Ouabain is a potent promoter of growth and activator of ERK1/2 in ouabain-resistant rat renal epithelial cells

Endogenous cardiotonic steroids (ECS) are putative ligands of the inhibitory binding site of the membrane sodium pump (Na+,K+-ATPase). There is growing evidence that cardiotonic steroids may promote the growth of cardiac and vascular myocytes, including evidence indicating growth stimulation at concentrations in the same range as circulating ECS concentrations. We investigated four parameters to determine whether ouabain, a proposed ECS, promotes growth of immortalized rat proximal tubule epithelial cells: cell count by hemocytometer; metabolic activity as reflected in the mitochondrial conversion of the tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, to its formazan product (MA); DNA synthesis reflected as bromodeoxyuridine incorporation (DNA); and mitosis reflected as histone phosphorylation state detected using anti-phosphohistone 3 antibody (HP). Maximum stimulatory responses were observed at 1 nM ouabain (MA, 20.3% increase, p < 0.01; DNA, 28.4% increase, p < 0.001; HP, maximum response at 0.5 h, 50% increase, p < 0.001). We observed that growth stimulation was associated with stimulation of ERK1/2 phosphorylation (ERKP), and both growth and ERK-P could be blocked by the MEK inhibitor (U0126, 100 nM). Western blot analysis revealed that the only α isoform of Na+,K+-ATPase that could be detected in these cultures was the highly ouabain-resistant α1 isoform. Measurement of ouabain inhibition of ion transport in these cultures using Rb-86(+) uptake revealed the predominance of the expected ouabain-resistant isoform (IC50 = 24 μM) and an additional minor (∼15%) ouabain-sensitive inhibition with IC50 ∼30 pM. Similar bimodal transport inhibition curves were obtained in freshly dissected rat proximal tubules. These results indicate that renal epithelial cells may be a sensitive target of the ERK1/2-activating and growth-promoting effects of ouabain even in the presence of ouabain-resistant Na+,K+-ATPase.