugs (flecainide: 15 episodes, p = 0.04; ranolazine: 21 episodes, p = 0.09). Concomitant treatment of the sodium channel blockers flecainide or ranolazine with digitalis seems to be proarrhythmic. Abbreviation of repolarization and refractoriness that can facilitate re-entry was found as underlying mechanism.

Two isoforms of a ligand-activated nuclear receptor, RORγ and RORγT, have been implicated in various physiological functions, including energy metabolism, circadian rhythm and immune system development. Using a stably transfected reporter cell line, we screened two chemical libraries and identified three cardenolides (natural, plant-derived pesticides) as activators of RORγ-dependent transcription. These compounds increased G6PC and NPAS2 expression in HepG2 cells, accompanied by increased occupancy of RORγ within the promoters of these genes. Further, strophanthidin, digoxigenin and dihydroouabain upregulated IL17A and IL17F expression and enhanced IL17 secretion in Th17 human lymphocytes. Molecular docking analyses of these compounds to the RORγ LBD showed favorable docking scores, suggesting that cardenolides may act as agonists of the receptor. Thus, our results provide new chemical structures for further development of RORγ-selective modulators with virtual therapeutic potential., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Aging listeners, even in the absence of overt hearing loss measured as changes in hearing thresholds, often experience impairments processing temporally complex sounds such as speech in noise. Recent evidence has shown that normal aging is accompanied by a progressive loss of synapses between inner hair cells and auditory nerve fibers. The role of this cochlear synaptopathy in degraded temporal processing with age is not yet understood. Here, we used population envelope following responses, along with other hair cell- and neural-based measures from an age-graded series of male and female CBA/CaJ mice to study changes in encoding stimulus envelopes. By comparing responses obtained before and after the application of the neurotoxin ouabain to the inner ear, we demonstrate that we can study changes in temporal processing on either side of the cochlear synapse. Results show that deficits in neural coding with age emerge at the earliest neural stages of auditory processing and are correlated with the degree of cochlear synaptopathy. These changes are seen before losses in neural thresholds and particularly affect the suprathreshold processing of sound. Responses obtained from more central sources show smaller differences with age, suggesting compensatory gain. These results show that progressive cochlear synaptopathy is accompanied by deficits in temporal coding at the earliest neural generators and contribute to the suprathreshold sound processing deficits observed with age. SIGNIFICANCE STATEMENT Aging listeners often experience difficulty hearing and understanding speech in noisy conditions. The results described here suggest that age-related loss of cochlear synapses may be a significant contributor to those performance declines. We observed aberrant neural coding of sounds in the early auditory pathway, which was accompanied by and correlated with an age-progressive loss of synapses between the inner hair cells and the auditory nerve. Deficits first appeared before changes in hearing thresholds and were largest at higher sound levels relevant to real world communication. The noninvasive tests described here may be adapted to detect cochlear synaptopathy in the clinical setting., (Copyright © 2018 the authors 0270-6474/18/387108-12$15.00/0.)

Round window membrane (RWM) application of ouabain is known to selectively destroy type I spiral ganglion cells (SGCs) in cochleas of several rodent species, while leaving hair cells intact. This protocol has been used in rats and Mongolian gerbils, but observations in the guinea pig are conflicting. This is why we reinvestigated the effect of ouabain on the guinea pig cochlea. Ouabain solutions of different concentrations were placed, in a piece of gelfoam, upon the RWM of the right cochleas. Auditory function was assessed using acoustically evoked auditory brainstem responses (aABR). Finally, cochleas were fixed and processed for histological examination. Due to variability within treatment groups, histological data was pooled and three categories based upon general histological observations were defined: cochleas without outer hair cell (OHC) and SGC loss (Category 1), cochleas with OHC loss only (Category 2), and cochleas with OHC and SGC loss (Category 3). Animals treated with 1 mM or 10 mM ouabain showed shifts in hearing thresholds, corresponding with varying histological changes in their cochleas. Most cochleas exhibited complete outer hair cell loss in the basal and middle turns, while some had no changes, together with either moderate or near-complete loss of SGCs. Neither loss of inner hair cells nor histological changes of the stria vascularis were observed in any of the animals. Cochleas in Category 1 had normal aABRs and morphology. On average, in Category 2 OHC loss was 46.0±5.7%, SGC loss was below threshold, ABR threshold shift was 44.9±2.7 dB, and ABR wave II amplitude was decreased by 17.1±3.8 dB. In Category 3 OHC loss was 68.3±6.9%, SGC loss was 49.4±4.3%, ABR threshold shift was 39.0±2.4 dB, and ABR amplitude was decreased by 15.8±1.6 dB. Our results show that ouabain does not solely destroy type I SGCs in the guinea pig cochlea.

Background: In contrast to mammals, zebrafish have the capacity to regenerate retinal neurons following a variety of injuries. Two types of glial cells, Müller glia (MG) and microglia, are known to exist in the zebrafish retina. Recent work has shown that MG give rise to regenerated retinal neurons, but the role of resident microglia, and the innate immune system more generally, during retinal regeneration is not well defined. Specifically, characteristics of the immune system and microglia following substantial neuron death and a successful regenerative response have not been documented., Methods: The neurotoxin ouabain was used to induce a substantial retinal lesion of the inner retina in zebrafish. This lesion results in a regenerative response that largely restores retinal architecture, neuronal morphologies, and connectivities, as well as recovery of visual function. We analyzed cryosections from damaged eyes following immunofluorescence and H&E staining to characterize the initial immune response to the lesion. Whole retinas were analyzed by confocal microscopy to characterize microglia morphology and distribution. Statistical analysis was performed using a two-tailed Student's t test comparing damaged to control samples., Results: We find evidence of early leukocyte infiltration to the retina in response to ouabain injection followed by a period of immune cell proliferation that likely includes both resident microglia and substantial numbers of proliferating, extra-retinally derived macrophages, leading to rapid accumulation upon retinal damage. Following immune cell proliferation, Müller glia re-enter the cell cycle. In retinas that have regenerated the layers lost to the initial injury (histologically regenerated), microglia retain morphological features of activation, suggesting ongoing functions that are likely essential to restoration of retinal function., Conclusions: Collectively, these results indicate that microglia and the immune system are dynamic during a successful regenerative response in the retina. This study provides an important framework to probe inflammation in the initiation of, and functional roles of microglia during retinal regeneration.

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.

Adult zebrafish ( Danio rerio ) are capable of regenerating retinal neurons that have been lost due to mechanical, chemical, or light damage. In the case of chemical damage, there is evidence that visually mediated behaviors are restored after regeneration, consistent with recovery of retinal function. However, the extent to which regenerated retinal neurons attain appropriate morphologies and circuitry after such tissue-disrupting lesions has not been investigated. Adult zebrafish of both sexes were subjected to intravitreal injections of ouabain, which destroys the inner retina. After retinal regeneration, cell-selective markers, confocal microscopy, morphometrics, and electrophysiology were used to examine dendritic and axonal morphologies, connectivities, and the diversities of each, as well as retinal function, for a subpopulation of regenerated bipolar neurons (BPs). Although regenerated BPs were reduced in numbers, BP dendritic spreads, dendritic tree morphologies, and cone-bipolar connectivity patterns were restored in regenerated retinas, suggesting that regenerated BPs recover accurate input pathways from surviving cone photoreceptors. Morphological measurements of bipolar axons found that numbers and types of stratifications were also restored; however, the thickness of the inner plexiform layer and one measure of axon branching were slightly reduced after regeneration, suggesting some minor differences in the recovery of output pathways to downstream partners. Furthermore, ERG traces from regenerated retinas displayed waveforms matching those of controls, but with reduced b-wave amplitudes. These results support the hypothesis that regenerated neurons of the adult zebrafish retina are capable of restoring complex morphologies and circuitry, suggesting that complex visual functions may also be restored. SIGNIFICANCE STATEMENT Adult zebrafish generate new retinal neurons after a tissue-disrupting lesion. Existing research does not address whether regenerated neurons of adults successfully reconnect with surrounding neurons and establish complex morphologies and functions. We report that, after a chemical lesion that ablates inner retinal neurons, regenerated retinal bipolar neurons (BPs), although reduced in numbers, reconnected to undamaged cone photoreceptors with correct wiring patterns. Regenerated BPs had complex morphologies similar to those within undamaged retina and a physiological measure of photoreceptor-BP connectivity, the ERG, was restored to a normal waveform. This new understanding of neural connectivity, morphology, and physiology suggests that complex functional processing is possible within regenerated adult retina and offers a system for the future study of synaptogenesis during adult retinal regeneration., (Copyright © 2018 the authors 0270-6474/18/380121-17$15.00/0.)

Background: The intracerebroventricular injection of ouabain, a specific inhibitor of the Na+/K+-adenosine-triphosphatase (Na+/K+-ATPase) enzyme, induces hyperactivity in rats in a putative animal model of mania. Several evidences have suggested that the protein kinase C signaling pathway is involved in bipolar disorder. In addition, it is known that protein kinase C inhibitors, such as lithium and tamoxifen, are effective in treating acute mania., Methods: In the present study, we investigated the effects of lithium and tamoxifen on the protein kinase C signaling pathway in the frontal cortex and hippocampus of rats submitted to the animal model of mania induced by ouabain. We showed that ouabain induced hyperlocomotion in the rats., Results: Ouabain increased the protein kinase C activity and the protein kinase C and MARCKS phosphorylation in frontal cortex and hippocampus of rats. Lithium and tamoxifen reversed the behavioral and protein kinase C pathway changes induced by ouabain. These findings indicate that the Na+/K+-ATPase inhibition can lead to protein kinase C alteration., Conclusions: The present study showed that lithium and tamoxifen modulate changes in the behavior and protein kinase C signalling pathway alterations induced by ouabain, underlining the need for more studies of protein kinase C as a possible target for treatment of bipolar disorder., (© The Author 2017. Published by Oxford University Press on behalf of CINP.)

Objective: Circulating levels of cardiotonic steroids (CTS) are elevated in various chronic inflammatory conditions, but the role of CTS in inflammation remains largely unknown. We have previously shown that the CTS ouabain stimulates proinflammatory responses in murine macrophages. In this study, we aim to explore the mechanism how CTS induce proinflammatory responses in primary murine and human macrophages., Approach and Results: Using both murine peritoneal macrophages and human monocyte-derived macrophages, we demonstrated that ouabain activated NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells), leading to proinflammatory cytokine (eg, MCP-1 [monocyte chemotactic protein 1], TNF-α [tumor necrosis factor-α], IL-1β [interleukin-1β], and IL-6) production. By applying siRNA techniques and murine peritoneal macrophages isolated from genetically modified mice, we showed that macrophages partially deficient in Na/K-ATPase, the receptor for CTS, or fully deficient in the scavenger receptor CD36 or TLR4 (Toll-like receptor) were resistant to ouabain-induced NF-κB activation, suggesting an indispensable role of these 3 receptors in this pathway. Mechanistically, this effect of ouabain was independent of the ion transport function of the Na/K-ATPase. Instead, ouabain stimulated a signaling complex, including Na/K-ATPase, CD36, and TLR4. Subsequently, TLR4 recruited MyD88 adaptor protein for NF-κB activation. Furthermore, intraperitoneal injection of ouabain into mice specifically recruited Ly6C + CCR2 + monocyte subtypes to the peritoneal cavities, indicating that the CTS ouabain triggers inflammation in vivo., Conclusions: CTS activate NF-κB leading to proinflammatory cytokine production in primary macrophages through a signaling complex, including CD36, TLR4, and Na/K-ATPase. These findings warrant further studies on endogenous CTS in chronic inflammatory diseases, such as atherosclerosis., (© 2017 American Heart Association, Inc.)

Toads are chemically defended by cardiotonic steroids known as bufadienolides. Resistance to the acute effects of bufadienolides in snakes that prey on toads is conferred by target-site insensitivity of the toxin's target enzyme, the Na + /K + -ATPase. Previous studies have focused largely on the molecular mechanisms of resistance but have not investigated the physiological mechanisms or consequences of exposure to the toxins. Adrenal enlargement in snakes often is associated with specialization on a diet of toads. These endocrine glands are partly composed of interrenal tissue, which produces the corticosteroids corticosterone and aldosterone. Corticosterone is the main hormone released in response to stress in reptiles, and aldosterone plays an important role in maintaining ion balance through upregulation of Na + /K + -ATPase. We tested the endocrine response of select species of snakes to acute cardiotonic steroid exposure by measuring circulating aldosterone and corticosterone concentrations. We found that Rhabdophis tigrinus, which specializes on a diet of toads, responds with lower corticosterone and higher aldosterone compared to other species that exhibit target-site resistance to the toxins but do not specialize on toads. We also found differences between sexes in R. tigrinus, with males generally responding with higher corticosterone and aldosterone than females. This study provides evidence of physiological adaptations, beyond target-site resistance, associated with tolerance of bufadienolides in a specialized toad-eating snake., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Ciguatoxins (CTXs) are emerging marine neurotoxins representing the main cause of ciguatera fish poisoning, an intoxication syndrome which configures a health emergency and constitutes an evolving issue constantly changing due to new vectors and derivatives of CTXs, as well as their presence in new non-endemic areas. The study applied the neuroblastoma cell model of human origin (SH-SY5Y) to evaluate species-specific mechanistic information on CTX toxicity. Metabolic functionality, cell morphology, cytosolic Ca 2+ i responses, neuronal cell growth and proliferation were assessed after short- (4-24h) and long-term exposure (10days) to P-CTX-3C. In SH-SY5Y, P-CTX-3C displayed a powerful cytotoxicity requiring the presence of both Veratridine and Ouabain. SH-SY5Y were very sensitive to Ouabain: 10 and 0.25nM appeared the optimal concentrations, for short- and long-term toxicity studies, respectively, to be used in co-incubation with Veratridine (25μM), simulating the physiological and pathological endogenous Ouabain levels in humans. P-CTX-3C cytotoxic effect, on human neurons co-incubated with OV (Ouabain+Veratridine) mix, was expressed starting from 100pM after short- and 25pM after long-term exposure. Notably, P-CTX-3C alone at 25nM induced cytotoxicity after 24h and prolonged exposure. This human brain-derived cell line appears a suitable cell-based-model to evaluate cytotoxicity of CTX present in marine food contaminated at low toxic levels and to characterize the toxicological profile of other/new congeners., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Cardiotoxicity is one of the most serious side effects of new drugs. Early detection of the drug induced cardiotoxicity based on the biomarkers provides an important preventative strategy for detecting potential cardiotoxicity of candidate drugs. In this study, we aim to identify the predictive genomics biomarkers for drug-induced cardiac toxicity based on the RTCA coupled with PCR Array technology in primary cells. Three prototypical cardiotoxic compounds (doxorubicin, isoproterenol, ouabain) with different mechanisms were firstly real-time monitored to diagnose the cytotoxicity by using the RTCA, while the functional alterations of cardiomyocytes were also monitored by analyzing the beating frequency of cardiomyocytes. Then cardiac specific toxicity gene expression changes were studied by using the technology of PCR Array, which can detect the changes of 84 cardiac functions related genes. Rps6kb1 was identified to be the common cardiac biomarkers by using multivariate statistical and integration analyses. The biomarker was further verified by selecting other drugs with or without cardiotoxicity, and the results showed that the gene exhibited specific changes in cardiac toxicity. Moreover, IPA was applied to combine relevant pathways of Rps6kb1, and identify the main types of cardiac toxicity. These results would further enrich the evaluating strategy of drug-induced cardiotoxicity in vitro, and Rps6kb1 could be used as the specific biomarker of cardiotoxcity during safety assessment of the novel drug candidates., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Our laboratory has recently demonstrated that low concentrations of ouabain increase blood pressure in rats associated with stimulation of NaK ATPase activity and activation of the Src signaling cascade in NHE1-dependent manner. Proteomic analysis of human kidney proximal tubule cells (HKC11) suggested that the Angiotensin II type 1 receptor (AT1R) as an ouabain-associating protein. We hypothesize that ouabain-induced stimulation of NaK ATPase activity is mediated through AT1R. To test this hypothesis, we examined the effect of ouabain on renal cell angiotensin II production, the effect of AT1R inhibition on ouabain-stimulated NKA activity, and the effect of ouabain on NKA-AT1R association. Ouabain increased plasma angiotensin II levels in rats treated with ouabain (1μg/kg body wt./day) for 9days and increased angiotensin II levels in cell culture media after 24h treatment with ouabain in human (HKC11), mouse (MRPT), and human adrenal cells. Ouabain 10pM stimulated NKA-mediated 86 Rb uptake and phosphorylation of EGFR, Src, and ERK1/2. These effects were prevented by the AT1R receptor blocker candesartan. FRET and TIRF microscopy using Bodipy-labeled ouabain and mCherry-NKA or mCherry-AT1R demonstrated association of ouabain with AT1R and NKA. Further our FRET and TIRF studies demonstrated increased association between AT1R and NKA upon treatment with low dose ouabain. We conclude that ouabain stimulates NKA in renal proximal tubule cells through an angiotensin/AT1R-dependent mechanism and that this pathway contributes to cardiac glycoside associated hypertension., Competing Interests: Conflict of Interest/Disclosure: No Conflicts/Disclosure to report, (Copyright © 2016 Elsevier B.V. All rights reserved.)

Neurons at higher stages of sensory processing can partially compensate for a sudden drop in peripheral input through a homeostatic plasticity process that increases the gain on weak afferent inputs. Even after a profound unilateral auditory neuropathy where >95% of afferent synapses between auditory nerve fibers and inner hair cells have been eliminated with ouabain, central gain can restore cortical processing and perceptual detection of basic sounds delivered to the denervated ear. In this model of profound auditory neuropathy, auditory cortex (ACtx) processing and perception recover despite the absence of an auditory brainstem response (ABR) or brainstem acoustic reflexes, and only a partial recovery of sound processing at the level of the inferior colliculus (IC), an auditory midbrain nucleus. In this study, we induced a profound cochlear neuropathy with ouabain and asked whether central gain enabled a compensatory plasticity in the auditory thalamus comparable to the full recovery of function previously observed in the ACtx, the partial recovery observed in the IC, or something different entirely. Unilateral ouabain treatment in adult mice effectively eliminated the ABR, yet robust sound-evoked activity persisted in a minority of units recorded from the contralateral medial geniculate body (MGB) of awake mice. Sound driven MGB units could decode moderate and high-intensity sounds with accuracies comparable to sham-treated control mice, but low-intensity classification was near chance. Pure tone receptive fields and synchronization to broadband pulse trains also persisted, albeit with significantly reduced quality and precision, respectively. MGB decoding of temporally modulated pulse trains and speech tokens were both greatly impaired in ouabain-treated mice. Taken together, the absence of an ABR belied a persistent auditory processing at the level of the MGB that was likely enabled through increased central gain. Compensatory plasticity at the level of the auditory thalamus was less robust overall than previous observations in cortex or midbrain. Hierarchical differences in compensatory plasticity following sensorineural hearing loss may reflect differences in GABA circuit organization within the MGB, as compared to the ACtx or IC.

Adaptation is defined as an evolutionary process allowing organisms to succeed in certain habitats or conditions. Chromosomal inversions have the potential to be key in the adaptation processes, since they can contribute to the maintenance of favoured combinations of adaptive alleles through reduced recombination between individuals carrying different inversions. We have analysed six genes (Pif1A, Abi, Sqd, Yrt, Atpα and Fmr1), located inside and outside three inversions of the O chromosome in European populations of Drosophila subobscura. Genetic differentiation was significant between inversions despite extensive recombination inside inverted regions, irrespective of gene distance to the inversion breakpoints. Surprisingly, the highest level of genetic differentiation between arrangements was found for the Atpα gene, which is located outside the O1 and O7 inversions. Two derived unrelated arrangements (O3+4+1 and O3+4+7) are nearly fixed for several amino acid substitutions at the Atpα gene that have been described to confer resistance in other species to the cardenolide ouabain, a plant toxin capable of blocking ATPases. Similarities in the Atpα variants, conferring ouabain resistance in both arrangements, may be the result of convergent substitution and be favoured in response to selective pressures presumably related to the presence of plants containing ouabain in the geographic locations where both inversions are present.

In this report an analysis of cochlear response harmonics is developed to derive a mathematical function to estimate the gross mechanics involved in the in vivo transfer of acoustic sound into neural excitation (f(Tr)). In a simulation it is shown that the harmonic distortion from a nonlinear system can be used to estimate the nonlinearity, supporting the next phase of the experiment: Applying the harmonic analysis to physiologic measurements to derive estimates of the unknown, in vivo f(Tr). From gerbil ears, estimates of f(Tr) were derived from cochlear response measurements made with an electrode at the round window niche from 85 Hz tone bursts. Estimates of f(Tr) before and after inducing auditory neuropathy-loss of auditory nerve responses with preserved hair cell responses from neurotoxic treatment with ouabain-showed that the neural excitation from low-frequency tones contributes to the magnitude of f(Tr) but not the sigmoidal, saturating, nonlinear morphology.

Ouabain is an endogenous Na(+)/K(+)-ATPase inhibitor whose chronic administration induces hypertension. Endogenous ouabain levels increase in human essential hypertension. On the other hand, Rho/Rho kinase (ROCK) pathway has been implicated in various animal models of hypertension. In the current work, we evaluated the possible involvement of Rho kinase in ouabain-induced hypertension. Ouabain was administered daily (20 µg/kg, i.p.) to Wistar rats for 6 weeks. After the ouabain treatment, we evaluated the possible changes in vascular responses to KCl and phenylephrine alone and in the presence of Rho kinase inhibitor Y27632. We also determined the expressions of ROCKs, Rho A and phosphorylation of myosin binding subunit of myosin light chain phosphatase (pMYPT) and activation of Rho A. Agonist-induced contractions in the presence of Y27632 are significantly decreased and Y27632-induced relaxations in aortas precontracted with phenylephrine are significantly enhanced with the chronic treatment of ouabain. Although the expressions of ROCK I and ROCK II remained unchanged, pMYPT expression was significantly increased in ouabain-treated group. Moreover, Rho A expression and activation were decreased after treatment with ouabain. Although Rho kinase expression did not change in aortas, increased basal Rho kinase activation may contribute to the development of ouabain-induced hypertension. Our current data present the first evidence that Rho kinase is involved in the development of ouabain-induced hypertension in rats.

In rodents, ubiquitous α1-Na(+), K(+)-ATPase is inhibited by ouabain and other cardiotonic steroids (CTS) at ~10(3)-fold higher concentrations than those effective in other mammals. To examine the specific roles of the CTS-sensitive α1S- and CTS-resistant α1R-Na(+), K(+)-ATPase isoforms, we compared the effects of ouabain on intracellular Na(+) and K(+) content, cell survival, and mitogen-activated protein kinases (MAPK) in human and rat vascular smooth muscle cells (HASMC and RASMC), human and rat endothelial cells (HUVEC and RAEC), and human and rat brain astrocytes. 6-h exposure of HASMC and HUVEC to 3 μM ouabain dramatically increased the intracellular [Na(+)]/[K(+)] ratio to the same extend as in RASMC and RAEC treated with 3000 μM ouabain. In 24, 3 μM ouabain triggered the death of all types of human cells used in this study. Unlike human cells, we did not detect any effect of 3000-5000 μM ouabain on the survival of rat cells, or smooth muscle cells from mouse aorta (MASMC). Unlike in the wild-type α1(R/R) mouse, ouabain triggered death of MASMC from α1(S/S) mouse expressing human α1-Na(+), K(+)-ATPase. Furthermore, transfection of HUVEC with rat α1R-Na(+), K(+)-ATPase protected them from the ouabain-induced death. In HUVEC, ouabain led to phosphorylation of p38 MAPK, whereas in RAEC it stimulated phosphorylation of ERK1/2. Overall, our results, demonstrate that the drastic differences in cytotoxic action of ouabain on human and rodent cells are caused by unique features of α1S/α1R-Na(+), K(+)-ATPase, rather than by any downstream CTS-sensitive/resistant components of the cell death machinery.

A stroke in humans may induce focal injury to the brain tissue resulting in various disabilities. Although motor deficits are the most discernible, cognitive impairments seem to be crucial for patients mental well-being. The current lack of effective treatments encourages scientists and clinicians to develop novel approaches. Before applying them in clinic, testing for safety and effectiveness in non-human models is necessary. Such animal model should include significant cognitive impairments resulting from brain lesion. We used ouabain stereotactic injection into the right dorsolateral striatum of male Wistar rats, and enriched environment housing. To confirm the brain injury before cognitive testing, rats were given a beam-walking task to evaluate the level of sensorimotor deficits. To determine the cognitive impairment after focal brain damage, rats underwent a set of selected tasks over an observation period of 30 days. Brain injury induced by ouabain significantly impaired the acquisition of the T-maze habit learning task, where 'win-stay' strategy rules were applied. The injured rats also showed significant deficits in the performance of the T-maze switching task, which involved shifting from multiple clues previously relevant to the only one important clue. Focal brain injury also significantly changed 'what--where' memory, tested in the object exploration task, in which a novel object consecutively appeared in the same place while the location of a familiar item was continuously changed. In conclusion, we developed an animal model of distinct cognitive impairments after focal brain injury that provides a convenient method to test the effectiveness of restorative therapies., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The present study aimed to investigate the effects of L-aspartic acid potassium salt (potassium aspartate, K-asp) on SH-SY5Y cells treated with ouabain and H2O2. An 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to investigate the effects of K-asp on SH-SY5Y cell death induced by ouabain. Nissl staining was used to demonstrate the morphological changes of the SH-SY5Y cells. Light microscopy and 4',6-diamidino-2-phenylindole (DAPI) staining were performed to visualize apoptosis in SH-SY5Y cells incubated with ouabain for 6, 24 and 48 h. Transmission electron microscopy was used to observe the effect of K-asp on ultrastructural changes of the SH-SY5Y cells following incubation with ouabain for 24 and 48 h. An annexin V-fluorescein isothiocyanate/propidium binding assay and flow cytometry were performed successively to investigate how K-asp affected the H2O2-induced cell apoptosis. The MTT assay demonstrated that K-asp attenuated the cytotoxicity of the SH-SY5Y cells following treatment with ouabain, in a dose-dependent manner. The cell survival rates following 48 h incubation in the K-asp (15 mM) and K-asp (25 mM) groups were higher compared with the KCl and MK801 groups. Nissl staining demonstrated that the severity of cell injury in the KCl and K-asp (25 mM) groups were alleviated. In the DAPI staining and transmission electron microscopy analyses, KCl and K-asp (25 mM) reduced the rate of ouabain-induced apoptosis. Flow cytometry revealed that K-asp (25 mM) reduced H2O2 -induced apoptosis. These results demonstrated that K-asp (25 mM) inhibited the ouabain and H2O2-induced SH-SY5Y cell damage and apoptosis, possibly by supplementing levels of intracellular K(+).

The present study aimed to investigate the effects of mood stabilizers, specifically lithium (Li) and valproate (VPA), on mitochondrial superoxide, lipid peroxidation, and proteins involved in cell death signaling pathways in the brains of rats subjected to the ouabain-induced animal model of mania. Wistar rats received Li, VPA, or saline twice a day for 13 days. On the 7th day of treatment, the animals received a single intracerebroventricular injection of ouabain or aCSF. After the ICV injection, the treatment with mood stabilizers continued for 6 additional days. The locomotor activity of rats was measured using the open-field test. In addition, we analyzed oxidative stress parameters, specifically levels of phosphorylated p53 (pp53), BAX and Bcl-2 in the brain of rats by immunoblot. Li and VPA reversed ouabain-related hyperactivity. Ouabain decreased Bcl-2 levels and increased the oxidative stress parameters BAX and pp53 in the brains of rats. Li and VPA improved these ouabain-induced cellular dysfunctions; however, the effects of the mood stabilizers were dependent on the protein and brain region analyzed. These findings suggest that the Na(+)/K(+)-ATPase can be an important link between oxidative damage and the consequent reduction of neuronal and glial density, which are both observed in BD, and that Li and VPA exert protective effects against ouabain-induced activation of the apoptosis pathway., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Bipolar Disorder (BD) is one of the most severe psychiatric disorders. Despite adequate treatment, patients continue to have recurrent mood episodes, residual symptoms, and functional impairment. Some preclinical studies have shown that histone deacetylase inhibitors may act on manic-like behaviors. Neurotrophins have been considered important mediators in the pathophysiology of BD. The present study aims to investigate the effects of lithium (Li), valproate (VPA), and sodium butyrate (SB), an HDAC inhibitor, on BDNF, NGF and GDNF in the brain of rats subjected to an animal model of mania induced by ouabain. Wistar rats received a single ICV injection of ouabain or artificial cerebrospinal fluid. From the day following ICV injection, the rats were treated for 6 days with intraperitoneal injections of saline, Li, VPA or SB twice a day. In the 7th day after ouabain injection, locomotor activity was measured using the open-field test. The BDNF, NGF and GDNF levels were measured in the hippocampus and frontal cortex by sandwich-ELISA. Li, VPA or SB treatments reversed ouabain-related manic-like behavior. Ouabain decreased BDNF, NGF and GDNF levels in hippocampus and frontal cortex of rats. The treatment with Li, VPA or SB reversed these impairment induced by ouabain. In addition, Li, VPA and SB per se increased NGF and GDNF levels in hippocampus of rats. Our data support the notion that neurotrophic factors play a role in BD and in the mechanisms of the action of Li, VPA and SB., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Studies have revealed alterations in mitochondrial complexes in the brains of bipolar patients. However, few studies have examined changes in the enzymes of the tricarboxylic acid cycle. Several preclinical studies have suggested that histone deacetylase inhibitors may have antimanic effects. The present study aims to investigate the effects of lithium, valproate and sodium butyrate, a histone deacetylase inhibitor, on the activity of tricarboxylic acid cycle enzymes in the brains of rats subjected to an animal model of mania induced by ouabain. Wistar rats received a single intracerebroventricular injection of ouabain or cerebrospinal fluid. Starting on the day following the intracerebroventricular injection, the rats were treated for 7days with intraperitoneal injections of saline, lithium, valproate or sodium butyrate. Risk-taking behavior, locomotor and exploratory activities were measured using the open-field test. Citrate synthase, succinate dehydrogenase, and malate dehydrogenase were examined in the frontal cortex and hippocampus. All treatments reversed ouabain-related risk-taking behavior and hyperactivity in the open-field test. Ouabain inhibited tricarboxylic acid cycle enzymes in the brain, and valproate and sodium butyrate but not lithium reversed this ouabain-induced dysfunction. Thus, protecting the tricarboxylic acid cycle may contribute to the therapeutic effects of histone deacetylase inhibitors., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Physiological effects of ouabain, an endogenous human hormone, are being intensively investigated. However, its role in regulation of integrin pattern in lung cancer is largely unknown. The switching in the expression pattern of integrins is recognized as an important factor facilitating metastasis of several cancers., Materials and Methods: Cytotoxicity and proliferative effects of ouabain on H460 lung cancer cells were evaluated by the MTT assay. The levels of integrin proteins in response to ouabain were determined by western blotting. Anchorage-independent growth and migration behaviors were performed by the wound healing assay and colony formation assay, respectively., Results: Herein, the results suggested that exposure of the lung cancer cells to physiological concentrations of ouabain significantly altered the level of integrins. Ouabain suppressed integrin α4, α5, αv, β3 and β4, whereas it had no significant effect on integrin β1 and β4. According to the switch patterns of integrins, ouabain treatment resulted in a dramatic reduction of cell colony size and inhibition of cancer cell migration. However, ouabain-induced integrin switch had only a slight effect on chemotherapeutic drug susceptibility., Conclusion: Ouabain may have a role in suppressing cancer metastasis via integrin regulation., (Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Sound-evoked compound action potential (CAP), which captures the synchronous activation of the auditory nerve fibers (ANFs), is commonly used to probe deafness in experimental and clinical settings. All ANFs are believed to contribute to CAP threshold and amplitude: low sound pressure levels activate the high-spontaneous rate (SR) fibers, and increasing levels gradually recruit medium- and then low-SR fibers. In this study, we quantitatively analyze the contribution of the ANFs to CAP 6 days after 30-min infusion of ouabain into the round window niche. Anatomic examination showed a progressive ablation of ANFs following increasing concentration of ouabain. CAP amplitude and threshold plotted against loss of ANFs revealed three ANF pools: 1) a highly ouabain-sensitive pool, which does not participate in either CAP threshold or amplitude, 2) a less sensitive pool, which only encoded CAP amplitude, and 3) a ouabain-resistant pool, required for CAP threshold and amplitude. Remarkably, distribution of the three pools was similar to the SR-based ANF distribution (low-, medium-, and high-SR fibers), suggesting that the low-SR fiber loss leaves the CAP unaffected. Single-unit recordings from the auditory nerve confirmed this hypothesis and further showed that it is due to the delayed and broad first spike latency distribution of low-SR fibers. In addition to unraveling the neural mechanisms that encode CAP, our computational simulation of an assembly of guinea pig ANFs generalizes and extends our experimental findings to different species of mammals. Altogether, our data demonstrate that substantial ANF loss can coexist with normal hearing threshold and even unchanged CAP amplitude., (Copyright © 2014 the American Physiological Society.)

Spiral ganglion neuron (SGN) injury is a generally accepted precursor of auditory neuropathy. Receptor-interacting protein 3 (RIP3) has been reported as an important necroptosis pathway mediator that can be blocked by necrostatin-1 (Nec-1). In our study, we sought to identify whether necroptosis participated in SGN injury. Ouabain was applied to establish an SGN injury model. We measured the auditory brain-stem response (ABR) threshold shift as an indicator of the auditory conditions. Positive β3-tubulin immunofluorescence staining indicated the surviving SGNs. RIP3 expression was evaluated using immunofluorescence, quantitative real-time polymerase chain reaction and western blot. SGN injury promoted an increase in RIP3 expression that could be suppressed by application of the necroptosis inhibitor Nec-1. A decreased ABR threshold shift and increased SGN density were observed when Nec-1 was administered with apoptosis inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD). These results demonstrated that necroptosis is an indispensable pathway separately from apoptosis leading to SGN death pathway, in which RIP3 plays an important role., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

The present study deals with the possible effects of selected environmental agents upon the uptake and metabolism of d-glucose in isolated acinar and ductal cells from the rat submandibular salivary gland. In acinar cells, the uptake of d-[U-(14) C]glucose and its non-metabolised analogue 3-O-[(14) C-methyl]-d-glucose was not affected significantly by phloridzin (0.1 mM) or substitution of extracellular NaCl (115 mM) by an equimolar amount of CsCl, whilst cytochalasin B (20 μM) decreased significantly such an uptake. In ductal cells, both phloridzin and cytochalasin B decreased the uptake of d-glucose and 3-O-methyl-d-glucose. Although the intracellular space was comparable in acinar and ductal cells, the catabolism of d-glucose (2.8 or 8.3 mM) was two to four times higher in ductal cells than in acinar cells. Phloridzin (0.1 mM), ouabain (1.0 mM) and cytochalasin B (20 μM) all impaired d-glucose catabolism in ductal cells. Such was also the case in ductal cells incubated in the absence of extracellular Ca(2+) or in media in which NaCl was substituted by CsCl. It is proposed that the ductal cells in the rat submandibular gland are equipped with several systems mediating the insulin-sensitive, cytochalasin B-sensitive and phloridzin-sensitive transport of d-glucose across the plasma membrane., (Copyright © 2014 John Wiley & Sons, Ltd.)

A growing number of researches have shown that ouabain can regulate mammalian sperm function and male reproduction by modulating the sperm motility, capacitation and acrosome reaction in vitro. This study further examined the relationship between ouabain and asthenozoospermia. In this study, the rat was intraperitoneally injected with ouabain at different concentrations (low-dose ouabain group: 12.5 μg/kg body weight per day, and high-dose ouabain group: 25 μg/kg body weight per day) for 30 days to establish the asthenozoospermia model. The sperms from 60 males with normal fertility were incubated with ouabain of gradient concentrations (10(-7)-10(-2) mol/L) for 4 h. The sperm motility was evaluated under a microscope. Moreover, the endogenous ouabain (EO) level was determined in seminal plasma of mild or severe asthenozoospermia patients and males with normal fertility by competitive inhibition ELISA. The results showed that the sperm motility was significantly diminished in the rats treated with different concentrations of ouabain. The number of motile sperms (grades a and b) was decreased greatly in a time- and dose-dependent manner in 10(-5)-10(-2) mol/L ouabain groups (P<0.01), while no obvious change in sperm motility was observed in 10(-7)-10(-6)mol/L groups even for 4-h incubation (P>0.05). Furthermore, the EO level was significantly increased in asthenozoospermia patients as compared with that in males with normal fertility (25.27±1.71 μg/L in mild asthenozoospermia patients, 26.52±1.82 μg/L in severe asthenozoospermia patients, 19.31±1.45 μg/L in normal fertility men) (P<0.01). In conclusion, rat asthenozoospermia was successfully established by intraperitoneal injection of ouabain, and 10(-5) mol/L ouabain was sufficient enough to inhibit sperm motility in vitro. Moreover, EO, a normal constituent of seminal plasma, was highly expressed in asthenozoospermia males as compared with normal fertility ones.

Intracerebroventricular (ICV) administration of ouabain, a specific Na-K-ATPase inhibitor, in rats mimics the manic phenotypes of bipolar disorder and thus has been proposed as one of the best animal models of mania. Bipolar mania has been known to be associated with dysfunctions of medial prefrontal cortex (mPFC), a brain area critically involved in mental functions; however, the exact mechanism underlying these dysfunctions is not yet clear. The present study investigated synaptic transmission, synaptic plasticity, and dopamine release in Sprague-Dawley rat mPFC following ICV administration of ouabain (5 μl of 1 mM ouabain). The electrophysiological results demonstrated that ouabain depressed the short- and the long-term synaptic plasticity, represented by paired-pulse facilitation and long-term potentiation, respectively, in the mPFC. These ouabain-induced alterations in synaptic plasticity can be prevented by pre-treatment with lithium (intraperitoneal injection of 47.5 mg/kg lithium, twice a day, 7 days), which acts as an effective mood stabilizer in preventing mania. The electrochemical results demonstrated that ICV administration of ouabain enhanced dopamine release in the mPFC, which did not be affected by pre-treatment with lithium. These findings suggested that alterations in synaptic plasticity and dopamine release in the mPFC might underlie the dysfunctions of mPFC accompanied with ouabain administration-induced bipolar mania.

Spiral ganglion neurons (SGNs) are poorly regenerated in the mammalian inner ear. Because of this, stem cell transplantation has been used to replace injured SGNs, and several studies have addressed this approach. However, the difficulty of delivering stem cells into the cochlea and encouraging their migration to Rosenthal's canal (RC), where the SGNs are located, severely restricts this therapeutic strategy. In this study, we attempted to establish a new stem cell transplantation route into the cochlea via the cochlear lateral wall (CLW). First, we tested the precision of this route by injecting Fluorogold into the CLW and next assessed its safety by mock surgeries. Then, using a degenerated SGN animal model, we transplanted neural stem cells (NSCs), derived from the olfactory bulb of C57BL/6-green fluorescent protein (GFP) mice, via the CLW route and examined the cells' distribution in the cochlea. We found the CLW transplantation route is precise and safe. In addition, NSCs migrated into RC with a high efficiency and differentiated into neurons in a degenerated SGN rat model after the CLW transplantation. This result revealed that the basilar membrane (BM) may have crevices permitting the migration of NSCs. The result of this study demonstrates a novel route for cell transplantation to the inner ear, which is important for the replacement of degenerated SGNs and may contribute to the treatment of sensorineural hearing loss., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Lithium (LiCl) and carbamazepine (CBZ), the common mood stabilizers, are thought to be effective treatments for bipolar disorder. The aim of the present study was to investigate whether LiCl as well as CBZ has similar effects on the bipolar disorder-associated cognitive dysfunctions in rats, particularly the spatial learning and depressive responses. Adult male Wistar rats were administered intracerebroventricularly with 5μl of 10(-3)M ouabain on session 1, and then received an intraperitoneal injection of LiCl or CBZ for 4 sessions (1 session/2days). For the behavioral tests, all rats were subjected to the water maze 15min for spatial learning and the forced swimming test 5min for depression on each session. The present results showed that ouabain resulted in increased latency and longer distance traveled to reach the hidden platform in the water maze, indicating that ouabain impaired the spatial learning. However, ouabain did not affect swimming velocity in the water maze and depressive responses in the forced swimming test. LiCl treatment decreased the ouabain-enhanced latency and the total distance, but not the velocity, swam to reach the hidden platform in the water maze task. Additionally, LiCl did not result in changes of any depressive indices, such as struggling behavior, swimming behavior, and floating behavior. Likewise, CBZ did not affect any behavioral indices of spatial learning and depression. A linear regression analysis suggested that LiCl, but not CBZ, could predict the decreased latency and total distance traveled except the velocity of swimming in the water maze and depressive behaviors. In summary, the present results suggested that lithium provided a better therapeutic effect than CBZ for ouabain-caused dysfunctions of spatial learning in a rat model of bipolar disorder., (Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.)

The aim of this work is to study cytoskeletal impairment during the development of ouabain-induced ventricular hypertrophy. Male Sprague-Dawley rats were treated with either ouabain or saline. Systolic blood pressure (SBP) was recorded weekly. At the end of the 3rd and 6th week, the rats were killed and cardiac mass index were measured. Hematoxylin-eosin and Sirius red staining were carried out and cardiac ultrastructure were studied using transmission electron microscopy. The mRNA level of Profilin-1, Desmin, PCNA, TGF-β(1) and ET-1 in the left ventricle were measured using real-time quantitative PCR while their protein levels were examined by Western blot or immunohistochemistry. After 3 weeks, there was no significant difference in the mean SBP, cardiac mass index, mRNA and protein expression of PCNA, TGF-β(1) and ET-1 between the two groups. However, ouabain-treated rats showed disorganized cardiac cytoskeleton with abnormal expression of Profilin-1 and Desmin. After 6 weeks, the cardiac mass index remained the same in the two groups while PCNA, TGF-β(1), and ET-1 have been upregulated in ouabain-treated rats. The cardiac cytoskeletal impairment was more severe in ouabain-treated rats with further changes of Profilin-1 and Desmin. Cytoskeletal abnormality is an ultra-early change during ouabain-induced ventricular hypertrophy, before the release of hypertrophic factors. Therapy for prevention of ouabain-induced hypertrophy should start at the early stage by preventing the cytoskeleton from disorganization.

This study was aimed to clarify if the bone marrow stromal cells (BMSCs) significantly improve functional outcome after lacunar stroke when stereotactically transplanted into the brain. Ouabain, a Na/K ATPase pump inhibitor, was stereotactically injected into the right striatum of Wistar rats. One week later, the superparamagnetic iron oxide (SPIO)-labeled rat BMSCs (n=7) or vehicle (n=8) were stereotactically transplanted into the left striatum. Using rotarod test, motor function was serially evaluated through the experiment. A 7.0-T MR apparatus was employed to serially monitor the migration of BMSCs in the host brain. Histological analysis was performed at 7 weeks after ouabain injection, i.e., 6 weeks after BMSC transplantation. Ouabain injection yielded the reproducible, focal lesion in the right striatum, causing continuous motor dysfunction throughout the experiment. BMSC transplantation significantly enhanced the recovery of motor function after ouabain injection. MR imaging demonstrated that the BMSCs aggressively migrated towards the lesion through the corpus callosum. Histological analysis supported the findings on MRI. The BMSCs significantly enhanced the neurogenesis in the subventricular zone (SVZ) on both sides. Some of them also expressed neuronal or astrocytic phenotypes in the neocortex, SVZ, corpus callosum, and peri-lesion area. These findings strongly suggest that the BMSCs may serve therapeutic impacts on lacunar stroke when stereotactically transplanted at clinically relevant timing.

Objective: To investigate the detrimental effects of ouabain on cochlear spiral ganglion neurons (SGCs) in vivo and in vitro., Methods: Seventy-five male SD rats were randomly divided into 5 groups. In addition to the normal control group, rats in other 4 groups received 0.01, 0.02, 0.05 mmol/L of Ouabain or saline through cochlear scala tympani drilling. Seven days after surgery, the hearing threshold was measured by distortion product otoacoustic emissions (DPOAE) and auditory brainstem response (ABR) in rats. In the in vitro study, SGNs were isolated from SD rats (E14) and treated with 1 × 10(-8) mmol/L of Ouabain. The damaged of SGCs were detected after ouabain treatment using immunohistochemistry, transmission electron microscope and scanning electron microscope in vitro., Results: After administration of Ouabain, DPOAE did not change significantly. No significant difference in the amplitude of DPOAE could be observed among all the groups (P > 0.05). Compared with saline and normal control, ABR threshold was significantly increased in the Ouabain treated groups (P < 0.05), which correlated with the concentration of Ouabain. Electron microscopy showed that after treated with Ouabain, SGCs presented degenerative changes, including collapse of organelle structures, the karyotheca dissolved, myelin sheath disintegrating. Ouabain could damage type I SGCs but not type II SGNs., Conclusions: Ouabain can damage SGCs, either in the in vivo or in vitro conditions.

Insulin has shown to have cardioprotective effect in diabetic patient after digoxin intoxication. The latter, prompted us to study whether insulin interacts directly with Na⁺/K⁺-ATPase. The interaction of insulin with Na⁺/K⁺-ATPase was explored using enzyme activity, Biacore and Western blot. We also used, flow cytometry, immunohistochemistry and chronotropy on both neonatal and adult rats cardiomyocytes. Insulin at concentration 1.7e⁻⁷ M blunted the effect of digoxin on Na⁺/K⁺-ATPase activity. In Western blot, the same insulin concentration decreased enzyme α subunit immunoreactivity. Insulin and digoxin decreased both enzyme α subunit immunoreactivity but insulin/digoxin co-treatment did not. Biacore confirmed a direct interaction between insulin and Na⁺/K⁺-ATPase. In neonatal rat cardiomyocytes, insulin plus digoxin induced cell apoptosis but not alone. In adult rat cardiomyocytes, insulin at optimal dose did not induce apoptosis but prevented the one induced by digoxin. In immunocytochemsitry both insulin and digoxin altered Na⁺/K⁺-ATPase α subunit immunoreactivity while their association did not. Finally, insulin increased the beating rate of neonatal rat cardiomyocytes (45±7 beats/min); so did digoxin (36±13 beats/min). The effect of insulin was prevented after pre-treated with digoxin. These results demonstrate that insulin interacts directly with Na⁺/K⁺-ATPase pump and alters the effect of digoxin. This would have important clinical relevance in cardiac complications related to type I and II diabetes., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Aim: To investigate the anti-arrhythmic effects of sulfamide analogues of changrolin and to characterize the sulfate of compound 6f (sulcardine sulfate, Sul) as a novel anti-arrhythmic agent., Methods: The anti-arrhythmic effects of compounds were studied against aconitine-induced arrhythmias in rats and ouabain-induced arrhythmias in guinea pigs. The effects of Sul on transmembrane action potentials were investigated in isolated rabbit sinoatrial nodes and guinea-pig papillary muscles using intracellular recording. With a whole-cell recording technique, the effects of Sul on sodium current, calcium current, and potassium currents were examined in isolated single guinea-pig ventricular myocytes., Results: In aconitine-induced arrhythmias of rats, sulfamide analogues of changrolin (4, 5, and 6a-6p) exhibited various anti-arrhythmic activities. The sulfate of compound 6f (Sul) increased the amount of aconitine required to induce arrhythmias in each treated animal. The ED₅₀ value of Sul in rats was 196 mg/kg. In ouabain-induced arrhythmias of guinea pigs, 25, 50, and 100 mg/kg doses of Sul increased the dose of ouabain required to induce VP, VT, and VF in a dose-dependent manner. In papillary preparations, Sul produced a concentration-dependent decrease in APA and V(max), prolonged APD(90) and ERP, whereas RP was unaffected. In the spontaneously beating sinus nodes, Sul reduced APA and V(max) in a concentration-dependent manner. The whole-cell recording studies revealed that Sul produced a reversible reduction in I(Na) (IC₅₀=26.9 μmol/L) and I(Ca,L)(IC₅₀=69.2 μmol/L), whereas the inward rectifier (I(K1)) and the delayed rectifier potassium currents (I(K)) were unaffected., Conclusion: As a multi-ion channel blocker, Sul may have potent efficacy in anti-atrial and ventricular arrhythmias.

The composition of different isoforms of Na+-K+-ATPase (NKA, Na/K pump) in ventricular myocytes is an important factor in determining the therapeutic effect and toxicity of cardiac glycosides (CGs) on heart failure. The mechanism whereby CGs cause these effects is still not completely clear. In the present study, we prepared two site-specific antibodies (SSA78 and WJS) against the H₁-H₂ domain of α₁ and α₂ isoforms of NKA in rat heart, respectively, and compared their influences on the effect of ouabain (OUA) in isolated rat ventricular myocytes. SSA78 or WJS, which can specifically bind with the α₁ or α₂ isoform, were assessed with enzyme linked immunosorbent assay (ELISA), Western blot and immunofluorescent staining methods. Preincubation of myocytes with SSA78 inhibited low OUA affinity pump current but not high OUA affinity pump current, reduced the rise in cytosolic calcium concentration ([Ca²⁺](i)), attenuated mitochondrial Ca²⁺ overload, restored mitochondrial membrane potential reduction, and delayed the decrease of the myocardial contractile force as well as the occurrence of arrhythmic contraction induced by high concentrations (1 mM) but not low concentrations (1 μM) of OUA. Similarly, preincubation of myocytes with WJS inhibited high OUA affinity pump current, reduced the increase of [Ca²⁺](i) and the contractility induced by 1 μM but not that induced by 1 mM OUA. These results indicate that the H₁-H₂ domain of the NKA α₁ isoform mediates OUA-induced cardiac toxicity in rat ventricular myocytes, and inhibitors for this binding site may be used as an adjunct to CGs treatment for cardiovascular disease., (Copyright © 2012 Elsevier Inc. All rights reserved.)

In addition to inotropic effects, cardiac glycosides exert deleterious effects on the heart which limit their use for cardiac therapeutics. In this study, we determined the possible contribution of ouabain-induced iNOS stimulation to the resultant hypertrophic as well as cytotoxic effects of the glycoside on cultured adult rat ventricular myocytes. Myocytes were treated with ouabain (50 μM) for up to 24 h. Ouabain significantly increased gene and protein levels of inducible nitric oxide synthase (iNOS) which was associated with significantly increased release of NO from myocytes as well as increased total release of reactive oxygen species (ROS), superoxide anion (O(2) (-)), and increased peroxynitrite formation as assessed by protein tyrosine nitration. Administration of ouabain was also associated with increased levels of myocyte toxicity as determined by myocyte morphology, trypan blue staining and lactate dehydrogenase (LDH) efflux. The nonspecific NOS inhibitor Nω-nitro-L: -arginine methyl ester and the more selective iNOS inhibitor 1400W both abrogated the increase in LDH release but had no significant effect on either morphology or trypan blue staining. Ouabain also significantly increased both myocyte surface area and expression of atrial natriuretic peptide indicating a hypertrophic response with both parameters being completely prevented by NOS inhibition. The effects of iNOS inhibitors were associated with diminished ouabain tyrosine nitration as well as abrogation of ouabain-induced p38 and ERK phosphorylation. Our study shows that ouabain is a potent inducer of NO formation, iNOS upregulation, and increased production of ROS. Inhibition of ouabain-dependent peroxynitrite formation may contribute to the antihypertrophic effect of iNOS inhibition possibly by preventing downstream MAPK activation.

Endogenous cardiotonic steroids (CTS) raise blood pressure (BP) via vascular sodium calcium exchange (NCX1.3) and transient receptor-operated channels (TRPCs). Circulating CTS are superelevated in pregnancy-induced hypertension and preeclampsia. However, their significance in normal pregnancy, where BP is low, is paradoxical. Here we test the hypothesis that vascular resistance to endogenous ouabain (EO) develops in normal pregnancy and is mediated by reduced expression of NCX1.3 and TRPCs. We determined plasma and adrenal levels of EO and the impact of exogenous ouabain in pregnancy on arterial expression of Na(+) pumps, NCX1.3, TRPC3, and TRPC6 and BP. Pregnant (embryonic day 4) and nonpregnant rats received infusions of ouabain or vehicle. At 14-16 days, tissues and plasma were collected for blotting and EO assay by radioimmunoassay (RIA), liquid chromatography (LC)-RIA, and LC-multidimensional mass spectrometry (MS3). BP (-8 mmHg; P < 0.05) and NCX1.3 expression fell (aorta -60% and mesenteric artery -30%; P < 0.001) in pregnancy while TRPC expression was unchanged. Circulating EO increased (1.14 ± 0.13 nM) vs. nonpregnant (0.6 ± 0.08 nM; P < 0.05) and was confirmed by LC-MS3 and LC-RIA. LC-MS3 revealed two previously unknown isomers of EO; one increased ∼90-fold in pregnancy. Adrenal EO but not isomers were increased in pregnancy. In nonpregnant rats, similar infusions of ouabain raised BP (+24 ± 3 mmHg; P < 0.001). In ouabain-infused rats, impaired fetal and placental growth occurred with no BP increase. In summary, normal pregnancy is an ouabain-resistant state associated with low BP, elevated circulating levels of EO, two novel steroidal EO isomers, and increased adrenal mass and EO content. Ouabain raises BP only in nonpregnant animals. Vascular resistance to the chronic pressor activity of endogenous and exogenous ouabain is mediated by suppressed NCX1.3 and reduced sensitivity of events downstream of Ca(2+) entry. The mechanisms of EO resistance and the impaired fetal and placental growth due to elevated ouabain may be important in pregnancy-induced hypertension (PIH) and preeclampsia (PE).

Palytoxin (PlTX) is a marine toxin originally isolated from the zoantharians of the genus Palythoa. It is considered to be one of the most lethal marine toxins that block the Na⁺/K⁺-ATPase. This study was designed to investigate the acute effects of PlTX and ouabain, also an Na⁺/K⁺-ATPase blocker, on the mammalian peripheral nervous system using an ex vivo electrophysiological preparation: the isolated mouse sciatic nerve. Amplitude of the evoked nerve compound action potential (nCAP) was used to measure the proper functioning of the sciatic nerve fibres. The half-vitality time of the nerve fibres (the time required to inhibit the nCAP to 50% of its initial value: IT₅₀) incubated in normal saline was 24.5 ± 0.40 h (n = 5). Nerves incubated continuously in 50.0, 10.0, 1.0, 0.5, 0.250 and 0.125 nM of PlTX had an IT₅₀ of 0.06 ± 0.00, 0.51 ± 0.00, 2.1 ± 0.10, 8.9 ± 0.30, 15.1 ± 0.30 h, and 19.5 ± 0.20 h, respectively (n = 5, 3, 4, 4, 10). PlTX was extremely toxic to the sciatic nerve fibres, with a minimum effective concentration (mEC) of 0.125 nM (n = 5) and inhibitory concentration to 50% (IC₅₀) of 0.32 ± 0.08 nM (incubation time 24 h). Ouabain was far less toxic, with a mEC of 250.0 μM (n = 5) and IC₅₀ of 370.0 ± 18.00 μM (incubation 24.5 h). Finally, when the two compounds were combined--e.g. pre-incubation of the nerve fibre in 250.0 μM ouabain for 1 h and then exposure to 1.0 nM PlTX--ouabain offered minor a neuroprotection of 9.1-17.6% against PlTX-induced neurotoxicity. Higher concentrations of ouabain (500.0 μM) offered no protection. The mouse sciatic nerve preparation is a simple and low-cost bioassay that can be used to assess and quantify the neurotoxic effects of standard PlTX or PlTX-like compounds, since it appears to have the same sensitivity as the haemolysis of erythrocytes assay--the standard ex vivo test for PlTX toxicity., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: Digitalis-induced Na(+) accumulation results in an increase in Ca(2+)(i) via the Na(+)/Ca(2+) exchanger, leading to enhanced sarcoplasmic reticulum (SR) Ca(2+) load, responsible for the positive inotropic and toxic arrhythmogenic effects of glycosides. A digitalis-induced increase in Ca(2+)(i) could also activate calcium-calmodulin kinase II (CaMKII), which has been shown to have proarrhythmic effects. Here, we investigate whether CaMKII underlies digitalis-induced arrhythmias and the subcellular mechanisms involved., Methods and Results: In paced rat ventricular myocytes (0.5 Hz), 50 μmol/L ouabain increased contraction amplitude by 160 ± 5%. In the absence of electric stimulation, ouabain promoted spontaneous contractile activity and Ca(2+) waves. Ouabain activated CaMKII (p-CaMKII), which phosphorylated its downstream targets, phospholamban (PLN) (Thr17) and ryanodine receptor (RyR) (Ser2814). Ouabain-induced spontaneous activity was prevented by inhibiting CaMKII with 2.5 μmol/L KN93 but not by 2.5 μmol/L of the inactive analog, KN92. Similar results were obtained using the CaMKII inhibitor, autocamtide-2 related inhibitory peptide (AIP) (1 to 2.5 μmol/L), and in myocytes from transgenic mice expressing SR-targeted AIP. Consistently, CaMKII overexpression exacerbated ouabain-induced spontaneous contractile activity. Ouabain was associated with an increase in SR Ca(2+) content and Ca(2+) spark frequency, indicative of enhanced SR Ca(2+) leak. KN93 suppressed the ouabain-induced increase in Ca(2+) spark frequency without affecting SR Ca(2+) content. Similar results were obtained with digoxin. In vivo, ouabain-induced arrhythmias were prevented by KN93 and absent in SR-AIP mice., Conclusions: These results show for the first time that CaMKII mediates ouabain-induced arrhythmic/toxic effects. We suggest that CaMKII-dependent phosphorylation of the RyR, resulting in Ca(2+) leak from the SR, is the underlying mechanism involved.