h-89 and Carcinoma--Ehrlich-Tumor

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.

Topics: Actins; Animals; Bumetanide; Carcinoma, Ehrlich Tumor; Cell Membrane; Cell Size; Cell-Free System; Cyclic AMP-Dependent Protein Kinase Type II; Cyclic AMP-Dependent Protein Kinases; Female; Hypertonic Solutions; Isoquinolines; Marine Toxins; Mice; Myosin Type II; Myosin-Light-Chain Kinase; Oxazoles; p38 Mitogen-Activated Protein Kinases; Phosphoprotein Phosphatases; Phosphorylation; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Rubidium Radioisotopes; Sodium Potassium Chloride Symporter Inhibitors; Sodium-Potassium-Chloride Symporters; Solute Carrier Family 12, Member 2; Staurosporine; Sulfonamides

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.

Topics: Animals; Azepines; Bradykinin; Carbazoles; Carcinoma, Ehrlich Tumor; Carrier Proteins; Chlorides; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Homeostasis; Hypertonic Solutions; Indoles; Isoquinolines; Kinetics; Marine Toxins; Mice; Myosin-Light-Chain Kinase; Naphthalenes; Osmolar Concentration; Oxazoles; Phosphoprotein Phosphatases; Potassium; Protein Kinases; Pyrroles; Sodium-Potassium-Chloride Symporters; Sulfonamides; Tumor Cells, Cultured