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

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-sensitive Rb-86(+) influx in isolated perfused rat hearts and isolated cardiomyocytes. Stimulation of alpha(1)-adrenoceptors (AR) by phenylephrine ( 30 muM) increased Rb-86(+) influx. The NKCC inhibitor bumetanide ( 50 muM) reduced the response to phenylephrine by 45 +/- 13% ( n = 12, P < 0.01). PD-98059 ( 10 mu M), 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 alpha(1)-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 alpha(1)-AR stimulation was shown by immunoprecipitation of the phosphoprotein from P-32(i) prelabeled 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 alpha(1)-AR stimulation through phosphorylation of this protein involving the ERK pathway.