Na+/K+/Cl- cotransporter activates MAP-kinase cascade downstream to protein kinase C, and upstream to MEK.
In this study, we demonstrated that the specific inhibitors of the Na+/K+/Cl- cotransporter (NKCC1), bumetanide and furosemide, inhibited extracellular regulated kinase (ERK) phosphorylation in Balb/c 3T3 fibroblasts, stimulated with a variety of mitogens. In addition to fibroblast growth factor (FGF) shown before, the various mitogens tested in the present study (endothelial growth factor (EGF), platelet-derived growth factor (PDGF), insulin, thrombin, and the phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA)). Enter, the Ras/Raf/MEK/ERK cascade via different growth factors receptors and through one of the two main routes. The results of the present study provide evidence that have led us to conclude that the target protein which is controlled by the Na+/K+/Cl- cotransporter, is downstream of tyrosine kinase receptors, as well as of the G-protein-coupled receptor (GPCR). Several additional lines of evidence supported the above conclusion: (i) furosemide inhibits phosphorylation of MAPK kinase (MEK) induced by receptor tyrosine kinase (RTK) ligands, such as PDGF, FGF, and EGF. (ii) Furosemide also inhibited ERK phosphorylation, induced by thrombin, a GPCR. (iii) Furosemide inhibited MEK and ERK phosphorylation even when ERK phosphorylation was induced by direct activation of protein kinase C (PKC) by TPA, which bypasses early steps of the mitogenic cascade. In addition, we found that furosemide did not affect PKC phosphorylation induced directly by TPA. Taken together, the results of the present study indicate that the signal transduction protein, controlled by the Na+/K+/Cl- cotransporter, must be downstream of the PKC, and at/or upstream to MEK in the Ras/Raf/MEK/ERK cascade.
Panet
Rivka
R
Department of Medical Biophysics and Nuclear Medicine, Hadassah Hebrew University Hospital, Jerusalem, Israel. panetr@yahoo.com
Eliash
Michal
M
Atlan
Henri
H
D000818:Animals; D041702:BALB 3T3 Cells; D002034:Bumetanide / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism; D005665:Furosemide / Q000494:pharmacology; D048369:MAP Kinase Kinase 1 / Q000378:metabolism; D048370:MAP Kinase Kinase 2 / Q000378:metabolism; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism*; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008934:Mitogens / Q000494:pharmacology; D008954:Models, Biological; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism*; D015398:Signal Transduction; D049994:Sodium Potassium Chloride Symporter Inhibitors / Q000494:pharmacology; D028021:Sodium-Potassium-Chloride Symporters / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D008934:Mitogens; D049994:Sodium Potassium Chloride Symporter Inhibitors; D028021:Sodium-Potassium-Chloride Symporters; D002034:Bumetanide; D004815:Epidermal Growth Factor; D005665:Furosemide; D066246:ErbB Receptors; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases; D020930:MAP Kinase Kinase Kinases; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; D013755:Tetradecanoylphorbol Acetate