Shrinkage insensitivity of NKCC1 in myosin II-depleted cytoplasts from Ehrlich ascites tumor cells.
Protein phosphorylation/dephosphorylation and cytoskeletal reorganization regulate the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) during osmotic shrinkage; however, the mechanisms involved are unclear. We show that in cytoplasts, plasma membrane vesicles detached from Ehrlich ascites tumor cells (EATC) by cytochalasin treatment, NKCC1 activity evaluated as bumetanide-sensitive (86)Rb influx was increased compared with the basal level in intact cells yet could not be further increased by osmotic shrinkage. Accordingly, cytoplasts exhibited no regulatory volume increase after shrinkage. In cytoplasts, cortical F-actin organization was disrupted, and myosin II, which in shrunken EATC translocates to the cortical region, was absent. Moreover, NKCC1 activity was essentially insensitive to the myosin light chain kinase (MLCK) inhibitor ML-7, a potent blocker of shrinkage-induced NKCC1 activity in intact EATC. Cytoplast NKCC1 activity was potentiated by the Ser/Thr protein phosphatase inhibitor calyculin A, partially inhibited by the protein kinase A inhibitor H89, and blocked by the broad protein kinase inhibitor staurosporine. Cytoplasts exhibited increased protein levels of NKCC1, Ste20-related proline- and alanine-rich kinase (SPAK), and oxidative stress response kinase 1, yet they lacked the shrinkage-induced plasma membrane translocation of SPAK observed in intact cells. The basal phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) was increased in cytoplasts compared with intact cells, yet in contrast to the substantial activation in shrunken intact cells, p38 MAPK could not be further activated by shrinkage of the cytoplasts. Together these findings indicate that shrinkage activation of NKCC1 in EATC is dependent on the cortical F-actin network, myosin II, and MLCK.
Hoffmann
Else K
EK
Department of Molecular Biology, University of Copenhagen, 13 Universitetsparken, Dk-2100 Copenhagen, Denmark. ekhoffmann@aki.ku.dk
Pedersen
Stine F
SF
D000199:Actins / Q000378:metabolism*; D000818:Animals; D002034:Bumetanide / Q000494:pharmacology; D002286:Carcinoma, Ehrlich Tumor / Q000378:metabolism* / Q000473:pathology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D048429:Cell Size; D002474:Cell-Free System; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D005260:Female; D006982:Hypertonic Solutions; D007546:Isoquinolines / Q000494:pharmacology; D008387:Marine Toxins; D051379:Mice; D024462:Myosin Type II / Q000172:deficiency / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*; D010080:Oxazoles / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012414:Rubidium Radioisotopes; D049994:Sodium Potassium Chloride Symporter Inhibitors / Q000494:pharmacology; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism*; D064506:Solute Carrier Family 12, Member 2; D019311:Staurosporine / Q000494:pharmacology; D013449:Sulfonamides / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D000199:Actins; D006982:Hypertonic Solutions; D007546:Isoquinolines; D008387:Marine Toxins; D010080:Oxazoles; D047428:Protein Kinase Inhibitors; D012414:Rubidium Radioisotopes; C578878:Slc12a2 protein, mouse; D049994:Sodium Potassium Chloride Symporter Inhibitors; D028021:Sodium-Potassium-Chloride Symporters; D064506:Solute Carrier Family 12, Member 2; D013449:Sulfonamides; D002034:Bumetanide; C059041:calyculin A; D017346:Protein Serine-Threonine Kinases; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases; D009219:Myosin-Light-Chain Kinase; D048051:p38 Mitogen-Activated Protein Kinases; D010749:Phosphoprotein Phosphatases; D024462:Myosin Type II; D019311:Staurosporine; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide