Absence Of Ouabain Research Articles

FTY720P inhibits hepatic Na(+)-K(+) ATPase via S1PR2 and PGE2.

Sphingosine-1-phosphate (S1P) was found previously to inhibit Na(+)-K(+) ATPase in HepG2 cells. Whether fingolimod (FTY720), a S1P receptor (S1PR) agonist, similarly inhibits the ATPase is a question that needs to be addressed. The aim of this work was to study the effect of FTY720P, the active form of the drug, on the activity of Na(+)-K(+) ATPase in HepG2 cells and determine its mechanism of action. The activity of the ATPase was assayed by measuring the amount of inorganic phosphate liberated in the presence and the absence of ouabain. FTY720-P (7.5 nmol/L, 15 min) significantly reduced the activity of the ATPase. This effect disappeared completely in the presence of JTE-013, which is a specific blocker of sphingosine-1-phosphate receptor 2 (S1PR2), as well as in the presence of calphostin and indomethacin, which are inhibitors of protein kinase C (PKC) and COX-2, respectively. The effect of FTY720P was mimicked by prostaglandin E2 (PGE2) and PMA, but abrogated by NF-κB inhibition. When NF-κB was inhibited, the effect of exogenous PGE2 still appeared, but that of PMA did not manifest, suggesting that NF-κB is upstream of PGE2 and downstream of PKC. It was concluded that FTY720P activates via S1PR2, PKC, and NF-κB. The latter induces PGE2 generation and inhibits Na(+)-K(+) ATPase.

Mediators of the Inhibitory Effect of Sphingosine‐1‐Phosphate on Hepatic Na+/K+ ATPase

We demonstrated previously an inhibitory effect of sphingosine‐1‐phosphate (S1P) on hepatic Na+/K+ ATPase. This work is an attempt to delineate the signaling pathway involved using its agonist FTY720 phosphate (FTY720‐P). HepG2 cells were used as a model and the activity of the Na+/K+ ATPased was assayed by measuring the amount of inorganic phosphate liberated in presence and absence of ouabain, a specific inhibitor of the ATPase. FTY720‐P (7.5 nM, 15 min) inhibited significantly the Na+/K+ ATPase. This effect disappeared completely in presence of JTE‐013, a specific blocker of S1P2 receptors. This inhibition was similarly abrogated by calphostin, indomethacin, RpcAMP, PTIO, and PD98059, respective inhibitors of protein kinase C (PKC), cyclooxygenase‐2(COX‐2), protein kinase A (PKA), nitric oxide synthase (NOS), and extracellular signal regulated kinase (ERK). Treatment with PGE2 or selective activators of these enzymes mimicked the effect of FTY720‐P. PGE2 was found to act via its EP2 receptor, and its effect was still manifested in the presence of calphostin, but disappeared completely in the presence of inhibitors of PKA, NOS and ERK. Furthermore, western blot analysis showed phosphorylation of ERK by dibutyryl cAMP, a PKA activator. This phosphorylation did not appear in the presence of PTIO, indicating that PKA is upstream of NO.It was concluded that FTY720‐P activates PKC via S1P2 receptors leading to PGE2 generation. PGE2 acts through its EP2 receptors and activates PKA which in turn activates NOS resulting in NO production and ERK activation. ERK then inhibits directly or indirectly the Na+/K+ ATPase.

Ouabain-induced cytoplasmic vesicles and their role in cell volume maintenance.

Cellular swelling is controlled by an active mechanism of cell volume regulation driven by a Na+/K+-dependent ATPase and by aquaporins which translocate water along the osmotic gradient. Na+/K+-pump may be blocked by ouabain, a digitalic derivative, by inhibition of ATP, or by drastic ion alterations of extracellular fluid. However, it has been observed that some tissues are still able to control their volume despite the presence of ouabain, suggesting the existence of other mechanisms of cell volume control. In 1977, by correlating electron microscopy observation with ion and water composition of liver slices incubated in different metabolic conditions in the presence or absence of ouabain, we observed that hepatocytes were able to control their volume extruding water and recovering ion composition in the presence of ouabain. In particular, hepatocytes were able to sequester ions and water in intracellular vesicles and then secrete them at the bile canaliculus pole. We named this “vesicular mechanism of cell volume control.” Afterward, this mechanism has been confirmed by us and other laboratories in several mammalian tissues. This review summarizes evidences regarding this mechanism, problems that are still pending, and questions that need to be answered. Finally, we shortly review the importance of cell volume control in some human pathological conditions.

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.

The influence of Ouabain on human dendritic cells maturation.

Although known as a Na,K-ATPase inhibitor, several other cellular and systemic actions have been ascribed to the steroid Ouabain (Oua). Particularly in the immune system, our group showed that Ouabain acts on decreasing lymphocyte proliferation, synergizing with glucocorticoids in spontaneous thymocyte apoptosis, and also lessening CD14 expression and blocking CD16 upregulation on human monocytes. However, Ouabain effects on dendritic cells (DCs) were not explored so far. Considering the peculiar plasticity and the importance of DCs in immune responses, the aim of our study was to investigate DC maturation under Ouabain influence. To generate immature DCs, human monocytes were cultured with IL-4 and GM-CSF (5 days). To investigate Ouabain role on DC activation, DCs were stimulated with TNF-α for 48 h in the presence or absence of Ouabain. TNF-induced CD83 expression and IL-12 production were abolished in DCs incubated with 100 nM Ouabain, though DC functional capacity concerning lymphocyte activation remained unaltered. Nevertheless, TNF-α-induced antigen capture downregulation, another maturation marker, occurred even in the presence of Ouabain. Besides, Ouabain increased HLA-DR and CD86 expression, whereas CD80 expression was maintained. Collectively, our results suggest that DCs respond to Ouabain maturating into a distinct category, possibly contributing to the balance between immunity and tolerance.

Effects of medium calcium, and agents affecting cytoskeletal function, on cellular volume and morphology in liver tissue in vitro

The possible role of an exocytotic, vesicular mechanism in cellular volume regulation under iso-osmotic conditions has been studied in slices of rat liver. The effects of incubation conditions and agents affecting the actin cytoskeleton were examined for changes of water, ionic composition, and ultrastructure. Slices were pre-incubated at 1°C in an iso-osmotic buffered medium to induce swelling. Upon restoration to 37°C in the same medium, tissue lost water. The Na+-K+ adenosine triphosphatase (ATPase) inhibitor ouabain inhibited water extrusion of about 50%, an effect that was accompanied by the formation of characteristic vesicles in the cytoplasmic region between the Golgi apparatus and the bile canaliculi. Water extrusion in the presence of ouabain was partially inhibited by trifluoroperazine and completely inhibited when the medium was free of Ca2+. In the presence of ouabain, brefeldin A caused a small reduction of water extrusion, whereas phalloidin and cytochalasins A, D, or E caused a marked inhibition. In these conditions there was a marked increase in size and number of cytoplasmic vesicles and a more widespread distribution of them within the cells, lacking the more specific orientation to the Golgi and canalicular regions that was seen in the presence of ouabain alone. Water extrusion was inhibited by phalloidin and cytochalasins in the absence of ouabain. In conclusion, our results are consistent with the hypothesis that iso-osmotic expulsion of water from hepatocytes can proceed partly through an accumulation of water in cytoplasmic vesicles, followed by exocytosis. This mechanism does not depend on Na+-K+ ATPase activity.

Evidence for a Na/K pump in nematocytes isolated from sea anemones

Nematocytes, the stinging cells of the primitive marine animals jellyfish and sea anemones respond with regulatory volume decrease (RVD) to osmotic swelling in 35% diluted artificial sea water (65% ASW, Marino et al., Cell Physiol Biochem 2010). Since RVD is attained through K+ and Cl‐efflux via channels and cotransporters, an active pump mechanism must be required to maintain the cellular K+ gradient and expel Na, which was here explored. Cells were isolated from acontia of the anemone Aiptasia mutabilis (Strait of Messina, Italy) by applying 605mM NaSCN plus 0.01mM Ca2+ and then exposed to isosmotic or 65% hyposmotic ASW in the presence and absence of ouabain, the specific Na/K pump inhibitor. Ouabain present at 10‐4 M, but not at 10‐6 and 10‐8M, induced significant cell swelling in isosmotic ASW suggesting Na/K pump inhibition and failure to extrude Na causing an osmotic influx of water. This effect was attenuated by replacing Na with choline in the ASW. Ouabain applied at 10‐4M prior to 65% ASW did not affect the initial osmotic swelling phase but inhibited RVD consistent with loss of K required for this process. Results suggest that nematocytes possess Na/K pumps with low ouabain affinity that actively extrude Na and maintain cellular K steady state levels, necessary to perform RVD.

Effects of cardiotonic steroids on trabecular meshwork cells: Search for mediator of ouabain-enhanced outflow facility

Lowering intraocular pressure (IOP) is currently the only strategy documented to slow the onset and progression of glaucomatous blindness. Ouabain, a cardiotonic glycoside inhibitor of Na+, K+-activated ATPase, was recently reported to enhance outflow facility in porcine anterior segments at concentrations as low as 30 nM for ≥4 h, suggesting a novel approach to lowering IOP. The underlying mechanism is unknown, but associated cytoskeletal changes were observed in porcine trabecular meshwork cells. We have previously found that changes in ATP release and subsequent ectoenzymatic conversion to adenosine may play a role in linking cytoskeletal remodeling with modulation of outflow resistance. We now tested whether altered ATP release might also be a mediator of ouabain's effect on outflow facility. ATP release from transformed human TM5 and explant-derived human trabecular meshwork cells was measured by the luciferin–luciferase reaction. Matrix metalloproteinases (MMPs) were studied by zymography, cell Na+ concentration by SBFI fluorometry, gene expression of ATP-release pathways by real-time PCR, cell volume by electronic cell sorting and cell viability by the LDH and MTT methods. Actin was examined by confocal microscopy of phalloidin-stained cells. Contrary to expectation, ouabain at concentrations ≥10 nM inhibited swelling-triggered ATP release from TM5 cells after ≥4 h of exposure. Inhibition was enhanced by increasing ouabain concentration and exposure time. Similar effects were produced by the reversible cardiac aglycone strophanthidin. Ouabain also inhibited swelling-activated ATP release from explant-derived native human TM cells. Ouabain (4 h, 30 nM and 100 nM) did not alter gene expression of the ATP-release pathways, and cell viability was unchanged by exposure to ouabain (30 nM–1 μM). Preincubation with 30 nM ouabain for 4 h did not detectably change Na+ level, the regulatory volume decrease (RVD) or the actin cytoskeleton of TM5 cells, but did inhibit hypotonicity-elicited ATP release. Moreover, even when N-methyl-d-glucosamine replaced Na+ in the extracellular fluid, ouabain still inhibited swelling-initiated ATP release at 100 nM. In the absence of ouabain, extracellular ATP stimulated MMP secretion, which was largely blocked by inhibiting conversion of ATP to adenosine, as expected. In contrast, ouabain reduced ATP release, but did not alter secretion of MMP-2 and MMP-9 from cells pretreated for ≤4 h. The results suggest that: (1) ouabain can trigger enhancement of outflow facility independent of its transport and actin-restructuring effects exerted at higher concentration and longer duration; (2) ouabain exerts parallel independent effects on ATP release and outflow facility; and (3) these effects likely reflect ouabain-induced changes in the scaffolding and/or signaling functions of Na+, K+-activated ATPase.

Impaired Stimulatory Effect of ETB Receptor on D3 Receptor in Immortalized Renal Proximal Tubule Cells of Spontaneously Hypertensive Rats

Background: Activation of renal D₃ receptor induces natriuresis and diuresis in Wistar-Kyoto (WKY) rats; in the presence of ETB receptor antagonist, the natriuretic effect of D₃ receptor in WKY rats is reduced. We hypothesize that ETB receptor activation may regulate D₃ receptor expression in renal proximal tubule (RPT) cells from WKY rats, which is impaired in RPT cells from spontaneously hypertensive rats (SHRs). Methods: D₃ receptor expression was determined by immunoblotting; the D₃/ETB receptor linkage was checked by coimmunoprecipitation; Na⁺-K⁺-ATPase activity was determined as the rate of inorganic phosphate released in the presence or absence of ouabain. Results: In RPT cells from WKY rats, the ETB receptor agonist BQ3020 increased D₃ receptor protein. In contrast, in RPT cells from SHRs, BQ3020 did not increase D₃ receptor. There was coimmunoprecipitation between D₃ and ETB receptors in RPT cells from WKY and SHRs. Activation of ETB receptor increased D₃/ETB coimmunoprecipitation in RPT cells from WKY rats, but not from SHRs. The basal levels of D₃/ETB receptor coimmunoprecipitation were greater in RPT cells from WKY rats than in those from SHRs. Stimulation of D₃ receptor inhibited Na⁺-K⁺-ATPase activity, which was augmented by the pretreatment with the ETB receptor agonist BQ3020 in WKY RPT cells, but not in SHR RPT cells. Conclusion: ETB receptors regulate and physically interact with D₃ receptors differently in WKY rats and SHRs. The impaired natriuretic effect in SHRs may be, in part, related to impaired ETB and D₃ receptor interactions.

Adducin- and Ouabain-Related Gene Variants Predict the Antihypertensive Activity of Rostafuroxin, Part 1: Experimental Studies

Essential hypertension is a complex, multifactorial disease associated with a high cardiovascular risk and whose genetic-molecular basis is heterogeneous and largely unknown. Although multiple antihypertensive therapies are available, the large individual variability in drug response results in only a modest reduction of the cardiovascular risk and unsatisfactory control of blood pressure in the hypertensive population as a whole. Two mechanisms, among others, are associated with essential hypertension and related organ damage: mutant α-adducin variants and high concentrations of endogenous ouabain. An antihypertensive agent, rostafuroxin, selectively inhibits these mechanisms in rodents. We investigated the molecular and functional effects of mutant α-adducin, ouabain, and rostafuroxin in hypertensive rats, human cells, and cell-free systems and demonstrated that both mutant α-adducin variants and the ouabain-Na,K-ATPase (Na(+)- and K(+)-dependent adenosine triphosphatase) complex can interact with the Src-SH2 (Src homology 2) domain, increasing Src activity and the Src-dependent Na,K-ATPase phosphorylation and activity. Wild-type α-adducin or Na,K-ATPase in the absence of ouabain showed no interaction with the Src-SH2 domain. Rostafuroxin disrupted the interactions between the Src-SH2 domain and mutant α-adducin or the ouabain-Na,K-ATPase complex and blunted Src activation and Na,K-ATPase phosphorylation, resulting in blood pressure normalization in the hypertensive rats. We have also shown the translatability of these data to humans in a pharmacogenomic clinical trial, as described in the companion paper.

Time-dependent increases in ouabain-sensitive Na +, K +-ATPase activity in aortas from diabetic rats: The role of prostanoids and protein kinase C

Aims Na +, K +-ATPase activity contributes to the regulation of vascular contractility and it has been suggested that vascular Na +, K +-ATPase activity may be altered during the progression of diabetes; however the mechanisms involved in the altered Na +, K +-ATPase activity changes remain unclear. Thus, the aim of the present study was to evaluate ouabain-sensitive Na +, K +-ATPase activity and the mechanism(s) responsible for any alterations on this activity in aortas from 1- and 4-week streptozotocin-pretreated (50 mg kg − 1 , i.v.) rats. Main methods Aortic rings were used to evaluate the relaxation induced by KCl (1–10 mM) in the presence and absence of ouabain (0.1 mmol/L) as an index of ouabain-sensitive Na +, K +-ATPase activity. Protein expression of COX-2 and p-PKC-βII in aortas were also investigated. Key findings Ouabain-sensitive Na +, K +-ATPase activity was unaltered following 1-week of streptozotocin administration, but was increased in the 4-week diabetic aorta (27%). Endothelium removal or nitric oxide synthase inhibition with l-NAME decreased ouabain-sensitive Na +, K +-ATPase activity only in control aortas. In denuded aortic rings, indomethacin, NS-398, ridogrel or Gö-6976 normalized ouabain-sensitive Na +, K +-ATPase activity in 4-week diabetic rats. In addition, COX-2 (51%) and p-PKC-βII (59%) protein expression were increased in 4-week diabetic aortas compared to controls. Significance In conclusion, diabetes led to a time-dependent increase in ouabain-sensitive Na +, K +-ATPase activity. The main mechanism involved in this activation is the release of TxA 2/PGH 2 by COX-2 in smooth muscle cells, linked to activation of the PKC pathway.

Na+/K+ATPase Activity in Sheep with Natural Babesiosis

The aim of this study was to determine the Na+/K+ATPase activity in the erythrocytes of sheep naturally infected withBabesia ovisbefore and after treatment. Seven diseased sheep and seven control animals were used for the study. Babesia infection was confirmed with Giemsa’s staining of blood smears. Na+/K+ATPase activity in erythrocyte was determined colorimetrically by the release of inorganic phosphate from ATP in the presence and absence of ouabain. A marked decrease of Na+/K+ATPase activity (p< 0.05) was obtained in infected sheep (0.81 ± 0.28 μmol inorganic phosphate/h.mg protein) compared to control animals (3.63 ± 0.72 μmol Pi/h.mg protein). Beside this decline, Na+/K+ATPase activity of treated animals (1.29 ± 0.81 μmol Pi/h.mg protein) were significantly (p< 0.05) decreased compared to control animals. It can be concluded that decreased erythrocyte Na+/K+ATPase activity inbabesia ovismay be due to the usage of new permeation pathways (NPPs) for the flux of ions instead of Na+/K+pump.

Expression of the Electrogenic Na+-HCO3--Cotransporter NBCe1 in Tumoral Insulin-Producing BRIN-BD11 Cells

Background/aims: The expression of the electrogenic Na⁺-HCO₃^--cotransporter NBCe1 was recently documented in rat pancreatic islet B-cells, it being speculated that such a protein participates in the extrusion of bicarbonate generated by the oxidative catabolism of nutrients from insulin-producing cells. Considering the prevalence of a Crabtree effect in tumoral insulin-producing cells, the possible presence of NBCe1 was now investigated in BRIN-BD11 cells, an insulin-producing cell line established by electrofusion of normal pancreatic B-cells with immortalized RINm5F cells. Methods: The possible presence of NBCe1 in BRIN-BD11 cells was investigated by RT-PCR, western blot analysis and immunocytochemistry. The release of insulin and net uptake of ²²Na⁺ were also measured in the BRIN-BD11 cells. Results: RT-PCR, western blot analysis and immunocytochemistry documented the presence of NBCe1 in BRIN-BD11 cells. A reported inhibitor of NBCe1, i.e. tenidap, (50-100 μM), inhibited basal and hypotonicity-induced insulin release from the BRIN-BD11 cells, whilst increasing the net uptake of ²²Na⁺ by the same cells. The latter effect was, in relative terms, more pronounced in the presence than absence of ouabain. Conclusion: BRIN-BD11 cells, like normal pancreatic islet B-cells, express NBCe1, with predominance of the B variant of this electrogenic Na⁺-HCO₃^--cotransporter.

Comparative study of the use of neuroblastoma cells (Neuro-2a) and neuroblastoma × glioma hybrid cells (NG108-15) for the toxic effect quantification of marine toxins

The suitability and sensitivity of two neural cell models, NG108-15 and Neuro-2a, to different marine toxins were evaluated under different incubation and exposure times and in the presence or absence of ouabain and veratridine (O/V). NG108-15 cells were more sensitive to pectenotoxin-2 than Neuro-2a cells. For saxitoxin, brevetoxin-3, palytoxin, okadaic acid and dinophysistoxin-1 both cell types proved to be sensitive and suitable for toxicity evaluation. For domoic acid preliminary results were presented. Setting incubation time and exposure time proved to be critical for the development of the assays. In order to reduce the duration of the assays, it was better to reduce cell time incubation previous to toxin exposure than exposure time. For palytoxin, after 24 h of growth, both cell types were sensitive in the absence of O/V. When growth time previous to toxin exposure was reduced, both cell types were unsensitive to palytoxin when O/V was absent. Although dinophysistoxin-1 and okadaic acid are both phosphatase inhibitors, these toxins did not respond similarly in front of the experimental conditions studied. Both cell types were able to identify Na-channel acting toxins and allowed to quantify the effect of saxitoxin, brevetoxin-3, palytoxin, okadaic acid, dinophysistoxin-1 and pectenotoxin-2 under different experimental conditions.

Brain Na +, K +-ATPase isoforms: Different hypothalamus and mesencephalon response to acute desipramine treatment

We studied Na +, K +-ATPase activity alpha isoforms by performing ouabain inhibition curves in rat hypothalamus and mesencephalon after acute administration of desipramine to rats. In hypothalamus, Ki values for high, intermediate and low affinity populations were 0.075 × 10 − 9 M, 0.58 × 10 − 6 M and 0.97 × 10 − 3 M, with isoform distribution of 55%, 28% and 17%, respectively. In mesencephalon, Ki values for high, intermediate and low affinity populations were 1.80 × 10 − 9 M, 0.56 × 10 − 6 M and 0.21 × 10 − 3 M, with isoform distribution of 28%, 46% and 21%, respectively. Three hours after acute administration of 10 mg/kg desipramine to rats, Na +, K +-ATPase activity in hypothalamus increased significantly 54%, 39% and 51% as assayed respectively in the absence of ouabain or in the presence of 1 × 10 − 9 M, or 5 × 10 − 6 M ouabain, whereas only a trend was recorded in the presence of 1 × 10 − 3 M ouabain. In such conditions, enzyme activity in mesencephalon increased significantly 73%, 54%, 30% and 271%, respectively. Present results showed that desipramine treatment enhances the activity of Na +, K +-ATPase alpha isoforms in rat hypothalamus and mesencephalon, but the extent of this increase differs according to the isoform and the anatomical area studied, suggesting a differential enzyme regulation in response to noradrenergic stimulation.

13 Effect of Erythropoietin on Fluid Movement Across the Respiratory Epithelium.

Erythropoietin has a wide range of nonhematopoietic effects in the body, including the regulation of certain enzymes in the lung. Clinically, its administration has been associated with a decrease in the number of days in oxygen for sick premature infants. If erythropoietin has a favorable effect on respiratory illness, one mechanism could be a reduction in pulmonary edema. In previous studies, we demonstrated that erythropoietin increases cellular uptake of sodium, a necessary first step in ion transport-linked fluid clearance (Pediatr Res 58:817A, 2005). To further characterize the effect on ion transport, we studied the effect of erythropoietin on the Na-K-ATPase, an enzyme important in sodium translocation. To accomplish this goal, we treated A549 cells, an immortal adenocarcinoma cell line with features of Type II cells, with erythropoietin and measured the uptake of 86Rb+ (a mimic of K+) in the presence and absence of ouabain, an inhibitor of Na-K-ATPase. We found that after 24h, erythropoietin (35 U/ml) increased ouabain-sensitive Rb uptake on average by 25% (n=8, control: 1.6 ± 0.6 vs erythropoietin: 2.0 ± 0.8 nmol Rb/million cells/min, p<0.05), indicating an increase in Na-K-ATPase activity. To confirm this finding, we disrupted the cell membrane and assessed Na-K-ATPase activity by an alternative method in which we measured the release of inorganic phosphate in the presence and absence of ouabain. Similar to our results in intact cells, erythropoietin (24h, 35 U/ml) increased the activity of the Na-K-ATPase (n=7, control: 0.9 ± 0.4 vs erythropoietin: 2.1 ± 1.3 mg phosphate/mg protein, p<0.05). In additional studies, we grew A549 cells on a semipermeable membrane and measured fluid movement across the monolayer by measuring the change in protein concentration in the supernatant as a reflection of fluid loss. After 24 h, erythropoietin increased by nearly 25% the volume of fluid loss from the supernatant (n=6, control: 132 ± 26 vs erythropoietin: 164 ± 34 uL/24 h, p<0.05). We then added amiloride (1mM), an inhibitor of Na channels important in the transepithelial movement of liquid, to monolayers treated with erythropoietin and found that fluid loss from the supernatant decreased to that of control cells (n=6, control: 126 ± 26 uL/24 h). Our findings show that erythropoietin increases Na-K-ATPase activity and transepithelial liquid movement in vitro. We speculate that if erythropoietin were to increase sodium transport and Na-K-ATPase activity in vivo, then lung water balance and respiratory difficulty might improve as well.

β-Adrenergic Receptor Stimulation and Adenoviral Overexpression of Superoxide Dismutase Prevent the Hypoxia-mediated Decrease in Na,K-ATPase and Alveolar Fluid Reabsorption

Hypoxia has been shown to cause lung edema and impair lung edema clearance. In the present study, we exposed isolated rat lungs to pO(2) = 40 mm Hg for 60 min or rats to 8% O(2) for up to 24 h and then measured changes in alveolar fluid reabsorption (AFR) and Na,K-ATPase function. Low levels of oxygen severely impaired AFR in both ex vivo and in vivo models. The decrease in AFR was associated with a decrease in Na,K-ATPase activity and protein abundance in the basolateral membranes from peripheral lung tissue of hypoxic rats. Beta-adrenergic agonists restored AFR in rats exposed to 8% O(2) (from 0.02 +/- 0.07 ml/h to 0.59 +/- 0.03 ml/h), which was associated with parallel increases in Na,K-ATPase protein abundance in the basolateral membrane. Hypoxia is associated with increased production of reactive oxygen species. Therefore, we examined whether overexpression of SOD2, manganese superoxide dismutase, would prevent the hypoxia-mediated decrease in AFR. Spontaneously breathing rats were infected with a replication-deficient human type 5 adenovirus containing cDNA for SOD2. An otherwise identical virus that contained no cDNA was used as a control (Adnull). Hypoxic Adnull rats had decreased rates of AFR (0.12 +/- 0.1 ml/h) as compared with hypoxic AdSOD2 and normoxic control rats (0.47 +/- 0.04 ml/h and 0.49 +/- 0.02 ml/h, respectively), with parallel changes in Na,K-ATPase.

FISH OSMOREGULATE WITH OUABAIN

Ouabain, a potent inhibitor of the enzyme Na+/K+-ATPase, is known to be a steroid hormone in mammals that is involved in blood pressure and volume regulation. Recently, a group from the University of Hawaii examined whether ouabain also acts as a hormone in fish and reported that ouabain is present in the plasma and various tissues of the euryhaline Mozambique tilapia, Oreochromis mossambicus. When the researchers transferred tilapia between fresh and salty water, they discovered that changes in plasma concentrations of ouabain in tilapia were well correlated with changes in plasma osmolality and cortisol, an important hormone during salinity change. Now, this same research group has followed up with an equally intriguing analysis of the functional role of ouabain in fish osmoregulation.The present study set out to establish whether ouabain interacts with growth hormone and prolactin (known osmoregulatory hormones in fish) in the same manner as cortisol. During seawater acclimation, cortisol stimulates Na+/K+-ATPase activity and the proliferation of chloride cells that are responsible for the active extrusion of Cl-. In addition, cortisol stimulates the release of growth hormone, which is also involved in seawater adaptation, and inhibits the release of prolactin, an important hormone in freshwater osmoregulation, from the pituitary gland. Prolactin release is dependent on calcium entering pituitary gland cells and the synthesis of cAMP, both of which are prevented by cortisol.To assess the role of ouabain in fish osmoregulation, Kajimura, Grau and the group examined the release of osmoregulatory hormones by tilapia pituitary glands. They isolated whole pituitary glands from tilapia and placed them individually in a 96-well plate containing 200 μl of incubation media. With this in vitro preparation, they measured prolactin and growth hormone release from tilapia pituitary glands in response to different doses of ouabain. To see if ouabain, like cortisol, inhibits prolactin release by preventing calcium uptake and cAMP synthesis, they measured prolactin release in response to calcium (using the calcium ionophore A23187) and to cAMP (using the analogue dbcAMP and the phosphodiesterase inhibitor IBMX) in the presence or absence of ouabain. The group found that at physiological concentrations,ouabain exerted a dose-dependent inhibitory effect on prolactin, whereas supra-physiological doses of ouabain stimulated prolactin release. By contrast, ouabain stimulated growth hormone release at lower concentrations but had an inhibitory effect at higher levels. Both A23187 and dbcAMP + IBMX had stimulatory effects on prolactin release, and these effects were inhibited by ouabain when it was also placed in the incubation media. This suggests that, like cortisol, ouabain regulates the mechanism of prolactin release. Furthermore, using microspectrofluorometry and a calcium-sensitive dye, the team was able to quantify `real-time' changes in calcium levels within pituitary cells. Their data indicated that at physiological concentrations,ouabain rapidly reduces intracellular calcium concentrations, just as cortisol does.The group has shown convincingly that, similar to the actions of cortisol during salinity change, ouabain plays an important role in fish osmoregulation by inhibiting the release of prolactin via the Ca2+ and cAMP signal transduction pathways. The team believes that ouabain in the hypothalamus of the brain may be involved in the regulation of pituitary prolactin release and hope to prove this hypothesis in the future by using a ouabain-specific antibody and immunocytochemistry. Until then, we will wait for more news about this exciting new fish hormone!

Reconstruction of the Complete Ouabain-binding Pocket of Na,K-ATPase in Gastric H,K-ATPase by Substitution of Only Seven Amino Acids

Although cardiac glycosides have been used as drugs for more than 2 centuries and their primary target, the sodium pump (Na,K-ATPase), has already been known for 4 decades, their exact binding site is still elusive. In our efforts to define the molecular basis of digitalis glycosides binding we started from the fact that a closely related enzyme, the gastric H,K-ATPase, does not bind glycosides like ouabain. Previously, we showed that a chimera of these two enzymes, in which only the M3-M4 and M5-M6 hairpins were of Na,K-ATPase, bound ouabain with high affinity (Koenderink, J. B., Hermsen, H. P. H., Swarts, H. G. P., Willems, P. H. G. M., and De Pont, J. J. H. H. M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 11209-11214). We also demonstrated that only three amino acids (Phe(783), Thr(797), and Asp(804)) present in the M5-M6 hairpin of Na,K-ATPase were sufficient to confer high affinity ouabain binding to a chimera which contained in addition the M3-M4 hairpin of Na,K-ATPase (Qiu, L. Y., Koenderink, J. B., Swarts, H. G., Willems, P. H., and De Pont, J. J. H. H. M. (2003) J. Biol. Chem. 278, 47240-47244). To further pinpoint the ouabain-binding site here we used a chimera-based loss-of-function strategy and identified four amino acids (Glu(312), Val(314), Ile(315), Gly(319)), all present in M4, as being important for ouabain binding. In a final gain-of-function study we showed that a gastric H,K-ATPase that contained Glu(312), Val(314), Ile(315), Gly(319), Phe(783), Thr(797), and Asp(804) of Na,K-ATPase bound ouabain with the same affinity as the native enzyme. Based on the E(2)P crystal structure of Ca(2+)-ATPase we constructed a homology model for the ouabain-binding site of Na,K-ATPase involving all seven amino acids as well as several earlier postulated amino acids.

Effect of Pentylenetetrazole and Carbodiimide on Oxidation Stress Markers in Rat Brain

A number of studies suggest that epileptic seizures are related to certain harmful functions of nitric oxide (Tsuda et al. 1998). This disorder can be induced with pentylenetetrazole, a drug that causes generalized seizures with abnormal levels of nitric oxide and serotonin (5-HT) (Balakrishnan et al. 1999). Analogous to pentylenetetrazole, carbodiimide participates in the biochemical metabolism of nitric oxide (Rotzingers et al. 1995), even though its mechanism of action remains unknown. Since nitric oxide is an unstable and gaseous free radical with a short half-life, its indirect measurement is possible as a result of the calculation of nitrites or nitrates. Diseases associated with epileptic seizures involve the role played by oxidative stress generated by the effect of free radicals (Bashkatova et al. 2003), where unknown biochemical mechanisms are related with lipid peroxidation levels (Ischiropoulos & Beckman 2003). The aim of the present study is to evaluate the effect of pentylenetetrazole and carbodiimide on some biochemical markers of oxidative stress as nitric oxide metabolites, 5HT, Naπ Kπ ATPase and thiobarbituric acid in rat brain. Twenty-four Wistar male adult rats (350 g) were divided into 3 groups of 8 animals: group I, control group 0.9% NaCl was given; group II treated with carbodiimide (20 mg/ kg), and group III, treated with pentylenetetrazole (40 mg/ kg), drugs were administered in a single intraperitoneal dose. After 10 min. the animals were decapitated and dissected in sagittal section: the tissue of left-sided section was used for the Naπ, Kπ-ATPase, and total ATPase assays, lipid peroxidation (TBARS), and levels of nitrites and nitrates. The right-sided section was used for determination of serotonin (5-HT). The inorganic phosphorus (Pi) content was measured using the method proposed by Balbonting et al. (1961), the difference of inorganic phosphorus content