α1-AR-mediated activation of NKCC in rat cardiomyocytes involves ERK-dependent phosphorylation of the cotransporter

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Abstract
We studied molecular and functional characteristics as well as hormonal regulation of the Na-K-2Cl cotransporter (NKCC) in the isolated rat heart and cardiomyocytes. NKCC activity was measured as bumetanide-sensitive86Rb+influx in isolated perfused rat hearts and isolated cardiomyocytes. Stimulation of α1-adrenoceptors (AR) by phenylephrine (30 μM) increased86Rb+influx. The NKCC inhibitor bumetanide (50 μM) reduced the response to phenylephrine by 45 ± 13% ( n = 12, P < 0.01). PD-98059 (10 μM), an inhibitor of the activation of the mitogen-activated protein kinases extracellular signal-regulated protein kinase 1 and 2 (ERK1/2), reduced the total response to phenylephrine by 51 ± 13% ( n = 10, P < 0.01) and eliminated the bumetanide-sensitive component, indicating that α1-AR mediated stimulation of NKCC is dependent on activation of ERK1/2. Inhibitors of protein kinase C or phosphatidylinositol 3-kinase had no effect. The presence of NKCC mRNA and protein was demonstrated in isolated rat cardiomyocytes. Phosphorylation of NKCC after α1-AR stimulation was shown by immunoprecipitation of the phosphoprotein from32Piprelabeled cardiomyocytes. Increased phosphorylation of the NKCC protein was also abolished by PD-98059. We conclude that the NKCC is present in rat cardiomyocytes and that ion transport by the cotransporter is regulated by α1-AR stimulation through phosphorylation of this protein involving the ERK pathway.