The translocation mechanisms involved in the α1-adrenoceptor-stimulated efflux of the potassium analog86Rb+were studied in isolated rat hearts. Phenylephrine (in the presence of a β-blocker) increased the efflux of86Rb+and42K+, and the Na-K-2Cl (or K-Cl) cotransport inhibitor bumetanide reduced the response by 42 ± 11%. Furosemide inhibited the response with a lower potency than that of bumetanide. The bumetanide-insensitive efflux was largely sensitive to the K+ channel inhibitor 4-aminopyridine. Inhibitors of the Na+/H+exchanger or the Na+-K+pump had no effect on the increased86Rb+efflux. The activation of the Na-K-2Cl cotransporter was dependent on the extracellular signal-regulated kinase (ERK) subgroup of the mitogen-activated protein (MAP) kinase family. Phenylephrine stimulation increased ERK activity 3.4-fold. PD-98059, an inhibitor of the ERK cascade, reduced both the increased86Rb+efflux and ERK activity. Specific inhibitors of protein kinase C and Ca2+/calmodulin-dependent kinase II had no effect. In conclusion, α1-adrenoceptor stimulation increases86Rb+efflux from the rat heart via K+channels and a Na-K-2Cl cotransporter. Activation of the Na-K-2Cl cotransporter is apparently dependent on the MAP kinase pathway.

nes. ␣ 1-AR-mediated activation of NKCC in rat cardiomyocytes involves ERK-dependent phosphorylation of the cotransporter. Am J Physiol Heart Circ Physiol 286: H1354-H1360, 2004. First published November 20, 2003 10.1152/ajpheart.00549.2003.-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 86 Rb ϩ influx in isolated perfused rat hearts and isolated cardiomyocytes. Stimulation of ␣ 1-adrenoceptors (AR) by phenylephrine (30 M) increased 86 Rb ϩ influx. The NKCC inhibitor bumetanide (50 M) reduced the response to phenylephrine by 45 Ϯ 13% (n ϭ 12, P Ͻ 0.01). PD-98059 (10 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 Ϯ 13% (n ϭ 10, P Ͻ 0.01) and eliminated the bumetanide-sensitive component, indicating that ␣ 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 ␣ 1-AR stimulation was shown by immunoprecipitation of the phosphoprotein from 32 Pi 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 ␣ 1-AR stimulation through phosphorylation of this protein involving the ERK pathway. 86 Rb ϩ influx; phenylephrine; calyculin A; bumetanide; mitogenactivated protein kinase; ␣ 1-adrenoceptors

The anti-p-azobenzenearsonic acid antibody will be referred to subsequently in this paper as arsonic antibody. Similarly, the anti-p-azophenyltrimethylammonium antibody is abbreviated to ammonium antibody. ' Zaki, A., and H. Fahim, J. Chem. Soc., 270 (1942). The radioactive compound was prepared by New England Nuclear Corp., Boston, Mass., on the basis of the methods developed in this laboratory.

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