Abstract
In addition to inhibition of the Na-K ATPase, ouabain activates a signal transduction function, triggering growth and proliferation of cultured cells even at nanomolar concentrations. An isomer of ouabain (EO) circulates in mammalians at subnanomolar concentrations, and increased levels are associated with cardiac hypertrophy and hypertension. We present here a study of cardiac and renal hypertrophy induced by ouabain infused into rats for prolonged periods and relate this effect to the recently described ouabain-induced activation of the Src-EGFr-ERK signaling pathway. Ouabain infusion into rats (15 microg/kg/day for 18 weeks) doubled plasma ouabain levels from 0.3 to 0.7 nm and increased blood pressure by 20 mm Hg (p < 0.001), cardiac left ventricle (+11%, p < 0.05), and kidney weight (+9%, p < 0.01). These effects in vivo are associated with a significant enrichment of alpha1, beta1, gammaa Na-K ATPase subunits together with Src and EGFr in isolated renal caveolae membranes and activation of ERK1/2. In caveolae, direct Na-K ATPase/Src interactions can be demonstrated by co-immunoprecipitation. The interaction is amplified by ouabain, at a high affinity binding site, detectable in caveolae but not in total rat renal membranes. The high affinity site for ouabain is associated with Src-dependent tyrosine phosphorylation of rat alpha1 Na-K ATPase. The antihypertensive compound, PST 2238, antagonized all ouabain-induced effects at 10 microg/kg/day in vivo or 10(-10)-10(-8) m in vitro. These findings provide a molecular mechanism for the in vivo pro-hypertrophic and hypertensinogenic activity of ouabain, or by analogy those of EO in humans. They also explain the pharmacological basis for PST 2238 treatment.
Highlights
Until recently, the main, if not unique, function ascribed to the integral membrane protein Na-K ATPase is the maintenance and regulation of the electrochemical gradient across the cell membrane in all tissues (1)
It has been shown that even subnanomolar ouabain concentrations stimulate growth of cultured rat tubular cells, via activation of an extracellular signal-regulated kinase (ERK)-dependent pathway and, surprisingly, in view of the fact that these rat cells contain only the ␣1 isoform of Na-K ATPase, ouabain inhibits a component of active 86Rb uptake with a high affinity (9)
Effect of Ouabain on ERK1/2 Activation—We further investigated whether the effect of ouabain infusion on Na-K ATPase, Src, epidermal growth factor receptor (EGFr) expression into caveolae might result in the activation of the ERK pathway and whether PST 2238 might reverse this effect
Summary
The main, if not unique, function ascribed to the integral membrane protein Na-K ATPase is the maintenance and regulation of the electrochemical gradient across the cell membrane in all tissues (1). In recent years, several studies have indicated that Na-K ATPase can act as a signal transducer in response to the interaction with its natural ligand ouabain (4) This finding originates mainly from studies carried out on cultured rat cardiomyocytes or renal tubular cells based on effects on cell growth and hypertrophy of ouabain in the micromolar range. General or hormonal receptors (20) involved in blood pressure regulation or hormonal steroid control, antagonizes the pressor effect of subnanomolar ouabain concentrations and normalizes the ouabain-induced renal Na-K ATPase up-regulation in rats and renal cells (20) This pharmacological tool has been used to substantiate the specificity of the ouabain/Na-K ATPase interaction in caveolae and to provide evidence on its molecular mechanism of action
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