Primary amino acid sequences of decapod (Na+, K+)-ATPase provide evolutionary insights into osmoregulatory mechanisms.

Decapod Crustacea exhibit a marine origin, but many taxa have occupied environments ranging from brackish to fresh water and terrestrial habitats, overcoming their inherent osmotic challenges. Osmotic and ionic regulation is achieved by the gill epithelia, driven by two active ATP-hydrolyzing ion transporters, the basal (Na+, K+)-ATPase and the apical V(H+)-ATPase. The kinetic characteristic of gill (Na+, K+)-ATPase and the mRNA expression of its α subunit have been widely studied in various decapod species under different salinity challenges. However, the evolution of the primary structure has not been explored, especially considering the functional modifications associated with decapod phylogeny. Here, we proposed a model for the topology of the decapod α subunit, identifying the sites and motifs involved in its function and regulation, as well as the patterns of its evolution assuming a decapod phylogeny. We also examined both the amino acid substitutions and their functional implications within the context of biochemical and physiological adaptation. The α-subunit of decapod crustaceans shows greater conservation (∼94% identity) compared to the ß-subunit (∼40%). While the binding sites for ATP and modulators are conserved in the decapod enzyme, the residues involved in the α-ß interaction are only partially conserved. In the phylogenetic context of the complete sequence of (Na+, K+)-ATPase α-subunit, most substitutions appear to be characteristic of the entire group, with specific changes for different subgroups, especially among brachyuran crabs. Interestingly, there was no consistent separation of α-subunit partial sequences related to habitat, suggesting that the convergent evolution for freshwater or terrestrial modes of life is not correlated with similar changes in the enzyme's primary amino acid sequence.

(Na+, K+)- ATPase kinetics in Macrobrachium pantanalense: highlighting intra- and interspecific variation within the Macrobrachium amazonicum complex.

The Macrobrachium amazonicum complex is composed of at least the Macrobrachium amazonicum and Macrobrachium pantanalense species, with the latter described from specimens originally identified as part of an endemic M. amazonicum population in the Brazilian Pantanal region. While there may be a reproductive barrier between these two Macrobrachium species, both are phylogenetically close, with small genetic distance. However, there is currently no available biochemical information of Macrobrachium pantanalense (Na+, K+)-ATPase. Here, we report the kinetic characteristics of the gill (Na+, K+)-ATPase in two populations of M. pantanalense from Baiazinha Lagoon (Miranda, MS, Brazil) and Araguari River (Uberlândia, MG, Brazil), and compare them with Macrobrachium amazonicum populations from the Paraná-Paraguay River Basin. (Na+, K+)-ATPase activities were 67.9 ± 3.4 and 93.3 ± 4.1 nmol Pi min-1 mg-1 protein for the Baiazinha Lagoon and Araguari River populations, respectively. Two ATP hydrolyzing sites were observed for the Araguari River population while a single ATP site was observed for the Baiazinha Lagoon shrimps. Compared to the Araguari River population, a 3-fold greater apparent affinity for Mg2+ and Na+ was estimated for the Baiazinha Lagoon population, but no difference in K+ affinity and ouabain inhibition was seen. The kinetic differences observed in the gill (Na+, K+)-ATPase between the two populations of M. pantanalense, compared with those of various M. amazonicum populations, highlight interspecific divergence within the Macrobrachium genus, now examined from a biochemical perspective.

Zika virus: Antiviral immune response, inflammation, and cardiotonic steroids as antiviral agents.

Zika virus (ZIKV) is a mosquito-borne virus first reported from humans in Nigeria in 1954. The first outbreak occurred in Micronesia followed by an outbreak in French Polynesia and another in Brazil when the virus was associated with numerous cases of severe neurological manifestations such as Guillain-Barre syndrome in adults and congenital zika syndrome in fetuses, particularly congenital microcephaly. Innate immunity is the first line of defense against ZIKV through triggering an antiviral immune response. Along with innate immune responses, a sufficient balance between anti- and pro-inflammatory cytokines and the amount of these cytokines are triggered to enhance the antiviral responses. Here, we reviewed the complex interplay between the mediators and signal pathways that coordinate antiviral immune response and inflammation as a key to understanding the development of the underlying diseases triggered by ZIKV. In addition, we summarize current and new therapeutic strategies for ZIKV infection, highlighting cardiotonic steroids as antiviral drugs for the development of this agent.

Ouabain's Influence on TRPV4 Channels of Epithelial Cells: An Exploration of TRPV4 Activity, Expression, and Signaling Pathways.

Ouabain, a substance originally obtained from plants, is now classified as a hormone because it is produced endogenously in certain animals, including humans. However, its precise effects on the body remain largely unknown. Previous studies have shown that ouabain can influence the phenotype of epithelial cells by affecting the expression of cell-cell molecular components and voltage-gated potassium channels. In this study, we conducted whole-cell clamp assays to determine whether ouabain affects the activity and/or expression of TRPV4 channels. Our findings indicate that ouabain has a statistically significant effect on the density of TRPV4 currents (dITRPV4), with an EC50 of 1.89 nM. Regarding treatment duration, dITRPV4 reaches its peak at around 1 h, followed by a subsequent decline and then a resurgence after 6 h, suggesting a short-term modulatory effect related to on TRPV4 channel activity and a long-term effect related to the promotion of synthesis of new TRPV4 channel units. The enhancement of dITRPV4 induced by ouabain was significantly lower in cells seeded at low density than in cells in a confluent monolayer, indicating that the action of ouabain depends on intercellular contacts. Furthermore, the fact that U73122 and neomycin suppress the effect caused by ouabain in the short term suggests that the short-term induced enhancement of dITRPV4 is due to the depletion of PIP2 stores. In contrast, the fact that the long-term effect is inhibited by PP2, wortmannin, PD, FR18, and IKK16 suggests that cSrc, PI3K, Erk1/2, and NF-kB are among the components included in the signaling pathways.

Ouabain Induces Transcript Changes and Activation of RhoA/ROCK Signaling in Cultured Epithelial Cells (MDCK).

Ouabain, an organic compound with the ability to strengthen the contraction of the heart muscle, was originally derived from plants. It has been observed that certain mammalian species, including humans, naturally produce ouabain, leading to its classification as a new type of hormone. When ouabain binds to Na+/K+-ATPase, it elicits various physiological effects, although these effects are not well characterized. Previous studies have demonstrated that ouabain, within the concentration range found naturally in the body (10 nmol/L), affects the polarity of epithelial cells and their intercellular contacts, such as tight junctions, adherens junctions, and gap junctional communication. This is achieved by activating signaling pathways involving cSrc and Erk1/2. To further investigate the effects of ouabain within the hormonally relevant concentration range (10 nmol/L), mRNA-seq, a high-throughput sequencing technique, was employed to identify differentially expressed transcripts. The discovery that the transcript encoding MYO9A was among the genes affected prompted an exploration of whether RhoA and its downstream effector ROCK were involved in the signaling pathways through which ouabain influences cell-to-cell contacts in epithelial cells. Supporting this hypothesis, this study reveals the following: (1) Ouabain increases the activation of RhoA. (2) Treatment with inhibitors of RhoA activation (Y27) and ROCK (C3) eliminates the enhancing effect of ouabain on the tight junction seal and intercellular communication via gap junctions. These findings further support the notion that ouabain acts as a hormone to emphasize the epithelial phenotype.

Eugenol delays the onset of ouabain-induced ventricular cardiac arrhythmias in guinea pigs.

Eugenol is an aromatic compound used in the manufacture of medicines, perfumes, cosmetics and as an anaesthetic due to the ability of the drug to block the neuronal isoform of voltage-gated Na+ channels (NaV ). Some arrhythmias are associated with gain of function in the sodium current (INa ) found in cardiomyocytes, and antiarrhythmic sodium channel blockers are commonly used in the clinical practice. This study sought to elucidate the potential mechanisms of eugenol's protection in the arrhythmic model of ouabain-induced arrhythmias in guinea pig heart. Ex vivo arrhythmias were induced using 50 µM of ouabain. The antiarrhythmic properties of eugenol were evaluated in the ex vivo heart preparation and isolated ventricular cardiomyocytes. The compound's effects on cardiac sodium current and action potential using the patch-clamp technique were evaluated. In all, eugenol decreased the ex vivo cardiac arrhythmias induced by ouabain. Furthermore, eugenol showed concentration dependent effect upon peak INa , left-shifted the stationary inactivation curve and delayed the recovery from inactivation of the INa . All these aspects are considered to be antiarrhythmic. Our findings demonstrate that eugenol has antiarrhythmic activity, which may be partially explained by the ability of eugenol to change de biophysical properties of INa of cardiomyocytes.

Folic acid does not have an anti-manic effect but protect the brain against oxidative stress in an animal model of mania induced by ouabain.

BACKGROUND: Bipolar disorder (BD) is a complex and severe mental disorder that affects 1-3 % of the world population. Studies have suggested the involvement of oxidative stress in the physiopathology of this psychiatry disorder. Folic acid (FA), a vitamin from the B complex, is a nutraceutical that has recently been researched as a possible treatment for BD since folate is reduced in patients with the disorder. The present study aimed to evaluate the effects of lithium (Li) and FA on behavioral changes and oxidative stress parameters in an animal model of mania induced by ouabain (OUA). METHODS: Wistar rats received a single intracerebroventricular (ICV) injection of OUA or artificial cerebrospinal fluid (aCSF). From the day following ICV injection, the rats were treated for seven days with gavage injections of Li (47.5 mg/kg/mL), FA (50 mg/kg/mL), or water (1 mL/kg). On the 7th day after OUA injection, locomotor activity was measured using the open-field test. In addition, the oxidative stress parameters were evaluated in rats' frontal cortex, striatum, and hippocampus. RESULTS: OUA induced mania-like behavior and oxidative stress in rats' brains, but Li could reverse these alterations. FA did not affect behavior parameters; however, it presents an antioxidant effect on the brain structures evaluated. LIMITATIONS: The study was only evaluated male rats and ICV injection is an invasive procedure. CONCLUSION: These results indicate that even though FA has an effect against the oxidative stress induced by OUA, this effect was not strong enough to interfere with behavior parameters.

Ouabain Reverts CUS-Induced Disruption of the HPA Axis and Avoids Long-Term Spatial Memory Deficits.

Ouabain (OUA) is a cardiotonic steroid that modulates Na+, K+ -ATPase activity. OUA has been identified as an endogenous substance that is present in human plasma, and it has been shown to be associated with the response to acute stress in both animals and humans. Chronic stress is a major aggravating factor in psychiatric disorders, including depression and anxiety. The present work investigates the effects of the intermittent administration of OUA (1.8 µg/kg) during the chronic unpredictable stress (CUS) protocol in a rat's central nervous system (CNS). The results suggest that the intermittent OUA treatment reversed CUS-induced HPA axis hyperactivity through a reduction in (i) glucocorticoids levels, (ii) CRH-CRHR1 expression, and by decreasing neuroinflammation with a reduction in iNOS activity, without interfering with the expression of antioxidant enzymes. These changes in both the hypothalamus and hippocampus may reflect in the rapid extinction of aversive memory. The present data demonstrate the ability of OUA to modulate the HPA axis, as well as to revert CUS-induced long-term spatial memory deficits.

Effect of Ouabain on Glutamate Transport in the Hippocampus of Rats with LPS-Induced Neuroinflammation.

A lipopolysaccharide (LPS)-induced neuroinflammation rat model was used to study the effects of ouabain (OUA) at low concentrations, which can interact with the Na,K-ATPase, causing the modulation of intracellular signalling pathways in the Central Nervous System. Our study aimed to analyse the effects of OUA on glutamate transport in the hippocampus of rats with LPS-induced neuroinflammation. Adult male Wistar rats were divided into four groups: OUA (1.8 µg/kg), saline (CTR), LPS (200 µg/kg), and OUA + LPS (OUA 20 min before LPS). The animals were sacrificed after 2 h, and the hippocampus was collected for analysis. After treatment, we determined the activities of Na,K-ATPase and glutamine synthetase (GS). In addition, expression of the α1, α2, and α3 isoforms of Na,K-ATPase and the glutamate transporters, EAAT1 and EAAT2, were also analysed. Treatment with OUA caused a specific increase in the α2 isoform expression (~20%), whereas LPS decreased its expression (~22%), and treatment with OUA before LPS prevented the effects of LPS. Moreover, LPS caused a decrease of approximately 50% in GS activity compared with that in the CTR group; however, OUA pre-treatment attenuated this effect of LPS. Notably, it was found that treatment with OUA caused an increase in the expression of EAAT1 (~30%) and EAAT2 (~25%), whereas LPS caused a decrease in the expression of EAAT1 (~23%) and EAAT2 (~25%) compared with that in the CTR group. When treated with OUA, the effects of LPS were abrogated. In conclusion, the OUA pre-treatment abolished the effect caused by LPS, suggesting that this finding may be related to the restoration of the interaction between FXYD2 and the studied membrane proteins.

Ouabain Effects on Human Anaplastic Thyroid Carcinoma 8505C Cells.

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

Consequences of the Lack of TNFR1 in Ouabain Response in the Hippocampus of C57BL/6J Mice.

Ouabain is a cardiac glycoside that has a protective effect against neuroinflammation at low doses through Na+/K+-ATPase signaling and that can activate tumor necrosis factor (TNF) in the brain. TNF plays an essential role in neuroinflammation and regulates glutamate receptors by acting on two different receptors (tumor necrosis factor receptor 1 [TNFR1] and TNFR2) that have distinct functions and expression. The activation of constitutively and ubiquitously expressed TNFR1 leads to the expression of pro-inflammatory cytokines. Thus, this study aimed to elucidate the effects of ouabain in a TNFR1 knockout (KO) mouse model. Interestingly, the hippocampus of TNFR1 KO mice showed a basal increase in both TNFR2 membrane expression and brain-derived neurotrophic factor (BDNF) release, suggesting a compensatory mechanism. Moreover, ouabain activated TNF-α-converting enzyme/a disintegrin and metalloprotease 17 (TACE/ADAM17), decreased N-methyl-D-aspartate (NMDA) receptor subunit 2A (NR2A) expression, and induced anxiety-like behavior in both genotype animals, independent of the presence of TNFR1. However, ouabain induced an increase in interleukin (IL)-1ß in the hippocampus, a decrease in IL-6 in serum, and an increase in NMDA receptor subunit 1 (NR1) only in wild-type (WT) mice, indicating that TNFR1 or TNFR2 expression may be important for some effects of ouabain. Collectively, our results indicate a connection between ouabain signaling and TNFR1, with the effect of ouabain partially dependent on TNFR1.

Ouabain-Induced Changes in the Expression of Voltage-Gated Potassium Channels in Epithelial Cells Depend on Cell-Cell Contacts.

Ouabain is a cardiac glycoside, initially isolated from plants, and currently thought to be a hormone since some mammals synthesize it endogenously. It has been shown that in epithelial cells, it induces changes in properties and components related to apical-basolateral polarity and cell-cell contacts. In this work, we used a whole-cell patch clamp to test whether ouabain affects the properties of the voltage-gated potassium currents (Ik) of epithelial cells (MDCK). We found that: (1) in cells arranged as mature monolayers, ouabain induced changes in the properties of Ik; (2) it also accelerated the recovery of Ik in cells previously trypsinized and re-seeded at confluence; (3) in cell-cell contact-lacking cells, ouabain did not produce a significant change; (4) Na+/K+ ATPase might be the receptor that mediates the effect of ouabain on Ik; (5) the ouabain-induced changes in Ik required the synthesis of new nucleotides and proteins, as well as Golgi processing and exocytosis, as evidenced by treatment with drugs inhibiting those processes; and (5) the signaling cascade included the participation of cSrC, PI3K, Erk1/2, NF-κB and ß-catenin. These results reveal a new role for ouabain as a modulator of the expression of voltage-gated potassium channels, which require cells to be in contact with themselves.

Effect of ouabain on calcium signaling in rodent brain: A systematic review of in vitro studies.

The Na+/K+-ATPase is an integral membrane ion pump, essential to maintaining osmotic balance in cells in the presence of cardiotonic steroids; more specifically, ouabain can be an endogenous modulator of the Na+/K+-ATPase. Here, we conducted a systematic review of the in vitro effects of cardiotonic steroids on Ca2+ in the brain of rats and mice. Methods: The review was carried out using the PubMed, Virtual Health Library, and EMBASE databases (between 12 June 2020 and 30 June 2020) and followed the guidelines described in the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA). Results: in total, 829 references were identified in the electronic databases; however, only 20 articles were considered, on the basis of the inclusion criteria. The studies demonstrated the effects of ouabain on Ca2+ signaling in synaptosomes, brain slices, and cultures of rat and mouse cells. In addition to the well-known cytotoxic effects of high doses of ouabain, resulting from indirect stimulation of the reverse mode of the Na+/Ca2+ exchanger and increased intracellular Ca2+, other effects have been reported. Ouabain-mediated Ca2+ signaling was able to act increasing cholinergic, noradrenergic and glutamatergic neurotransmission. Furthermore, ouabain significantly increased intracellular signaling molecules such as InsPs, IP3 and cAMP. Moreover treatment with low doses of ouabain stimulated myelin basic protein synthesis. Ouabain-induced intracellular Ca2+ increase may promote the activation of important cell signaling pathways involved in cellular homeostasis and function. Thus, the study of the application of ouabain in low doses being promising for application in neurological diseases. Systematic Review Registration: https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42020204498, identifier CRD42020204498.

Neuroinflammation and Neutrophils: Modulation by Ouabain.

Cardiotonic steroids are natural compounds that present many physiological and pharmacological functions. They bind Na+/K+-ATPase (NKA) modifying cellular ion concentration and trigger cell signaling mechanisms without altering ion balance. These steroids are known to modulate some immune responses, including cytokine production, neutrophil migration, and inflammation (peripherally and in the nervous system). Inflammation can occur in response to homeostasis perturbations and is related to the development of many diseases, including immune-mediated diseases and neurodegenerative disorders. Considering the neutrophils role in the general neuroinflammatory response and that these cells can be modulated by cardiac steroids, this work aims to review the possible regulation of neutrophilic neuroinflammation by the cardiac steroid ouabain.

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

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

Ouabain-Na+/K+-ATPase Signaling Regulates Retinal Neuroinflammation and ROS Production Preventing Neuronal Death by an Autophagy-Dependent Mechanism Following Optic Nerve Axotomy In Vitro.

Ouabain is a classic Na+K+ATPase ligand and it has been described to have neuroprotective effects on neurons and glial cells at nanomolar concentrations. In the present work, the neuroprotective and immunomodulatory potential of ouabain was evaluated in neonatal rat retinal cells using an optic nerve axotomy model in vitro. After axotomy, cultured retinal cells were treated with ouabain (3 nM) at different periods. The levels of important inflammatory receptors in the retina such as TNFR1/2, TLR4, and CD14 were analyzed. We observed that TNFR1, TLR4, and CD14 were decreased in all tested periods (15 min, 45 min, 24 h, and 48 h). On the other hand, TNFR2 was increased after 24 h, suggesting an anti-inflammatory potential for ouabain. Moreover, we showed that ouabain also decreased Iba-1 (microglial marker) density. Subsequently, analyses of retrograde labeling of retinal ganglion cells (RGC) were performed after 48 h and showed that ouabain-induced RGC survival depends on autophagy. Using an autophagy inhibitor (3-methyladenine), we observed a complete blockage of the ouabain effect. Western blot analyses showed that ouabain increases the levels of autophagy proteins (LC3 and Beclin-1) coupled to p-CREB transcription factor and leads to autophagosome formation. Additionally, we found that the ratio of cleaved/pro-caspase-3 did not change after ouabain treatment; however, p-JNK density was enhanced. Also, ouabain decreased reactive oxygen species production immediately after axotomy. Taken together, our results suggest that ouabain controls neuroinflammation in the retina following optic nerve axotomy and promotes RGC neuroprotection through activation of the autophagy pathway.

Effects of renal denervation on endogenous ouabain in spontaneously hypertensive rats

Purpose: To investigate the role of renal denervation (RDN) on endogenous ouabain (EO) secretion in spontaneously hypertensive rats (SHR). Methods: Sixteen 12-week-old male SHR were randomly separated into the renal denervation group (RDNX group) and sham operation group (sham group), and eight age-matched Wistar Kyoto rats (WKY) were served as control group. EO concentrations, the Na+- K+-ATPaseactivity, and the expression of Na+-K+-ATPase were assessed. Results: EO levels in serum, kidneys and hypothalamus of sham group were higher than in RDNX group (p < 0.05). Renal Na+-K+-ATPase activity subjected to denervation surgery showed significantly reduction when compared with the sham groups (p < 0.05). A positive correlation existed between norepinephrine (NE) content and Na+-K+-ATPase activity in the kidney (r2 = 0.579). Renal Na+-K+-ATPase α1 subunit mRNA expression was down-regulated in the RDNX group compared with the sham group (P < 0.05), while renal Na+-K+-ATPase α1 subunit mRNA expression was no statistical significance between the groups (P = 0.63). Immunohistochemical analysis showed that there were significant differences in the renal expression of Na+-K+-ATPasebetween the three groups (P < 0.05). Conclusions: These experiments demonstrate that RDN exerted an anti-hypertensive effect with reduction of EO levels and Na+-K+-ATPase activity and Na+-K+-ATPase α1 subunit expression of kidney in SHR.

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.

Ouabain Enhances Gap Junctional Intercellular Communication by Inducing Paracrine Secretion of Prostaglandin E2.

Ouabain is a cardiac glycoside that has been described as a hormone, with interesting effects on epithelial physiology. We have shown previously that ouabain induces gap junctional intercellular communication (GJIC) in wild, sensitive cells (MDCK-S), but not in cells that have become insensitive (MDCK-I) by modifying their Na+-K+-ATPase. We have also demonstrated that prostaglandin E2 (PGE2) is able to induce increased GJIC by a mechanism other than ouabain, that does not depend on Na+-K+-ATPase. In this work we show, by dye transfer assays, that when MDCK-S and MDCK-I are randomly mixed, to form monolayers, the latter stablish GJIC, because of stimulation by a compound released to the extracellular media, by MDCK-S cells, after treatment with ouabain, as evidenced by the fact that monolayers of only MDCK-I cells, treated with a conditioned medium (CM) that is obtained after incubation of MDCK-S monolayers with ouabain, significantly increase their GJIC. The further finding that either (1) pre-treatment with COX-2 inhibitors or (2) addition to CM of antagonists of EP2 receptor abolish CM's ability to induce GJIC in MDCK-I monolayers indicate that PGE2 is the GJIC-inducing compound. Therefore, these results indicate that, in addition to direct stimulation, mediated by Na+-K+-ATPase, ouabain enhances GJIC indirectly through the paracrine production of PGE2.

Chronic cyclooxygenase-2 inhibition prevents the worsening of hypertension and endothelial dysfunction induced by ouabain in resistance arteries of spontaneously hypertensive rats.

AIM: Previous studies raise cyclooxygenase (COX) activation as a possible mechanism involved in the pathophysiology of ouabain-induced hypertension. We hypothesized that inhibition of COX-2 activity might prevent ouabain-induced vascular dysfunction and worsening of hypertension in spontaneously hypertensive rats (SHR). METHODS: SHR were exposed to ouabain or vehicle and treated or not with the selective COX-2 inhibitor nimesulide for 5 weeks. Systolic blood pressure was measured by plethysmography. Vascular reactivity by wire myograph and protein expression by Western-blot were assessed in mesenteric resistance arteries (MRA) of groups. Thromboxane A2 (TXA2) production by ELISA was evaluated in MRA supernatants of groups. RESULTS: Noradrenaline-induced maximal contraction (Emax) was greater in MRA from SHR receiving ouabain than those of vehicle group. In situ inhibition of COX-2, TXA2 synthase, or TP receptor reduced the Emax to noradrenaline in MRA of ouabain to vehicle levels. TXA2 production was higher in ouabain than in vehicle group. Ouabain enhanced expression of cytoplasmic tyrosine kinase Src (c-Src)/ERK1/2/COX-2/TXA2 synthase/TP receptor in SHR MRA, but did not change NFkB/iKB ratio. Anticontractile effect of nitric oxide (NO) was smaller in MRA from ouabain- than vehicle-treated SHR, as well as eNOS and nNOS expression. Nimesulide co-treatment prevented the ouabain-induced worsening of hypertension and noradrenaline MRA hypercontractility in SHR. CONCLUSION: Ouabain worsen hypertension and induce MRA hypercontractility in SHR associated with upregulated c-Src/ERK1/2/COX-2/TXA2 synthase/TXA2/TP receptor axis. These effects were prevented by COX-2 inhibition.