Nanomolar concentrations of ouabain stimulate Na‐K ATPase through sodium hydrogen exchanger‐1 (NHE1) dependent mechanism.

Abstract

Recent investigations have shown increased Na‐H exchanger activity and plasma levels of endogenous cardiotonic glycosides, such as ouabain in spontaneously hypertensive rats. At high concentrations, ouabain inhibits pump activity, and at lower concentrations, it increases pump activity and induces cell proliferation. Stimulation of NHE1 and Na‐K ATPase activity involve protein kinase B (AKT), suggesting that both NHE1 and Na‐K ATPase are mediated by the same proteins. We hypothesize that the activity of NHE1 and Na‐K ATPase are linked. In the present study we aim to 1) determine the role of activation of NHE1 in ouabain‐sensitive stimulation of Na‐K ATPase, 2) determine if NHE1 and Na‐K ATPase association increases when cells were treated with NH4Cl or ouabain in various kidney proximal tubule cell lines (OK, HK2, HK5, and HK11). Our data suggests that NH4Cl stimulation of NHE1 increases ouabain sensitive 86Rb uptake in all cell lines examined.Treatment with ethylisopropylamiloride (EIPA) a NHE1 specific inhibitor decreased Na‐K ATPase activity in HK5 and HK11 cells and prevented nanomolar ouabain‐stimulated Na‐K ATPase activity in OK cells. Immunoblot analysis indicated that NH4Cl increased membrane expression of NHE1 and Na‐K ATPase in OK and HK11 cells. Surprisingly, treatment with EIPA alone increased membrane expression of NHE1 and Na‐K ATPase. The reason for increased presence of these proteins in the membrane is unknown, however, tyrosine phosphorylation of the catalytic subunit of Na‐K ATPase, believed to be an indication of Na‐K ATPase activation, is decreased under these same conditions. These results suggest that activation of NHE1 is critical for ouabain stimulated increase in Na‐K ATPase activity, and that upon activation, NHE1 and Na‐K ATPase associate, perhaps to form a signalosome.