When probed with antibodies against total p38, the 38 kDa band showed no change at the investigated time Idelalisib in vivo points of OUA treatment, in comparison with that observed in the lysate of untreated cells (Figure 4c). Thus, OUA 100 nM activates p38 MAPK in U937 cells. Then, we investigated the involvement of NCX in the phosphorylation of p38. However, we did not detect a difference in the band of phospho-p38 in the lysate of cells pretreated with KBR and

then with OUA, in comparison with the band observed in the lysate of OUA treated cells (Figure 4c). Thus, these results suggest that, although p38 plays a pro-survival role in OUA treated cells, its activation is NCX independent. Discussion The first aim of our investigation was to Selleck RAD001 evaluate if OUA is cytotoxic for U937 cells and we detected that at concentrations ≥500 nM it causes ROS generation and a large increase of [Ca++]i followed by cell death. We did not explore the link between ROS generation, Ca++ increase and cell demise, as it is not surprising that this intracellular milieu can lead to cell death. We were surprised by the

survival pathway sparked by lower doses of OUA in which a modest rise of Ca++ seems to play an important role. Indeed, U937 cells exposed to ouabain 100 nM were growth arrested in G1 cell cycle phase and escaped from death by activation of a survival pathway, in which were involved the Na+/Ca++-exchanger active in the Ca++ influx mode and p38 MAPK. It is widely accepted that partial inhibition of the cardiac myocyte Na+/K+-ATPase by cardiac glycosides causes a modest increase of [Na+ i, which in turn affects the plasma membrane Na+/Ca++-exchanger, leading to a significant increase of [Ca++ i and in the force of contraction [4–9]. In the present investigation we show that in U937 cells OUA leads to a rise of [Ca++ i through NCX active in the Ca++ influx mode because this event could be prevented by KBR, an inhibitor known to affect only this type of NCX activity [30, 31]. Moreover, OUA became largely cytotoxic after NCX inhibition and not after block of L-type

Ca++ channel by nifedipine. These conclusions were confirmed treating the cells with the Na+ ionophore monensin which, similarly to OUA, causes an increase of [Ca++ Adenosine i through NCX active in the Ca++ influx mode. Finally, the endoplasmic reticulum stressor tunicamycin, not affecting NCX, proved to be a good control because it induced cell death in a low proportion of cells, not increased by KBR. MAPK are central mediators of cellular survival and death pathways [33–36]. To investigate their involvement in the survival pathway activated by OUA, we pretreated the cells with inhibitors at concentrations affecting specifically one MAPK and then analyzed cell viability. These experiments indicated that p38 plays a pro-survival role in OUA treated cells.