Administration Of Ouabain Research Articles

Long-term Intracerebroventricular Administration of Ouabain Causes Motor Impairments in C57Bl/6 Mice

Introduction. Cardiac glycosides are natural ligands of Na+/K+-ATPase, which regulate its activity and signaling. Intracerebroventricular administration of ouabain has been previously shown to induce hyperlocomotion in C57Bl/6 mice via a decrease in the rate of dopamine reuptake from the synaptic cleft.
 Materials and methods. This study involved forty C57BL/6 mice. 1.5 μL of 50 μM ouabain was administered daily into the left lateral cerebral ventricle over the course of 4 days. On day 5, open field, beam balance, and ladder rung walking tests were performed to assess the locomotor activity and motor impairments in the mice. We evaluated changes in the activation of signaling cascades, ratios of proapoptotic and antiapoptotic proteins, and the amount of α1 and α3 isoforms of the Na+/K+-ATPase α-subunit in brain tissue using Western blotting. Na+/K+-ATPase activity was evaluated in the crude synaptosomal fractions of the brain tissues.
 Results. We observed hyperlocomotion and stereotypic behavior during the open field test 24 hours after the last injection of ouabain. On day 5, the completion time and the number of errors made in the beam balance and ladder rung walking tests increased in the mice that received ouabain. Akt kinase activity decreased in the striatum, whereas the ratio of proapoptotic and antiapoptotic proteins and the number of Na+/K+-ATPase α-subunits did not change. Na+/K+-ATPase activity increased in the striatum and decreased in the brainstem.
 Conclusions. Long-term exposure to ouabain causes motor impairments mediated by changes in the activation of signaling cascades in dopaminergic neurons.

Ouabain enhances renal cyst growth in a slowly progressive mouse model of autosomal dominant polycystic kidney disease.

Renal cyst progression in autosomal dominant polycystic kidney disease (ADPKD) is highly dependent on agents circulating in blood. We have previously shown, using different in vitro models, that one of these agents is the hormone ouabain. By binding to Na+-K+-ATPase (NKA), ouabain triggers a cascade of signal transduction events that enhance ADPKD cyst progression by stimulating cell proliferation, fluid secretion, and dedifferentiation of the renal tubular epithelial cells. Here, we determined the effects of ouabain in vivo. We show that daily administration of ouabain to Pkd1RC/RC ADPKD mice for 1-5 mo, at physiological levels, augmented kidney cyst area and number compared with saline-injected controls. Also, ouabain favored renal fibrosis; however, renal function was not significantly altered as determined by blood urea nitrogen levels. Ouabain did not have a sex preferential effect, with male and female mice being affected equally. By contrast, ouabain had no significant effect on wild-type mice. In addition, the actions of ouabain on Pkd1RC/RC mice were exacerbated when another mutation that increased the affinity of NKA for ouabain was introduced to the mice (Pkd1RC/RCNKAα1OS/OS mice). Altogether, this work highlights the role of ouabain as a procystogenic factor in the development of ADPKD in vivo, that the ouabain affinity site on NKA is critical for this effect, and that circulating ouabain is an epigenetic factor that worsens the ADPKD phenotype.NEW & NOTEWORTHY This work shows that the hormone ouabain enhances the progression of autosomal dominant polycystic kidney disease (ADPKD) in vivo. Ouabain augments the size and number of renal cysts, the kidney weight to body weight ratio, and kidney fibrosis in an ADPKD mouse model. The Na+-K+-ATPase affinity for ouabain plays a critical role in these effects. In addition, these outcomes are independent of the sex of the mice.

Abstract 12449: Inhibition of the Sodium Potassium ATPase Alpha 3 (ATP1A3) Pump Function Alters Repolarization Time and is Proarrhythmic in Cardiac Myocytes

Introduction: Inhibition of Na + /K + -ATPase (NKA) is arrhythmogenic, but the specific effect of alpha 3 NKA isoform (ATP1A3) inhibition is unknown. Variants in human ATP1A3 can lead to ventricular arrhythmias, yet the mechanism remains unknown. Hypothesis: Loss of NKA function results in shortening of QT interval and increased arrhythmia susceptibility due to intracellular Na+ and Ca2+ overload. Pharmacological inhibition of sodium-calcium exchanger (NCX) can rescue ECG parameter changes and arrhythmic phenotypes by reducing Ca2+ overload in the cell. Methods: Whole-cell patch clamp action potential recordings were conducted on induced pluripotent stem cell cardiomyocytes (iPSC-CMs) from an ostensibly healthy individual to measure action potential duration (APD) and presence of delayed after depolarizations (DADs). Cells were exposed to 10 -7 M ouabain and 10 -5 M ORM-10103 (a selective inhibitor of NCX). ECGs and in vivo intracardiac electrophysiology studies were performed in mice at 12-14 weeks of age after intraperitoneal drug administration of 5 μg/g ouabain and 10 μg/g ORM-10103. Results: APD recordings of iPSC-CMs demonstrated both shortening and lengthening of APD with ouabain exposure. In both models, DADs were found in 6 of 18 cells with ouabain treatment. These findings were rescued with pretreatment using ORM-10103, as 0 of 8 cells developed DADs. On mouse ECG recordings, ouabain led to QRS and QT prolongation. During programmed electrical stimulation, no arrhythmia was noted with vehicle-treated mice. Following ouabain administration, 2 in 12 mice developed induced monomorphic ventricular tachycardia. Pretreatment of ORM-10103 in mice did not rescue QRS and QT prolongation with ouabain administration. Conclusions: Pharmacologic inhibition of ATP1A3 variably altered cardiac repolarization time in a species-specific manner, yet was associated with a substrate for triggered arrhythmias. The arrhythmogenic phenotype was rescued with inhibition of NCX in human cardiac myocytes, suggesting that NCX plays a role in the arrhythmogenic mechanism of ATP1A3. However, these effects were not seen in murine cardiac myocytes. Exploration of specifies-specific differences in NKA isoform expression and cardiac repolarization is needed.

Imipramine Can Be Effective on Depressive-Like Behaviors, but Not on Neurotrophic Factor Levels in an Animal Model for Bipolar Disorder Induced by Ouabain.

Despite possible risks of mania switching with the long-term use of antidepressants in patients with bipolar disorder (BD), these drugs may help in depressive episodes. Alterations in neurotrophic factor levels seem to be involved in the pathophysiology of BD. The present study aimed to evaluate the effect of acute treatment of imipramine on behavior and neurotrophic levels in rats submitted to the animal model for BD induced by ouabain. Wistar rats received a single intracerebroventricular (ICV) injection of artificial cerebrospinal fluid or ouabain (10-3M). Following the ICV administration, the rats were treated for 14days with saline (NaCl 0.9%, i.p.), lithium (47.5mg/kg, i.p.), or valproate (200mg/kg, i.p.). On the 13th and 14th days of treatment, the animals received an additional injection of saline or imipramine (10mg/kg, i.p.). Behavior tests were evaluated 7 and 14days after ICV injection. Adrenal gland weight and concentrations of ACTH were evaluated. Levels of neurotrophins BDNF, NGF, NT-3, and GDNF were measured in the frontal cortex and hippocampus by ELISA test. The administration of ouabain induced mania- and depressive-like behavior in the animals 7 and 14days after ICV, respectively. The treatment with lithium and valproate reversed the mania-like behavior. All treatments were able to reverse most of the depressive-like behaviors induced by ouabain. Moreover, ouabain increased HPA-axis parameters in serum and decreased the neurotrophin levels in the frontal cortex and hippocampus. All treatments, except imipramine, reversed these alterations. It can be suggested that acute administration of imipramine alone can be effective on depressive-like symptoms but not on neurotrophic factor alterations present in BD.

Chronic Ouabain Prevents Radiation-Induced Reduction in the α2 Na,K-ATPase Function in the Rat Diaphragm Muscle

The damaging effect of ionizing radiation (IR) on skeletal muscle Na,K-ATPase is an open field of research. Considering a therapeutic potential of ouabain, a specific ligand of the Na,K-ATPase, we tested its ability to protect against the IR-induced disturbances of Na,K-ATPase function in rat diaphragm muscle that co-expresses the α1 and α2 isozymes of this protein. Male Wistar rats (n = 26) were subjected to 6-day injections of vehicle (0.9% NaCl) or ouabain (1 µg/kg/day). On the fourth day of injections, rats were exposed to one-time total-body X-ray irradiation (10 Gy), or a sham irradiation. The isolated muscles were studied 72 h post-irradiation. IR decreased the electrogenic contribution of the α2 Na,K-ATPase without affecting its protein content, thereby causing sarcolemma depolarization. IR increased serum concentrations of ouabain, IL-6, and corticosterone, decreased lipid peroxidation, and changed cellular redox status. Chronic ouabain administration prevented IR-induced depolarization and loss of the α2 Na,K-ATPase electrogenic contribution without changing its protein content. This was accompanied with an elevation of ouabain concentration in circulation and with the lack of IR-induced suppression of lipid peroxidation. Given the crucial role of Na,K-ATPase in skeletal muscle performance, these findings may have therapeutic implications as countermeasures for IR-induced muscle pathology.

Depressive-like behavior accompanies neuroinflammation in an animal model of bipolar disorder symptoms induced by ouabain

IntroductionA previous study from our Laboratory showed no alteration in inflammatory parameters seven days after ouabain (OUA) administration, a Na+K+ATPase inhibitor, which was previously considered only a mania model. However, the administration of OUA in rats was recently validated as a model of bipolar disorder (BD) symptoms, demonstrating that 14 days after single intracerebroventricular (ICV) administration, OUA also induces depressive-like behavior. Therefore, it is important to investigate the long-term effect of OUA on inflammatory parameters since this mechanism seems to play a key role in BD physiopathology. MethodsAdult male Wistar rats received a single ICV administration of OUA or artificial cerebrospinal fluid (aCSF). From the fourth day after the ICV infusion, the rats received saline or Lithium (Li) for 14 days. The open-field test was performed on the 7th day after OUA. On the 14th day, locomotion was re-evaluated, and the forced swimming test (FST) was used to evaluate depressive-like behavior. Inflammatory parameters were assessed in the frontal cortex and hippocampus. ResultsOUA increased the locomotion of rats after seven days, considered a mania-like behavior. In the FST, OUA increased the time of immobility on the 14th day, considered a depressive-like behavior. Li reversed the mania-like behavior and partially reversed the depressive-like behavior. Furthermore, OUA increased the levels of interleukin (IL)-1β, IL-6, IL-10, TNF-α, and CINC-1 in the frontal cortex and hippocampus. Li treatment reverses all these inflammatory alterations. ConclusionThis study suggests that the long-term Na+K+ATPase inhibition effects induce depressive-like behavior, which was accompanied by inflammation in the BD symptoms model.

GLP-1 agonist liraglutide improves ouabain-induced mania and depressive state via GSK-3β pathway

Bipolar disorder (BD) is a severe mental illness characterized by aberrant mood changes between hypomania and mania or mixed states and depression. Metabolic changes also accompany disease progression and cause significant morbidity. Symptomatic treatment options are available, but asymptomatic patients and poor drug responders are significant problems. Based on the most common pharmacological agent that is used in the treatment, lithium and its main mechanisms of action, oxidative stress, and glycogen synthase kinase-3β (GSK-3β) signaling are extensively investigated. However, knowledge about the effects of compounds that positively affect oxidative stress and GSK-3β signaling, such as glucagon-like peptide-1 (GLP-1) mimetics, liraglutide, is still missing. Therefore, in this study, we aimed to investigate the effects of liraglutide on the ouabain-induced bipolar disease model in rats. After intracerebroventricular single dose ouabain administration, animals were treated with 100, 200, and 400 µg/kg liraglutide (s.c.) and valproic acid (200 mg/kg, i.p.) for 10 d. The locomotion and depressive states of animals were assessed by an open field, forced swimming test, and sucrose preference tests. Serum total antioxidant (TAS) and oxidant states (TOS) and glutathione, malonyl dialdehyde (MDA) levels in the brain tissue were determined. GSK-3β phosphorylation was evaluated by western blotting. Our results demonstrated that liraglutide attenuated ouabain-induced hyperlocomotion and depressive state. Additionally, liraglutide prevented oxidative stress after ouabain administration. Decreased GSK-3β phosphorylation due to the ouabain insult was alleviated by liraglutide treatment. These findings indicate that the manic and depressive-like behaviors are ameliorated by liraglutide, which exerted antioxidant action, possibly improving GSK-3β phosphorylation.

The extent of involvement of ouabain, hippocampal expression of Na+/K+-ATPase, and corticosterone/melatonin receptors ratio in modifying stress-induced behavior differs according to the stressor in context.

The aim of this study was to assess the effect of two types of stressors, regarding the extent of involvement of ouabain (OUA), hippocampal sodium/potassium ATPase (NKA) expression, and the hippocampal corticosterone receptors (CR)/melatonin receptors (MR) expression ratio, on the behavioral and cardiovascular responses and on the hippocampal cornu ammonis zone 3 (CA3) and dentate gyrus (DG). Thirty adult male Wistar albino rats aged 7-8 months were exposed to either chronic immobilization or a disturbed dark/light cycle and treated with either ouabain or vehicle. In the immobilized group, in the absence of hippocampal corticosterone (CORT) changes, rats were non-responsive to stress, despite experiencing increased pulse rate, downregulated hippocampal sodium/potassium pump, and enhanced hippocampal CR/MR expression ratio. Prolonged darkness precipitated a reduced upright attack posture, with elevated CORT against hippocampal MR downregulation. Both immobilization and, to a lesser extent, prolonged darkness stress resulted in histopathological and ultrastructural neurodegenerative changes in the hippocampus. OUA administration did not change the behavioral resilience in restrained rats, despite persistence of the underlying biochemical derangements, added to decreased CORT. On the contrary, with exposure to short photoperiods, OUA reverted the behavior towards a combative reduction of inactivity, with unvaried CR/MR and CORT, while ameliorating hippocampal neuro-regeneration, with co-existing NKA and MR repressions. Therefore, the extent of OUA, hippocampal NKA expression, and CR/MR expression, and subsequent behavioral and cardiac responses and hippocampal histopathology, differ according to the type of stressor, whether immobilization or prolonged darkness.

Role of TRPV1 channels on glycogen synthase kinase-3β and oxidative stress in ouabain-induced bipolar disease

Bipolar disorder (BD) is a multifactorial chronic and refractory disease characterized by manic, depressive, and mixed mood episodes. Although epidemiological, and pathophysiological studies demonstrated a strong correlation between bipolar disorder and oxidative stress, precise etiology is still missing. Recent studies suggested the possible role of transient receptor potential channels (TRP) in the BD but, current knowledge is limited. Therefore, the current study investigates the possible role of TRPV1 in the ouabain-induced model of BD. The model was created with intracerebroventricular single dose ouabain (10−3 M) administration. Animals were treated with capsaicin, capsazepine, and lithium for seven days. Mania and depressive-like states were investigated with open-field, sucrose preference, and elevated plus maze tests. Oxidative stress was assessed by measuring total antioxidant and oxidant states, spectrophotometrically. The phosphorylation Glycogen synthase kinase-3β (GSK-3β) evaluated by western blotting. Our results demonstrated that capsaicin dose-dependently inhibited the ouabain-induced hyperlocomotion and depression. Although capsazepine exacerbated behavioral impairment, it did not show a significant effect on the antioxidant and oxidant states, and the effects of capsazepine on behaviors were abolished by combination with capsaicin. Additionally, capsaicin potently prevented the ouabain-induced decrease in GSK-3β phosphorylation. In contrast, capsazepine potentiated ouabain-induced decrease in GSK-3β phosphorylation and combination with capsaicin, suppressed the effect of capsazepine on GSK-3β phosphorylation. The effects of TRPV1 activation on oxidative stress and mania-like behaviors in the ouabain-induced BD model might be regulated by GSK-3β phosphorylation.

Chronic Ouabain Prevents Na,K-ATPase Dysfunction and Targets AMPK and IL-6 in Disused Rat Soleus Muscle.

Sustained sarcolemma depolarization due to loss of the Na,K-ATPase function is characteristic for skeletal muscle motor dysfunction. Ouabain, a specific ligand of the Na,K-ATPase, has a circulating endogenous analogue. We hypothesized that the Na,K-ATPase targeted by the elevated level of circulating ouabain modulates skeletal muscle electrogenesis and prevents its disuse-induced disturbances. Isolated soleus muscles from rats intraperitoneally injected with ouabain alone or subsequently exposed to muscle disuse by 6-h hindlimb suspension (HS) were studied. Conventional electrophysiology, Western blotting, and confocal microscopy with cytochemistry were used. Acutely applied 10 nM ouabain hyperpolarized the membrane. However, a single injection of ouabain (1 µg/kg) prior HS was unable to prevent the HS-induced membrane depolarization. Chronic administration of ouabain for four days did not change the α1 and α2 Na,K-ATPase protein content, however it partially prevented the HS-induced loss of the Na,K-ATPase electrogenic activity and sarcolemma depolarization. These changes were associated with increased phosphorylation levels of AMP-activated protein kinase (AMPK), its substrate acetyl-CoA carboxylase and p70 protein, accompanied with increased mRNA expression of interleikin-6 (IL-6) and IL-6 receptor. Considering the role of AMPK in regulation of the Na,K-ATPase, we suggest an IL-6/AMPK contribution to prevent the effects of chronic ouabain under skeletal muscle disuse.

Ouabain induces memory impairment and alter the BDNF signaling pathway in an animal model of bipolar disorder: Cognitive and neurochemical alterations in BD model

BackgroundThe present study aims to evaluate the effects of ouabain on memory and neurotrophic parameters in the brains of rats. MethodsWistar 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. ResultsOuabain 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. ConclusionOuabain-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.

Preconditioning with ouabain reduces the neurological deficit in rats caused by compression-induced cerebral ischemia

Ischaemic brain damage is a major neurobiological and medical social problem, making experimental research of the pathogenesis of cerebral ischemia and the search for ways to minimize its consequences particularly relevant. The aim of the study was to determine the possibility of reducing the neurological deficit and functional limb asymmetry in laboratory rats through ischaemic tolerance using ouabain, a Na + /K + - ATPase inhibitor. Materials and methods. Cerebral ischemia was modeled using 20-minute focal compression of the left sensorimotor cortex in the rat brain. To induce tolerance, laboratory animals were given a single intravenous injection of 0.7 mg/kg of the Na + /K + -ATPase inhibitor ouabain 24 or 72 hours before the ischaemic event. Functional impairment was assessed with tests for neurological deficits in the limbs and a test for forelimb performance in laboratory animals. Results. Preliminary ouabain administration prevented the development of functional impairment due to compression-induced ischemia of the sensorimotor cortex, with a decrease in limb asymmetry and the severity of motor dysfunction. Conclusion. In animals, pharmacological preconditioning with ouabain increases the brain's resistance to subsequent compression-induced ischemia, preventing functional asymmetry and improving both right and left limb function. The obtained data expand the possibilities of using Na + /K + -ATPase inhibitors to treat cerebral ischemia.

P.719 Investigation of long-term effects of ouabain administration in mice, and analysis of ouabain-induced gene expression changes in a neuronal culture

The relationship between different physical parameters of various polymers (e.g., CR-39, Lexan, and PET) used as Nuclear Track Detectors (NTDs) and their intrinsic detection thresholds were investigated. A strong correlation was found between the electrical resistivity and detection thresholds of the NTDs and from that an empirical relationship between the two established. The predicted value of the detection threshold for Perspex® obtained from that empirical formula is in good agreement with its experimentally determined value.

Ouabain Protects Nephrogenesis in Rats Experiencing Intrauterine Growth Restriction and Partially Restores Renal Function in Adulthood.

Intrauterine growth restriction (IUGR) is, in general, accompanied by a reduction of the nephron number, which increases the risk of hypertension and renal dysfunction. Studies have revealed that ouabain can partially restore the number of nephrons during IUGR. However, there is limited information regarding the melioration of nephric structure and function. We used maternal malnutrition to induce an IUGR model in rats. Subsequently, we used a mini-pump to administer ouabain to IUGR rats during pregnancy. Male offspring were divided randomly into two groups. One group was fed a normal diet, whereas the other was fed an isocaloric 8% high-salt diet. Maternal malnutrition led to a reduction in the birth weight and number of nephrons in offspring. At the end of a 40-week follow-up period, offspring from the IUGR group had high blood pressure and abnormal excretion of urinary protein; these parameters were exacerbated in offspring fed a high-salt diet. However, ouabain administration during pregnancy could partially restore the number of nephrons in IUGR offspring, normalize blood pressure, and reduce urinary protein excretion, even when challenged with a high-salt diet. Pathology findings revealed that IUGR, particularly following feeding of a high-salt diet, damaged the ultrastructure of glomeruli, but these harmful effects were ameliorated in offspring treated with ouabain. Collectively, our data suggest that ouabain could rescue nephrogenesis in IUGR newborns and protect (at least in part) the structure and function of the kidney during adulthood even when encountering unfavorable environmental challenges in subsequent life.

Na+/K+-ATPase α4 regulates sperm hyperactivation while Na+/K+-ATPase α1 regulates basal motility in hamster spermatozoa

Recently, it was reported that hamster sperm hyperactivation is regulated by extracellular Na+. Two types of catalytic Na+/K+-ATPase (NKA) α subunits (α1 and α4) are present in spermatozoa. In this work, the contribution of these NKA subunits to the regulation of hamster sperm hyperactivation was investigated using the specific inhibitor ouabain. When 10−6 M ouabain was added to the modified Tyrode’s albumin lactate pyruvate medium (mTALP) medium, hyperactivation was significantly inhibited, whereas 10−5-10−4 M ouabain was needed to significantly reduce the amount of motile spermatozoa. When a more detailed analysis of flagellar movement was performed, 10−6 M ouabain suppressed the hyperactivation-associated change in the patterns of flagellar motion without affecting the sliding velocity of microtubules. Since 10−6 M ouabain specifically inhibits the α4 subunit while 10−5-10−4 M ouabain inhibits both the α1 and α4 subunits, these results suggest the α1 subunit is necessary for the maintenance of motility while the α4 subunit is necessary for the hyperactivation-associated change in flagellar movement. Ouabain did not inhibit tyrosine phosphorylation, and activation of tyrosine phosphorylation-dependent signaling had no effect on the inhibition of hyperactivation by ouabain. The immediate recovery of hyperactivation was observed when ouabain was washed out after a 3-h incubation. whereas the administration of ouabain after the onset of hyperactivation significantly inhibited hyperactivation. These results suggest ouabain inhibited hyperactivation in a manner that was independent of time-requiring phosphorylation-mediated signaling.

Skeletal Muscle Na,K-ATPase as a Target for Circulating Ouabain.

While the role of circulating ouabain-like compounds in the cardiovascular and central nervous systems, kidney and other tissues in health and disease is well documented, little is known about its effects in skeletal muscle. In this study, rats were intraperitoneally injected with ouabain (0.1–10 µg/kg for 4 days) alone or with subsequent injections of lipopolysaccharide (1 mg/kg). Some rats were also subjected to disuse for 6 h by hindlimb suspension. In the diaphragm muscle, chronic ouabain (1 µg/kg) hyperpolarized resting potential of extrajunctional membrane due to specific increase in electrogenic transport activity of the α2 Na,K-ATPase isozyme and without changes in α1 and α2 Na,K-ATPase protein content. Ouabain (10–20 nM), acutely applied to isolated intact diaphragm muscle from not injected rats, hyperpolarized the membrane to a similar extent. Chronic ouabain administration prevented lipopolysaccharide-induced (diaphragm muscle) or disuse-induced (soleus muscle) depolarization of the extrajunctional membrane. No stimulation of the α1 Na,K-ATPase activity in human red blood cells, purified lamb kidney and Torpedo membrane preparations by low ouabain concentrations was observed. Our results suggest that skeletal muscle electrogenesis is subjected to regulation by circulating ouabain via the α2 Na,K-ATPase isozyme that could be important for adaptation of this tissue to functional impairment.

Intracerebroventricular injection of ouabain causes mania-like behavior in mice through D2 receptor activation

Intracerebroventricular (ICV) administration of ouabain, an inhibitor of the Na, K-ATPase, is an approach used to study the physiological functions of the Na, K-ATPase and cardiotonic steroids in the central nervous system, known to cause mania-like hyperactivity in rats. We describe a mouse model of ouabain-induced mania-like behavior. ICV administration of 0.5 µl of 50 µM (25 pmol, 14.6 ng) ouabain into each lateral brain ventricle results in increased locomotor activity, stereotypical behavior, and decreased anxiety level an hour at minimum. Fast-scan cyclic voltammetry showed that administration of 50 µM ouabain causes a drastic drop in dopamine uptake rate, confirmed by elevated concentrations of dopamine metabolites detected in the striatum 1 h after administration. Ouabain administration also caused activation of Akt, deactivation of GSK3β and activation of ERK1/2 in the striatum of ouabain-treated mice. All of the abovementioned effects are attenuated by haloperidol (70 µg/kg intraperitoneally). Observed effects were not associated with neurotoxicity, since no dystrophic neuron changes in brain structures were demonstrated by histological analysis. This newly developed mouse model of ouabain-induced mania-like behavior could provide a perspective tool for studying the interactions between the Na,K-ATPase and the dopaminergic system.

Low Ouabain Doses and AMP-Activated Protein Kinase as Factors Supporting Electrogenesis in Skeletal Muscle.

Many motor disorders are associated with depolarization of the membrane of skeletal muscle fibers due to the impaired functioning of Na,K-ATPase. Here, we studied the role of ouabain (specific Na,K-ATPase ligand) and AMP-activated protein kinase (key regulator of muscle metabolism) in the maintenance of muscle electrogenesis; the levels of these endogenous factors are directly related to the motor activity. After 4-day intraperitoneal administration of ouabain (1 µg/kg daily), a hyperpolarization of sarcolemma was registered in isolated rat diaphragm muscles due to an increase in the electrogenic activity of Na,K-ATPase. In acute experiments, addition of nanomolar ouabain concentrations to the bathing solution resulted in the muscle membrane hyperpolarization within 15 min. The effect of ouabain reversed to membrane depolarization with the increase in the external potassium concentration. It is possible that Na,K-ATPase activation by ouabain may be regulated by such factors as specific subcellular location, interaction with molecular partners, and changes in the ionic balance. Preventive administration of the AMP-activated protein kinase activator AICAR (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside; 400 mg/kg body weight daily for 7 days) in chronic experiments resulted in the stabilization of the endplate structure and abolishment of depolarization of the rat soleus muscle membrane caused by the motor activity cessation. The obtained data can be useful for creating approaches for correction of muscle dysfunction, especially at the early stages, prior to the development of muscle atrophy.

Effects of Aspirin in Rats With Ouabain Intracerebral Treatment—Possible Involvement of Inflammatory Modulation?

Bipolar disorder (BD) is a chronic and refractory disease with high probability of morbidity and mortality. Although epidemiological studies have established a strong association between BD and immune dysfunction, the precise etiology is still debatable, and the underpinning mechanism remains poorly investigated and understood. In the present study, manic-like symptoms of BD were induced in rats after intracerebroventricular administration of ouabain. Aspirin, a commonly used anti-inflammatory agent, was used to treat the induced manic-like symptoms and inflammation. Concentrations of a spectrum of inflammatory cytokines were examined by enzyme-linked immunosorbent assay in both plasma and brain tissues, and expression of Toll-like receptors 3 and 4 were determined in rat brains. Locomotor activity was monitored with open-field test to assess the effects of ouabain challenge and to evaluate the treatment efficacy of aspirin. Ouabain administration recapitulated many mania-like features such as increased stereotypic counts, traveling distance in open-field test, and decreased expression of brain-derived neurotrophic factor, interferon gamma, and Toll-like receptor 3, which were frequently found in patients with BD. These abnormalities could be partially reversed by aspirin. Our findings suggest that aspirin could be used as a promising adjunctive therapy for BD.

Na+/K+-ATPase is involved in the regulation of food intake in rainbow trout but apparently not through brain glucosensing mechanisms

To assess the hypothesis that Na+/K+-ATPase (NKA) is involved in the central regulation of food intake in fish, we observed in a first experiment with rainbow trout (Oncorhynchus mykiss) that intracerebroventricular (ICV) treatment with ouabain decreased food intake. We hypothesized that this effect relates to modulation of glucosensing mechanisms in brain areas (hypothalamus, hindbrain, and telencephalon) involved in food intake control. Therefore, we evaluated in a second experiment, the effect of ICV administration of ouabain, in the absence or in the presence of glucose, on NKA activity, mRNA abundance of different NKA subunits, parameters related to glucosensing, transcription factors, and appetite-related neuropeptides in brain areas involved in the control of food intake. NKA activity and mRNA abundance of nkaα1a and nkaα1c in brain were inhibited by ouabain treatment and partially by glucose. The anorectic effect of ouabain is opposed to the orexigenic effect reported in mammals. The difference might relate to the activity of glucosensing as well as downstream mechanisms involved in food intake regulation. Ouabain inhibited glucosensing mechanisms, which were activated by glucose in hypothalamus and telencephalon. Transcription factors and neuropeptides displayed responses comparable to those elicited by glucose when ouabain was administered alone, but not when glucose and ouabain were administered simultaneously. Ouabain might therefore affect other processes, besides glucosensing mechanisms, generating changes in membrane potential and/or intracellular pathways finally modulating transcription factors and neuropeptide mRNA abundance leading to modified food intake.