Shrinkage insensitivity of NKCC1 in myosin II-depleted cytoplasts from Ehrlich ascites tumor cells.

Else K Hoffmann; Stine F Pedersen

doi:10.1152/ajpcell.00474.2006

Shrinkage insensitivity of NKCC1 in myosin II-depleted cytoplasts from Ehrlich ascites tumor cells.

Else K Hoffmann, Stine F Pedersen

Cell Biology and Physiology

13 Citations (Scopus)

Abstract

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.

Original language	English
Journal	American Journal of Physiology: Cell Physiology
Volume	292
Issue number	5
Pages (from-to)	C1854-66
ISSN	0363-6143
DOIs	https://doi.org/10.1152/ajpcell.00474.2006
Publication status	Published - 2007

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@article{3c977cf0990511dd86a6000ea68e967b,

title = "Shrinkage insensitivity of NKCC1 in myosin II-depleted cytoplasts from Ehrlich ascites tumor cells.",

abstract = "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.",

author = "Hoffmann, {Else K} and Pedersen, {Stine F}",

note = "Keywords: 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; Mice; Myosin Type II; Myosin-Light-Chain Kinase; Oxazoles; Phosphoprotein Phosphatases; Phosphorylation; Protein Kinase Inhibitors; Protein-Serine-Threonine Kinases; Rubidium Radioisotopes; Sodium Potassium Chloride Symporter Inhibitors; Sodium-Potassium-Chloride Symporters; Staurosporine; Sulfonamides; p38 Mitogen-Activated Protein Kinases",

year = "2007",

doi = "10.1152/ajpcell.00474.2006",

language = "English",

volume = "292",

pages = "C1854--66",

journal = "American Journal of Physiology: Cell Physiology",

issn = "0363-6143",

publisher = "American Physiological Society",

number = "5",

}

TY - JOUR

T1 - Shrinkage insensitivity of NKCC1 in myosin II-depleted cytoplasts from Ehrlich ascites tumor cells.

AU - Hoffmann, Else K

AU - Pedersen, Stine F

N1 - Keywords: 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; Mice; Myosin Type II; Myosin-Light-Chain Kinase; Oxazoles; Phosphoprotein Phosphatases; Phosphorylation; Protein Kinase Inhibitors; Protein-Serine-Threonine Kinases; Rubidium Radioisotopes; Sodium Potassium Chloride Symporter Inhibitors; Sodium-Potassium-Chloride Symporters; Staurosporine; Sulfonamides; p38 Mitogen-Activated Protein Kinases

PY - 2007

Y1 - 2007

N2 - 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.

AB - 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.

U2 - 10.1152/ajpcell.00474.2006

DO - 10.1152/ajpcell.00474.2006

M3 - Journal article

C2 - 17229812

SN - 0363-6143

VL - 292

SP - C1854-66

JO - American Journal of Physiology: Cell Physiology

JF - American Journal of Physiology: Cell Physiology

IS - 5

ER -

Shrinkage insensitivity of NKCC1 in myosin II-depleted cytoplasts from Ehrlich ascites tumor cells.

Abstract

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