Na+-K+-2Cl- cotransport in Ehrlich cells: regulation by protein phosphatases and kinases.
To identify protein kinases (PK) and phosphatases (PP) involved in regulation of the Na+-K+-2Cl- cotransporter in Ehrlich cells, the effect of various PK and PP inhibitors was examined. The PP-1, PP-2A, and PP-3 inhibitor calyculin A (Cal-A) was a potent activator of Na+-K+-2Cl- cotransport (EC50 = 35 nM). Activation by Cal-A was rapid (<1 min) but transient. Inactivation is probably due to a 10% cell swelling and/or the concurrent increase in intracellular Cl- concentration. Cell shrinkage also activates the Na+-K+-2Cl- cotransport system. Combining cell shrinkage with Cal-A treatment prolonged the cotransport activation compared with stimulation with Cal-A alone, suggesting PK stimulation by cell shrinkage. Shrinkage-induced cotransport activation was pH and Ca2+/calmodulin dependent. Inhibition of myosin light chain kinase by ML-7 and ML-9 or of PKA by H-89 and KT-5720 inhibited cotransport activity induced by Cal-A and by cell shrinkage, with IC50 values similar to reported inhibition constants of the respective kinases in vitro. Cell shrinkage increased the ML-7-sensitive cotransport activity, whereas the H-89-sensitive activity was unchanged, suggesting that myosin light chain kinase is a modulator of the Na+-K+-2Cl- cotransport activity during regulatory volume increase.
Department of Biochemistry, The August Krogh Institute, University of Copenhagen, DK-2100 Copenhagen, Denmark.
T
T
Krarup
L D
LD
Jakobsen
B S
BS
Jensen
E K
EK
Hoffmann
D000818:Animals; D001381:Azepines / Q000494:pharmacology; D001920:Bradykinin / Q000494:pharmacology; D002227:Carbazoles; D002286:Carcinoma, Ehrlich Tumor / Q000378:metabolism* / Q000503:physiopathology; D002352:Carrier Proteins / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006706:Homeostasis; D006982:Hypertonic Solutions; D007211:Indoles / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology; D007700:Kinetics; D008387:Marine Toxins; D051379:Mice; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors; D009281:Naphthalenes / Q000494:pharmacology; D009994:Osmolar Concentration; D010080:Oxazoles / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D011188:Potassium / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D011758:Pyrroles / Q000494:pharmacology; D028021:Sodium-Potassium-Chloride Symporters; D013449:Sulfonamides; D014407:Tumor Cells, Cultured
D001381:Azepines; D002227:Carbazoles; D002352:Carrier Proteins; D002712:Chlorides; D004791:Enzyme Inhibitors; D006982:Hypertonic Solutions; D007211:Indoles; D007546:Isoquinolines; D008387:Marine Toxins; D009281:Naphthalenes; D010080:Oxazoles; D011758:Pyrroles; D028021:Sodium-Potassium-Chloride Symporters; D013449:Sulfonamides; C056218:ML 9; C070571:ML 7; C057416:KT 5720; C059041:calyculin A; D011494:Protein Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D009219:Myosin-Light-Chain Kinase; D010749:Phosphoprotein Phosphatases; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; D011188:Potassium; D001920:Bradykinin