Mechanisms of ouabain toxicity

Valente, Raphael C; Capella, Luiz Sabbatini; Monteiro, Robson Q.; Rumjanek, Vivian M.; Lopes, Anibal Gil; Capella, Marcia A. M.

doi:10.1096/fj.02-0937fje

Cited by 48 publications

(46 citation statements)

References 59 publications

(102 reference statements)

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“…We have also shown that detachment may not be ascribed to the ensuing decrease of K content (8) and that a decrease of K content provoked by incubation of R or W cells in media with only 0.1 mM K ϩ (instead of the regular 4.0 mM) does not cause cell detachment (8). This finding is in keeping with the fact that changes in reduced͞oxidized glutathione modify the degree of ouabain resistance without reducing the cell K (10,11).…”

supporting

confidence: 76%

Contacts and cooperation between cells depend on the hormone ouabain

Larre

Ponce

Fiorentino

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Cell adhesion is a crucial step in proliferation, differentiation, migration, apoptosis, and metastasis. In previous works we have shown that cell adhesion is modulated by ouabain, a highly specific inhibitor of Na ؉ ,K ؉ -ATPase, recently found to be a hormone. In the present work we pursue the investigation of the effect of ouabain on a special type of cell-cell interaction: the rescue of ouabainsensitive MDCK cells (W) by ouabain-resistant cells (R). In cultured monolayers of pure W cells, ouabain triggers the ''P3 A mechanism'' (from pump͞adhesion) consisting of a cascade of phosphorylations that retrieves adhesion-associated molecules occludin and ␤-catenin and results in detachment of the cell. When W cells are instead cocultured with R cells, the P3 A reaction is blocked, and W cells are rescued. Furthermore, in these R͞W cocultures ouabain promotes cell-cell communication by means of gap junctions by specifically enhancing the expression of connexin 32 and addressing this molecule to the plasma membrane. Ouabain also promotes the internalization of the ␤-subunit of the Na ؉ ,K ؉ -ATPase. These observations open the possibility that the crucial processes mentioned at the beginning would be under the control of the hormone ouabain.ardiotonic steroids started in the 18th century as unknown active principles when William Withering used foxglove tea (Digitalis purpurea) to treat dropsy (congestive heart failure). Once ouabain was isolated, the demonstration that it specifically inhibits the Na ϩ ,K ϩ pump (1) transformed ouabain into a crucial tool to identify the pump with the membrane enzyme Na ϩ ,K ϩ -ATPase (2) and unravel its intrinsic mechanisms. Ouabain was recently recognized to be a hormone (3) whose blood level increases in physiological (e.g., exercise) as well as pathological (e.g., arterial hypertension, chronic cardiac and renal failure, preeclampsia, etc.) conditions (4-7). Furthermore, we found that ouabain specifically decreases cell adhesion by triggering a P3A mechanism that involves the activation of protein tyrosine kinases and extracellular signal-regulated kinases, decreases the cell content of the small GTP-binding protein RhoA, and changes the degree of phosphorylation, internalization, and degradation of adhesion molecules such as occludin, E-cadherin, etc. (8, 9) (D. Flores-Benítez, personal communication). We have also shown that detachment may not be ascribed to the ensuing decrease of K content (8) and that a decrease of K content provoked by incubation of R or W cells in media with only 0.1 mM K ϩ (instead of the regular 4.0 mM) does not cause cell detachment (8). This finding is in keeping with the fact that changes in reduced͞oxidized glutathione modify the degree of ouabain resistance without reducing the cell K (10, 11).Because in the meanwhile ouabain was shown to be a hormone (3), in the present work we pursue the study of its effect on a special type of cell-cell interaction: metabolic cooperation (12). A typical example of cooperation is provided by R cells that have low...

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supporting

confidence: 76%

Contacts and cooperation between cells depend on the hormone ouabain

Larre

Ponce

Fiorentino

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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“…Aizman et al [29] have demonstrated that low concentrations of ouabain induce cross-talk inside the cell, involving highly complex signaling cascades that lead to the formation of ROS. In line with these results, Valente et al [30] identified O 2 – · as one of the ROS formed by ouabain treatment in Madin-Darby canine kidney epithelial cells. In the present study, incubation with SOD decreased noradrenaline-induced contraction in resistance arteries from 20-week ouabain-treated rats, indicating a role for O 2 – · in the augmented noradrenaline-induced contraction observed in these vessels.…”

Section: Discussionmentioning

confidence: 61%

Long-Term Ouabain Treatment Impairs Vascular Function in Resistance Arteries

et al. 2011

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Background/Aims: The purpose of this study was to examine the cardiovascular effects of long-term ouabain treatment at different time points. Methods: Systolic blood pressure (SBP) was measured by tail-cuff method in male Wistar rats treated with ouabain (approx. 8.0 µg·day^–1) or vehicle for 5, 10 and 20 weeks. Afterwards, vascular function was assessed in mesenteric resistance arteries (MRA) using a wire myograph. ROS production and COX-1 and COX-2, TNF-α, and IL-6 protein expression were investigated. Results: SBP was increased by ouabain treatment up to the 6th week and remained stable until the 20th week. However, noradrenaline-induced contraction increased only in MRA in rats treated with ouabain for 20 weeks. NOS inhibition and endothelium removal increased the noradrenaline response, but to a smaller magnitude in MRA in the ouabain group. Moreover, inhibition of COX-2 or incubation with superoxide dismutase restores noradrenaline-induced contraction in the 20-week ouabain group to control levels. ROS production as well as COX-2, IL-6 and TNF-α protein expression increased in MRA in this group. Conclusion: Although ouabain treatment induced hypertension in all groups, a larger noradrenaline induced contraction was observed over 20 weeks of treatment. This vascular dysfunction was related to COX-2-derived prostanoids and oxidative stress, increased pro- inflammatory cytokines and reduced NO bioavailability.

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“…Although these inhibitors alone did not significantly alter mCD14 expression (data not shown), the data presented in figure 4 show that incubation with either p38 MAPK inhibitor or EGFR inhibitor reverted ouabain-induced mCD14 downregulation, increasing the number of mCD14 high cells in approximately 25 and 19%, respectively, compared to cells only treated with 100 n M ouabain. Additionally, several other inhibitors implicated in ouabain-induced cell signaling events were tested such as genistein (tyrosine kinase inhibitor), Ly 294002 (PI3K inhibitor), chelerythrine chloride (selective PKC inhibitor), PD98059 (MEK inhibitor), Jun amino-terminal kinase inhibitor and reduced glutathione, which was shown to abolish ouabain-induced signaling related to cell death in epithelial cells [31,32] . However, none of these compounds significantly hampered the ouabaininduced alteration in mCD14 expression (data not shown).…”

Section: Resultsmentioning

confidence: 99%

“…It has also been described that in parallel to the ERK1/2 activation there is an increment in GTPase protein Ras levels and phosphorylation of tyrosine residues in several proteins [32,38] , which may, in turn, lead to a cell-type-dependent activation of p38 MAPK, PKC and phospholipase C. These multiple signaling modules culminate with the activation of several transcription factors like activator protein-1 and nuclear factor-B, which might induce the regulation of several genes.…”

Section: Discussionmentioning

confidence: 99%

“…To analyze how ouabain exerts its effects on mCD14 expression, several periods of incubation were chosen. Moreover, to understand the cellular mechanisms involved in the modulation of mCD14 expression by ouabain, a number of inhibitors of cell signaling were employed whose pathways had been shown to be directly activated by ouabain in other cell types [31,32] . The present work used the p38 MAPK inhibitor SB202190 (20 M ) and reduced glutathione (a compound that was shown to modulate ouabain actions in other models, 2 m M ), both from Sigma; the PKC ␦ inhibitor rottlerin (2 M ), the EGFR inhibitor tyrphostin A23 (2.5 M ), the tyrosine kinase inhibitor genistein (10 M ), and Jun amino-terminal kinase inhibitor (1 M ), all obtained from Calbiochem, EUA; the selective PKC inhibitor chelerythrine chloride (1.25 M ), purchased from RBI, Natick, Mass., USA, the ERK kinase (MEK) inhibitor PD98059 (50 M ), purchased from Biosource, Wayne, Pa., USA, and the Src kinase inhibitor PP1 (1 M ) and the phosphatidylinositol 3-kinase (PI3K) inhibitor Ly 294002 (25 M ), both from Biomol, USA.…”

Section: Incubation With Ouabain and Inhibitors Of Cell-signaling Patmentioning

confidence: 99%

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mCD14 Expression in Human Monocytes Is Downregulated by Ouabain via Transactivation of Epithelial Growth Factor Receptor and Activation of p38 Mitogen-Activated Protein Kinase

Valente¹,

Nascimento

Araújo

et al. 2009

Neuroimmunomodulation

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Background and Aims: The steroid ouabain is found in plasma and in many mammalian tissues, and is now considered as a hormone. In the immune system, ouabain regulates a number of lymphocyte functions, but little is known about its effects on monocyte function. Monocytes are important for adequate immune responses. The aim of this work was to analyze the effect of ouabain on mCD14 expression, a surface molecule involved in the response against Gram-negative bacteria and phagocytosis. Methods: Human peripheral blood mononuclear cells obtained from healthy donors were separated by density gradient centrifugation. Monocytes were separated by adherence and treated for 24 h with 100 nM ouabain. mCD14, CD1a and P-p38 expression was analyzed by flow cytometry. Inhibitors of cell-signaling pathways, i.e. SB202190, reduced glutathione, rottlerin, tyrphostin A23, genistein, chelerythrine chloride, PD98059, PP1 and Ly 294002, were used concomitantly with ouabain to observe their effect on mCD14 expression. Results: Ouabain induced a significant decrease in mCD14 expression. This feature was not related to receptor endocytosis or cell death. Furthermore, mCD14 downregulation did not reflect a shift in differentiation into dendritic cells because this hormone failed to induce CD1a expression. Amongst several inhibitors of cell-signaling pathways triggered by ouabain, only epidermal growth factor receptor (EGFR) and p38 mitogen-activated protein kinase (MAPK) inhibitors (tyrphostin A23 and SB202109) significantly reverted the effect of ouabain on mCD14 expression. Accordingly, the levels of P-p38 were increased on monocytes after ouabain treatment. However, incubation with epidermal growth factor did not alter mCD14 expression. Conclusion: These findings suggest that ouabain downregulates mCD14 expression on monocytes through EGFR transactivation and p38 MAPK activation.

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Mechanisms of ouabain toxicity

Cited by 48 publications

References 59 publications

Contacts and cooperation between cells depend on the hormone ouabain

Contacts and cooperation between cells depend on the hormone ouabain

Long-Term Ouabain Treatment Impairs Vascular Function in Resistance Arteries

mCD14 Expression in Human Monocytes Is Downregulated by Ouabain via Transactivation of Epithelial Growth Factor Receptor and Activation of p38 Mitogen-Activated Protein Kinase

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