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Mechanisms of ouabain toxicity¹

RAPHAEL C. VALENTE^*, LUIZ S. CAPELLA, ROBSON Q. MONTEIRO^*, VIVIAN M. RUMJANEK^*, ANÍBAL G. LOPES and MÁRCIA A. M. CAPELLA^*,,2

^* Departamento de Bioquímica Médica, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro (UFRJ);
Departamento de Clínica Médica, Universidade do Rio de Janeiro (UNI-RIO); and
Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro (UFRJ) Rio de Janeiro, Brazil

²Correspondence: Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, CCS-Bloco G, 21949-900, Rio de Janeiro, RJ, Brazil. E-mail: capella{at}bioqmed.ufrj.br; mcapella{at}biof.ufrj.br

SPECIFIC AIMS

We aimed to study the mechanisms of ouabain-induced cytotoxicity and the role of glutathione in avoiding the toxic effects of ouabain. Two cell lines were used: the ouabain-sensitive MDCK cells and the ouabain-resistant Ma104 cells.

PRINCIPAL FINDINGS

1. Ouabain-induced plasma membrane depolarization is not related to cell death
It is known that the blockage of Na⁺,K⁺-ATPase by ouabain leads to plasma membrane depolarization (PMD); this in turn has been associated with cell death. We have previously shown that glutathione (GSH) protects MDCK cells from ouabain toxicity, but the mechanism involved in this protection is unknown. The present study evaluated whether the protective effect of GSH was related to ouabain-induced PMD. To address this question, cells were treated for 30 min with ouabain in the presence of DiBAC₄(3), an anionic oxonol dye that enters cells upon PMD, increasing cell fluorescence. Figure 1 shows representative experiments of ouabain-induced PMD in MDCK (upper panel) and Ma104 (lower panel) cells; inserts show the means and standard deviations of the percentage of cells gated in region M1 of three experiments for each cell line. The treatment with ouabain induced significant PMD in both cell lines (28.1% of MDCK cells and 47.9% of Ma104 cells were gated in region M1, against 11.8% and 14.6% of the respective controls), but unexpectedly this effect was more remarkable in Ma104 cells. The preincubation of Ma104 cells with an inhibitor of GSH synthesis, DL-buthionine-(S,R)-sulfoximine (BSO), did not significantly alter their membrane polarization (8.6% of cells gated in region M1) but blocked the ouabain-induced depolarization (12.2% of cells gated in region M1). On the other hand, incubation with GSH alone induced plasma membrane depolarization in MDCK cells (42.4% of cells gated in region M1) and further increased the number of ouabain-sensitive cells depolarized after ouabain treatment (71.0%).

View larger version (30K): [in this window] [in a new window]   Figure 1. Changes in plasma membrane potential measured by DiBAC4(3). Cells were treated with 10 µM ouabain for 30 min in the presence of DiBAC4(3). In the flow cytometric analysis, a region M1 was delimited, representing cells undergoing plasma membrane depolarization. Upper panel: MDCK cells; lower panel: Ma104 cells. Black lines: incubation with DiBAC4(3) alone (control); dashed lines: incubation with DiBAC4(3) and 10 µM ouabain. Inserts: means and standard deviations of the percentage of cells gated in the region M1, obtained from 3 different experiments. aSatistically different from the control; bstatistically different from each other.

Our results suggest, surprisingly, that ouabain-induced depolarization is not harmful per se and that the ability of cells to depolarize their plasma membrane after ouabain-treatment may be a protection against ouabain-induced cell death.

2. Sustained increase in tyrosine phosphorylation and Ras expression are related to ouabain-induced cell death
There are presently two mechanisms described for the action of ouabain: 1) the classical inhibition of Na⁺,K⁺-ATPase, which leads to an increase in cytosolic calcium; and 2) the signal transduction pathway, leading to altered expression of several genes. It has been shown that ouabain causes a sustained increase in P-Tyr in MDCK cells but not in Ma104 cells. However, the relationship between P-Tyr and ouabain toxicity is not well determined. To address this issue, we examined the effects of GSH and BSO in ouabain-induced sustained tyrosine phosphorylation in these two cell lines (Fig. 2 ). Ouabain (10 µM) induced a sustained increase in P-Tyr in MDCK cells and GSH almost completely reverted this effect. For Ma104 cells, even 100 µM ouabain was insufficient to increase substantially P-Tyr, but preincubation with BSO for 24 h augmented ouabain-induced tyrosine phosphorylation in these cells.

View larger version (40K): [in this window] [in a new window]   Figure 2. Effect of GSH and BSO on ouabain-induced protein tyrosine phosphorylation in MDCK and Ma104 cells. Cells seeded in coverslips were incubated for 3 h with 10 µM ouabain in the presence or absence of 2 mM GSH (MDCK cells) or 100 µM ouabain after preincubation for 24 h with 2 mM BSO (Ma104 cells). Fluorescence was observed under an Axiovert 100 fluorescence microscope, using an oil immersion 40x fluor objective. In each experiment, 3 to 5 images obtained from different fields of the same coverslip were used. The bars represent the average of pixels above background. Upper panel: MDCK cells; lower panel: Ma104 cells. Inserts: representative photographs of the experiments. Values are expressed as the mean of at least 3 different experiments for each cell line. aStatistically different from the control; bstatistically different from each other.

Ouabain (10 µM) also increased the expression of Ras protein in MDCK cells and GSH completely avoided this increase. On the other hand, no significant induction of Ras expression could be detected in Ma104 cells treated with 100 µM ouabain, but the preincubation with BSO increased Ras expression after treatment with ouabain. These results suggest that the sustained increase in P-Tyr and increased Ras expression are related to ouabain cytotoxicity.

3. Ouabain induced the formation of superoxide anion
Although two different groups have reported the production of ROS by ouabain, those authors could not identify the type of ROS formed due to insufficient selectivity of the probe used. By using proxyl-fluorescamine, a probe specific for superoxide radical, we were able to identify this anion as one of the ROS formed by ouabain treatment. The induction of O₂^•- was seen only in the ouabain-sensitive cells. As O₂^•- is involved in the Ras pathway, these results further substantiate the relationship of the pathway Ras-ROS with ouabain cytotoxicity.

CONCLUSIONS

Although it has long been known that ouabain may be cytotoxic, the mechanisms of its toxicity are only now being uncovered. The suggested involvement of ouabain in hypertension raised the need for a better understanding of its cellular actions. The results obtained in the present study have three major implications: first, there is no direct correlation between membrane depolarization and ouabain-induced cell death; second, ouabain toxicity is related to signaling transduction, more specifically to the Ras-ROS pathway, and is not directly related to its classical action in Na⁺,K⁺-ATPase inhibition; and third, GSH plays a major role in ouabain-induced signaling transduction and cell death (Fig. 3 ). This role of GSH needs more thorough investigation, because it is known that plasma levels of GSH are reduced and those of ouabain are increased in animal and human hypertension.

View larger version (36K): [in this window] [in a new window]   Figure 3. Scheme showing the events following ouabain binding to Na+,K+-ATPase and the effects of GSH on cell survival. GSH increases the cell population undergoing plasma membrane depolarization (PMD) and inhibits tyrosine phosphorylation (P-Tyr), leading to cell survival. Opposing, GSH depletion blocks PMD in the majority of the population and increases P-Tyr and Ras expression, leading to cell death.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0937fje; doi: 10.1096/fj.02-0937fje

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