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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.

BACKGROUND: enhancement of the sustained component of the sodium current (INaL) is a major factor contributing to myocardial damage. A beneficial effect of the INaL blocker Ranolazine (RAN) on glycemic control was recently described in the MERLIN-TIMI 36 trial in diabetic patients. However, the mechanism underlying this finding hasn't been elucidated yet.AIM: To characterize INaL in insulin-secreting cells (INS-1E) and evaluate its role in glucose-stimulated insulin secretion (GSIS).METHODS: INaL, identified as the steady-state current blocked by 10 μM RAN (IRAN) or 0.5 μM TTX (ITTX), and its impact on membrane potential (Vm) were assessed by patch clamp. Veratridine (VERA) was used as INaL enhancer. INaL-dependent intracellular Ca2+ changes were detected by Fluo-4AM. To simulate hyperglycemic stress (CHG), INS-1E cells were exposed to 33 mM glucose for 24 hrs. GSIS was measured by HTRF assay.RESULTS: Glucose- and tolbutamide-triggered action potentials were suppressed by TTX, but not by RAN. VERA caused depolarization, countered by INaL blockade. The reversal potentials of ITTX and IRAN were negative to Na+ equilibrium potential, but they approached it when K+-channels were blocked. This revealed INaL coupling to Na+-activated K+ current (IKNa); expression of IKNa channels (Slo2.1/2.2) was confirmed by transcript analysis. INaL blockade blunted cytosolic Ca2+ response to depolarization. CHG enhanced INaL. Whereas acute INaL enhancement (VERA) increased GSIS, chronic one (CHG or VERA) depressed GSIS, which was partially restored by RAN.CONCLUSIONS: INaL is expressed in insulin-secreting cells, is coupled to IKNa , affects Ca2+ signalling and, when enhanced acutely, increases GSIS. However, constitutive INaL enhancement, induced by chronic hyperglycemic stress, and leads to GSIS depression instead. Overall, chronic INaL enhancement may represent a mechanism of damage progression common to myocardial and insulin-secreting cells.