Na‐K ATPase Regulation by Cardioglycosides: Role of AT1R

Cardioglycosides are specific inhibitors of Na‐K ATPase; however, at low doses increase BP and Na‐K ATPase activity in kidney basolateral membranes. TIRF and FRET microscopy suggested that 10 pM ouabain associates with Na‐K only at cell‐cell junctions. We hypothesized that low and high molar ouabain interacts with divergent sets of surface proteins in kidney. To address this hypothesis, we treated HK11 cells with 100nM or 10pM ouabain. Cell lysates were immunoprecipitated using anti‐ouabain antibodies. Unique proteins identified by proteomics in 10pM ouabain treated samples include Ang II type 1 receptor (AT1R), ankyrin 2 and 3, Akt1, and RTK erbB‐4. Unique proteins identified from 100nM ouabain cells include RTK erbB‐2, HK ATPase, and SLC4A4 isoform 2. Na‐K ATPase, NHE1, Src kinase, and EGFR were identified at both 10pM and 100nM ouabain. Based on these data we hypothesized that 10pM ouabain stimulates Na‐K ATPase through an AT1R dependent mechanism. To address this hypothesis cells were treated with 10pM ouabain in the presence or absence of candesartan, an AT1R antagonist. Ouabain‐induced stimulation of Na‐K ATPase activity, expression, phosphorylation, and Src signaling were prevented by candesartan. These data suggest that low concentrations of ouabain may increase BP through novel interactions with proteins of the renin angiotensin system. Supported by AHA and VA