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Low dose ouabain regulation of Na‐K ATPase requires NHE‐1 scaffolding properties.

Plasma cardioglycosides (ouabain) levels increase in rat models of hypertension and in humans with heart and kidney failure. Ouabain activates Src, ERK, and Akt, resulting in increased 86Rb uptake and cell proliferation. NHE1 is also activated by Akt, suggesting coordinate regulation of NHE1 and Na‐K ATPase (Na‐K). We hypothesize that ouabain regulates Na‐K through an NHE1 dependent mechanism. In the present study we determined the role of ouabain in NHE1‐mediated pH recovery and regulation of NHE1 and Na‐K association and phosphorylation. Pretreatment of OK cells with 10nM ouabain, 5uM EIPA (NHE1inhibitor), or both inhibited intracellular pH recovery in a bicarbonate‐free buffer. Ouabain (10nM) stimulated Akt phosphorylation, 86Rb uptake, and phosphorylation and association of Na‐K with NHE1. These effects of ouabain were prevented by EIPA. In HKC‐2 cells that lack NHE‐1 expression, 10pM ouabain had no effect on 86Rb uptake and Akt phosphorylation. Transfection with wild type NHE1 but not with a mutant NHE1 that prevents NHE‐1 association with cytoskeleton, into HKC‐2 cells rescued ouabain‐mediated Akt phosphorylation. These results suggest that NHE1 plays a critical role in ouabain‐mediated increase in Na‐K activity and Akt phosphorylation through its scaffolding function. The role of NHE1 in regulation of proximal renal tubule intracellular pH is not clear.