Abstract
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 (ERK-P), and both growth and ERK-P could be blocked by the MEK inhibitor (U0126, 100 nm). Western blot analysis revealed that the only alpha isoform of Na+, K+-ATPase that could be detected in these cultures was the highly ouabain-resistant alpha1 isoform. Measurement of ouabain inhibition of ion transport in these cultures using 86Rb+ uptake revealed the predominance of the expected ouabain-resistant isoform (IC50 = 24 microm) and an additional minor ( approximately 15%) ouabain-sensitive inhibition with IC50 approximately 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.
Highlights
There is evidence that cardiotonic steroids (CS)1 are a normal product of the mammalian adrenal cortex
The major finding of this study is that the cardiotonic steroid-glycoside, ouabain, which has been proposed to be an endogenous mammalian steroid product of the adrenal cortex (40 – 42), induces renal epithelial cell growth and proliferation via the mitogen-activated protein kinase cascade (ERK1/2) at concentrations corresponding to the reported circulating level of ouabain [22, 23]
These effects occur in cells that express exclusively the rat Naϩ,Kϩ-ATPase ␣1 isoform that is highly resistant to inhibition by ouabain
Summary
Rat renal proximal convoluted tubule epithelial cells (PCT) were originally obtained from Dr Ulrich Hopfer, Case Western Reserve Medical School, and we have maintained them for the past 5 years. The epithelial phenotype of the cell line is well documented, and the cells demonstrate numerous morphological and functional characteristics of differentiated PCT cells [24, 25]. Cells were cultured in Dulbecco’s modified Eagle’s medium/F12 (1:1) supplemented with 15 mM HEPES, This paper is available on line at http://www.jbc.org
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