Cardiotonic steroids trigger non-classical testosterone signaling in Sertoli cells via the α4 isoform of the sodium pump.

The α4 isoform of the Na(+),K(+)-ATPase (sodium pump) is known to be expressed in spermatozoa and to be critical for their motility. In the investigation presented here, we find that the rat-derived Sertoli cell line 93RS2 also expresses considerable amounts of the α4 isoform in addition to the α1 isoform. Since Sertoli cells are not motile, one can assume that the function of the α4 isoform in these cells must differ from that in spermatozoa. Thus, we assessed a potential involvement of this isoform in signaling pathways that are activated by the cardiotonic steroid (CTS) ouabain, a highly specific sodium pump ligand. Treatment of 93RS2 cells with ouabain leads to activation of the c-Src/c-Raf/Erk1/2 signaling cascade. Furthermore, we show for the first time that the activation of this cascade by ouabain results in phosphorylation and activation of the transcription factor CREB. This signaling cascade is induced at low nanomolar concentrations of ouabain, consistent with the involvement of the α4 isoform. This is further supported by experiments involving siRNA: silencing of α4 expression entirely blocks ouabain-induced activation of Erk1/2 whereas silencing of α1 has no effect. The findings of this study unveil new aspects in CTS/sodium pump interactions by demonstrating for the first time ouabain-induced signaling through the α4 isoform. The c-Src/c-Raf/Erk1/2/CREB cascade activated by ouabain is identical to the so-called non-classical signaling cascade that is normally triggered in Sertoli cells by testosterone. Taking into consideration that CTS are produced endogenously, our results may help to gain new insights into the physiological mechanisms associated with male fertility and reproduction.

Sodium pump alpha1 and alpha3 subunit isoforms mediate distinct responses to ouabain and are both essential for survival of human neuroblastoma.

Using SK-N-AS human neuroblastoma cells, which co-express the alpha1 and alpha3 isoforms of the sodium pump alpha subunit, we selectively silenced either the alpha1 or alpha3 subunit by means of transfection with small interfering RNA, and investigated cell survival and the cellular response to ouabain. We found that both of the alpha subunits are essential for cell survival, indicating that substitution of one subunit for the other is not sufficient. In the presence of both alpha subunits, ouabain causes sustained activation of extracellular signal-regulated kinases 1 and 2 (Erk1/2). This activation is not affected when the alpha1 subunit is silenced. However, when alpha3 expression is silenced, ouabain-induced activation of Erk1/2 does not occur, even at a high concentration of ouabain (1 microM). Thus, ouabain-induced Erk1/2 activation is mediated in SK-N-AS cells by alpha3 only, and alpha1 does not participate in this event. This is a clear demonstration of selective involvement of a specific sodium pump alpha subunit isoform in ouabain-induced signaling.

Ouabain activates signaling pathways associated with cell death in human neuroblastoma.

Cardiotonic steroids (CTS) like ouabain are not only specific inhibitors of the sodium pump (Na(+),K(+)-ATPase), they also can influence various cytosolic signaling events in a hormone-like manner. In the neuroblastoma cell line SH-SY5Y ouabain triggers multiple signaling pathways. Within 30 min of incubation with 1 or 10 microM ouabain, SH-SY5Y cells generate reactive oxygen species to a level approximately 50% above control and show a modest but significant elevation in cytosolic [Ca(2+)] of about 25%. After 6 h of exposure, ouabain stimulates a series of anti-apoptotic actions in SH-SY5Y cells, including concentration-dependent phosphorylation of Erk1/2, Akt, and Bad. Nevertheless, at the same time this CTS also induces a series of events that inhibit retinoic acid-induced neuritogenesis and promote cell death. Both of these latter phenomena are possibly associated with the observed ouabain-induced reduction in the abundance of the anti-apoptotic proteins Bcl-XL and Bcl-2. In addition, ouabain treatment results in cytochrome c release into the cytosol and induces activation of caspase 3, events that point towards the stimulation of apoptotic pathways that are probably enhanced by the stimulation of p53 phosphorylation at Ser15 also observed in this study. These pathways may eventually lead to cell death: treatment with 10 nM ouabain results in a 20% decrease in cell number after 4 days of incubation and treatment with 1 microM ouabain decreases cells number by about 75%. The results obtained here emphasize the importance of further research in order to elucidate the various signalling cascades triggered by ouabain and possibly other CTS that are used in the treatment of heart failure and to identify their primary receptor(s).

Signaling pathways involving the sodium pump stimulate NO production in endothelial cells.

The cardiac steroid ouabain, a known inhibitor of the sodium pump (Na+, K+ -ATPase), has been shown to release endothelin from endothelial cells when used at concentrations below those that inhibit the pump. The present study addresses the question of which signaling pathways are activated by ouabain in endothelial cells. Our findings indicate that ouabain, applied at low concentrations to human umbilical cord endothelial cells (HUAECs), induces a reaction cascade that leads to translocation of endothelial nitric oxide synthase (eNOS) and to activation of phosphatidylinositol 3-kinase (PI3K). These events are followed by phosphorylation of Akt (also known as protein kinase B, or PKB) and activation of eNOS by phosphorylation. This signaling pathway, which results in increased nitric oxide (NO) production in HUAECs, is inhibited by the PI3K-specific inhibitor LY294002. Activation of the reaction cascade is not due to endothelin-1 (ET-1) binding to the ET-1 receptor B (ETB), since application of the ETB-specific antagonist BQ-788 did not have any effect on Akt or eNOS phosphorylation. The results shown here indicate that ouabain binding to the sodium pump results in the activation of the proliferation and survival pathways involving PI3K, Akt activation, stimulation of eNOS, and production of NO in HUAECs. Together with results from previous publications, the current investigation implies that the sodium pump is involved in vascular tone regulation.