Ouabain Research Articles

Ouabain attenuates oxidative stress and modulates lipid composition in hippocampus of rats in lipopolysaccharide-induced hypocampal neuroinflammation in rats.

Our study aimed to analyze the effect of ouabain (OUA) administration on lipopolysaccharide (LPS)-induced changes in hippocampus of rats. Oxidative parameters were analyzed in Wistar rats after intraperitoneal injection of OUA (1.8 µg/kg), LPS (200 µg/kg), or OUA plus LPS or saline. To reach our goal, activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX), in addition to levels of reduced glutathione (GSH), protein carbonyl (PCO) and lipid peroxidation (LPO) were evaluated. We also analyzed the membrane lipid profile and some important lipids for the nervous system, such as phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidic acid and sphingomyelin. The group that received only LPS showed increased oxidative stress, as evidenced by an increase in LPO (about twice), PCO (about three times) levels, and CAT activity (80%). Conversely, administration of LPS decreased GSH levels (55%), and GPx activity (30%), besides a reduction in the amount of PI (60%) and PC (45%). By other side, OUA alone increased the amount of PI (45%), PE (85%), and PC (70%). All harmful effects recorded were attenuated by OUA, suggesting a protective effect against LPS-induced oxidative stress. The relevance of our results extends beyond changes in oxidative parameters induced by LPS, because nanomolar doses of OUA may be useful in neurodegenerative models. Other studies on other cardenolides and substances related issues, as well as the development of new molecules derived from OUA, could also be useful in general oxidative and/or cellular stress, a condition favoring the appearance of neuronal pathologies.

Ouabain increases neuronal branching in hippocampus and improves spatial memory

Previous research shows Ouabain (OUA) to bind Na, K-ATPase, thereby triggering a number of signaling pathways, including the transcription factors NFᴋB and CREB. These transcription factors play a key role in the regulation of BDNF and WNT-β-catenin signaling cascades, which are involved in neuroprotection and memory regulation. This study investigated the effects of OUA (10 nM) in the modulation of the principal signaling pathways involved in morphological plasticity and memory formation in the hippocampus of adult rats. The results show intrahippocampal injection of OUA 10 nM to activate the Wnt/β-Catenin signaling pathway and to increase CREB/BDNF and NFᴋB levels. These effects contribute to important changes in the cellular microenvironment, resulting in enhanced levels of dendritic branching in hippocampal neurons, in association with an improvement in spatial reference memory and the inhibition of long-term memory extinction.

Inhibition of GSK-3β on Behavioral Changes and Oxidative Stress in an Animal Model of Mania.

The present study evaluated the effects of AR-A014418 on behavioral and oxidative stress parameters of rats submitted to the animal model of mania induced by ouabain (OUA). Wistar rats were submitted to stereotaxic surgery and received a single intracerebroventricular (ICV) injection of artificial cerebrospinal fluid (aCSF), OUA, or AR-A014418. After 7days, the animals were submitted to open-field test. After behavioral analysis, the brains were dissected in frontal cortex and hippocampus to the evaluation of oxidative stress. The OUA induced manic-like behavior in rats, which was reversed by AR-A014418 treatment. The ICV administration of OUA increases the levels of superoxide in submitochondrial particles, lipid hydroperoxide (LPH), 4-hydroxynonenal (4-HNE), 8-isoprostane, protein carbonyl, 3-nitrotyrosine, and activity of superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione reductase (GR) in both structures evaluated. In general, the treatment with AR-A014418 reversed these effects of OUA on the submitochondrial particles, LPH, 4-HNE, 8-isoprostane, protein carbonyl, 3-nitrotyrosine levels, and SOD activity. Furthermore, the injection of OUA decreased the catalase activity, and AR-A014418 promoted an increase in activity of this enzyme in the brain structures. These results suggest that GSK-3β inhibition can modulate manic-like behaviors. Also, it can be suggested that inhibition of GSK-3β can be effective against oxidative stress. However, more studies are needed to better elucidate these mechanisms. Graphical Abstract The effects of AR-A014418 on the behavioral and oxidative stress parameters in the animal model of mania induced by ouabain. Superoxide = superoxide production in submitochondrial particles; LPH = lipid hydroperoxide; 4-HNE = 4-hydroxynonenal; SOD = superoxide dismutase; GPx = glutathione peroxidase; GR = glutathione reductase.

ROSTAFUROXIN AMELIORATES THE INWARD REMODELING AND STIFFNESS IN RESISTANCE ARTERIES FROM DEOXYCORTICOSTERONE ACETATE-SALT HYPERTENSIVE RATS

Objective: We have shown that rostafuroxin (ROSTA), an antagonist of endogenous ouabain (OUA), block the biding of OUA to Na+ K+ -ATPase and inhibits the cSRC downstream activation in mesenteric resistance arteries (MRA), which ameliorates endothelial dysfunction and oxidative stress, as reduces hypertension in deoxycorticosterone acetate (DOCA)-salt rats. In addition, MRA of DOCA-salt rats also present the hypertrophic inward remodeling with stiffness. Moreover, the chronic treatment with OUA induces hypotrophic inward remodeling with stiffness in MRA. In line with these results we investigate the role of endogenous OUA on remodeling and stiffness in MRA of DOCA-salt rats. Design and method: A hypertensive model was established in uninephrectomized Wistar rats using DOCA-salt. After 5 weeks DOCA-salt treatment, with high systolic blood pressure (SBP) stabilization, this hypertensive model was divided into two groups: DOCA-salt and DOCA-salt co-treated with ROSTA (1 mg/kg per day gavage, 3 weeks). The SBP was measured using the tail-cuff method, and vascular remodeling was assessed in MRA using a pressure myograph. Histological and western blot analysis were performed in MRA. Results: Our results reinforce that ROSTA treatment reduces SBP in DOCA-salt rats, but did not restore it to control levels. For the first time, the results suggest that ROSTA ameliorates the remodeling and stiffness observed in MRA of DOCA-salt rats, enhancing the luminal diameter and distensibility, reducing the cross-section area and wall/lumen ratio as well as shifting to the right the stress-strain curve in MRA. ROSTA restores collagen type I/III ratio, internal elastic lamina thickness, tyrosine kinase Src phosphorylation (Tyr418), as well EGFR, cRaf, ERK, p38MAPK, TGFβ1, CTGF, and collagen I protein expression in MRA from DOCA-salt rats. Conclusions: The results suggested that ROSTA reduces hypertension, the hypertrophic inward remodeling and the augmented wall/lumen ratio in MRA of DOCA-salt rats. In addition, endogenous OUA emerges as an important mechanism to induce vascular stiffness via Na+ K+ -ATPase/ cSRC/ EGFR/ cRas/ ERK/ p38MAPK/ CTGF/ TGFβ1 pathway in MRA. These results reinforce the idea that endogenous OUA is a putative target for the treatment of vascular adjustments present in hypertension.

Long‐term Ouabain treatment promotes ATP1A1‐dependent IL‐6 expression in a fetal human lens epithelial cell line (FHL124)

The cardiac glycoside ouabain (OUA) selectively inhibits Na+/K+‐ATPase (NKA) governing the steady state maintenance of the electro‐chemical gradient of Na+ and K+ across the cell membranes. OUA, following binding to the NAK, activates several growth‐related signaling pathways: the EGFR/Src–Ras–ERK pathway and the PI3K1A–PDK–Akt pathway (Xie and Askari, Eur J Biochem 269(10):2434–2439, 2002). OUA also influences pro‐ and anti‐inflammation (Cavalcante‐Silva et al., Frontiers in Physiology, 8:895, 2017) and cell proliferation depending on OUA concentration and incubation time, and cell type (Kulikov et al., Biochim et Biophys Acta 1768: 1691–1702, 2007 and Nguyen et al. JASN 18 (1): 46–57, 2007). Our group proposed and showed the direct interaction between NKA and BcL2 family proteins, which promote or inhibit apoptosis in human fetal lens epithelial cell line (FHL124) (Lauf et al., Cell Physiol Biochem 31:257–276, 2013; Lauf et al., Am J Physiol. Cell Physiol. 308, C51‐60, 2015). Here, we aimed to study the time dependence of OUA action at a concentration of 10−7M on cell viability and transcriptional gene regulation of various pro‐apoptotic and pro‐inflammatory related genes in FHL124 cells. Cells were incubated with either 1–100 μM OUA in 7 μM DMSO or 7 μM DMSO alone (solvent control) relative to an untreated control for 1, 24 and 48 h. The OUA‐treated cells cultured for 24 h revealed a slightly decreased cell viability, however, 48 h incubation dramatically decreased the number of cells by 75–80% while DMSO alone reduced cell viability by 40–50% compared to untreated cells. Like all epithelial cells, FHL124 cells possess α1 (atp1a1) isoform of NAK. The 5′‐untranslated region (5′UTR) of the atp1a1 gene contains two p53 and three NF‐kB (transcription factors nuclear factor‐kappa‐B) response elements. To test for the functional expression of the p53 and NFKB target genes, we selected mdm2, which negatively regulates the p53 gene and IL‐6 (interleukin‐6), which is induced by NF‐KB. Quantitative polymerase chain reaction (qPCR) method, using TaqMan probes, was employed to measure comparative gene expression of the atp1a1, mdm2, and IL‐6 genes. Cells OUA‐treated for 48 h promoted an increase of the IL‐6 expression by ~eight fold and the atp1a1 expression by two fold, but not the mdm2 expression. These findings suggest that OUA, in addition to utilizing existing signaling pathways mentioned above, may act either in complex with cytosolically internalized NKA (see Stricker et al., this FASEB meeting 2018) or derived therefrom through to be determined gene regulation factors proximal to the NF‐kB response elements. Further studies are aimed to confirm NKA‐dependent IL‐6 expression after long‐treatment with OUA.Support or Funding InformationSupported by the P&T Graduate program and WSU Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mechanisms of Sodium Transport in Plants-Progresses and Challenges.

Understanding the mechanisms of sodium (Na+) influx, effective compartmentalization, and efflux in higher plants is crucial to manipulate Na+ accumulation and assure the maintenance of low Na+ concentration in the cytosol and, hence, plant tolerance to salt stress. Na+ influx across the plasma membrane in the roots occur mainly via nonselective cation channels (NSCCs). Na+ is compartmentalized into vacuoles by Na+/H+ exchangers (NHXs). Na+ efflux from the plant roots is mediated by the activity of Na+/H+ antiporters catalyzed by the salt overly sensitive 1 (SOS1) protein. In animals, ouabain (OU)-sensitive Na+, K+-ATPase (a P-type ATPase) mediates sodium efflux. The evolution of P-type ATPases in higher plants does not exclude the possibility of sodium efflux mechanisms similar to the Na+, K+-ATPase-dependent mechanisms characteristic of animal cells. Using novel fluorescence imaging and spectrofluorometric methodologies, an OU-sensitive sodium efflux system has recently been reported to be physiologically active in roots. This review summarizes and analyzes the current knowledge on Na+ influx, compartmentalization, and efflux in higher plants in response to salt stress.

Abstract P437: The Extracellular Domain of Na + /K + ATPase Serves as a Receptor for Cyclic GMP in Mediating Natriuresis

Previous studies from our laboratory have shown that extracellular renal interstitial (RI) cyclic guanosine 3’5’-monophosphate (cGMP) inhibits renal proximal tubule (RPT) sodium reabsorption and induces natriuesis via activation of Src family kinase in vivo . The cellular mechanisms by which extracellular cGMP regulates this process are unknown. Previous immunofluorescence competitive binding studies in isolated RPTs from normal rat kidneys demonstrated that cGMP and ouabain (OUA) compete for binding to Na + /K + -ATPase (NKA). We hypothesized that cGMP binds to the extracellular domain of the α1-subunit of NKA on basolateral membranes of RPT cells thereby inhibiting Na + transport. In the present study, we cross-linked cGMP to isolated RPTs from normal rat kidneys (N=6). We incubated RPT cells with 4-N 3 ;-PET-8-Biotin-11-cGMP (azido cGMP) or 8-N 3 -6-Biotin-10-cAMP (azido cAMP; both at 2 μM) in the presence or absence of UV light to induce cross linking; azido cAMP served as a negative control. Immunoprecipitation was then performed using strepavidin beads followed by Western blot analysis probing for NKA. RPTs with cross linked azido cGMP exhibited a strong signal for NKA that was significantly weaker for non-cross linked azido cGMP (33.4 ± 6%; P<0.0001) and cross linked (45.8 ± 8%; P<0.001) or non-cross linked (35.2 ± 6%; P<0.001) azido cAMP samples. We further demonstrated that in the presence of OUA (10 μM) NKA was reduced in cross linked azido cGMP RPTs, providing confirmation for previous immunofluorescence competition studies. Azido cGMP cross linked samples (N=3) were subsequently separated in a Tris-HCl gel and stained with Coomassie Blue. The protein band corresponding to NKA was excised and processed for mass spectrometry. NKA was identified as the second most ubiquitous protein in that band with 50 unique peptides for NKA represented covering 47% of all the amino acids in NKA. Together, these data demonstrate that cGMP competes with OUA for binding on NKA and that NKA serves as a receptor for cGMP thereby mediating natriuresis.

Ouabain Protects Human Renal Cells against the Cytotoxic Effects of Shiga Toxin Type 2 and Subtilase Cytotoxin

Hemolytic uremic syndrome (HUS) is one of the most common causes of acute renal failure in children. The majority of cases are associated with Shiga toxin (Stx)-producing Escherichia coli (STEC). In Argentina, HUS is endemic and presents the highest incidence rate in the world. STEC strains expressing Stx type 2 (Stx2) are responsible for the most severe cases of this pathology. Subtilase cytotoxin (SubAB) is another STEC virulence factor that may contribute to HUS pathogenesis. To date, neither a licensed vaccine nor effective therapy for HUS is available for humans. Considering that Ouabain (OUA) may prevent the apoptosis process, in this study we evaluated if OUA is able to avoid the damage caused by Stx2 and SubAB on human glomerular endothelial cells (HGEC) and the human proximal tubule epithelial cell (HK-2) line. HGEC and HK-2 were pretreated with OUA and then incubated with the toxins. OUA protected the HGEC viability from Stx2 and SubAB cytotoxic effects, and also prevented the HK-2 viability from Stx2 effects. The protective action of OUA on HGEC and HK-2 was associated with a decrease in apoptosis and an increase in cell proliferation. Our data provide evidence that OUA could be considered as a therapeutic strategy to avoid the renal damage that precedes HUS.

Alpha 2 Na+,K+-ATPase silencing induces loss of inflammatory response and ouabain protection in glial cells

Ouabain (OUA) is a cardiac glycoside that binds to Na+,K+-ATPase (NKA), a conserved membrane protein that controls cell transmembrane ionic concentrations and requires ATP hydrolysis. At nM concentrations, OUA activates signaling pathways that are not related to its typical inhibitory effect on the NKA pump. Activation of these signaling pathways protects against some types of injury of the kidneys and central nervous system. There are 4 isoforms of the alpha subunit of NKA, which are differentially distributed across tissues and may have different physiological roles. Glial cells are important regulators of injury and inflammation in the brain and express the α1 and α2 NKA isoforms. This study investigated the role of α2 NKA in OUA modulation of the neuroinflammatory response induced by lipopolysaccharide (LPS) in mouse primary glial cell cultures. LPS treatment increased lactate dehydrogenase release, while OUA did not decrease cell viability and blocked LPS-induced NF-κB activation. Silencing α2 NKA prevented ERK and NF-κB activation by LPS. α2 NKA also regulates TNF-α and IL-1β levels. The data reported here indicate a significant role of α2 NKA in regulating central LPS effects, with implications in the associated neuroinflammatory processes.

Effects of cardiotonic steroids on isolated perfused kidney and NHE3 activity in renal proximal tubules

Cardiotonic steroids (CS) are known as modulators of sodium and water homeostasis. These compounds contribute to the excretion of sodium under overload conditions due to its natriuretic property related to the inhibition of the renal Na+/K+-ATPase (NKA) pump α1 isoform. NHE3, the main route for Na+ reabsorption in the proximal tubule, depends on the Na+ gradient generated by the NKA pump. In the present study we aimed to investigate the effects of marinobufagin (MBG) and telocinobufagin (TBG) on the renal function of isolated perfused rat kidney and on the inhibition of NKA activity. Furthermore, we investigated the mechanisms for the cardiotonic steroid-mediated natriuretic effect, by evaluating and comparing the effects of bufalin (BUF), ouabain (OUA), MBG and TBG on NHE3 activity in the renal proximal tubule in vivo. TBG significantly increased GFR, UF, natriuresis and kaliuresis in isolated perfused rat kidney, and inhibits the activity of NKA at a much higher rate than MBG. By stationary microperfusion technique, the perfusion with BUF, OUA, TBG or MBG promoted an inhibitory effect on NHE3 activity, whereas BUF was the most effective agent, and demonstrated a dose-dependent response, with maximal inhibition at 50nM. Furthermore, our data showed the role of NKA-Src kinase pathway in the inhibition of NHE3 by CS. Finally, a downstream step, MEK1/2-ERK1/2 was also investigated, and, similar to Src inhibition, the MEK1/2 inhibitor (U0126) suppressed the BUF effect. Our findings indicate the involvement of NKA-SRc-Kinase-Ras-Raf-ERK1/2 pathway in the downregulation of NHE3 by cardiotonic steroids in the renal proximal tubule, promoting a reduction of proximal sodium reabsorption and natriuresis.

Evaluation of Se-phenyl-thiazolidine-4-carboselenoate protective activity against oxidative and behavioral stress in the maniac model induced by ouabain in male rats

This study investigates Se-phenyl-thiazolidine-4-carboselenoate (Se-PTC) protective activity against oxidative and behavioral stress in the model of mania induced by ouabain (OUA) in male rats. The compound used was Se-PTC (50mg/kg) and the positive control LiCl (45mg/kg) was administered for intragastric route (i.g.) 30min prior to administration of OUA (10−5M). OUA was dissolved in artificial cerebrospinal fluid (aCSF) and administered at the 5μl through an intracerebroventricular (i.c.v) cannula. The pretreatment with Se-PTC was effective in preventing the increase in locomotor activity induced by OUA, however the positive control LiCl is capable to block crossing augmentation induced by OUA. Na+/K+-ATPase activity was significantly reduced in OUA group and the Se-PTC to normalize Na+/K+−ATPase activity. Pretreatment with Se-PTC protect against the increase in catalase activity and thiobarbituric acid reactive species (TBARS) content in the brain caused by OUA. Therefore, Se-PTC is effective against OUA-induced hyperactivity and alterations in brain oxidative status of rats.

Knockdown of Apolipoprotein E Enhanced Sensitivity of Hep3B Cells to Cardiac Steroids via Regulating Na+/K+-ATPase Signalosome.

This study compared the sensitivity of human hepatoma Hep3B, SK-HEP-1, SMMC-7721, and BEL-7402 cells to cardiac steroids, including bufalin (BF), a bufalin derivative (BF211), ouabain (OUA), and digitoxin (DIG). Hep3B cells exhibited relatively low sensitivity to cardiac steroids. Expression levels of subunits of Na+/K+-ATPase were high in Hep3B cells. However, colocalization of Na+/K+-ATPase and caveolin was nearly undetectable in Hep3B cells. By using RNA-Seq technology, we found a total of 36 genes to be differentially expressed between Hep3B cells and SK-HEP-1 cells, which are highly sensitive to cardiac steroids. Our bioinformatics analysis determined that these genes were mostly comprised of extracellular space, protein binding, and extracellular region. Among these 36 genes, apolipoprotein E (APOE) played a critical role, as knockdown APOE expression induced colocalization of Na+/K+-ATPase and caveolin and increased sensitivity of Hep3B cells to both proliferation-inhibiting and cytotoxic effects of BF or BF211. Also, the effects of BF on PI3K/AKT/GSK3β and apoptosis signal cascades were enhanced in APOE knockdown cells. The results of our study confirmed the role of Na+/K+-ATPase signalosome in cytotoxicity of cardiac steroids and suggested that APOE regulated the sensitivity of cells to cardiac steroids by affecting formation and function of Na+/K+-ATPase signalosome. In addition, intercellular interaction with high level of Na+/K+-ATPase β1 subunit may be also a factor in the low sensitivity of Hep3B cells to cardiac steroids. Mol Cancer Ther; 15(12); 2955-65. ©2016 AACR.

Lithium and valproate act on the GSK-3β signaling pathway to reverse manic-like behavior in an animal model of mania induced by ouabain

The present study aimed to investigate the effects of mood stabilizers, specifically lithium (Li) and valproate (VPA), on the PI3K/Akt signaling pathway in the brains of rats subjected to the ouabain (OUA)-induced animal model of mania. In addition, the effects of AR-A014418, a GSK-3β inhibitor, on manic-like behavior induced by OUA were evaluated. In the first experimental protocol Wistar rats received a single ICV injection of OUA or artificial cerebrospinal fluid (aCSF). From the day following ICV injection, the rats were treated for 6 days with intraperitoneal injections of saline, Li or VPA twice a day. In the second experimental protocol, rats received OUA, aCSF, OUA plus AR-A014418, or aCSF plus AR-A014418. On the 7th day after OUA injection, locomotor activity was measured using the open-field test. In addition, we analyzed the levels of p-PI3K, p-MAPK, p-Akt, and p-GSK-3β in the brain of rats by immunoblot. Li and VPA reversed OUA-related hyperactivity. OUA decreased p-PI3K, p-Akt and p-GSK-3β levels. Li and VPA improved these OUA-induced cellular dysfunctions; however, the effects of the mood stabilizers were dependent on the protein and brain region analyzed. In addition, AR-A014418 reversed the manic-like behavior induced by OUA. These findings suggest that the manic-like effects of ouabain are associated with the activation of GSK-3β, and that Li and VPA exert protective effects against OUA-induced inhibition of the GSK-3β pathway.

The Na+/Ca2+ exchanger: A possible link between oxidative stress and endogenous ouabain in hypertension

Oxidative stress-induced hypertension involves a number of membrane transport proteins that play a critical role in maintaining the cytosolic homeostasis of Na + and Ca 2+ . These transport proteins include Na + /K + -ATPase, the Na + /H + exchanger, Na channels, and the Na + /Ca 2+ exchanger. The exact link between these membrane transporters is not exactly known but appears to involve their location in microdomains called PLasmERosomes, oxidative stress and circulating ouabain levels. Oxidative stress stimulates the generation of ouabain from the adrenal glands which elevates circulating endogenous ouabain levels. At concentrations above 1nM, ouabain inhibits the activity of the Na + /K + -ATPase. At 1nM or lower concentrations, the Na + /K + -ATPase acts as a transducer which induces a cascade of events that begins with phosphorylation of the epidermal growth factor receptor (EGFR) by Src and activation of Ras, Raf, mitogen activated protein kinase kinase (MEK), mitogen activated protein kinase (MAPK), extracellular signal-regulated kinase1/2 (ERK1/2), and p90 ribosomal S6 kinase (p90RSK). p90RSK activates the Na + /H + exchanger through phosphorylation. Ouabain-mediated inhibition and activation of the Na + /K + -ATPase and Na + /H + exchanger, respectively, leads to elevation of Na + in the PLasmERosome which stimulates the Na + /Ca 2+ exchanger to extrude Na + in exchange for Ca 2+ . This elevates Ca 2+ in the PLasmERosome which stimulates junctional ER to release Ca 2+ . Ca 2+ influx through the Na + /Ca 2+ exchanger and release from junctional ER leads to Ca 2+ overload which can lead to a number of pathologies including increased vascular tone and hypertension.

Dynamics of murine B lymphocytes is modulated by in vivo treatment with steroid ouabain

Ouabain (OUA) is a steroid hormone capable of inhibiting the protein Na+K+ATPase present in the plasma membrane of cells. Ouabain was initially extracted from the roots of African trees such as Acocanthera ouabaio and Strophantus gratus seeds and later described as an endogenous component found in higher mammals. The adrenal gland is the main site of synthesis of ouabain and it is released in stressful situations, conditions similar to those where there is secretion of corticosteroids. Immunological functions have been shown to be regulated by ouabain. In order to understand the effects of ouabain on B lymphocyte populations in different lymphoid organs, mice received intraperitoneal injections of ouabain for 3 consecutive days. Twenty-four hours after the last injection, cells were analyzed by flow cytometry. In the spleen, ouabain modulated especially follicular B cells, inducing a significant decrease in the percentage and absolute numbers of those cells. Ouabain also reduced the absolute number of marginal zone B lymphocytes. No difference in the percentage or absolute number of B lymphocytes in the spleen forty-eight hours after the last injection was observed. An increase in the number of B cells was seen in mesenteric lymph nodes and this retention appears to be directly related to increased expression of CXCR5 chemokine receptor and reduction of CD62L, which also explains the observed reduction of B cells in the spleen. Our results indicate that ouabain regulates the dynamics of B lymphocytes in peripheral organs but production of total IgM and IgG in the serum of animals treated in vivo with ouabain was not affected.

Ouabain Modulates the Lipid Composition of Hippocampal Plasma Membranes from Rats with LPS-induced Neuroinflammation.

The effects of ouabain (OUA) and lipopolysaccharide (LPS) in vivo on hippocampal membranes (RHM) of Wistar male rats aged 3 months were analyzed. After intraperitoneal (i.p.) injection of OUA only, LPS only, OUA plus LPS, or saline, the content of proteins, phospholipids, cholesterol and gangliosides from RHM was analyzed. The total protein and cholesterol contents of RHM were not significantly affected by OUA or LPS for the experimentally paired groups. In contrast, total phospholipids and gangliosides were strongly modulated by either OUA or LPS treatments. LPS reduced the total phospholipids (roughly 23 %) and increased the total gangliosides (approximately 40 %). OUA alone increased the total phospholipids (around 23 %) and also the total gangliosides (nearly 34 %). OUA pretreatment compensated the LPS-induced changes, preserving the total phospholipids and gangliosides around the same levels of the control. Thus, an acute treatment with OUA not only modulated the composition of hippocampal membranes from 3-month-old rats, but also was apparently able to counteract membrane alterations resulting from LPS-induced neuroinflammation. This study demonstrates for the first time that the OUA capacity modulates the lipid composition of hippocampal plasma membranes from rats with LPS-induced neuroinflammation.

A p38 mitogen-activated protein kinase inhibitor attenuates cardiotonic steroids-induced apoptotic and stress signaling in a Sw-71 cytotrophoblast cell line

IntroductionPreeclampsia (preE) is characterized by abnormal placentation. Marinobufagenin (MBG), a cardiotonic steroid (CTS), inhibits the function of cytotrophoblast cells (CTBs). We demonstrated that CTSs induce anti-angiogenic and anti-proliferative effects in Sw-71 CTBs. This study tests that CTSs induce apoptotic and stress signaling. MethodsHuman extravillous Sw-71 CTBs were incubated with 0, 0.1, 1, 10, and 100 nM of each of three CTSs (MBG, cinobufatalin (CINO) and ouabain (OUB)) for 48 h. Some cells were pretreated with 10 μM p38 mitogen-activated protein kinase (p38 MAPK) inhibitor (SB203580) for 2 h prior to CTSs treatment. We analyzed p38 MAPK phosphorylation, expression of pro-inflammatory protein cyclooxygenase-2 (Cox-2) and ratio of pro-apoptotic Bcl-2-associated X protein (Bax) to anti-apoptotic Bcl-2 protein by western blot in CTSs-treated CTBs lysates. Levels of vascular endothelial growth factor (VEGF), placental growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng) were measured in culture media using ELISA kits. Statistical comparisons were performed using analysis of variance with Duncan's post hoc test. Resultsp38 MAPK phosphorylation, expression of Cox-2 and Bax/Bcl-2 was upregulated (*p < 0.05) in CTBs exposed to ≥ 0.1 nM CTSs. Secretion of sFlt-1 and sEng were increased while VEGF and PIGF were decreased in Sw-71 CTBs treated ≥1 nM of each CTSs (*p < 0.01 for each). The SB203580 pretreatment of CTBs significantly attenuated CTS-induced effects. DiscussionExposure of Sw-71 CTBs to CTSs induced apoptotic and stress signaling and causing anti-angiongenic effect. The observed diminution of CTS-induced signaling by SB203580 pretreatment implicates p38 MAPK as a regulator of these pathways.

Cardiotonic Steroids Inhibit NHE3 in Renal Proximal Tubules by a Src Kinase Dependent Pathway

Cardiotonic steroids (CE) are known as sodium‐pump inhibitors. The natriuretic property of these compounds is related to the inhibition of the α1 isoform of Na+/K+ATPase (NKA) in renal tubules. NHE3, the main route for Na+ and bicarbonate reabsorption in proximal tubules, depends on the Na+ gradient generated by the NKA. The present study aimed to investigate and compare the effects and mechanisms of 50 nM bufalin (BUF), telocinobufagenin (TBG), marinobufagenin (MBG) and ouabain (OUA) on NHE3 activity by the stationary renal microperfusion technique. By this method we are able to measure the H+ secretion in nephron segments through H+ sensitive microelectrodes. We observed that all compounds studied promoted a inhibition of proximal H+ secretion / bicarbonate reabsorption when compared to the control, being BUF the most potent compound (JHCO3‐,nmol.cm‐2.s‐1–control ethanol ‐2,11±0,16(10); BUF 1.04 ± 0.07 (8)*; TBG 0.077 ± 1.36(10)*; OUA 1.48 ± 0.10 (11)* and MBG 1.63 ± 0.08 (10)*. Our data also showed that this effect involved the inhibition of NHE3, since in the presence of NHE3 inhibitor (S3226), BUF did not promote additional inhibition. Furthermore, we demonstrated the involvement of NKA‐Src kinase pathway in the action signaling mechanism of these steroids on NHE3 activity, since in the presence of Src kinase specific inhibitor (SU6656), the inhibitory effect promoted by the CE was abolished.

OUABAIN IMPROVES THE RESPONSE TO MELANOMA (B16) IN MICE BY MODULATING B AND T LYMPHOCYTES

Event Abstract Back to Event OUABAIN IMPROVES THE RESPONSE TO MELANOMA (B16) IN MICE BY MODULATING B AND T LYMPHOCYTES Joyle M. Silva1*, Augusto Das Neves Azevedo1, Raul Corrêa Aleixo1, Arthur Mendes Clemente1, Vinicius Ribeiro Cabral2 and Luciana S. Paiva1 1 Universidade Federal Fluminense, Instituto de Biologia, Laboratório de Imunorregulação, Brazil 2 Universidade Federal Fluminense, Faculdade de Educação, Departamento de Fundamentos Pedagógicos, Brazil Ouabain (OUA) is a steroid endogenous produced mainly by the adrenal gland in superior mammals. It has been suggested that the ouabain is released together with other glucocorticoids during stress situations and can regulate several immune functions. Recently, our group observed that the in vivo treatment with ouabain was able to reduce the regulatory T cells in spleen of mice. This result is important because regulatory T cells are involved in downregulation of the immune response to tumor. Here, we studied in vivo ouabain role in the modulation of B and T lymphocytes in melanoma mouse model (B16). We used C57BL/6 mice injected intraperitoneally with 0.56mg/Kg ouabain for 3 consecutive days. In the fourth day, some animals were euthanized and others were injected with 10^6 cells of syngenic melanoma in the presence or absence of ouabain. The B and T lymphocytes of spleen were analyzed by flow cytometry twenty four hours after the last injection of ouabain or in the twenty-first day after the injection with melanoma cells. We observed a significant reduction in the absolute number of B lymphocytes in animals injected with melanoma alone. The treatment with ouabain protects B cells keeping them in numbers similar to control animals. In the case of T lymphocytes, there was a decrease in the absolute number of regulatory T cells in the groups treated with ouabain alone, injected with melanoma cells and treated and injected. The macroscopic observation of the peritoneal cavity reveals that in the group injected with melanoma cells, the metastases is larger than in the group injected with melanoma and treated with ouabain, although the reduction of metastases is not directly related to increased survival of treated. Together, these findings contribute to understanding of the study of the modulation of the immune system by ouabain in the presence of melanoma (B16). Acknowledgements PROPPI/UFF, FAPERJ, CNPq Keywords: Ouabain, B lymphocytes, T lymphocytes, B16 melanoma, Immune Regulation Conference: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología, Medellin, Colombia, 13 Oct - 16 Oct, 2015. Presentation Type: Poster Presentation Topic: Tumor immunology Citation: Silva JM, Das Neves Azevedo A, Corrêa Aleixo R, Mendes Clemente A, Ribeiro Cabral V and Paiva LS (2015). OUABAIN IMPROVES THE RESPONSE TO MELANOMA (B16) IN MICE BY MODULATING B AND T LYMPHOCYTES. Front. Immunol. Conference Abstract: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología. doi: 10.3389/conf.fimmu.2015.05.00066 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 15 May 2015; Published Online: 14 Sep 2015. * Correspondence: Mrs. Joyle M Silva, Universidade Federal Fluminense, Instituto de Biologia, Laboratório de Imunorregulação, Niterói, RJ, Brazil, joylemoreira@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Joyle M Silva Augusto Das Neves Azevedo Raul Corrêa Aleixo Arthur Mendes Clemente Vinicius Ribeiro Cabral Luciana S Paiva Google Joyle M Silva Augusto Das Neves Azevedo Raul Corrêa Aleixo Arthur Mendes Clemente Vinicius Ribeiro Cabral Luciana S Paiva Google Scholar Joyle M Silva Augusto Das Neves Azevedo Raul Corrêa Aleixo Arthur Mendes Clemente Vinicius Ribeiro Cabral Luciana S Paiva PubMed Joyle M Silva Augusto Das Neves Azevedo Raul Corrêa Aleixo Arthur Mendes Clemente Vinicius Ribeiro Cabral Luciana S Paiva Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Intracerebral Administration of BDNF Protects Rat Brain Against Oxidative Stress Induced by Ouabain in an Animal Model of Mania.

Several studies have suggested that alterations in brain-derived neurotrophic factor (BDNF) and increased oxidative stress have a central role in bipolar disorder (BD). Intracerebroventricular (ICV) injection of ouabain (OUA) in rats alters oxidative stress parameters and decreases BDNF levels in the brain. In this context, the present study aims to investigate the effects of BDNF ICV administration on BDNF levels and oxidative stress parameters in brains of rats submitted to animal model of mania induced by OUA. Wistar rats received an ICV injection of OUA, artificial cerebrospinal fluid (ACSF), OUA plus BDNF, or ACSF plus BDNF. Locomotor activity and risk-taking behavior in the rats were measured using the open-field test. In addition, we analyzed the BDNF levels and oxidative stress parameters (TBARS, Carbonyl, CAT, SOD, GR, and GPx) in the frontal cortex and hippocampus of rats. The BDNF was unable to reverse the ouabain-induced hyperactivity and risk-taking behavior. Nevertheless, BDNF treatment increased BDNF levels, modulated the antioxidant enzymes, and protected the OUA-induced oxidative damage in the brain of rats. These results suggest that BDNF alteration observed in BD patients may be associated with oxidative damage, both seen in this disorder.