Alpha1-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 (86)Rb(+) influx in isolated perfused rat hearts and isolated cardiomyocytes. Stimulation of alpha(1)-adrenoceptors (AR) by phenylephrine (30 microM) increased (86)Rb(+) influx. The NKCC inhibitor bumetanide (50 microM) reduced the response to phenylephrine by 45 +/- 13% (n = 12, P < 0.01). PD-98059 (10 microM), 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 (32)P(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.
Andersen
Geir Øystein
GØ
Department of Pharmacology, University of Oslo, Oslo, Norway. g.o.andersen@labmed.uio.no
Skomedal
Tor
T
Enger
Mette
M
Fidjeland
Astrid
A
Brattelid
Trond
T
Levy
Finn Olav
FO
Osnes
Jan-Bjørn
JB
D000818:Animals; D001711:Biotransformation; D002034:Bumetanide / Q000494:pharmacology; D002469:Cell Separation; D004232:Diuretics / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015151:Immunoblotting; D066298:In Vitro Techniques; D008297:Male; D008387:Marine Toxins; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology*; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000201:enzymology*; D010080:Oxazoles / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017208:Rats, Wistar; D018340:Receptors, Adrenergic, alpha-1 / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012414:Rubidium Radioisotopes; D012964:Sodium / Q000502:physiology; D049994:Sodium Potassium Chloride Symporter Inhibitors; D028021:Sodium-Potassium-Chloride Symporters / Q000096:biosynthesis / Q000378:metabolism*; D064466:Solute Carrier Family 12, Member 1
D002034:Bumetanide; D004232:Diuretics; D004791:Enzyme Inhibitors; D008387:Marine Toxins; D020928:Mitogen-Activated Protein Kinases; D010080:Oxazoles; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D012333:RNA, Messenger; D018340:Receptors, Adrenergic, alpha-1; D012414:Rubidium Radioisotopes; D012964:Sodium; D049994:Sodium Potassium Chloride Symporter Inhibitors; D028021:Sodium-Potassium-Chloride Symporters; D064466:Solute Carrier Family 12, Member 1; C578716:Slc12a1 protein, rat; C059041:calyculin A